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Sample records for isogenic yeast dna

  1. Direct cloning of isogenic murine DNA in yeast and relevance of isogenicity for targeting in embryonic stem cells.

    PubMed

    Andréasson, Claes; Schick, Anna J; Pfeiffer, Susanne M; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A

    2013-01-01

    Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. PMID:24058528

  2. Direct Cloning of Isogenic Murine DNA in Yeast and Relevance of Isogenicity for Targeting in Embryonic Stem Cells

    PubMed Central

    Pfeiffer, Susanne M.; Sarov, Mihail; Stewart, Francis; Wurst, Wolfgang; Schick, Joel A.

    2013-01-01

    Efficient gene targeting in embryonic stem cells requires that modifying DNA sequences are identical to those in the targeted chromosomal locus. Yet, there is a paucity of isogenic genomic clones for human cell lines and PCR amplification cannot be used in many mutation-sensitive applications. Here, we describe a novel method for the direct cloning of genomic DNA into a targeting vector, pRTVIR, using oligonucleotide-directed homologous recombination in yeast. We demonstrate the applicability of the method by constructing functional targeting vectors for mammalian genes Uhrf1 and Gfap. Whereas the isogenic targeting of the gene Uhrf1 showed a substantial increase in targeting efficiency compared to non-isogenic DNA in mouse E14 cells, E14-derived DNA performed better than the isogenic DNA in JM8 cells for both Uhrf1 and Gfap. Analysis of 70 C57BL/6-derived targeting vectors electroporated in JM8 and E14 cell lines in parallel showed a clear dependence on isogenicity for targeting, but for three genes isogenic DNA was found to be inhibitory. In summary, this study provides a straightforward methodological approach for the direct generation of isogenic gene targeting vectors. PMID:24058528

  3. Slow-growing cells within isogenic populations have increased RNA polymerase error rates and DNA damage.

    PubMed

    van Dijk, David; Dhar, Riddhiman; Missarova, Alsu M; Espinar, Lorena; Blevins, William R; Lehner, Ben; Carey, Lucas B

    2015-01-01

    Isogenic cells show a large degree of variability in growth rate, even when cultured in the same environment. Such cell-to-cell variability in growth can alter sensitivity to antibiotics, chemotherapy and environmental stress. To characterize transcriptional differences associated with this variability, we have developed a method--FitFlow--that enables the sorting of subpopulations by growth rate. The slow-growing subpopulation shows a transcriptional stress response, but, more surprisingly, these cells have reduced RNA polymerase fidelity and exhibit a DNA damage response. As DNA damage is often caused by oxidative stress, we test the addition of an antioxidant, and find that it reduces the size of the slow-growing population. More generally, we find a significantly altered transcriptome in the slow-growing subpopulation that only partially resembles that of cells growing slowly due to environmental and culture conditions. Slow-growing cells upregulate transposons and express more chromosomal, viral and plasmid-borne transcripts, and thus explore a larger genotypic--and so phenotypic--space. PMID:26268986

  4. Slow-growing cells within isogenic populations have increased RNA polymerase error rates and DNA damage

    PubMed Central

    van Dijk, David; Dhar, Riddhiman; Missarova, Alsu M.; Espinar, Lorena; Blevins, William R.; Lehner, Ben; Carey, Lucas B.

    2015-01-01

    Isogenic cells show a large degree of variability in growth rate, even when cultured in the same environment. Such cell-to-cell variability in growth can alter sensitivity to antibiotics, chemotherapy and environmental stress. To characterize transcriptional differences associated with this variability, we have developed a method—FitFlow—that enables the sorting of subpopulations by growth rate. The slow-growing subpopulation shows a transcriptional stress response, but, more surprisingly, these cells have reduced RNA polymerase fidelity and exhibit a DNA damage response. As DNA damage is often caused by oxidative stress, we test the addition of an antioxidant, and find that it reduces the size of the slow-growing population. More generally, we find a significantly altered transcriptome in the slow-growing subpopulation that only partially resembles that of cells growing slowly due to environmental and culture conditions. Slow-growing cells upregulate transposons and express more chromosomal, viral and plasmid-borne transcripts, and thus explore a larger genotypic—and so phenotypic — space. PMID:26268986

  5. Characterization of environmental chemicals with potential for DNA damage using isogenic DNA repair-deficient chicken DT40 cell lines

    PubMed Central

    Yamamoto, Kimiyo N.; Hirota, Kouji; Kono, Koichi; Takeda, Shunichi; Sakamuru, Srilatha; Xia, Menghang; Huang, Ruili; Austin, Christopher P.; Witt, Kristine L.; Tice, Raymond R.

    2012-01-01

    Included among the quantitative high throughput screens (qHTS) conducted in support of the U.S. Tox21 program are those being evaluated for the detection of genotoxic compounds. One such screen is based on the induction of increased cytotoxicity in 7 isogenic chicken DT40 cell lines deficient in DNA repair pathways compared to the parental DNA repair-proficient cell line. To characterize the utility of this approach for detecting genotoxic compounds and identifying the type(s) of DNA damage induced, we evaluated nine of 42 compounds identified as positive for differential cytotoxicity in qHTS (actinomycin D, adriamycin, alachlor, benzotrichloride, diglycidyl resorcinol ether, lovastatin, melphalan, trans-1,4-dichloro-2-butene, tris(2,3-epoxypropyl)isocyanurate) and one non-cytotoxic genotoxic compound (2-aminothiamine) for (1) clastogenicity in mutant and wild-type cells; (2) the comparative induction of γH2AX positive foci by melphalan; (3) the extent to which a 72-hr exposure duration increased assay sensitivity or specificity; (4) the use of 10 additional DT40 DNA repair-deficient cell lines to better analyze the type(s) of DNA damage induced; and (5) the involvement of reactive oxygen species in the induction of DNA damage. All compounds but lovastatin and 2-aminothiamine were more clastogenic in at least one DNA repair-deficient cell line than the wild-type cells. The differential responses across the various DNA repair-deficient cell lines provided information on the type(s) of DNA damage induced. The results demonstrate the utility of this DT40 screen for detecting genotoxic compounds, for characterizing the nature of the DNA damage, and potentially for analyzing mechanisms of mutagenesis. PMID:21538559

  6. Assessment of Chromosomal DNA Fragmentation by Quinolones in an Isogenic Collection of Escherichia coli with Defined Resistance Mechanisms.

    PubMed

    Rodríguez-Martínez, José-Manuel; Santiso, Rebeca; Machuca, Jesús; Bou, Germán; Pascual, Álvaro; Fernández, José Luis

    2016-07-01

    The aim of this study was to investigate the potential usefulness of DNA fragmentation as a quick and simple procedure for detecting resistance to fluoroquinolones (FQ) in isogenic Escherichia coli strains harboring defined and multiple quinolone resistance mechanisms, including low-level quinolone resistance (LLQR) phenotypes. DNA fragmentation assay (Micromax(®)) was evaluated for detecting resistance to FQ in 71 isogenic strains of E. coli harboring specific quinolone resistance mechanisms frequently found in clinical isolates. These isogenic strains represent a consistent and reliable model of increasing minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP), ranging from 0.004 to 16 mg/L. According to CLSI criteria, the assay correctly identified all CIP-resistant strains (MIC ≥4 mg/L). As regards susceptible strains, 96% of bacterial strains were correctly assigned as susceptible to CIP. Moreover, the procedure enabled LLQR phenotypes to be efficiently identified; this subset may show different levels of DNA damage depending on the strain, even with similar MIC. Interestingly, despite increasing the dose according to the MIC, a lower response to quinolones occurs in strains with higher MIC values. This is a simple, rapid, and reliable test for evaluating susceptibility to FQ of E. coli, including the detection of strains harboring LLQR mechanisms. PMID:26890225

  7. Size and Structure of Yeast Chromosomal DNA

    PubMed Central

    Petes, Thomas D.; Byers, Breck; Fangman, Walton L.

    1973-01-01

    Electron microscopic analysis indicates that yeast nuclear DNA can be isolated as linear molecules ranging in size from 50 μm (1.2 × 108 daltons) to 355 μm (8.4 × 108 daltons). Analysis indicates the data is consistent with the hypothesis that each yeast chromosome contains a single, linear DNA duplex. Mitochondrial DNA molecules have a contour length of 21 ± 2 μm and are mostly linear. Images PMID:4594033

  8. Visualization of yeast chromosomal DNA

    NASA Technical Reports Server (NTRS)

    Lubega, Seth

    1990-01-01

    The DNA molecule is the most significant life molecule since it codes the blue print for other structural and functional molecules of all living organisms. Agarose gel electrophoresis is now being widely used to separate DNA of virus, bacteria, and lower eukaryotes. The task was undertaken of reviewing the existing methods of DNA fractionation and microscopic visualization of individual chromosonal DNA molecules by gel electrophoresis as a basis for a proposed study to investigate the feasibility of separating DNA molecules in free fluids as an alternative to gel electrophoresis. Various techniques were studied. On the molecular level, agarose gel electrophoresis is being widely used to separate chromosomal DNA according to molecular weight. Carl and Olson separate and characterized the entire karyotype of a lab strain of Saccharomyces cerevisiae. Smith et al. and Schwartz and Koval independently reported the visualization of individual DNA molecules migrating through agarose gel matrix during electrophoresis. The techniques used by these researchers are being reviewed in the lab as a basis for the proposed studies.

  9. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome

    PubMed Central

    DeKelver, Russell C.; Choi, Vivian M.; Moehle, Erica A.; Paschon, David E.; Hockemeyer, Dirk; Meijsing, Sebastiaan H.; Sancak, Yasemin; Cui, Xiaoxia; Steine, Eveline J.; Miller, Jeffrey C.; Tam, Phillip; Bartsevich, Victor V.; Meng, Xiangdong; Rupniewski, Igor; Gopalan, Sunita M.; Sun, Helena C.; Pitz, Kathleen J.; Rock, Jeremy M.; Zhang, Lei; Davis, Gregory D.; Rebar, Edward J.; Cheeseman, Iain M.; Yamamoto, Keith R.; Sabatini, David M.; Jaenisch, Rudolf; Gregory, Philip D.; Urnov, Fyodor D.

    2010-01-01

    Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a “safe harbor” locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells. PMID:20508142

  10. Extrachromosomal circular DNA is common in yeast

    PubMed Central

    Møller, Henrik D.; Parsons, Lance; Jørgensen, Tue S.; Botstein, David; Regenberg, Birgitte

    2015-01-01

    Examples of extrachromosomal circular DNAs (eccDNAs) are found in many organisms, but their impact on genetic variation at the genome scale has not been investigated. We mapped 1,756 eccDNAs in the Saccharomyces cerevisiae genome using Circle-Seq, a highly sensitive eccDNA purification method. Yeast eccDNAs ranged from an arbitrary lower limit of 1 kb up to 38 kb and covered 23% of the genome, representing thousands of genes. EccDNA arose both from genomic regions with repetitive sequences ≥15 bases long and from regions with short or no repetitive sequences. Some eccDNAs were identified in several yeast populations. These eccDNAs contained ribosomal genes, transposon remnants, and tandemly repeated genes (HXT6/7, ENA1/2/5, and CUP1-1/-2) that were generally enriched on eccDNAs. EccDNAs seemed to be replicated and 80% contained consensus sequences for autonomous replication origins that could explain their maintenance. Our data suggest that eccDNAs are common in S. cerevisiae, where they might contribute substantially to genetic variation and evolution. PMID:26038577

  11. Mechanism for priming DNA synthesis by yeast DNA Polymerase α

    PubMed Central

    Perera, Rajika L; Torella, Rubben; Klinge, Sebastian; Kilkenny, Mairi L; Maman, Joseph D; Pellegrini, Luca

    2013-01-01

    The DNA Polymerase α (Pol α)/primase complex initiates DNA synthesis in eukaryotic replication. In the complex, Pol α and primase cooperate in the production of RNA-DNA oligonucleotides that prime synthesis of new DNA. Here we report crystal structures of the catalytic core of yeast Pol α in unliganded form, bound to an RNA primer/DNA template and extending an RNA primer with deoxynucleotides. We combine the structural analysis with biochemical and computational data to demonstrate that Pol α specifically recognizes the A-form RNA/DNA helix and that the ensuing synthesis of B-form DNA terminates primer synthesis. The spontaneous release of the completed RNA-DNA primer by the Pol α/primase complex simplifies current models of primer transfer to leading- and lagging strand polymerases. The proposed mechanism of nucleotide polymerization by Pol α might contribute to genomic stability by limiting the amount of inaccurate DNA to be corrected at the start of each Okazaki fragment. DOI: http://dx.doi.org/10.7554/eLife.00482.001 PMID:23599895

  12. Recent advances in yeast molecular biology: recombinant DNA. [Lead abstract

    SciTech Connect

    Not Available

    1982-09-01

    Separate abstracts were prepared for the 25 papers presented at a workshop focusing on chromosomal structure, gene regulation, recombination, DNA repair, and cell type control, that have been obtained by experimental approaches incorporating the new technologies of yeast DNA transformation, molecular cloning, and DNA sequence analysis. (KRM)

  13. Isolation of Circular DNA from a Mitochondrial Fraction from Yeast

    PubMed Central

    Clark-Walker, G. D.

    1972-01-01

    Breakage and fractionation of respiratory competent yeast in the presence of ethidium bromide, and subsequent centrifugation of a detergent lysate of the mitochondrial fraction by the dye-buoyant-density technique, results in the isolation of closed-circular DNA. After removal of bound dye, this DNA has two components when analyzed by equilibrium buoyant density in the analytical ultracentrifuge. A minor component has a buoyant density of 1.684 g/cm3, which is characteristic of mitochondrial DNA, but the major component has a buoyant density of 1.701 g/cm3. This species of DNA is also present in yeast that have been mutagenized to respiratory deficiency in the presence of the highest concentration of ethidium bromide compatible with cell growth. The closed-circular DNA of buoyant density 1.701 g/cm3, and free of linear DNA, is associated with the sole particulate band obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-deficient cells. Two particulate bands are obtained on sucrose gradient centrifugation of a mitochondrial preparation from respiratory-competent cells, the upper band containing DNA of buoyant density 1.701 g/cm3 and the lower band DNA of buoyant density 1.684 g/cm3. The suggestion is advanced, in view of the reputed sedimentation behaviour of yeast peroxisomes, that the closed-circular DNA of buoyant density 1.701 g/cm3 may be located in peroxisomes. Images PMID:4551142

  14. Modeling the Control of DNA Replication in Fission Yeast

    NASA Astrophysics Data System (ADS)

    Novak, Bela; Tyson, John J.

    1997-08-01

    A central event in the eukaryotic cell cycle is the decision to commence DNA replication (S phase). Strict controls normally operate to prevent repeated rounds of DNA replication without intervening mitoses (``endoreplication'') or initiation of mitosis before DNA is fully replicated (``mitotic catastrophe''). Some of the genetic interactions involved in these controls have recently been identified in yeast. From this evidence we propose a molecular mechanism of ``Start'' control in Schizosaccharomyces pombe. Using established principles of biochemical kinetics, we compare the properties of this model in detail with the observed behavior of various mutant strains of fission yeast: wee1- (size control at Start), cdc13Δ and rum1OP (endoreplication), and wee1- rum1Δ (rapid division cycles of diminishing cell size). We discuss essential features of the mechanism that are responsible for characteristic properties of Start control in fission yeast, to expose our proposal to crucial experimental tests.

  15. DNA sequence of the yeast transketolase gene.

    PubMed

    Fletcher, T S; Kwee, I L; Nakada, T; Largman, C; Martin, B M

    1992-02-18

    Transketolase (EC 2.2.1.1) is the enzyme that, together with aldolase, forms a reversible link between the glycolytic and pentose phosphate pathways. We have cloned and sequenced the transketolase gene from yeast (Saccharomyces cerevisiae). This is the first transketolase gene of the pentose phosphate shunt to be sequenced from any source. The molecular mass of the proposed translated protein is 73,976 daltons, in good agreement with the observed molecular mass of about 75,000 daltons. The 5'-nontranslated region of the gene is similar to other yeast genes. There is no evidence of 5'-splice junctions or branch points in the sequence. The 3'-nontranslated region contains the polyadenylation signal (AATAAA), 80 base pairs downstream from the termination codon. A high degree of homology is found between yeast transketolase and dihydroxyacetone synthase (formaldehyde transketolase) from the yeast Hansenula polymorpha. The overall sequence identity between these two proteins is 37%, with four regions of much greater similarity. The regions from amino acid residues 98-131, 157-182, 410-433, and 474-489 have sequence identities of 74%, 66%, 83%, and 82%, respectively. One of these regions (157-182) includes a possible thiamin pyrophosphate (TPP) binding domain, and another (410-433) may contain the catalytic domain. PMID:1737042

  16. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  17. The Yeast Copper Response Is Regulated by DNA Damage

    PubMed Central

    Dong, Kangzhen; Addinall, Stephen G.; Lydall, David

    2013-01-01

    Copper is an essential but potentially toxic redox-active metal, so the levels and distribution of this metal are carefully regulated to ensure that it binds to the correct proteins. Previous studies of copper-dependent transcription in the yeast Saccharomyces cerevisiae have focused on the response of genes to changes in the exogenous levels of copper. We now report that yeast copper genes are regulated in response to the DNA-damaging agents methyl methanesulfonate (MMS) and hydroxyurea by a mechanism(s) that requires the copper-responsive transcription factors Mac1 and AceI, copper superoxide dismutase (Sod1) activity, and the Rad53 checkpoint kinase. Furthermore, in copper-starved yeast, the response of the Rad53 pathway to MMS is compromised due to a loss of Sod1 activity, consistent with the model that yeast imports copper to ensure Sod1 activity and Rad53 signaling. Crucially, the Mac1 transcription factor undergoes changes in its redox state in response to changing levels of copper or MMS. This study has therefore identified a novel regulatory relationship between cellular redox, copper homeostasis, and the DNA damage response in yeast. PMID:23959798

  18. Mitochondrial DNA repairs double-strand breaks in yeast chromosomes.

    PubMed

    Ricchetti, M; Fairhead, C; Dujon, B

    1999-11-01

    The endosymbiotic theory for the origin of eukaryotic cells proposes that genetic information can be transferred from mitochondria to the nucleus of a cell, and genes that are probably of mitochondrial origin have been found in nuclear chromosomes. Occasionally, short or rearranged sequences homologous to mitochondrial DNA are seen in the chromosomes of different organisms including yeast, plants and humans. Here we report a mechanism by which fragments of mitochondrial DNA, in single or tandem array, are transferred to yeast chromosomes under natural conditions during the repair of double-strand breaks in haploid mitotic cells. These repair insertions originate from noncontiguous regions of the mitochondrial genome. Our analysis of the Saccharomyces cerevisiae mitochondrial genome indicates that the yeast nuclear genome does indeed contain several short sequences of mitochondrial origin which are similar in size and composition to those that repair double-strand breaks. These sequences are located predominantly in non-coding regions of the chromosomes, frequently in the vicinity of retrotransposon long terminal repeats, and appear as recent integration events. Thus, colonization of the yeast genome by mitochondrial DNA is an ongoing process. PMID:10573425

  19. DNA Compaction by Yeast Mitochondrial Protein ABF2p

    SciTech Connect

    Friddle, R W; Klare, J E; Noy, A; Corzett, M; Balhorn, R; Baskin, R J; Martin, S S; Baldwin, E P

    2003-05-09

    We used high resolution Atomic Force Microscopy (AFM) to image compaction of linear and circular DNA by the yeast mitochondrial protein ABF2p , which plays a major role in maintaining mitochondrial DNA. AFM images show that protein binding induces drastic bends in the DNA backbone for both linear and circular DNA. At high concentration of ABF2p DNA collapses into a tight globular structure. We quantified the compaction of linear DNA by measuring the end-to-end distance of the DNA molecule at increasing concentrations of ABF2p. We also derived a polymer statistical mechanics model that gives quantitative description of compaction observed in our experiments. This model shows that a number of sharp bends in the DNA backbone is often sufficient to cause DNA compaction. Comparison of our model with the experimental data showed excellent quantitative correlation and allowed us to determine binding characteristics for ABF2. Our studies indicate that ABF2 compacts DNA through a novel mechanism that involves bending of DNA backbone. We discuss the implications of such a mechanism for mitochondrial DNA maintenance.

  20. Mitochondrial DNA size diversity in the Dekkera/Brettanomyces yeasts.

    PubMed

    McArthur, C R; Clark-Walker, G D

    1983-03-01

    Restriction endonuclease digestion of mitocondrial DNAs from the nine Dekkera/Brettanomyces yeasts have revealed that three separate pairs of species, namely D. bruxellensis/B. lambicus; B. abstinens/B. custersii and B. anomalus/B. clausenii have identical genomes. This observation suggests that such analysis of mtDNA could be an important procedure for yeast taxonomy. Sizes of mtDNAs showed a graded range from the 28 kbp molecule in B. custersianus to the 100 kbp molecule in B. custersii. Furthermore, although the mtDNAs from D. intermedia (72 kbp) and D. bruxellensis (82 kbp) differ in size by 10 kbp the restriction enzyme fragmentation patterns are generally similar. The differences are reminiscent of mtDNA polymorphisms found in strains of Saccharomyces cervisiae which result from insertions or deletions, chiefly within genic sequences. By analogy, the two Dekkera species may, on further analysis, be revealed as variants of a single species. PMID:24173115

  1. Structural Basis of High-Fidelity DNA Synthesis by Yeast DNA Polymerase δ

    SciTech Connect

    Swan, M.; Johnson, R; Prakash, L; Prakash, S; Aggarwal, A

    2009-01-01

    DNA polymerase ? (Pol ?) has a crucial role in eukaryotic replication. Now the crystal structure of the yeast DNA Pol ? catalytic subunit in complex with template primer and incoming nucleotide is presented at 2.0-A resolution, providing insight into its high fidelity and a framework to understand the effects of mutations involved in tumorigenesis.

  2. Single-stranded-DNA-binding protein-dependent DNA unwinding of the yeast ARS1 region.

    PubMed Central

    Matsumoto, K; Ishimi, Y

    1994-01-01

    DNA unwinding of autonomously replicating sequence 1 (ARS1) from the yeast Saccharomyces cerevisiae was investigated. When a negatively supercoiled plasmid DNA containing ARS1 was digested with single-strand-specific mung bean nuclease, a discrete region in the vector DNA was preferentially digested. The regions containing the core consensus A domain and the 3'-flanking B domain of ARS1 were weakly digested. When the DNA was incubated with the multisubunit single-stranded DNA-binding protein (SSB, also called RPA [replication protein A]) from human and yeast cells prior to mung bean nuclease digestion, the cleavage in the A and B domains was greatly increased. Furthermore, a region corresponding to the 5'-flanking C domain of ARS1 was digested. These results indicate that three domains of ARS1, each of which is important for replication in yeast cells, closely correspond to the regions where the DNA duplex is easily unwound by torsional stress. SSB may stimulate the unwinding of the ARS1 region by its preferential binding to the destabilized three domains. Mung bean nuclease digestion of the substitution mutants with mutations of ARS1 (Y. Marahrens and B. Stillman, Science 255:817-823, 1992) revealed that the sequences in the B2 and A elements are responsible for the unwinding of the B domain and the region containing the A domain, respectively. Images PMID:8007967

  3. Quantitative PCR for detection of DNA damage in mitochondrial DNA of the fission yeast Schizosaccharomyces pombe.

    PubMed

    Senoo, Takanori; Yamanaka, Mayumi; Nakamura, Atori; Terashita, Tomoki; Kawano, Shinji; Ikeda, Shogo

    2016-08-01

    Quantitative polymerase chain reaction (QPCR) has been employed to detect DNA damage and repair in mitochondrial DNA (mtDNA) of human and several model organisms. The assay also permits the quantitation of relative mtDNA copy number in cells. Here, we developed the QPCR assay primers and reaction conditions for the fission yeast Schizosaccharomyces pombe, an important model of eukaryote biology, not previously described. Under these conditions, long targets (approximately 10kb) in mtDNA were quantitatively amplified using 0.1ng of crude DNA templates without isolation of mitochondria and mtDNA. Quantitative detection of oxidative DNA damage in mtDNA was illustrated by using a DNA template irradiated with UVA in the presence of riboflavin. The damage to mtDNA in S. pombe cells treated with hydrogen peroxide and paraquat was also quantitatively measured. Finally, we found that mtDNA copy number in S. pombe cells increased after transition into a stationary phase and that the damage to mtDNA due to endogenous cellular processes accumulated during chronological aging. PMID:27236021

  4. Inaccurate DNA Synthesis in Cell Extracts of Yeast Producing Active Human DNA Polymerase Iota

    PubMed Central

    Makarova, Alena V.; Grabow, Corinn; Gening, Leonid V.; Tarantul, Vyacheslav Z.; Tahirov, Tahir H.; Bessho, Tadayoshi; Pavlov, Youri I.

    2011-01-01

    Mammalian Pol ι has an unusual combination of properties: it is stimulated by Mn2+ ions, can bypass some DNA lesions and misincorporates “G” opposite template “T” more frequently than incorporates the correct “A.” We recently proposed a method of detection of Pol ι activity in animal cell extracts, based on primer extension opposite the template T with a high concentration of only two nucleotides, dGTP and dATP (incorporation of “G” versus “A” method of Gening, abbreviated as “misGvA”). We provide unambiguous proof of the “misGvA” approach concept and extend the applicability of the method for the studies of variants of Pol ι in the yeast model system with different cation cofactors. We produced human Pol ι in baker's yeast, which do not have a POLI ortholog. The “misGvA” activity is absent in cell extracts containing an empty vector, or producing catalytically dead Pol ι, or Pol ι lacking exon 2, but is robust in the strain producing wild-type Pol ι or its catalytic core, or protein with the active center L62I mutant. The signature pattern of primer extension products resulting from inaccurate DNA synthesis by extracts of cells producing either Pol ι or human Pol η is different. The DNA sequence of the template is critical for the detection of the infidelity of DNA synthesis attributed to DNA Pol ι. The primer/template and composition of the exogenous DNA precursor pool can be adapted to monitor replication fidelity in cell extracts expressing various error-prone Pols or mutator variants of accurate Pols. Finally, we demonstrate that the mutation rates in yeast strains producing human DNA Pols ι and η are not elevated over the control strain, despite highly inaccurate DNA synthesis by their extracts. PMID:21304950

  5. Cloning, characterization, and sequence of the yeast DNA topoisomerase I gene.

    PubMed Central

    Thrash, C; Bankier, A T; Barrell, B G; Sternglanz, R

    1985-01-01

    The structural gene for yeast DNA topoisomerase I (TOP1) has been cloned from two yeast genomic plasmid banks. Integration of a plasmid carrying the gene into the chromosome and subsequent genetic mapping shows that TOP1 is identical to the gene previously called MAK1. Seven top1 (mak1) mutants including gene disruptions are viable, demonstrating that DNA topoisomerase I is not essential for viability in yeast. A 3787-base-pair DNA fragment including the gene has been sequenced. The protein predicted from the DNA sequence has 769 amino acids and a molecular weight of 90,020. Images PMID:2989818

  6. Structural basis of high-fidelity DNA synthesis by yeast DNA polymerase [delta

    SciTech Connect

    Swan, Michael K.; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2009-09-25

    DNA polymerase {delta} (Pol {delta}) is a high-fidelity polymerase that has a central role in replication from yeast to humans. We present the crystal structure of the catalytic subunit of yeast Pol {delta} in ternary complex with a template primer and an incoming nucleotide. The structure, determined at 2.0-{angstrom} resolution, catches the enzyme in the act of replication, revealing how the polymerase and exonuclease domains are juxtaposed relative to each other and how a correct nucleotide is selected and incorporated. The structure also reveals the 'sensing' interactions near the primer terminus, which signal a switch from the polymerizing to the editing mode. Taken together, the structure provides a chemical basis for the bulk of DNA synthesis in eukaryotic cells and a framework for understanding the effects of cancer-causing mutations in Pol {delta}.

  7. Repair of UV damaged DNA, genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1991-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, as well as studying the human homologs of yeast excision repair and postreplication repair proteins. In addition to its single-stranded DNA-dependent A TPase and DNA helicase activities, we have found that RAD3 protein also possesses DNA-RNA helicase activity, and that like RAD3, the Schizosaccharomyces pombe RAD3 homolog, rhp3{sup +}, is an essential gene. We have overexpressed the human RAD3 homolog, ERCC2, in yeast to facilitate its purification. The RAD10 protein was purified to homogeneity and shown to bind DNA. ERCC3y, the yeast homolog of the human ERCC-3/XP-B gene, has been sequenced and shown to be essential for viability. The Drosophila and human homologs of RAD6, required for postreplication repair and UV induced mutagenesis, were shown to complement the rad6 {Delta} mutation of yeast. Since defective DNA repair and enhanced neoplasia characterize several human genetic diseases, and repair proteins are highly conserved between yeast and man, a thorough understanding of the molecular mechanisms of DNA repir in yeast should provide a better understanding of the causes of carcinogenesis.

  8. Rif1: A Conserved Regulator of DNA Replication and Repair Hijacked by Telomeres in Yeasts

    PubMed Central

    Mattarocci, Stefano; Hafner, Lukas; Lezaja, Aleksandra; Shyian, Maksym; Shore, David

    2016-01-01

    Rap1-interacting factor 1 (Rif1) was originally identified in the budding yeast Saccharomyces cerevisiae as a telomere-binding protein that negatively regulates telomerase-mediated telomere elongation. Although this function is conserved in the distantly related fission yeast Schizosaccharomyces pombe, recent studies, both in yeasts and in metazoans, reveal that Rif1 also functions more globally, both in the temporal control of DNA replication and in DNA repair. Rif1 proteins are large and characterized by N-terminal HEAT repeats, predicted to form an elongated alpha-helical structure. In addition, all Rif1 homologs contain two short motifs, abbreviated RVxF/SILK, that are implicated in recruitment of the PP1 (yeast Glc7) phosphatase. In yeasts the RVxF/SILK domains have been shown to play a role in control of DNA replication initiation, at least in part through targeted de-phosphorylation of proteins in the pre-Replication Complex. In human cells Rif1 is recruited to DNA double-strand breaks through an interaction with 53BP1 where it counteracts DNA resection, thus promoting repair by non-homologous end-joining. This function requires the N-terminal HEAT repeat-containing domain. Interestingly, this domain is also implicated in DNA end protection at un-capped telomeres in yeast. We conclude by discussing the deployment of Rif1 at telomeres in yeasts from both an evolutionary perspective and in light of its recently discovered global functions. PMID:27066066

  9. Extraction of genomic DNA from yeasts for PCR-based applications.

    PubMed

    Lõoke, Marko; Kristjuhan, Kersti; Kristjuhan, Arnold

    2011-05-01

    We have developed a quick and low-cost genomic DNA extraction protocol from yeast cells for PCR-based applications. This method does not require any enzymes, hazardous chemicals, or extreme temperatures, and is especially powerful for simultaneous analysis of a large number of samples. DNA can be efficiently extracted from different yeast species (Kluyveromyces lactis, Hansenula polymorpha, Schizosaccharomyces pombe, Candida albicans, Pichia pastoris, and Saccharomyces cerevisiae). The protocol involves lysis of yeast colonies or cells from liquid culture in a lithium acetate (LiOAc)-SDS solution and subsequent precipitation of DNA with ethanol. Approximately 100 nanograms of total genomic DNA can be extracted from 1 × 10(7) cells. DNA extracted by this method is suitable for a variety of PCR-based applications (including colony PCR, real-time qPCR, and DNA sequencing) for amplification of DNA fragments of ≤ 3500 bp. PMID:21548894

  10. Amplification of a Zygosaccharomyces bailii DNA Segment in Wine Yeast Genomes by Extrachromosomal Circular DNA Formation

    PubMed Central

    Galeote, Virginie; Bigey, Frédéric; Beyne, Emmanuelle; Novo, Maite; Legras, Jean-Luc; Casaregola, Serge; Dequin, Sylvie

    2011-01-01

    We recently described the presence of large chromosomal segments resulting from independent horizontal gene transfer (HGT) events in the genome of Saccharomyces cerevisiae strains, mostly of wine origin. We report here evidence for the amplification of one of these segments, a 17 kb DNA segment from Zygosaccharomyces bailii, in the genome of S. cerevisiae strains. The copy number, organization and location of this region differ considerably between strains, indicating that the insertions are independent and that they are post-HGT events. We identified eight different forms in 28 S. cerevisiae strains, mostly of wine origin, with up to four different copies in a single strain. The organization of these forms and the identification of an autonomously replicating sequence functional in S. cerevisiae, strongly suggest that an extrachromosomal circular DNA (eccDNA) molecule serves as an intermediate in the amplification of the Z. bailii region in yeast genomes. We found little or no sequence similarity at the breakpoint regions, suggesting that the insertions may be mediated by nonhomologous recombination. The diversity between these regions in S. cerevisiae represents roughly one third the divergence among the genomes of wine strains, which confirms the recent origin of this event, posterior to the start of wine strain expansion. This is the first report of a circle-based mechanism for the expansion of a DNA segment, mediated by nonhomologous recombination, in natural yeast populations. PMID:21423766

  11. Repair of uv damaged DNA: Genes and proteins of yeast and human

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  12. Gene sequence analyses and other DNA-based methods for yeast species recognition

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA sequence analyses, as well as other DNA-based methodologies, have transformed the way in which yeasts are identified. The focus of this chapter will be on the resolution of species using various types of DNA comparisons. In other chapters in this book, Rozpedowska, Piškur and Wolfe discuss mul...

  13. Mitotic entry in the presence of DNA damage is a widespread property of aneuploidy in yeast

    PubMed Central

    Blank, Heidi M.; Sheltzer, Jason M.; Meehl, Colleen M.; Amon, Angelika

    2015-01-01

    Genetic instability is a hallmark of aneuploidy in budding and fission yeast. All aneuploid yeast strains analyzed to date harbor elevated levels of Rad52-GFP foci, a sign of DNA damage. Here we investigate how continuously elevated levels of DNA damage affect aneuploid cells. We show that Rad52-GFP foci form during S phase, consistent with the observation that DNA replication initiation and elongation are impaired in some aneuploid yeast strains. We furthermore find that although DNA damage is low in aneuploid cells, it nevertheless has dramatic consequences. Many aneuploid yeast strains adapt to DNA damage and undergo mitosis despite the presence of unrepaired DNA leading to cell death. Wild-type cells exposed to low levels of DNA damage exhibit a similar phenotype, indicating that adaptation to low levels of unrepaired DNA is a general property of the cell's response to DNA damage. Our results indicate that by causing low levels of DNA damage, whole-chromosome aneuploidies lead to DNA breaks that persist into mitosis. Such breaks provide the substrate for translocations and deletions that are a hallmark of cancer. PMID:25694455

  14. RNA∶DNA hybrids initiate quasi-palindrome-associated mutations in highly transcribed yeast DNA.

    PubMed

    Kim, Nayun; Cho, Jang-Eun; Li, Yue C; Jinks-Robertson, Sue

    2013-11-01

    RNase H enzymes promote genetic stability by degrading aberrant RNA:DNA hybrids and by removing ribonucleotide monophosphates (rNMPs) that are present in duplex DNA. Here, we report that loss of RNase H2 in yeast is associated with mutations that extend identity between the arms of imperfect inverted repeats (quasi-palindromes or QPs), a mutation type generally attributed to a template switch during DNA synthesis. QP events were detected using frameshift-reversion assays and were only observed under conditions of high transcription. In striking contrast to transcription-associated short deletions that also are detected by these assays, QP events do not require Top1 activity. QP mutation rates are strongly affected by the direction of DNA replication and, in contrast to their elevation in the absence of RNase H2, are reduced when RNase H1 is additionally eliminated. Finally, transcription-associated QP events are limited by components of the nucleotide excision repair pathway and are promoted by translesion synthesis DNA polymerases. We suggest that QP mutations reflect either a transcription-associated perturbation of Okazaki-fragment processing, or the use of a nascent transcript to resume replication following a transcription-replication conflict. PMID:24244191

  15. DNA synthesis in yeast cell-free extracts dependent on recombinant DNA plasmids purified from Escherichia coli.

    PubMed Central

    Jong, A Y; Scott, J F

    1985-01-01

    In our attempts to establish a cell-free DNA replication system for the yeast Saccharomyces cerevisiae, we have observed that recombinant DNA plasmids purified from Escherichia coli by a common procedure (lysozyme-detergent lysis and equilibrium banding in cesium chloride ethidium bromide gradients) often serve as templates for DNA synthesis by elongation enzymes. The templates could be elongated equally well by enzymes present in the yeast cell-free extracts, by the large proteolytic fragment of E. coli DNA polymerase I or by T4 DNA polymerase. The template activity of the purified plasmids was dependent on the presence of heterologous DNA segments in the bacterial vectors. The template activity could be diminished by treatment with alkali. We propose that the ability of recombinant plasmids isolated from bacterial hosts to serve as elongation templates may lead to erroneous conclusions when these plasmids are used as templates for in vitro replication or transcription reactions. Images PMID:3889851

  16. Problem-Solving Test: Analysis of DNA Damage Recognizing Proteins in Yeast and Human Cells

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2013-01-01

    The experiment described in this test was aimed at identifying DNA repair proteins in human and yeast cells. Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [alpha-[superscript 32]P]ATP, [gamma-[superscript…

  17. Assembly of long DNA sequences using a new synthetic Escherichia coli-yeast shuttle vector.

    PubMed

    Hou, Zheng; Zhou, Zheng; Wang, Zonglin; Xiao, Gengfu

    2016-04-01

    Synthetic biology is a newly developed field of research focused on designing and rebuilding novel biomolecular components, circuits, and networks. Synthetic biology can also help understand biological principles and engineer complex artificial metabolic systems. DNA manipulation on a large genome-wide scale is an inevitable challenge, but a necessary tool for synthetic biology. To improve the methods used for the synthesis of long DNA fragments, here we constructed a novel shuttle vector named pGF (plasmid Genome Fast) for DNA assembly in vivo. The BAC plasmid pCC1BAC, which can accommodate large DNA molecules, was chosen as the backbone. The sequence of the yeast artificial chromosome (YAC) regulatory element CEN6-ARS4 was synthesized and inserted into the plasmid to enable it to replicate in yeast. The selection sequence HIS3, obtained by polymerase chain reaction (PCR) from the plasmid pBS313, was inserted for screening. This new synthetic shuttle vector can mediate the transformation-associated recombination (TAR) assembly of large DNA fragments in yeast, and the assembled products can be transformed into Escherichia coli for further amplification. We also conducted in vivo DNA assembly using pGF and yeast homologous recombination and constructed a 31-kb long DNA sequence from the cyanophage PP genome. Our findings show that this novel shuttle vector would be a useful tool for efficient genome-scale DNA reconstruction. PMID:27113243

  18. A single protocol for extraction of gDNA from bacteria and yeast.

    PubMed

    Vingataramin, Laurie; Frost, Eric H

    2015-03-01

    Guanidine thiocyanate breakage of microorganisms has been the standard initial step in genomic DNA (gDNA) extraction of microbial DNA for two decades, despite the requirement for pretreatments to extract DNA from microorganisms other than Gram-negative bacteria. We report a quick and low-cost gDNA extraction protocol called EtNa that is efficient for bacteria and yeast over a broad range of concentrations. EtNa is based on a hot alkaline ethanol lysis. The solution can be immediately centrifuged to yield a crude gDNA extract suitable for PCR, or it can be directly applied to a silica column for purification. PMID:25757544

  19. Structural Basis for Error-free Replication of Oxidatively Damaged DNA by Yeast DNA Polymerase eta

    SciTech Connect

    T Silverstein; R Jain; R Johnson; L Prakash; S Prakash; A Aggarwal

    2011-12-31

    7,8-dihydro-8-oxoguanine (8-oxoG) adducts are formed frequently by the attack of oxygen-free radicals on DNA. They are among the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxoG(anti) with dCTP, 8-oxoG in the syn conformation can base pair with dATP, causing G to T transversions. We provide here for the first time a structural basis for the error-free replication of 8-oxoG lesions by yeast DNA polymerase {eta} (Pol{eta}). We show that the open active site cleft of Pol{eta} can accommodate an 8-oxoG lesion in the anti conformation with only minimal changes to the polymerase and the bound DNA: at both the insertion and post-insertion steps of lesion bypass. Importantly, the active site geometry remains the same as in the undamaged complex and provides a basis for the ability of Pol to prevent the mutagenic replication of 8-oxoG lesions in cells.

  20. Relationships between rDNA, Nop1 and Sir complex in biotechnologically relevant distillery yeasts.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Potocki, Leszek; Kuna, Ewelina; Kaplan, Jakub; Pabian, Sylwia; Kwiatkowska, Aleksandra; Lewinska, Anna; Wnuk, Maciej

    2016-09-01

    Distillery yeasts are poorly characterized physiological group among the Saccharomyces sensu stricto complex. As industrial yeasts are under constant environmental stress during fermentation processes and the nucleolus is a stress sensor, in the present study, nucleolus-related parameters were evaluated in 22 commercially available distillery yeast strains. Distillery yeasts were found to be a heterogeneous group with a variable content and length of rDNA and degree of nucleolus fragmentation. The levels of rDNA were negatively correlated with Nop1 (r = -0.59, p = 0.0038). Moreover, the protein levels of Sir transcriptional silencing complex and longevity regulators, namely Sir1, Sir2, Sir3 and Fob1, were studied and negative correlations between Sir2 and Nop1 (r = -0.45, p = 0.0332), and between Sir2 and Fob1 (r = -0.49, p = 0.0211) were revealed. In general, S. paradoxus group of distillery yeasts with higher rDNA pools and Sir2 level than S. bayanus group was found to be more tolerant to fermentation-associated stress stimuli, namely mild cold/heat stresses and KCl treatment. We postulate that rDNA state may be considered as a novel factor that may modulate a biotechnological process. PMID:27329282

  1. The yeast telomere length regulator TEL2 encodes a protein that binds to telomeric DNA.

    PubMed Central

    Kota, R S; Runge, K W

    1998-01-01

    TEL2 is required for telomere length regulation and viability in Saccharomyces cerevisiae. To investigate the mechanism by which Tel2p regulates telomere length, the majority (65%) of the TEL2 ORF was fused to the 3'-end of the gene for maltose binding protein, expressed in bacteria and the purified protein used in DNA binding studies. Rap1p, the major yeast telomere binding protein, recognizes a 13 bp duplex site 5'-GGTGTGTGGGTGT-3' in yeast telomeric DNA with high affinity. Gel shift experiments revealed that the MBP-Tel2p fusion binds the double-stranded yeast telomeric Rap1p site in a sequence-specific manner. Analysis of mutated sites showed that MBP-Tel2p could bind 5'-GTGTGTGG-3' within this 13 bp site. Methylation interference analysis revealed that Tel2p contacts the 5'-terminal guanine in the major groove. MBP-Tel2p did not bind duplex telomeric DNA repeats from vertebrates, Tetrahymena or Oxytricha. These results suggest that Tel2p is a DNA binding protein that recognizes yeast telomeric DNA. PMID:9490802

  2. DNA polymerase γ and disease: what we have learned from yeast

    PubMed Central

    Lodi, Tiziana; Dallabona, Cristina; Nolli, Cecilia; Goffrini, Paola; Donnini, Claudia; Baruffini, Enrico

    2015-01-01

    Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (PEO), Alpers' syndrome, and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissues with variable degrees of severity. In this review, we summarize the genetic and biochemical knowledge published on yeast mitochondrial DNA polymerase from 1989, when the MIP1 gene was first cloned, up until now. The role of yeast is particularly emphasized in (i) validating the pathological mutations found in human POLG and modeled in MIP1, (ii) determining the molecular defects caused by these mutations and (iii) finding the correlation between mutations/polymorphisms in POLGA and mtDNA toxicity induced by specific drugs. We also describe recent findings regarding the discovery of molecules able to rescue the phenotypic defects caused by pathological mutations in Mip1, and the construction of a model system in which the human Pol γ holoenzyme is expressed in yeast and complements the loss of Mip1. PMID:25852747

  3. The Yeast Mitochondrial RNA Polymerase and Transcription Factor Complex Catalyzes Efficient Priming of DNA Synthesis on Single-stranded DNA.

    PubMed

    Ramachandran, Aparna; Nandakumar, Divya; Deshpande, Aishwarya P; Lucas, Thomas P; R-Bhojappa, Ramanagouda; Tang, Guo-Qing; Raney, Kevin; Yin, Y Whitney; Patel, Smita S

    2016-08-01

    Primases use single-stranded (ss) DNAs as templates to synthesize short oligoribonucleotide primers that initiate lagging strand DNA synthesis or reprime DNA synthesis after replication fork collapse, but the origin of this activity in the mitochondria remains unclear. Herein, we show that the Saccharomyces cerevisiae mitochondrial RNA polymerase (Rpo41) and its transcription factor (Mtf1) is an efficient primase that initiates DNA synthesis on ssDNA coated with the yeast mitochondrial ssDNA-binding protein, Rim1. Both Rpo41 and Rpo41-Mtf1 can synthesize short and long RNAs on ssDNA template and prime DNA synthesis by the yeast mitochondrial DNA polymerase Mip1. However, the ssDNA-binding protein Rim1 severely inhibits the RNA synthesis activity of Rpo41, but not the Rpo41-Mtf1 complex, which continues to prime DNA synthesis efficiently in the presence of Rim1. We show that RNAs as short as 10-12 nt serve as primers for DNA synthesis. Characterization of the RNA-DNA products shows that Rpo41 and Rpo41-Mtf1 have slightly different priming specificity. However, both prefer to initiate with ATP from short priming sequences such as 3'-TCC, TTC, and TTT, and the consensus sequence is 3'-Pu(Py)2-3 Based on our studies, we propose that Rpo41-Mtf1 is an attractive candidate for serving as the primase to initiate lagging strand DNA synthesis during normal replication and/or to restart stalled replication from downstream ssDNA. PMID:27311715

  4. Evidence that a single DNA ligase is involved in replication and recombination in yeast.

    PubMed Central

    Fabre, F; Roman, H

    1979-01-01

    The possible existence in yeast of different nuclear DNA ligase enzymes led us to ask whether induced recombination (gene conversion) involves the same ligase as that involved in DNA replication. The conditional cdc9 mutant is known to be defective, under restrictive conditions, in the rejoining of Okazaki fragments. We show here that under the same conditions, x-ray-induced convertants within the cdc9 locus are produced with kinetics indicating that most, if not all, of the conversion events require the participation of the cdc9-controlled ligase. Thus, the same DNA ligase is involved in DNA replication and in induced gene conversion. PMID:388446

  5. Heavy ion induced DNA-DSB in yeast and mammalian cells

    NASA Technical Reports Server (NTRS)

    Loebrich, M.; Ikpeme, S.; Kiefer, J.

    1994-01-01

    Molecular changes at the DNA are assumed to be the main cause for radiation effects in a number of organisms. During the course of the last decades techniques have been developed for measuring DNA double-strand breaks (dsb), generally assumed to be the most critical DNA lesions. The outcome of all those different approaches portrays a collection of data useful for a theoretical description of radiation action mechanisms. However, in the case of heavy ion induced DNA dsb the picture is not quite clear yet and further projects and strategies have to be developed. The biological systems studied in our group are yeast and mammalian cells. While in the case of yeast cells technical and methodical reasons highlight these organisms mammalian cells reach greater importance when dsb repair studies are performed. In both types of organisms the technique of pulsed-field gel electrophoresis (PFGE) is applied, although with different modifications and evaluation procedures mainly due to the different genome sizes.

  6. GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA

    PubMed Central

    Blount, Benjamin A.; Driessen, Maureen R. M.; Ellis, Tom

    2016-01-01

    Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb. PMID:27240644

  7. GC Preps: Fast and Easy Extraction of Stable Yeast Genomic DNA.

    PubMed

    Blount, Benjamin A; Driessen, Maureen R M; Ellis, Tom

    2016-01-01

    Existing yeast genomic DNA extraction methods are not ideally suited to extensive screening of colonies by PCR, due to being too lengthy, too laborious or yielding poor quality DNA and inconsistent results. We developed the GC prep method as a solution to this problem. Yeast cells from colonies or liquid cultures are lysed by vortex mixing with glass beads and then boiled in the presence of a metal chelating resin. In around 12 minutes, multiple samples can be processed to extract high yields of genomic DNA. These preparations perform as effectively in PCR screening as DNA purified by organic solvent methods, are stable for up to 1 year at room temperature and can be used as the template for PCR amplification of fragments of at least 8 kb. PMID:27240644

  8. High-resolution DNA-binding specificity analysis of yeast transcription factors

    PubMed Central

    Zhu, Cong; Byers, Kelsey J.R.P.; McCord, Rachel Patton; Shi, Zhenwei; Berger, Michael F.; Newburger, Daniel E.; Saulrieta, Katrina; Smith, Zachary; Shah, Mita V.; Radhakrishnan, Mathangi; Philippakis, Anthony A.; Hu, Yanhui; De Masi, Federico; Pacek, Marcin; Rolfs, Andreas; Murthy, Tal; LaBaer, Joshua; Bulyk, Martha L.

    2009-01-01

    Transcription factors (TFs) regulate the expression of genes through sequence-specific interactions with DNA-binding sites. However, despite recent progress in identifying in vivo TF binding sites by microarray readout of chromatin immunoprecipitation (ChIP-chip), nearly half of all known yeast TFs are of unknown DNA-binding specificities, and many additional predicted TFs remain uncharacterized. To address these gaps in our knowledge of yeast TFs and their cis regulatory sequences, we have determined high-resolution binding profiles for 89 known and predicted yeast TFs, over more than 2.3 million gapped and ungapped 8-bp sequences (“k-mers”). We report 50 new or significantly different direct DNA-binding site motifs for yeast DNA-binding proteins and motifs for eight proteins for which only a consensus sequence was previously known; in total, this corresponds to over a 50% increase in the number of yeast DNA-binding proteins with experimentally determined DNA-binding specificities. Among other novel regulators, we discovered proteins that bind the PAC (Polymerase A and C) motif (GATGAG) and regulate ribosomal RNA (rRNA) transcription and processing, core cellular processes that are constituent to ribosome biogenesis. In contrast to earlier data types, these comprehensive k-mer binding data permit us to consider the regulatory potential of genomic sequence at the individual word level. These k-mer data allowed us to reannotate in vivo TF binding targets as direct or indirect and to examine TFs' potential effects on gene expression in ∼1700 environmental and cellular conditions. These approaches could be adapted to identify TFs and cis regulatory elements in higher eukaryotes. PMID:19158363

  9. Identification of food and beverage spoilage yeasts from DNA sequence analyses

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Detection, identification, and classification of yeasts has undergone a major transformation in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of th...

  10. DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

    The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast. PMID:10861204

  11. Proficient Replication of the Yeast Genome by a Viral DNA Polymerase.

    PubMed

    Stodola, Joseph L; Stith, Carrie M; Burgers, Peter M

    2016-05-27

    DNA replication in eukaryotic cells requires minimally three B-family DNA polymerases: Pol α, Pol δ, and Pol ϵ. Pol δ replicates and matures Okazaki fragments on the lagging strand of the replication fork. Saccharomyces cerevisiae Pol δ is a three-subunit enzyme (Pol3-Pol31-Pol32). A small C-terminal domain of the catalytic subunit Pol3 carries both iron-sulfur cluster and zinc-binding motifs, which mediate interactions with Pol31, and processive replication with the replication clamp proliferating cell nuclear antigen (PCNA), respectively. We show that the entire N-terminal domain of Pol3, containing polymerase and proofreading activities, could be effectively replaced by those from bacteriophage RB69, and could carry out chromosomal DNA replication in yeast with remarkable high fidelity, provided that adaptive mutations in the replication clamp PCNA were introduced. This result is consistent with the model that all essential interactions for DNA replication in yeast are mediated through the small C-terminal domain of Pol3. The chimeric polymerase carries out processive replication with PCNA in vitro; however, in yeast, it requires an increased involvement of the mutagenic translesion DNA polymerase ζ during DNA replication. PMID:27072134

  12. DNA replication components as regulators of epigenetic inheritance--lesson from fission yeast centromere.

    PubMed

    He, Haijin; Gonzalez, Marlyn; Zhang, Fan; Li, Fei

    2014-06-01

    Genetic information stored in DNA is accurately copied and transferred to subsequent generations through DNA replication. This process is accomplished through the concerted actions of highly conserved DNA replication components. Epigenetic information stored in the form of histone modifications and DNA methylation, constitutes a second layer of regulatory information important for many cellular processes, such as gene expression regulation, chromatin organization, and genome stability. During DNA replication, epigenetic information must also be faithfully transmitted to subsequent generations. How this monumental task is achieved remains poorly understood. In this review, we will discuss recent advances on the role of DNA replication components in the inheritance of epigenetic marks, with a particular focus on epigenetic regulation in fission yeast. Based on these findings, we propose that specific DNA replication components function as key regulators in the replication of epigenetic information across the genome. PMID:24691906

  13. Analysis of ssDNA Gaps and DSBs in Genetically Unstable Yeast Cultures

    PubMed Central

    Peng, Jie; Raghuraman, M.K.; Feng, Wenyi

    2015-01-01

    DNA replication defects are an underlying cause of genome instability, which could stem from alterations in replication intermediates such as extensive single-stranded DNA (ssDNA). Under replication stress, ssDNA is a precursor of the ultimate double-strand breaks (DSBs). Indeed, mutations that render the cell incapable of mediating and protecting the replication forks produce ssDNA genome-wide at high frequency and cause lethality when encountering DNA damage or replication perturbation. Here we describe two related microarray-based methods to query genetically unstable yeast cultures, such as the mec1 and rad53 mutants. These mutants are defective in central protein kinases in the checkpoint pathway. To induce ssDNA and DSB formation in these mutants, we utilize hydroxyurea, a drug that causes nucleotide shortage in the cell. PMID:24906332

  14. A modified procedure for isolation of yeast mitochondrial DNA.

    PubMed

    Nedeva, Trayana; Petrova, Ventzislava; Hristozova, Tsonka; Kujumdzieva, Anna

    2002-01-01

    A modified, rapid and inexpensive method for preparation of mitochondrial DNA (mtDNA), suitable for molecular analysis is proposed. It comprises batch cultivation of Saccharomyces cerevisiae strain NBIMCC 583 on a simple nutrient medium at 28 degrees C; permeabialization of cells from late exponential growth phase with cetyltrimethylamonnium bromide, mechanical disintegration of the cell wall; preparation of a mitochondrial fraction and subsequent isolation and purification of mtDNA. The amount and the purity of the obtained mtDNA have been checked and its application for molecular analysis proven. The main advantages of the proposed procedure for isolation of mtDNA are introduction of simple nutrient medium, replacement of the enzymatic lysis of the cell wall by the cheaper mechanical one, avoidance of ultracentrifugation steps and use of harmful chemical substances. PMID:12440743

  15. Transcription-dependent DNA transactions in the mitochondrial genome of a yeast hypersuppressive petite mutant.

    PubMed

    Van Dyck, E; Clayton, D A

    1998-05-01

    Mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae contains highly conserved sequences, called rep/ori, that are associated with several aspects of its metabolism. These rep/ori sequences confer the transmission advantage exhibited by a class of deletion mutants called hypersuppressive petite mutants. In addition, because they share features with the mitochondrial leading-strand DNA replication origin of mammals, rep/ori sequences have also been proposed to participate in mtDNA replication initiation. Like the mammalian origins, where transcription is used as a priming mechanism for DNA synthesis, yeast rep/ori sequences contain an active promoter. Although transcription is required for maintenance of wild-type mtDNA in yeast, the role of the rep/ori promoter as a cis-acting element involved in the replication of wild-type mtDNA is unclear, since mitochondrial deletion mutants need neither transcription nor a rep/ori sequence to maintain their genome. Similarly, transcription from the rep/ori promoter does not seem to be necessary for biased inheritance of mtDNA. As a step to elucidate the function of the rep/ori promoter, we have attempted to detect transcription-dependent DNA transactions in the mtDNA of a hypersuppressive petite mutant. We have examined the mtDNA of the well-characterized petite mutant a-1/1R/Z1, whose repeat unit shelters the rep/ori sequence ori1, in strains carrying either wild-type or null alleles of the nuclear genes encoding the mitochondrial transcription apparatus. Complex DNA transactions were detected that take place around GC-cluster C, an evolutionarily conserved GC-rich sequence block immediately downstream from the rep/ori promoter. These transactions are strictly dependent upon mitochondrial transcription. PMID:9566917

  16. The Strictly Aerobic Yeast Yarrowia lipolytica Tolerates Loss of a Mitochondrial DNA-Packaging Protein

    PubMed Central

    Bakkaiova, Jana; Arata, Kosuke; Matsunobu, Miki; Ono, Bungo; Aoki, Tomoyo; Lajdova, Dana; Nebohacova, Martina; Nosek, Jozef; Miyakawa, Isamu

    2014-01-01

    Mitochondrial DNA (mtDNA) is highly compacted into DNA-protein structures termed mitochondrial nucleoids (mt-nucleoids). The key mt-nucleoid components responsible for mtDNA condensation are HMG box-containing proteins such as mammalian mitochondrial transcription factor A (TFAM) and Abf2p of the yeast Saccharomyces cerevisiae. To gain insight into the function and organization of mt-nucleoids in strictly aerobic organisms, we initiated studies of these DNA-protein structures in Yarrowia lipolytica. We identified a principal component of mt-nucleoids in this yeast and termed it YlMhb1p (Y. lipolytica mitochondrial HMG box-containing protein 1). YlMhb1p contains two putative HMG boxes contributing both to DNA binding and to its ability to compact mtDNA in vitro. Phenotypic analysis of a Δmhb1 strain lacking YlMhb1p resulted in three interesting findings. First, although the mutant exhibits clear differences in mt-nucleoids accompanied by a large decrease in the mtDNA copy number and the number of mtDNA-derived transcripts, its respiratory characteristics and growth under most of the conditions tested are indistinguishable from those of the wild-type strain. Second, our results indicate that a potential imbalance between subunits of the respiratory chain encoded separately by nuclear DNA and mtDNA is prevented at a (post)translational level. Third, we found that mtDNA in the Δmhb1 strain is more prone to mutations, indicating that mtHMG box-containing proteins protect the mitochondrial genome against mutagenic events. PMID:24972935

  17. DNA strand annealing is promoted by the yeast Rad52 protein.

    PubMed Central

    Mortensen, U H; Bendixen, C; Sunjevaric, I; Rothstein, R

    1996-01-01

    The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8855248

  18. Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes.

    PubMed

    Joshi, Ricky S; Piña, Benjamin; Roca, Joaquim

    2010-02-17

    How DNA helical tension is constrained along the linear chromosomes of eukaryotic cells is poorly understood. In this study, we induced the accumulation of DNA (+) helical tension in Saccharomyces cerevisiae cells and examined how DNA transcription was affected along yeast chromosomes. The results revealed that, whereas the overwinding of DNA produced a general impairment of transcription initiation, genes situated at <100 kb from the chromosomal ends gradually escaped from the transcription stall. This novel positional effect seemed to be a simple function of the gene distance to the telomere: It occurred evenly in all 32 chromosome extremities and was independent of the atypical structure and transcription activity of subtelomeric chromatin. These results suggest that DNA helical tension dissipates at chromosomal ends and, therefore, provides a functional indication that yeast chromosome extremities are topologically open. The gradual escape from the transcription stall along the chromosomal flanks also indicates that friction restrictions to DNA twist diffusion, rather than tight topological boundaries, might suffice to confine DNA helical tension along eukaryotic chromatin. PMID:20057354

  19. Positional dependence of transcriptional inhibition by DNA torsional stress in yeast chromosomes

    PubMed Central

    Joshi, Ricky S; Piña, Benjamin; Roca, Joaquim

    2010-01-01

    How DNA helical tension is constrained along the linear chromosomes of eukaryotic cells is poorly understood. In this study, we induced the accumulation of DNA (+) helical tension in Saccharomyces cerevisiae cells and examined how DNA transcription was affected along yeast chromosomes. The results revealed that, whereas the overwinding of DNA produced a general impairment of transcription initiation, genes situated at <100 kb from the chromosomal ends gradually escaped from the transcription stall. This novel positional effect seemed to be a simple function of the gene distance to the telomere: It occurred evenly in all 32 chromosome extremities and was independent of the atypical structure and transcription activity of subtelomeric chromatin. These results suggest that DNA helical tension dissipates at chromosomal ends and, therefore, provides a functional indication that yeast chromosome extremities are topologically open. The gradual escape from the transcription stall along the chromosomal flanks also indicates that friction restrictions to DNA twist diffusion, rather than tight topological boundaries, might suffice to confine DNA helical tension along eukaryotic chromatin. PMID:20057354

  20. Profiling DNA damage-induced phosphorylation in budding yeast reveals diverse signaling networks.

    PubMed

    Zhou, Chunshui; Elia, Andrew E H; Naylor, Maria L; Dephoure, Noah; Ballif, Bryan A; Goel, Gautam; Xu, Qikai; Ng, Aylwin; Chou, Danny M; Xavier, Ramnik J; Gygi, Steven P; Elledge, Stephen J

    2016-06-28

    The DNA damage response (DDR) is regulated by a protein kinase signaling cascade that orchestrates DNA repair and other processes. Identifying the substrate effectors of these kinases is critical for understanding the underlying physiology and mechanism of the response. We have used quantitative mass spectrometry to profile DDR-dependent phosphorylation in budding yeast and genetically explored the dependency of these phosphorylation events on the DDR kinases MEC1, RAD53, CHK1, and DUN1. Based on these screens, a database containing many novel DDR-regulated phosphorylation events has been established. Phosphorylation of many of these proteins has been validated by quantitative peptide phospho-immunoprecipitation and examined for functional relevance to the DDR through large-scale analysis of sensitivity to DNA damage in yeast deletion strains. We reveal a link between DDR signaling and the metabolic pathways of inositol phosphate and phosphatidyl inositol synthesis, which are required for resistance to DNA damage. We also uncover links between the DDR and TOR signaling as well as translation regulation. Taken together, these data shed new light on the organization of DDR signaling in budding yeast. PMID:27298372

  1. Repair of uv damaged DNA: Genes and proteins of yeast and human. Progress report, November 1, 1991--April 15, 1992

    SciTech Connect

    Prakash, L.

    1992-04-01

    Our objectives are to determine the molecular mechanism of the incision step of excision repair of ultraviolet (UV) light damaged DNA in eukaryotic organisms, using the yeast Saccharomyces cerevisiae as a model system, and to study the human homologs of yeast excision repair and postreplication repair proteins progress is described.

  2. A Novel Mutation in DNA Topoisomerase I of Yeast Causes DNA Damage and Rad9-Dependent Cell Cycle Arrest

    PubMed Central

    Levin, N. A.; Bjornsti, M. A.; Fink, G. R.

    1993-01-01

    DNA topoisomerases, enzymes that alter the superhelicity of DNA, have been implicated in such critical cellular functions as transcription, DNA replication, and recombination. In the yeast Saccharomyces cerevisiae, a null mutation in the gene encoding topoisomerase I (TOP1) causes elevated levels of mitotic recombination in the ribosomal DNA (rDNA), but has little effect on growth. We have isolated a missense mutation in TOP1 that causes mitotic hyper-recombination not only in the rDNA, but also at other loci, in addition to causing a number of other unexpected phenotypes. This topoisomerase I mutation (top1-103) causes slow growth, constitutive expression of DNA damage-inducible genes, and inviability in the absence of the double-strand break repair system. Overexpression of top1-103 causes RAD9-dependent cell cycle arrest in G(2). We show that the Top1-103 enzyme nicks DNA in vitro, suggesting that it damages DNA directly. We propose that Top1-103 mimics the action of wild-type topoisomerase I in the presence of the anti-tumor drug, camptothecin. PMID:8385050

  3. Satellite DNA from the brine shrimp Artemia affects the expression of a flanking gene in yeast.

    PubMed

    Maiorano, D; Cece, R; Badaracco, G

    1997-04-11

    We have previously revealed that in the brine shrimp Artemia franciscana an AluI DNA family of repeats, 113 bp in length, is the major component of the constitutive heterochromatin and that this repetitive DNA shows a stable curvature that confers a solenoidal geometry on the double helix in vitro. It was suggested that this particular structure may play a relevant role in determining the condensation of the heterochromatin. In this report we have cloned hexamers of highly-repetitive sequence (AluI-satellite DNA) in proximity to a yeast lacZ reporter gene on a plasmid. We find that the expression of the reporter gene is affected by the presence of this DNA in a dose- and orientation-dependent manner in the yeast, S. cerevisiae. We show that this effect is not dependent on under-replication or re-arrangements of the repetitive DNA in the cell but is due to decreased expression of the reporter gene. Our results indicate that the AluI-satellite DNA of Artemia per se is able to influence gene expression. PMID:9161405

  4. Crystal Structure of Yeast DNA Polymerase ε Catalytic Domain

    PubMed Central

    Jain, Rinku; Rajashankar, Kanagalaghatta R.; Buku, Angeliki; Johnson, Robert E.; Prakash, Louise; Prakash, Satya; Aggarwal, Aneel K.

    2014-01-01

    DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2G:C). Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2. PMID:24733111

  5. Fission Yeast Pxd1 Promotes Proper DNA Repair by Activating Rad16XPF and Inhibiting Dna2

    PubMed Central

    Zhang, Jia-Min; Liu, Xiao-Man; Ding, Yue-He; Xiong, Liang-Yao; Ren, Jing-Yi; Zhou, Zhi-Xiong; Wang, Hai-Tao; Zhang, Mei-Jun; Yu, Yang; Dong, Meng-Qiu; Du, Li-Lin

    2014-01-01

    Structure-specific nucleases play crucial roles in many DNA repair pathways. They must be precisely controlled to ensure optimal repair outcomes; however, mechanisms of their regulation are not fully understood. Here, we report a fission yeast protein, Pxd1, that binds to and regulates two structure-specific nucleases: Rad16XPF-Swi10ERCC1 and Dna2-Cdc24. Strikingly, Pxd1 influences the activities of these two nucleases in opposite ways: It activates the 3′ endonuclease activity of Rad16-Swi10 but inhibits the RPA-mediated activation of the 5′ endonuclease activity of Dna2. Pxd1 is required for Rad16-Swi10 to function in single-strand annealing, mating-type switching, and the removal of Top1-DNA adducts. Meanwhile, Pxd1 attenuates DNA end resection mediated by the Rqh1-Dna2 pathway. Disabling the Dna2-inhibitory activity of Pxd1 results in enhanced use of a break-distal repeat sequence in single-strand annealing and a greater loss of genetic information. We propose that Pxd1 promotes proper DNA repair by differentially regulating two structure-specific nucleases. PMID:25203555

  6. Yeast telomeres exert a position effect on recombination between internal tracts of yeast telomeric DNA

    PubMed Central

    Stavenhagen, Jeffrey B.; Zakian, Virginia A.

    1998-01-01

    In Saccharomyces cerevisiae, proximity to a telomere affects both transcription and replication of adjacent DNA. In this study, we show that telomeres also impose a position effect on mitotic recombination. The rate of recombination between directly repeated tracts of telomeric C1–3A/TG1–3 DNA was reduced severely by proximity to a telomere. In contrast, recombination of two control substrates was not affected by telomere proximity. Thus, unlike position effects on transcription or replication, inhibition of recombination was sequence specific. Moreover, the repression of recombination was not under the same control as transcriptional repression (telomere position effect; TPE), as mutations in genes essential for TPE did not alleviate telomeric repression of recombination. The reduction in recombination between C1–3A/TG1–3 tracts near the telomere was caused by an absence of Rad52p-dependent events as well as a reduction in Rad1p-dependent events. The sequence-specific repression of recombination near the telomere was eliminated in cells that overexpressed the telomere-binding protein Rap1p, a condition that also increased recombination between C1–3A/TG1–3 tracts at internal positions on the chromosome. We propose that the specific inhibition between C1–3A/TG1–3 tracts near the telomere occurs through the action of a telomere-specific end-binding protein that binds to the single-strand TG1–3 tail generated during the processing of recombination intermediates. The recombination inhibitor protein may also block recombination between endogenous telomeres. PMID:9765206

  7. Replication dynamics in fission and budding yeasts through DNA polymerase tracking.

    PubMed

    Vázquez, Enrique; Antequera, Francisco

    2015-10-01

    The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof-read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions. PMID:26293347

  8. Astragalin from Cassia alata induces DNA adducts in vitro and repairable DNA damage in the yeast Saccharomyces cerevisiae.

    PubMed

    Saito, Samuel; Silva, Givaldo; Santos, Regineide Xavier; Gosmann, Grace; Pungartnik, Cristina; Brendel, Martin

    2012-01-01

    Reverse phase-solid phase extraction from Cassia alata leaves (CaRP) was used to obtain a refined extract. Higher than wild-type sensitivity to CaRP was exhibited by 16 haploid Saccharomyces cerevisiae mutants with defects in DNA repair and membrane transport. CaRP had a strong DPPH free radical scavenging activity with an IC(50) value of 2.27 μg mL(-1) and showed no pro-oxidant activity in yeast. CaRP compounds were separated by HPLC and the three major components were shown to bind to DNA in vitro. The major HPLC peak was identified as kampferol-3-O-β-d-glucoside (astragalin), which showed high affinity to DNA as seen by HPLC-UV measurement after using centrifugal ultrafiltration of astragalin-DNA mixtures. Astragalin-DNA interaction was further studied by spectroscopic methods and its interaction with DNA was evaluated using solid-state FTIR. These and computational (in silico) docking studies revealed that astragalin-DNA binding occurs through interaction with G-C base pairs, possibly by intercalation stabilized by H-bond formation. PMID:22489129

  9. Identification of food and beverage spoilage yeasts from DNA sequence analyses.

    PubMed

    Kurtzman, Cletus P

    2015-11-20

    Detection, identification and classification of yeasts have undergone major changes in the last decade and a half following application of gene sequence analyses and genome comparisons. Development of a database (barcode) of easily determined DNA sequences from domains 1 and 2 (D1/D2) of the nuclear large subunit rRNA gene and from ITS now permits many laboratories to identify species quickly and accurately, thus replacing the laborious and often inaccurate phenotypic tests previously used. Phylogenetic analysis of gene sequences has resulted in a major revision of yeast systematics resulting in redefinition of nearly all genera. This new understanding of species relationships has prompted a change of rules for naming and classifying yeasts and other fungi, and these new rules are presented in the recently implemented International Code of Nomenclature for algae, fungi, and plants (Melbourne Code). The use of molecular methods for species identification and the impact of Code changes on classification will be discussed, especially in the context of food and beverage spoilage yeasts. PMID:26051959

  10. Genome-Wide Profiling of Yeast DNA:RNA Hybrid Prone Sites with DRIP-Chip

    PubMed Central

    Lu, Phoebe Y. T.; Luo, Zongli; Hamza, Akil; Kobor, Michael S.; Stirling, Peter C.; Hieter, Philip

    2014-01-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  11. Genome-wide profiling of yeast DNA:RNA hybrid prone sites with DRIP-chip.

    PubMed

    Chan, Yujia A; Aristizabal, Maria J; Lu, Phoebe Y T; Luo, Zongli; Hamza, Akil; Kobor, Michael S; Stirling, Peter C; Hieter, Philip

    2014-04-01

    DNA:RNA hybrid formation is emerging as a significant cause of genome instability in biological systems ranging from bacteria to mammals. Here we describe the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation (DRIP) followed by hybridization on tiling microarray. These profiles show that DNA:RNA hybrids preferentially accumulated at rDNA, Ty1 and Ty2 transposons, telomeric repeat regions and a subset of open reading frames (ORFs). The latter are generally highly transcribed and have high GC content. Interestingly, significant DNA:RNA hybrid enrichment was also detected at genes associated with antisense transcripts. The expression of antisense-associated genes was also significantly altered upon overexpression of RNase H, which degrades the RNA in hybrids. Finally, we uncover mutant-specific differences in the DRIP profiles of a Sen1 helicase mutant, RNase H deletion mutant and Hpr1 THO complex mutant compared to wild type, suggesting different roles for these proteins in DNA:RNA hybrid biology. Our profiles of DNA:RNA hybrid prone loci provide a resource for understanding the properties of hybrid-forming regions in vivo, extend our knowledge of hybrid-mitigating enzymes, and contribute to models of antisense-mediated gene regulation. A summary of this paper was presented at the 26th International Conference on Yeast Genetics and Molecular Biology, August 2013. PMID:24743342

  12. Delineating Rearrangements in Single Yeast Artificial Chromosomes by Quantitative DNA Fiber Mapping

    SciTech Connect

    Weier, Heinz-Ulrich G.; Greulich-Bode, Karin M.; Wu, Jenny; Duell, Thomas

    2009-09-18

    Cloning of large chunks of human genomic DNA in recombinant systems such as yeast or bacterial artificial chromosomes has greatly facilitated the construction of physical maps, the positional cloning of disease genes or the preparation of patient-specific DNA probes for diagnostic purposes. For this process to work efficiently, the DNA cloning process and subsequent clone propagation need to maintain stable inserts that are neither deleted nor otherwise rearranged. Some regions of the human genome; however, appear to have a higher propensity than others to rearrange in any host system. Thus, techniques to detect and accurately characterize such rearrangements need to be developed. We developed a technique termed 'Quantitative DNA Fiber Mapping (QDFM)' that allows accurate tagging of sequence elements of interest with near kilobase accuracy and optimized it for delineation of rearrangements in recombinant DNA clones. This paper demonstrates the power of this microscopic approach by investigating YAC rearrangements. In our examples, high-resolution physical maps for regions within the immunoglobulin lambda variant gene cluster were constructed for three different YAC clones carrying deletions of 95 kb and more. Rearrangements within YACs could be demonstrated unambiguously by pairwise mapping of cosmids along YAC DNA molecules. When coverage by YAC clones was not available, distances between cosmid clones were estimated by hybridization of cosmids onto DNA fibers prepared from human genomic DNA. In addition, the QDFM technology provides essential information about clone stability facilitating closure of the maps of the human genome as well as those of model organisms.

  13. Surface Etching and DNA Damage Induced by Low-Energy Ion Irradiation in Yeast

    NASA Astrophysics Data System (ADS)

    Liu, Xuelan; Xu, An; Dai, Yin; Yuan, Hang; Yu, Zengliang

    2011-06-01

    Bio-effects of survival and etching damage on cell surface and DNA strand breaks were investigated in the yeast saccharomyces cerevisiae after exposure by nitrogen ion with an energy below 40 keV. The result showed that 16% of trehalose provided definite protection for cells against vacuum stress compared with glycerol. In contrast to vacuum control, significant morphological damage and DNA strand breaks were observed, in yeast cells bombarded with low-energy nitrogen, by scanning electron microscopy (SEM) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) immunofluorescence assays. Moreover, PI (propidium iodide) fluorescent staining indicated that cell integrity could be destroyed by ion irradiation. Cell damage eventually affected cell viability and free radicals were involved in cell damage as shown by DMSO (dimethyl sulfoxide) rescue experiment. Our primary experiments demonstrated that yeast cells can be used as an optional experimental model to study the biological effects of low energy ions and be applied to further investigate the mechanism(s) underlying the bio-effects of eukaryotic cells.

  14. Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.

    PubMed

    Bankmann, M; Prakash, L; Prakash, S

    1992-02-01

    Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines. PMID:1741034

  15. Induction and isolation of DNA transformation mutants in the yeast Saccharomyces cerevisiae

    SciTech Connect

    Hegerich, P.A.; Bruschi, C.V.

    1987-01-01

    The objective of this research was to induce and isolate mutants of the yeast Saccharomyces cerevisiae which have become transformable by purified plasmid DNA. Non-transformable yeast cells were mutagenized by ultraviolet light using a 65% lethal dose (480 ergs/mm/sup 2/). After a period of overnight liquid holding recovery, the irradiated cells were subjected to DNA transformation using our CaCl/sub 2/ protocol with the multi-marker shuttle plasmid pBB carrying the LEU 2 leucine gene. Following transformation the colonies that grew on selective leucineless medium were identified and subjected to further genetic analysis. From a total of 1 x 10/sup 9/ cells the authors have isolated 7 colonies deriving from putative mutants that have acquired the capability to uptake plasmid DNA. The transformants were cured from the plasmid by its mitotic loss on non-selective medium, then re-transformed to verify their genetic competence to give rise to a number of transformants comparable to transformable strains. We have identified and isolated one mutant, coded trs-1, which is able to reproduce a frequency of transformation comparable with the tranformable control. They, therefore, conclude that this mutant is specific for plasmid DNA transformation and that the mutation is mitotically stable.

  16. Genetic transformation of the yeast Dekkera/Brettanomyces bruxellensis with non-homologous DNA.

    PubMed

    Miklenić, Marina; Štafa, Anamarija; Bajić, Ana; Žunar, Bojan; Lisnić, Berislav; Svetec, Ivan-Krešimir

    2013-05-01

    Yeast Dekkera/Brettanomyces bruxellensis is probably the most common contaminant in wineries and ethanol production processes. The considerable economic losses caused by this yeast, but also its ability to produce and tolerate high ethanol concentrations, make it an attractive subject for research with potential for industrial applications. Unfortunately, efforts to understand the biology of D. bruxellensis and facilitate its broader use in industry are hampered by the lack of adequate procedures for delivery of exogenous DNA into this organism. Here we describe the development of transformation protocols (spheroplast transformation, LiAc/PEG method, and electroporation) and report the first genetic transformation of yeast D. bruxellensis. A linear heterologous DNA fragment carrying the kanMX4 sequence was used for transformation, which allowed transformants to be selected on plates containing geneticin. We found the spheroplast transformation method using 1M sorbitol as osmotic stabilizer to be inappropriate because sorbitol strikingly decreases the plating efficiency of both D. bruxellensis spheroplast and intact cells. However, we managed to modify the LiAc/ PEG transformation method and electroporation to accommodate D. bruxellensis transformation, achieving efficiencies of 0.6-16 and 10-20 transformants/microg DNA, respectively. The stability of the transformants ranged from 93.6% to 100%. All putative transformants were analyzed by Southern blot using the kanMX4 sequence as a hybridization probe, which confirmed that the transforming DNA fragment had integrated into the genome. The results of the molecular analysis were consistent with the expected illegitimate integration of a heterologous transforming fragment. PMID:23648858

  17. In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse.

    PubMed

    Morselli, Marco; Pastor, William A; Montanini, Barbara; Nee, Kevin; Ferrari, Roberto; Fu, Kai; Bonora, Giancarlo; Rubbi, Liudmilla; Clark, Amander T; Ottonello, Simone; Jacobsen, Steven E; Pellegrini, Matteo

    2015-01-01

    Methylation of cytosines (5(me)C) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5(me)C levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo. PMID:25848745

  18. Connections between RNA splicing and DNA intron mobility in yeast mitochondria: RNA maturase and DNA endonuclease switching experiments.

    PubMed Central

    Goguel, V; Delahodde, A; Jacq, C

    1992-01-01

    The intron-encoded proteins bI4 RNA maturase and aI4 DNA endonuclease can be faithfully expressed in yeast cytoplasm from engineered forms of their mitochondrial coding sequences. In this work we studied the relationships between these two activities associated with two homologous intron-encoded proteins: the bI4 RNA maturase encoded in the fourth intron of the cytochrome b gene and the aI4 DNA endonuclease (I-SceII) encoded in the fourth intron of the gene coding for the subunit I of cytochrome oxidase. Taking advantage of both the high recombinogenic properties of yeast and the similarities between the two genes, we constructed in vivo a family of hybrid genes carrying parts of both RNA maturase and DNA endonuclease coding sequences. The presence of a sequence coding for a mitochondrial targeting peptide upstream from these hybrid genes allowed us to study the properties of their translation products within the mitochondria in vivo. We thus could analyze the ability of the recombinant proteins to complement RNA maturase deficiencies in different strains. Many combinations of the two parental intronic sequences were found in the recombinants. Their structural and functional analysis revealed the following features. (i) The N-terminal half of the bI4 RNA maturase could be replaced in total by its equivalent from the aI4 DNA endonuclease without affecting the RNA maturase activity. In contrast, replacing the C-terminal half of the bI4 RNA maturase with its equivalent from the aI4 DNA endonuclease led to a very weak RNA maturase activity, indicating that this region is more differentiated and linked to the maturase activity. (ii) None of the hybrid proteins carrying an RNA maturase activity kept the DNA endonuclease activity, suggesting that the latter requires the integrity of the aI4 protein. These observations are interesting because the aI4 DNA endonuclease is known to promote the propagation, at the DNA level, of the aI4 intron, whereas the bI4 RNA maturase

  19. Yeast DNA-repair gene RAD14 encodes a zinc metalloprotein with affinity for ultraviolet-damaged DNA

    SciTech Connect

    Guzder, S.N.; Sung, P.; Prakash, S. ); Prakash, L. )

    1993-06-15

    Xeroderma pigmentosum (XP) patients suffer from a high incidence of skin cancers due to a defect in excision repair of UV light-damaged DNA. Of the seven XP complementation groups, A--G, group A represents a severe and frequent form of the disease. The Saccharomyces cerevisiae RAD14 gene is a homolog of the XP-A correcting (XPAC) gene. Like XP-A cells, rad14-null mutants are defective in the incision step of excision repair of UV-damaged DNA. The authors have purified RAD14 protein to homogeneity from extract of a yeast strain genetically tailored to overexpress RAD14. As determined by atomic emission spectroscopy, RAD14 contains one zinc atom. They also show in vitro that RAD14 binds zinc but does not bind other divalent metal ions. In DNA mobility-shift assays, RAD14 binds specifically to UV-damaged DNA. Removal of cyclobutane pyrimidine dimers from damaged DNA by enzymatic photoreactivation has no effect on binding, strongly suggesting that RAD14 recognizes pyrimidine(6-4)pyrimidone photoproduct sites. These findings indicate that RAD14 functions in damage recognition during excision repair. 37 refs., 4 figs.

  20. Separation of chromosomal DNA molecules from yeast by orthogonal-field-alternation gel electrophoresis.

    PubMed Central

    Carle, G F; Olson, M V

    1984-01-01

    A simple agarose-gel apparatus has been developed that allows the separation of DNA molecules in the size range from 50 kb to well over 750 kb, the largest size for which size standards were available. The apparatus is based on the recent discovery that large DNA molecules are readily fractionated on agarose gels if they are alternately subjected to two approximately orthogonal electric fields. The switching time, which was on the order of 20-50 sec in our experiments, can be adjusted to optimize fractionation in a given size range. The resolution of the technique is sufficient to allow the fractionation of a sample of self-ligated lambda DNA into a ladder of approximately 15 bands, spaced at 50 kb intervals. We have applied the technique to the fractionation of yeast DNA into 11 distinct bands, several of which have been shown by DNA-DNA hybridization to hybridize uniquely to different chromosome-specific hybridization probes. In this paper, we describe the design of the apparatus, the electrophoretic protocol, and the sample-handling procedures that we have employed. Images PMID:6379602

  1. Two polypeptide chains in yeast transcription factor tau interact with DNA

    SciTech Connect

    Gabrielsen, O.S.; Marzouki, N.; Ruet, A.; Sentenac, A.; Fromageot, P.

    1989-05-05

    Yeast transcription factor tau interacts with the A and B blocks of the intragenic promoter of tRNA genes. The structure of tau was investigated by identifying the polypeptide chains specifically complexed to the tRNA3Glu gene. Highly purified factor, obtained by an improved purification procedure, contained several polypeptide chains, four of which (Mr = 145,000, 135,000, 100,000 and 65,000) comigrated with tau-DNA complex by polyacrylamide gel electrophoresis. Antibodies raised against the 145- and 100-kDa components altered the migration of tau-DNA complexes in band shift assays and inhibited tRNA synthesis in a reconstituted transcription system. These components are immunologically unrelated proteins. By UV cross-linking to /sup 32/P-body-labeled tDNA followed by extensive DNase treatment, two polypeptides of the same size (145 and 100 kDa) were found to be radioactively labeled. Factor tau, therefore, appears to be a multisubunit DNA-binding protein with two distinct polypeptides contributing to DNA recognition. Limited proteolysis of tau generated a protease-resistant tau B (tau B) domain that binds solely to the B block. tau B-tDNA complexes were recognized by anti-145 IgG and contained a 120-kDa polypeptide that could originate from the 145-kDa component by proteolysis. These results strongly suggest that the 145-kDa polypeptide belongs to tau B and is responsible for B block binding.

  2. Effect of storage and processing on plasmid, yeast and plant genomic DNA stability in juice from genetically modified oranges.

    PubMed

    Weiss, Julia; Ros-Chumillas, Maria; Peña, Leandro; Egea-Cortines, Marcos

    2007-01-30

    Recombinant DNA technology is an important tool in the development of plant varieties with new favourable features. There is strong opposition towards this technology due to the potential risk of horizontal gene transfer between genetically modified plant material and food-associated bacteria, especially if genes for antibiotic resistance are involved. Since horizontal transfer efficiency depends on size and length of homologous sequences, we investigated the effect of conditions required for orange juice processing on the stability of DNA from three different origins: plasmid DNA, yeast genomic DNA and endogenous genomic DNA from transgenic sweet orange (C. sinensis L. Osb.). Acidic orange juice matrix had a strong degrading effect on plasmid DNA which becomes apparent in a conformation change from supercoiled structure to nicked, linear structure within 5h of storage at 4 degrees C. Genomic yeast DNA was degraded during exposure to acidic orange juice matrix within 4 days, and also the genomic DNA of C. sinensis suffered degradation within 2 days of storage as indicated by amplification results from transgene markers. Standard pasteurization procedures affected DNA integrity depending on the method and time used. Our data show that the current standard industrial procedures to pasteurize orange juice as well as its acidic nature causes a strong degradation of both yeast and endogenous genomic DNA below sizes reported to be suitable for horizontal gene transfer. PMID:17064805

  3. A direct droplet digital PCR method for quantification of residual DNA in protein drugs produced in yeast cells.

    PubMed

    Hussain, Musaddeq; Fantuzzo, Rebecca; Mercorelli, Suzanne; Cullen, Constance

    2016-05-10

    Yeast cells, in particular Pichia pastoris, are the host cell of choice for manufacturing several protein therapeutic agents in the biopharmaceutical industry. Host cell DNA is an impurity of such manufacturing process and the residual DNA after the purification process of the drug must be monitored to ensure drug purity and safety. Currently, real-time PCR (qPCR) based methods are widely employed for quantification of host residual DNA. At the same time the digital PCR technology is coming into prominence with promise of higher sensitivity. Here we report a method where the protein drug is directly added to the droplet digital PCR (ddPCR) reaction including yeast-specific primers and fluorescent-tagged probe and nanoliter-sized droplets are generated. The droplets are then subjected to PCR followed by analysis for fluorescence. This Pichia residual DNA direct ddPCR method for yeast can be used to test higher amount of drug compared to the corresponding qPCR method thereby increasing sensitivity, retaining high precision and accuracy and has a wide linear range of determination. The method has been successfully tested with three batches of a recombinant human IgG1-Fc-based drug (RP-1) and with commercially available human insulin, both manufactured in yeast cells. This method simplifies the residual DNA quantification protocol by eliminating DNA extraction or protease digestion and eliminates use of DNA standards in day-to-day running of the method. PMID:26896631

  4. The DNA damage checkpoint allows recombination between divergent DNA sequences in budding yeast

    PubMed Central

    George, Carolyn M.; Lyndaker, Amy M.; Alani, Eric

    2011-01-01

    In the early steps of homologous recombination, single-stranded DNA (ssDNA) from a broken chromosome invades homologous sequence located in a sister or homolog donor. In genomes that contain numerous repetitive DNA elements or gene paralogs, recombination can potentially occur between non-allelic/divergent (homeologous) sequences that share sequence identity. Such recombination events can lead to lethal chromosomal deletions or rearrangements. However, homeologous recombination events can be suppressed through rejection mechanisms that involve recognition of DNA mismatches in heteroduplex DNA by mismatch repair factors, followed by active unwinding of the heteroduplex DNA by helicases. Because factors required for heteroduplex rejection are hypothesized to be targets and/or effectors of the DNA damage response (DDR), a cell cycle control mechanism that ensures timely and efficient repair, we tested whether the DDR, and more specifically, the RAD9 gene, had a role in regulating rejection. We performed these studies using a DNA repair assay that measures repair by single-strand annealing (SSA) of a double-strand break (DSB) using homeologous DNA templates. We found that repair of homeologous DNA sequences, but not identical sequences, induced a RAD9- dependent cell cycle delay in the G2 stage of the cell cycle. Repair through a divergent DNA template occurred more frequently in RAD9 compared to rad9Δ strains. However, repair in rad9Δ mutants could be restored to wild-type levels if a G2 delay was induced by nocodazole. These results suggest that cell cycle arrest induced by the Rad9-dependent DDR allows repair between divergent DNA sequences despite the potential for creating deleterious genome rearrangements, and illustrates the importance of additional cellular mechanisms that act to suppress recombination between divergent DNA sequences. PMID:21978436

  5. Stable yeast transformants that secrete functional. cap alpha. -amylase encoded by cloned mouse pancreatic cDNA

    SciTech Connect

    Filho, S.A.; Galembeck, E.V.; Faria, J.B.; Frascino, A.C.S.

    1986-04-01

    Mouse pancreatic ..cap alpha..-amylase complementary DNA was inserted into a yeast shuttle vector after the Saccharomyces cerevisiae MF..cap alpha..1 promoter and secretion signals coding sequences. When transformed with the recombinant plasmid, S. cerevisiae cells were able to synthesize and secrete functional ..cap alpha..-amylase, efficiently hydrolyzing starch present in the culture medium. Stable amylolytic cells were obtained from different yeast strains. This work represents a significant step towards producing yeast that can convert starchy materials directly to ethanol.

  6. DNA topoisomerase-targeting antitumor drugs can be studied in yeast.

    PubMed Central

    Nitiss, J; Wang, J C

    1988-01-01

    The antitumor drugs camptothecin and an anilinoacridine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), which act on DNA topoisomerase I and II, respectively, are shown to inhibit the growth of Saccharomyces cerevisiae mutants selected for their permeability to other inhibitors. In addition to growth inhibition, these drugs induce high levels of homologous recombination and induce the expression of a DNA damage-inducible gene DIN3. Cytotoxicity of the drugs is more pronounced in strains that also carry a rad52 mutation. An analog of mAMSA), which is ineffective as an inhibitor of DNA topoisomerase II in mammalian cells, is also ineffective in eliciting physiological responses in these yeast strains. The physiological effects of camptothecin, but not those of mAMSA, disappear if the TOP1 gene encoding DNA topoisomerase I is disrupted. This shows that DNA topoisomerase I is the sole target of camptothecin cytotoxicity and illustrates that a nonessential enzyme can nevertheless be the target for a cytotoxic drug. PMID:2845409

  7. The role of recombination and RAD52 in mutation of chromosomal DNA transformed into yeast.

    PubMed Central

    Larionov, V; Graves, J; Kouprina, N; Resnick, M A

    1994-01-01

    While transformation is a prominent tool for genetic analysis and genome manipulation in many organisms, transforming DNA has often been found to be unstable relative to established molecules. We determined the potential for transformation-associated mutations in a 360 kb yeast chromosome III composed primarily of unique DNA. Wild-type and rad52 Saccharomyces cerevisiae strains were transformed with either a homologous chromosome III or a diverged chromosome III from S. carlsbergensis. The host strain chromosome III had a conditional centromere allowing it to be lost on galactose medium so that recessive mutations in the transformed chromosome could be identified. Following transformation of a RAD+ strain with the homologous chromosome, there were frequent changes in the incoming chromosome, including large deletions and mutations that do not lead to detectable changes in chromosome size. Based on results with the diverged chromosome, interchromosomal recombinational interactions were the source of many of the changes. Even though rad52 exhibits elevated mitotic mutation rates, the percentage of transformed diverged chromosomes incapable of substituting for the resident chromosome was not increased in rad52 compared to the wild-type strain, indicating that the mutator phenotype does not extend to transforming chromosomal DNA. Based on these results and our previous observation that the incidence of large mutations is reduced during the cloning of mammalian DNA into a rad52 as compared to a RAD+ strain, a rad52 host is well-suited for cloning DNA segments in which gene function must be maintained. Images PMID:7937151

  8. Yeast Rad17/Mec3/Ddc1: A sliding clamp for the DNA damage checkpoint

    PubMed Central

    Majka, Jerzy; Burgers, Peter M. J.

    2003-01-01

    The Saccharomyces cerevisiae Rad24 and Rad17 checkpoint proteins are part of an early response to DNA damage in a signal transduction pathway leading to cell cycle arrest. Rad24 interacts with the four small subunits of replication factor C (RFC) to form the RFC-Rad24 complex. Rad17 forms a complex with Mec3 and Ddc1 (Rad17/3/1) and shows structural similarities with the replication clamp PCNA. This parallelism with a clamp-clamp loader system that functions in DNA replication has led to the hypothesis that a similar clamp-clamp loader relationship exists for the DNA damage response system. We have purified the putative checkpoint clamp loader RFC-Rad24 and the putative clamp Rad17/3/1 from a yeast overexpression system. Here, we provide experimental evidence that, indeed, the RFC-Rad24 clamp loader loads the Rad17/3/1 clamp around partial duplex DNA in an ATP-dependent process. Furthermore, upon ATP hydrolysis, the Rad17/3/1 clamp is released from the clamp loader and can slide across more than 1 kb of duplex DNA, a process which may be well suited for a search for damage. Rad17/3/1 showed no detectable exonuclease activity. PMID:12604797

  9. Yeast RNA polymerase II transcription of circular DNA at different degrees of supercoiling.

    PubMed Central

    Pedone, F; Filetici, P; Ballario, P

    1982-01-01

    Purified yeast RNA polymerase II was tested for transcriptional activity as a function of the degree of circular DNA supercoiling. Chimaeric plasmids P30 and P31 both containing inserts from the yeast transposable element TY1 cloned in pBR322 and the vector pBR322 were used as templates. For pBR322 the transcriptional activity increases about 4 fold from the fully relaxed covalently closed circles to the native supercoiled forms, further supercoiling having no effect on transcription. P30 shows a 5 fold increase of transcriptional activity reaching a plateau at the native supercoiled conformation. However, at an intermediate degree of supercoiling (sigma = 0.024), transcription decreases to a value close to zero. P31 too exhibits a conformation (sigma = 0.014) in which there is a drop of transcriptional activity. Furthermore, a 10 fold increase of transcription is obtained at the higher values of superhelix density. Both kinetic and autoradiographic experiments confirm the existence of DNA conformations that can inhibit "in vitro" transcription. Images PMID:6292834

  10. Calcium-mediated DNA adsorption to yeast cells and kinetics of cell transformation by electroporation.

    PubMed Central

    Neumann, E; Kakorin, S; Tsoneva, I; Nikolova, B; Tomov, T

    1996-01-01

    Detailed kinetic data suggest that the direct transfer of plasmid DNA (YEp 351, 5.6 kbp, supercoiled, Mr approximately 3.5 x 10(6)) by membrane electroporation of yeast cells (Saccharomyces cerevisiae, strain AH 215) is mainly due to electrodiffusive processes. The rate-limiting step for the cell transformation, however, is a bimolecular DNA-binding interaction in the cell interior. Both the adsorption of DNA, directly measured with [32P]dCTP DNA, and the number of transformants are collinearly enhanced with increasing total concentrations [Dt] and [Cat] of DNA and of calcium, respectively. At [Cat] = 1 mM, the half-saturation or equilibrium constant is KD = 15 +/- 1 nM at 293 K (20 degrees C). The optimal transformation frequency is TFopt = 4.1 +/- 0.4 X 10(-5) if a single exponential pulse of initial field strength E0 = 4 kV cm-1 and decay time constant tauE = 45 ms is applied at [Dt] = 2.7 nM and 10(8) cells in 0.1 ml. The dependence of TF on [Cat] yields the equilibrium constants KCazero = 1.8 +/- 0.2 mM (in the absence of DNA) and K'Ca (at 2.7 nM DNA), comparable with and derived from electrophoresis data. In yeast cells, too, the appearance of a DNA molecule in its whole length in the cell interior is clearly an after-field event. At Eo = 4.0 kV cm-1 and T = 293 K, the flow coefficient of DNA through the porous membrane patches is Kto = 7.0 +/- 0.7 x 10(3)S-1 and the electrodiffusion of DNA is approximately 10 times more effective than simple diffusion: D/D0 approximately 10.3. The mean radius of these pores is rp = 0.39 +/- 0.05 nm, and the mean number of pores per cell (of size ø approximately 5.5 microns) is Np = 2.2 +/- 0.2 x 10(4). The maximal membrane area that is involved in the electrodiffusive penetration of adsorbed DNA into the outer surface of the electroporated cell membrane patches is only 0.023% of the total cell surface. The surface penetration is followed either by additional electrodiffusive or by passive (after-field) diffusive

  11. Yeast phenotype classifies mammalian protein kinase C cDNA mutants.

    PubMed Central

    Riedel, H; Su, L; Hansen, H

    1993-01-01

    The phorbol ester receptor protein kinase C (PKC) gene family encodes essential mediators of eukaryotic cellular signals. Molecular dissection of their mechanisms of action has been limited in part by the lack of random mutagenesis approaches and by the complexity of signaling pathways in mammalian cells which involve multiple PKC isoforms. Here we present a rapid screen which permits the quantification of mammalian PKC activity phenotypically in the yeast Saccharomyces cerevisiae. Bovine PKC alpha cDNA is functionally expressed in S. cerevisiae. This results in a phorbol ester response: a fourfold increase in the cell doubling time and a substantial decrease in yeast colony size on agar plates. We have expressed pools of bovine PKC alpha cDNAs mutagenized by Bal 31 deletion of internal, amino-terminal, or carboxyl-terminal sequences and have identified three classes of mutants on the basis of their distinct yeast phenotypes. Representatives of each class were analyzed. An internal deletion of amino acids (aa) 172 to 225 displayed ligand-dependent but reduced catalytic activity, an amino-terminal truncation of aa 1 to 153 displayed elevated and ligand-independent activity, and a carboxyl-terminal 26-aa truncation (aa 647 to 672) lacked activity under any conditions. Additional mutations confirmed the distinct functional characteristics of these classes. Our data show that deletion of the V1 and C1 regions results in elevated basal catalytic activity which is still Ca2+ responsive. Internal deletions in the V2 and C2 regions do not abolish phorbol ester or Ca2+ regulation of PKC activity, suggesting that most of the C2 domain is not essential for phorbol ester stimulation and most of the regulatory domain is dispensable for Ca2+ regulation of PKC activity. These distinct activities od the PKC mutants correlate with a specific and proportional yeast phenotype and are quantified on agar plates by yeast colony size. This provides a phenotypic screen which is suitable

  12. Characterization of a DNA uptake reaction through the nuclear membrane of isolated yeast nuclei. [Saccharomyces cerevisiae

    SciTech Connect

    Tsuchiya, E.; Shakuto, S.; Miyakawa, T.; Fukui, S.

    1988-02-01

    Isolated yeast nuclei were able to incorporate /sup 3/H-labeled pJDB219 DNA in vitro in the presence of ATP and Mg/sup 2 +/. The number of plasmid molecules incorporated into each nucleus was calculated to be 60 under the conditions we used. Enzyme-histochemical staining of the incorporated biotinylated pJDB219 with streptavidin-biotinylated-peroxidase complex indicated a uniform distribution of the incorporated plasmids within each nucleus. After intranuclear incorporation, substrate pJDB219 DNAs (open and closed circular forms) were changed to the linear form and were weakly digested over the longer incubation period (over 60 min). Facile release of the once-incorporated plasmid DNA was never observable; discharge of the incorporated (/sup 3/H)pJDB219 during a 60-min incubation was less than 5%. The addition of adenylyl-imidodiphosphate, N,N'-dicyclohexylcarbodiimide (DCCD), or quercetin inhibited in vitro DNA uptake reaction. DCCD and quercetin inhibited the nuclear ATPase and apparent protein kinase, respectively; hence, the involvement of these enzymes in the nuclear DNA transport system was suggested.

  13. The fission yeast CENP-B protein Abp1 prevents pervasive transcription of repetitive DNA elements.

    PubMed

    Daulny, Anne; Mejía-Ramírez, Eva; Reina, Oscar; Rosado-Lugo, Jesus; Aguilar-Arnal, Lorena; Auer, Herbert; Zaratiegui, Mikel; Azorin, Fernando

    2016-10-01

    It is well established that eukaryotic genomes are pervasively transcribed producing cryptic unstable transcripts (CUTs). However, the mechanisms regulating pervasive transcription are not well understood. Here, we report that the fission yeast CENP-B homolog Abp1 plays an important role in preventing pervasive transcription. We show that loss of abp1 results in the accumulation of CUTs, which are targeted for degradation by the exosome pathway. These CUTs originate from different types of genomic features, but the highest increase corresponds to Tf2 retrotransposons and rDNA repeats, where they map along the entire elements. In the absence of abp1, increased RNAPII-Ser5P occupancy is observed throughout the Tf2 coding region and, unexpectedly, RNAPII-Ser5P is enriched at rDNA repeats. Loss of abp1 also results in Tf2 derepression and increased nucleolus size. Altogether these results suggest that Abp1 prevents pervasive RNAPII transcription of repetitive DNA elements (i.e., Tf2 and rDNA repeats) from internal cryptic sites. PMID:27345571

  14. Infectious RNA transcripts from full-length dengue virus type 2 cDNA clones made in yeast.

    PubMed Central

    Polo, S; Ketner, G; Levis, R; Falgout, B

    1997-01-01

    The dengue virus type 2 genomic RNA was amplified by reverse transcription-PCR and cloned as four cDNA fragments. We could not assemble these four fragments into full-length cDNA in Escherichia coli. The full-length dengue virus cDNA was constructed by homologous recombination in yeast, either as part of a yeast artificial chromosome or in a yeast-E. coli shuttle vector. Full-length cDNA clones were propagated once in E. coli to prepare useful quantities of DNA. In vitro transcription of these clones produced full-length RNA transcripts. Introduction of these transcripts into LLC-MK2 cells produced typical dengue infection, as judged by cytopathic effects and indirect immunofluorescence. Infectivity was sensitive to RNase digestion and was dependent on the presence of cap analog in the transcription reaction mixture. Virus in the medium was passaged on C6-36 cells to produce stocks, and these stocks had titers and plaque morphologies similar to those of the parental dengue virus type 2. Intracellular dengue virus RNA from cells infected with transcript-derived virus contained an introduced BstEII site, proving that infectivity was derived from RNA transcripts and not from contamination with parental dengue virus. Transcript-derived virus was comparable to dengue virus type 2 for growth and protein expression in tissue culture cells. Sequence analysis of the dengue virus cDNA in one full-length clone revealed only one unexpected silent mutation. By using yeast technology, it will be easy to introduce specific mutations into the dengue virus cDNA, allowing analysis of the virus phenotype in cells transfected with mutant transcripts. PMID:9188607

  15. Physical properties of naked DNA influence nucleosome positioning and correlate with transcription start and termination sites in yeast

    PubMed Central

    2011-01-01

    Background In eukaryotic organisms, DNA is packaged into chromatin structure, where most of DNA is wrapped into nucleosomes. DNA compaction and nucleosome positioning have clear functional implications, since they modulate the accessibility of genomic regions to regulatory proteins. Despite the intensive research effort focused in this area, the rules defining nucleosome positioning and the location of DNA regulatory regions still remain elusive. Results Naked (histone-free) and nucleosomal DNA from yeast were digested by microccocal nuclease (MNase) and sequenced genome-wide. MNase cutting preferences were determined for both naked and nucleosomal DNAs. Integration of their sequencing profiles with DNA conformational descriptors derived from atomistic molecular dynamic simulations enabled us to extract the physical properties of DNA on a genomic scale and to correlate them with chromatin structure and gene regulation. The local structure of DNA around regulatory regions was found to be unusually flexible and to display a unique pattern of nucleosome positioning. Ab initio physical descriptors derived from molecular dynamics were used to develop a computational method that accurately predicts nucleosome enriched and depleted regions. Conclusions Our experimental and computational analyses jointly demonstrate a clear correlation between sequence-dependent physical properties of naked DNA and regulatory signals in the chromatin structure. These results demonstrate that nucleosome positioning around TSS (Transcription Start Site) and TTS (Transcription Termination Site) (at least in yeast) is strongly dependent on DNA physical properties, which can define a basal regulatory mechanism of gene expression. PMID:21981773

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

    PubMed

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

    2014-07-01

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

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

    PubMed Central

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

    2014-01-01

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

  18. Human DNA-Damage-Inducible 2 Protein Is Structurally and Functionally Distinct from Its Yeast Ortholog

    PubMed Central

    Sivá, Monika; Svoboda, Michal; Veverka, Václav; Trempe, Jean-François; Hofmann, Kay; Kožíšek, Milan; Hexnerová, Rozálie; Sedlák, František; Belza, Jan; Brynda, Jiří; Šácha, Pavel; Hubálek, Martin; Starková, Jana; Flaisigová, Iva; Konvalinka, Jan; Šašková, Klára Grantz

    2016-01-01

    Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization. PMID:27461074

  19. Insertion mutagenesis of the yeast Candida famata (Debaryomyces hansenii) by random integration of linear DNA fragments.

    PubMed

    Dmytruk, Kostyantyn V; Voronovsky, Andriy Y; Sibirny, Andriy A

    2006-09-01

    The feasibility of using random insertional mutagenesis to isolate mutants of the flavinogenic yeast Candida famata was explored. Mutagenesis was performed by transformation of the yeast with an integrative plasmid containing the Saccharomyces cerevisiae LEU2 gene as a selective marker. The addition of restriction enzyme together with the plasmid (restriction enzyme-mediated integration, REMI) increased the transformation frequency only slightly. Integration of the linearized plasmid occurred randomly in the C. famata genome. To investigate the potential of insertional mutagenesis, it was used for tagging genes involved in positive regulation of riboflavin synthesis in C. famata. Partial DNA sequencing of tagged genes showed that they were homologous to the S. cerevisiae genes RIB1, MET2, and SEF1. Intact orthologs of these genes isolated from Debaryomyces hansenii restored the wild phenotype of the corresponding mutants, i.e., the ability to overproduce riboflavin under iron limitation. The Staphylococcus aureus ble gene conferring resistance to phleomycin was used successfully in the study as a dominant selection marker for C. famata. The results obtained indicate that insertional mutagenesis is a powerful tool for tagging genes in C. famata. PMID:16770625

  20. Human DNA-Damage-Inducible 2 Protein Is Structurally and Functionally Distinct from Its Yeast Ortholog.

    PubMed

    Sivá, Monika; Svoboda, Michal; Veverka, Václav; Trempe, Jean-François; Hofmann, Kay; Kožíšek, Milan; Hexnerová, Rozálie; Sedlák, František; Belza, Jan; Brynda, Jiří; Šácha, Pavel; Hubálek, Martin; Starková, Jana; Flaisigová, Iva; Konvalinka, Jan; Šašková, Klára Grantz

    2016-01-01

    Although Ddi1-like proteins are conserved among eukaryotes, their biological functions remain poorly characterized. Yeast Ddi1 has been implicated in cell cycle regulation, DNA-damage response, and exocytosis. By virtue of its ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains, it has been proposed to serve as a proteasomal shuttle factor. All Ddi1-like family members also contain a highly conserved retroviral protease-like (RVP) domain with unknown substrate specificity. While the structure and biological function of yeast Ddi1 have been investigated, no such analysis is available for the human homologs. To address this, we solved the 3D structures of the human Ddi2 UBL and RVP domains and identified a new helical domain that extends on either side of the RVP dimer. While Ddi1-like proteins from all vertebrates lack a UBA domain, we identify a novel ubiquitin-interacting motif (UIM) located at the C-terminus of the protein. The UIM showed a weak yet specific affinity towards ubiquitin, as did the Ddi2 UBL domain. However, the full-length Ddi2 protein is unable to bind to di-ubiquitin chains. While proteomic analysis revealed no activity, implying that the protease requires other factors for activation, our structural characterization of all domains of human Ddi2 sets the stage for further characterization. PMID:27461074

  1. Quantitative epigenetics through epigenomic perturbation of isogenic lines.

    PubMed

    Johannes, Frank; Colomé-Tatché, Maria

    2011-05-01

    Interindividual differences in chromatin states at a locus (epialleles) can result in gene expression changes that are sometimes transmitted across generations. In this way, they can contribute to heritable phenotypic variation in natural and experimental populations independent of DNA sequence. Recent molecular evidence shows that epialleles often display high levels of transgenerational instability. This property gives rise to a dynamic dimension in phenotypic inheritance. To be able to incorporate these non-Mendelian features into quantitative genetic models, it is necessary to study the induction and the transgenerational behavior of epialleles in controlled settings. Here we outline a general experimental approach for achieving this using crosses of epigenomically perturbed isogenic lines in mammalian and plant species. We develop a theoretical description of such crosses and model the relationship between epiallelic instability, recombination, parent-of-origin effects, as well as transgressive segregation and their joint impact on phenotypic variation across generations. In the limiting case of fully stable epialleles our approach reduces to the classical theory of experimental line crosses and thus illustrates a fundamental continuity between genetic and epigenetic inheritance. We consider data from a panel of Arabidopsis epigenetic recombinant inbred lines and explore estimates of the number of quantitative trait loci for plant height that resulted from a manipulation of DNA methylation levels in one of the two isogenic founder strains. PMID:21385727

  2. Coordination of DNA damage tolerance mechanisms with cell cycle progression in fission yeast

    PubMed Central

    Callegari, A. John; Kelly, Thomas J.

    2016-01-01

    ABSTRACT DNA damage tolerance (DDT) mechanisms allow cells to synthesize a new DNA strand when the template is damaged. Many mutations resulting from DNA damage in eukaryotes are generated during DDT when cells use the mutagenic translesion polymerases, Rev1 and Polζ, rather than mechanisms with higher fidelity. The coordination among DDT mechanisms is not well understood. We used live-cell imaging to study the function of DDT mechanisms throughout the cell cycle of the fission yeast Schizosaccharomyces pombe. We report that checkpoint-dependent mitotic delay provides a cellular mechanism to ensure the completion of high fidelity DDT, largely by homology-directed repair (HDR). DDT by mutagenic polymerases is suppressed during the checkpoint delay by a mechanism dependent on Rad51 recombinase. When cells pass the G2/M checkpoint and can no longer delay mitosis, they completely lose the capacity for HDR and simultaneously exhibit a requirement for Rev1 and Polζ. Thus, DDT is coordinated with the checkpoint response so that the activity of mutagenic polymerases is confined to a vulnerable period of the cell cycle when checkpoint delay and HDR are not possible. PMID:26652183

  3. The C-Terminal Domain of Yeast PCNA Is Required for Physical And Functional Interactions With Cdc9 DNA Ligase

    SciTech Connect

    Vijayakumar, S.; Chapados, B.R.; Schmidt, K.H.; Kolodner, R.D.; Tainer, J.A.; Tomkinson, A.E.

    2007-07-13

    There is compelling evidence that proliferating cell nuclear antigen (PCNA), a DNA sliding clamp, co-ordinates the processing and joining of Okazaki fragments during eukaryotic DNA replication. However, a detailed mechanistic understanding of functional PCNA:ligase I interactions has been incomplete. Here we present the co-crystal structure of yeast PCNA with a peptide encompassing the conserved PCNA interaction motif of Cdc9, yeast DNA ligase I. The Cdc9 peptide contacts both the inter-domain connector loop (IDCL) and residues near the C-terminus of PCNA. Complementary mutational and biochemical results demonstrate that these two interaction interfaces are required for complex formation both in the absence of DNA and when PCNA is topologically linked to DNA. Similar to the functionally homologous human proteins, yeast RFC interacts with and inhibits Cdc9 DNA ligase whereas the addition of PCNA alleviates inhibition by RFC. Here we show that the ability of PCNA to overcome RFC-mediated inhibition of Cdc9 is dependent upon both the IDCL and the C-terminal interaction interfaces of PCNA. Together these results demonstrate the functional significance of the {beta}-zipper structure formed between the C-terminal domain of PCNA and Cdc9 and reveal differences in the interactions of FEN-1 and Cdc9 with the two PCNA interfaces that may contribute to the coordinated, sequential action of these enzymes.

  4. Expression cloning in yeast of a cDNA encoding a broad specificity amino acid permease from Arabidopsis thaliana.

    PubMed Central

    Frommer, W B; Hummel, S; Riesmeier, J W

    1993-01-01

    To study amino acid transport in plants at the molecular level, we have isolated an amino acid permease cDNA from Arabidopsis thaliana by complementation of a yeast mutant defective in proline uptake with a cDNA. The predicted polypeptide of 53 kDa is highly hydrophobic with 12 putative membrane-spanning regions and shows no significant homologies to other known transporters. Expression of the cDNA enables the yeast mutant to take up L-[14C]proline. Competition studies argue for a broad but stereospecific substrate recognition by the permease, which resembles neutral or general amino acid transport systems from Chlorella and higher plants. Both pH dependence and inhibition by protonophores are consistent with a proton symport mechanism. Images Fig. 1 PMID:8327465

  5. Replication of single-stranded DNA templates by primase-polymerase complexes of the yeast, Saccharomyces cerevisiae.

    PubMed Central

    Biswas, E E; Biswas, S B

    1988-01-01

    A partially purified primase-polymerase complex from the yeast, Saccharomyces cerevisiae, was capable of replicating a single stranded circular phage DNA into a replicative form with high efficiency. The primase-polymerase complex exhibited primase activity and polymerase activity on singly primed circular ssDNA as well as on gapped DNA. In addition, it was able to replicate an unprimed, single-stranded, circular phage DNA through a coupled primase-polymerase action. On Biogel A-O.5m filtration the primase-polymerase activities appeared in the void volume, demonstrating a mass of greater than 500 kilodaltons. Primase and various primase-polymerase complexes synthesized unique primers on single stranded DNA templates and the size distribution of primers was dependent on the structure of the DNA and the nature of the primase-polymerase assembly. Images PMID:3041377

  6. A role for DNA polymerase alpha in epigenetic control of transcriptional silencing in fission yeast.

    PubMed

    Nakayama Ji; Allshire, R C; Klar, A J; Grewal, S I

    2001-06-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase alpha (pol(alpha)) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that pol(alpha) mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polalpha and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Pol(alpha) in vitro. Moreover, silencing-defective mutant Pol(alpha) displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Pol(alpha), in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  7. Identification of S-phase DNA damage-response targets in fission yeast reveals conservation of damage-response networks.

    PubMed

    Willis, Nicholas A; Zhou, Chunshui; Elia, Andrew E H; Murray, Johanne M; Carr, Antony M; Elledge, Stephen J; Rhind, Nicholas

    2016-06-28

    The cellular response to DNA damage during S-phase regulates a complicated network of processes, including cell-cycle progression, gene expression, DNA replication kinetics, and DNA repair. In fission yeast, this S-phase DNA damage response (DDR) is coordinated by two protein kinases: Rad3, the ortholog of mammalian ATR, and Cds1, the ortholog of mammalian Chk2. Although several critical downstream targets of Rad3 and Cds1 have been identified, most of their presumed targets are unknown, including the targets responsible for regulating replication kinetics and coordinating replication and repair. To characterize targets of the S-phase DDR, we identified proteins phosphorylated in response to methyl methanesulfonate (MMS)-induced S-phase DNA damage in wild-type, rad3∆, and cds1∆ cells by proteome-wide mass spectrometry. We found a broad range of S-phase-specific DDR targets involved in gene expression, stress response, regulation of mitosis and cytokinesis, and DNA replication and repair. These targets are highly enriched for proteins required for viability in response to MMS, indicating their biological significance. Furthermore, the regulation of these proteins is similar in fission and budding yeast, across 300 My of evolution, demonstrating a deep conservation of S-phase DDR targets and suggesting that these targets may be critical for maintaining genome stability in response to S-phase DNA damage across eukaryotes. PMID:27298342

  8. Use of yeast nuclear DNA sequences to define the mitochondrial RNA polymerase promoter in vitro.

    PubMed Central

    Marczynski, G T; Schultz, P W; Jaehning, J A

    1989-01-01

    We have extended an earlier observation that the TATA box for the nuclear GAL10 gene serves as a promoter for the mitochondrial RNA polymerase in in vitro transcription reactions (C. S. Winkley, M. J. Keller, and J. A. Jaehning, J. Biol. Chem. 260:14214-14223, 1985). In this work, we demonstrate that other nuclear genes also have upstream sequences that function in vitro as mitochondrial RNA polymerase promoters. These genes include the GAL7 and MEL1 genes, which are regulated in concert with the GAL10 gene, the sigma repetitive element, and the 2 microns plasmid origin of replication. We used in vitro transcription reactions to test a large number of nuclear DNA sequences that contain critical mitochondrial promoter sequences as defined by Biswas et al. (T. K. Biswas, J. C. Edwards, M. Rabinowitz, and G. S. Getz, J. Biol. Chem. 262:13690-13696, 1987). The results of these experiments allowed us to extend the definition of essential promoter elements. This extended sequence, -ACTATAAACGatcATAG-, was frequently found in the upstream regulatory regions of nuclear genes. On the basis of these observations, we hypothesized that either (i) a catalytic RNA polymerase related to the mitochondrial enzyme functions in the nucleus of the yeast cell or (ii) a DNA sequence recognition factor is shared by the two genetic compartments. By using cells deficient in the catalytic core of the mitochondrial RNA polymerase (rpo41-) and sensitive assays for transcripts initiating from the nuclear promoter sequences, we have conclusively ruled out a role for the catalytic RNA polymerase in synthesizing transcripts from all of the nuclear sequences analyzed. The possibility that a DNA sequence recognition factor functions in both the nucleus and the mitochondria remains to be tested. Images PMID:2677667

  9. Prevalence and Dynamics of Ribosomal DNA Micro-heterogeneity Are Linked to Population History in Two Contrasting Yeast Species

    PubMed Central

    James, Stephen A.; West, Claire; Davey, Robert P.; Dicks, Jo; Roberts, Ian N.

    2016-01-01

    Despite the considerable number and taxonomic breadth of past and current genome sequencing projects, many of which necessarily encompass the ribosomal DNA, detailed information on the prevalence and evolutionary significance of sequence variation in this ubiquitous genomic region are severely lacking. Here, we attempt to address this issue in two closely related yet contrasting yeast species, the baker’s yeast Saccharomyces cerevisiae and the wild yeast Saccharomyces paradoxus. By drawing on existing datasets from the Saccharomyces Genome Resequencing Project, we identify a rich seam of ribosomal DNA sequence variation, characterising 1,068 and 970 polymorphisms in 34 S. cerevisiae and 26 S. paradoxus strains respectively. We discover the two species sets exhibit distinct mutational profiles. Furthermore, we show for the first time that unresolved rDNA sequence variation resulting from imperfect concerted evolution of the ribosomal DNA region follows a U-shaped allele frequency distribution in each species, similar to loci that evolve under non-concerted mechanisms but arising through rather different evolutionary processes. Finally, we link differences between the shapes of these allele frequency distributions to the two species’ contrasting population histories. PMID:27345953

  10. Prevalence and Dynamics of Ribosomal DNA Micro-heterogeneity Are Linked to Population History in Two Contrasting Yeast Species.

    PubMed

    James, Stephen A; West, Claire; Davey, Robert P; Dicks, Jo; Roberts, Ian N

    2016-01-01

    Despite the considerable number and taxonomic breadth of past and current genome sequencing projects, many of which necessarily encompass the ribosomal DNA, detailed information on the prevalence and evolutionary significance of sequence variation in this ubiquitous genomic region are severely lacking. Here, we attempt to address this issue in two closely related yet contrasting yeast species, the baker's yeast Saccharomyces cerevisiae and the wild yeast Saccharomyces paradoxus. By drawing on existing datasets from the Saccharomyces Genome Resequencing Project, we identify a rich seam of ribosomal DNA sequence variation, characterising 1,068 and 970 polymorphisms in 34 S. cerevisiae and 26 S. paradoxus strains respectively. We discover the two species sets exhibit distinct mutational profiles. Furthermore, we show for the first time that unresolved rDNA sequence variation resulting from imperfect concerted evolution of the ribosomal DNA region follows a U-shaped allele frequency distribution in each species, similar to loci that evolve under non-concerted mechanisms but arising through rather different evolutionary processes. Finally, we link differences between the shapes of these allele frequency distributions to the two species' contrasting population histories. PMID:27345953

  11. Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids.

    PubMed

    Bakkaiova, Jana; Marini, Victoria; Willcox, Smaranda; Nosek, Jozef; Griffith, Jack D; Krejci, Lumir; Tomaska, Lubomir

    2016-01-01

    Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species. PMID:26647378

  12. Yeast mitochondrial HMG proteins: DNA-binding properties of the most evolutionarily divergent component of mitochondrial nucleoids

    PubMed Central

    Bakkaiova, Jana; Marini, Victoria; Willcox, Smaranda; Nosek, Jozef; Griffith, Jack D.; Krejci, Lumir; Tomaska, Lubomir

    2015-01-01

    Yeast mtDNA is compacted into nucleoprotein structures called mitochondrial nucleoids (mt-nucleoids). The principal mediators of nucleoid formation are mitochondrial high-mobility group (HMG)-box containing (mtHMG) proteins. Although these proteins are some of the fastest evolving components of mt-nucleoids, it is not known whether the divergence of mtHMG proteins on the level of their amino acid sequences is accompanied by diversification of their biochemical properties. In the present study we performed a comparative biochemical analysis of yeast mtHMG proteins from Saccharomyces cerevisiae (ScAbf2p), Yarrowia lipolytica (YlMhb1p) and Candida parapsilosis (CpGcf1p). We found that all three proteins exhibit relatively weak binding to intact dsDNA. In fact, ScAbf2p and YlMhb1p bind quantitatively to this substrate only at very high protein to DNA ratios and CpGcf1p shows only negligible binding to dsDNA. In contrast, the proteins exhibit much higher preference for recombination intermediates such as Holliday junctions (HJ) and replication forks (RF). Therefore, we hypothesize that the roles of the yeast mtHMG proteins in maintenance and compaction of mtDNA in vivo are in large part mediated by their binding to recombination/replication intermediates. We also speculate that the distinct biochemical properties of CpGcf1p may represent one of the prerequisites for frequent evolutionary tinkering with the form of the mitochondrial genome in the CTG-clade of hemiascomycetous yeast species. PMID:26647378

  13. The expression of the open reading frame of "Arabidopsis" CAX1, but not its cDNA, confers metal tolerance in yeast

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The biochemical properties and regulation of several plant CAX (CAtion eXchanger)-type vacuolar Ca (2+)/H (+) exchangers have been extensively analyzed in yeast expression assays. In the present study, we compare and contrast the phenotypes of yeast cells expressing the CAX1 cDNA and open reading fr...

  14. A chimeric vacuolar Na(+)/H(+) antiporter gene evolved by DNA family shuffling confers increased salt tolerance in yeast.

    PubMed

    Wu, Guangxia; Wang, Gang; Ji, Jing; Li, Yong; Gao, Hailing; Wu, Jiang; Guan, Wenzhu

    2015-06-10

    The vacuolar Na(+)/H(+) antiporter plays an important role in maintaining ionic homeostasis and the osmotic balance of the cell with the environment by sequestering excessive cytoplasmic Na(+) into the vacuole. However, the relatively low Na(+)/H(+) exchange efficiency of the identified Na(+)/H(+) antiporter could limit its application in the molecular breeding of salt tolerant crops. In this study, DNA family shuffling was used to create chimeric Na(+)/H(+) antiporters with improved transport activity. Two homologous Na(+)/H(+) antiporters from halophytes Salicornia europaea (SeNHX1) and Suaeda salsa (SsNHX1) were shuffled to generate a diverse gene library. Using a high-throughput screening system of yeast complementation, a novel chimeric protein SseNHX1 carrying 12 crossover positions and 2 point mutations at amino acid level was selected. Expression of SseNHX1 in yeast mutant exhibited approximately 46% and 22% higher salt tolerance ability in yeast growth test than that of SsNHX1and SeNHX1, respectively. Measurements of the ion contents demonstrated that SseNHX1 protein in yeast cells accumulated more Na(+) and slightly more K(+) than the parental proteins did. Furthermore, this chimera also conferred increased tolerance to LiCl and a similar tolerance to hygromycin B compared with the parental proteins in yeast. PMID:25784157

  15. RAP1 stimulates single- to double-strand association of yeast telomeric DNA: implications for telomere-telomere interactions.

    PubMed Central

    Gilson, E; Müller, T; Sogo, J; Laroche, T; Gasser, S M

    1994-01-01

    Repressor Activator Protein 1 (RAP1) of Saccharomyces cerevisiae is an abundant nuclear protein implicated in telomere length maintenance, transactivation, and in the establishment of silent chromatin domains. The RAP1 binding site 5' of the yeast HIS4 gene is also a region of hyperrecombination in meiosis. We report here that as RAP1 binds its recognition consensus, it appears to untwist double-stranded DNA, which we detect as the introduction of a negative supercoil in circularization assays. Coincident with the RAP1-dependent untwisting, we observe stimulation of the association of a single-stranded yeast telomeric sequence with its homologous double-stranded sequence in a supercoiled plasmid. This unusual distortion of the DNA double helix by RAP1 may contribute to the RAP1-dependent enhancement of recombination rates and promote non-duplex strand interactions at telomeres. Images PMID:7816621

  16. HRR25, a putative protein kinase from budding yeast: Association with repair of damaged DNA

    SciTech Connect

    Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.; Burbee, D.G. ); Liskay, R.M. ); Heffron, F. )

    1991-08-30

    In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH{sub 2}-terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily.

  17. Cloning yeast actin cDNA leads to an investigative approach for the molecular biology laboratory.

    PubMed

    Black, Michael W; Tuan, Alice; Jonasson, Erin

    2008-05-01

    The emergence of molecular tools in multiple disciplines has elevated the importance of undergraduate laboratory courses that train students in molecular biology techniques. Although it would also be desirable to provide students with opportunities to apply these techniques in an investigative manner, this is generally not possible in the classroom because of the preparation, expense, and logistics involved in independent student projects. The authors have designed a 10-week lab series that mimics the research environment by tying separate fundamental lab techniques to a common goal: to build a plasmid with yeast actin cDNA cloned in a particular orientation. In the process of completing this goal, a problem arises in that students are unable to obtain the target plasmid and instead only recover the gene cloned in the opposite orientation. To address this problem, students identify four plausible hypotheses and work in teams to address them by designing and executing experiments. This project reinforces the utility and flexibility of techniques covered earlier in the class and serves to develop their skills in experimental design and analysis. As the project is focused on one problem, the diversity of experimental approaches is limited and may be prepared in advance with little additional expense in reagents or technical support. PMID:21591194

  18. Identification of barriers to rotation of DNA segments in yeast from the topology of DNA rings excised by an inducible site-specific recombinase.

    PubMed Central

    Gartenberg, M R; Wang, J C

    1993-01-01

    Controlled excision of DNA segments to yield intracellular DNA rings of well-defined sequences was utilized to study the determinants of transcriptional supercoiling of closed circular DNA in the yeast Saccharomyces cerevisiae. In delta top1 top2ts strains of S. cerevisiae expressing Escherichia coli DNA topoisomerase I, accumulation of positive supercoils in intracellular DNA normally occurs upon thermal inactivation of DNA topoisomerase II because of the simultaneous generation of positively and negatively supercoiled domains by transcription and the preferential relaxation of the latter by the bacterial enzyme. Positive supercoil accumulation in DNA rings is shown to depend on the presence of specific sequence elements; one likely cause of this dependence is that the persistence of oppositely supercoiled domains in an intracellular DNA ring requires the presence of barriers to rotation of the DNA segments connecting the domains. Analysis of the S. cerevisiae 2-microns plasmid partition system by this approach suggests that the plasmid-encoded REP1 and REP2 proteins are involved in forming such a barrier in DNA containing the REP3 sequence. Images Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:8248138

  19. RNA Polymerase I and Fob1 contributions to transcriptional silencing at the yeast rDNA locus.

    PubMed

    Buck, Stephen W; Maqani, Nazif; Matecic, Mirela; Hontz, Robert D; Fine, Ryan D; Li, Mingguang; Smith, Jeffrey S

    2016-07-27

    RNA polymerase II (Pol II)-transcribed genes embedded within the yeast rDNA locus are repressed through a Sir2-dependent process called 'rDNA silencing'. Sir2 is recruited to the rDNA promoter through interactions with RNA polymerase I (Pol I), and to a pair of DNA replication fork block sites (Ter1 and Ter2) through interaction with Fob1. We utilized a reporter gene (mURA3) integrated adjacent to the leftmost rDNA gene to investigate localized Pol I and Fob1 functions in silencing. Silencing was attenuated by loss of Pol I subunits or insertion of an ectopic Pol I terminator within the adjacent rDNA gene. Silencing left of the rDNA array is naturally attenuated by the presence of only one intact Fob1 binding site (Ter2). Repair of the 2nd Fob1 binding site (Ter1) dramatically strengthens silencing such that it is no longer impacted by local Pol I transcription defects. Global loss of Pol I activity, however, negatively affects Fob1 association with the rDNA. Loss of Ter2 almost completely eliminates localized silencing, but is restored by artificially targeting Fob1 or Sir2 as Gal4 DNA binding domain fusions. We conclude that Fob1 and Pol I make independent contributions to establishment of silencing, though Pol I also reinforces Fob1-dependent silencing. PMID:27060141

  20. Analysis of repair and mutagenesis of chromium-induced DNA damage in yeast, mammalian cells, and transgenic mice.

    PubMed Central

    Cheng, L; Liu, S; Dixon, K

    1998-01-01

    Chromium (Cr) is a widespread environmental contaminant and a known human carcinogen. We have used shuttle vector systems in yeast, mammalian cells, and transgenic mice to characterize the mutational specificity and premutational DNA damage induced by Cr(VI) and its reduction intermediates in order to elucidate the mechanism by which Cr induces mutations. In the yeast system, treatment of vector-containing cells with Cr(VI) results in a dose-dependent increase in mutations in the SUP4-o target gene of the vector; mutagenesis is enhanced in an apn-1 yeast mutant, deficient in the capacity to repair oxidative-type DNA damage. In vector-containing mammalian cells, treatment with Cr(VI) also results in a dose-dependent increase in mutations in the vector target gene supF. The Cr-induced mutations in supF occurred mostly at G:C base pairs and were widely distributed across the gene, a pattern similar to those observed with ionizing radiation or hydrogen peroxide. These results support the hypothesis that Cr(VI)-induced oxidative-type DNA damage is responsible for Cr mutagenesis in the cell. Recently these studies were extended into the Big Blue transgenic mouse system in which Cr-induced mutagenesis was observed in the lung, the target organ for Cr carcinogenesis in humans. Analysis of the spectrum of these mutations will test whether Cr mutagenesis occurs by similar mechanisms in the intact animal as in cell culture systems and yeast. Images Figure 2 Figure 3 PMID:9703488

  1. Shared Genetic Pathways Contribute to the Tolerance of Endogenous and Low-Dose Exogenous DNA Damage in Yeast

    PubMed Central

    Lehner, Kevin; Jinks-Robertson, Sue

    2014-01-01

    DNA damage that escapes repair and blocks replicative DNA polymerases is tolerated by bypass mechanisms that fall into two general categories: error-free template switching and error-prone translesion synthesis. Prior studies of DNA damage responses in Saccharomyces cerevisiae have demonstrated that repair mechanisms are critical for survival when a single, high dose of DNA damage is delivered, while bypass/tolerance mechanisms are more important for survival when the damage level is low and continuous (acute and chronic damage, respectively). In the current study, epistatic interactions between DNA-damage tolerance genes were examined and compared when haploid yeast cells were exposed to either chronic ultraviolet light or chronic methyl methanesulfonate. Results demonstrate that genes assigned to error-free and error-prone bypass pathways similarly promote survival in the presence of each type of chronic damage. In addition to using defined sources of chronic damage, rates of spontaneous mutations generated by the Pol ζ translesion synthesis DNA polymerase (complex insertions in a frameshift-reversion assay) were used to infer epistatic interactions between the same genes. Similar epistatic interactions were observed in analyses of spontaneous mutation rates, suggesting that chronic DNA-damage responses accurately reflect those used to tolerate spontaneous lesions. These results have important implications when considering what constitutes a safe and acceptable level of exogenous DNA damage. PMID:25060101

  2. DNA-dependent DNA polymerase from yeast mitochondria. Dependence of enzyme activity on conditions of cell growth, and properties of the highly purified polymerase.

    PubMed

    Wintersberger, U; Blutsch, H

    1976-09-01

    The activity of DNA polymerase was determined in gradient-purified mitochondria from yeast cells grown under a variety of conditions. The specific enzyme activity was found to be dependent on the degree of aeration of the cells, and on the carbon source used for the medium. It was sensitive to glucose repression, and was enhanced about two-fold by the growth of yeast cells in the presence of ethidium bromide. Mitochondria DNA polymerase was highly purified and several properties were determined. Sucrose density gradient centrifugation, and dodecylsulfate-polyacylamide gel electrophoresis revealed the following structure: a monomer of molecular weight around 60 000 aggregated under relatively high salt concentration (0.2 M phosphate buffer) to a dimer of about 120 000 which under low salt concentration (0.2 M Tris-HCl buffer) formed higher aggregates. For optimal activity an Mg2+ ion concentration of 50 mM was found necessary, Mn ions did not promote activity at any concentration tested (0.5--50 mM). Indeed, if added to Mg2+-containing assays, Mn2+ strongly inhibited enzyme activity at low concentrations. This might be an explanation for the inducation of mitochondrial mutants in yeast cells grown in the presence of Mn2+ ions. Mitochondrial DNA polymerase activity was strongly inhibited by low concentrations of the -SH reagent p-chloromercuribenzoate, the nucleotide analogue cytosine arabinoside triphosphate also exerted an inhibitory effect. An about 50% decrease of activity was observed in the presence of 1 mM o-phenanthroline in assay mixture containing DNA at about the Km concentration. The enzyme preferred a gapped template primer, poly(dA) - (dT)10, over nicked DNA and was unable to use a polyribonucleotide template, poly(rA) - (dT)10. In the purest preparations no exonuclease activity could be detected. PMID:786635

  3. DNA Replication Forks Pause at Silent Origins near the HML Locus in Budding Yeast

    PubMed Central

    Wang, Yangzhou; Vujcic, Marija; Kowalski, David

    2001-01-01

    Chromosomal replicators in budding yeast contain an autonomously replicating sequence (ARS) that functions in a plasmid, but certain ARSs are silent as replication origins in their natural chromosomal context. In chromosome III, the HML ARS cluster (ARS302-ARS303-ARS320) and ARS301 flank the transcriptionally silent mating-type locus HML, and all of these ARSs are silent as replication origins. ARS301 and ARS302 function in transcriptional silencing mediated by the origin recognition complex (ORC) and a heterochromatin structure, while the functions of ARS303 and ARS320 are not known. In this work, we discovered replication fork pause sites at the HML ARS cluster and ARS301 by analyzing DNA replication intermediates from the chromosome via two-dimensional gel electrophoresis. The replication fork pause at the HML ARS cluster was independent of cis- and trans-acting mutations that abrogate transcriptional silencing at HML. Deletion of the HML ARS cluster led to loss of the pause site. Insertion of a single, heterologous ARS (ARS305) in place of the HML ARS cluster reconstituted the pause site, as did multiple copies of DNA elements (A and B1) that bind ORC. The orc2-1 mutation, known to alter replication timing at origins, did not detectably affect the pause but activated the silent origin at the HML ARS cluster in a minority of cells. Delaying the time of fork arrival at HML led to the elimination of the pause sites at the HML ARS cluster and at the copy of ARS305 inserted in place of the cluster. Loss of the pause sites was accompanied by activation of the silent origins in the majority of cells. Thus, replication fork movement near HML pauses at a silent origin which is competent for replication initiation but kept silent through Orc2p, a component of the replication initiator. Possible functions for replication fork pause sites in checkpoints, S-phase regulation, mating-type switching, and transcriptionally silent heterochromatin are discussed. PMID:11438651

  4. Isolation and DNA-binding characteristics of a protein involved in transcription activation of two divergently transcribed, essential yeast genes.

    PubMed Central

    Halfter, H; Müller, U; Winnacker, E L; Gallwitz, D

    1989-01-01

    We have identified a protein, BAF1, which has two oppositely oriented, partially overlapping binding sites within a symmetrical sequence located midway between and upstream of the divergently transcribed YPT1 and TUB2 genes of the yeast Saccharomyces cerevisiae. The 120 kd BAF1 protein was purified to near homogeneity and used to delineate the two binding sites and to identify apparent protein contact sites by the missing contact technique, methylation interference and by site-directed mutagenesis. The BAF1-recognition sequence contains a conserved TCN7ACG element recently identified at autonomously replicating sequences (ARS) and in the 5' and 3' flanking region of other yeast genes. The symmetrical sequence of the YPT1/TUB2 intergene region seems not to be involved in DNA replication but activates transcription in an orientation-independent fashion. Images PMID:2684633

  5. Chl1p, a DNA helicase-like protein in budding yeast, functions in sister-chromatid cohesion.

    PubMed Central

    Skibbens, Robert V

    2004-01-01

    From the time of DNA replication until anaphase onset, sister chromatids remain tightly paired along their length. Ctf7p/Eco1p is essential to establish sister-chromatid pairing during S-phase and associates with DNA replication components. DNA helicases precede the DNA replication fork and thus will first encounter chromatin sites destined for cohesion. In this study, I provide the first evidence that a DNA helicase is required for proper sister-chromatid cohesion. Characterizations of chl1 mutant cells reveal that CHL1 interacts genetically with both CTF7/ECO1 and CTF18/CHL12, two genes that function in sister-chromatid cohesion. Consistent with genetic interactions, Chl1p physically associates with Ctf7p/Eco1p both in vivo and in vitro. Finally, a functional assay reveals that Chl1p is critical for sister-chromatid cohesion. Within the budding yeast genome, Chl1p exhibits the highest degree of sequence similarity to human CHL1 isoforms and BACH1. Previous studies revealed that human CHLR1 exhibits DNA helicase-like activities and that BACH1 is a helicase-like protein that associates with the tumor suppressor BRCA1 to maintain genome integrity. Our findings document a novel role for Chl1p in sister-chromatid cohesion and provide new insights into the possible mechanisms through which DNA helicases may contribute to cancer progression when mutated. PMID:15020404

  6. Molecular visualization of the yeast Dmc1 protein ring and Dmc1-ssDNA nucleoprotein complex.

    PubMed

    Chang, Yuan-Chih; Lo, Yu-Hui; Lee, Ming-Hui; Leng, Chih-Hsiang; Hu, Su-Ming; Chang, Chia-Seng; Wang, Ting-Fang

    2005-04-26

    Saccharomyces cerevisiae Dmc1, a meiosis-specific homologue of RecA, catalyzes homologous pairing and strand exchange during meiotic DNA recombination. The purified budding yeast Dmc1 (ScDmc1) protein exhibits much weaker recombinase activity in vitro as compared to that of the Escherichia coli RecA protein. Using atomic force microscopy (AFM) with carbon nanotube tips, we found ScDmc1 forms rings with an external diameter of 18 nm and a central cavity of 4 nm. In the presence of single-stranded DNA (ssDNA), the majority of the ScDmc1 protein (90%) bound DNA as protein rings; only a small faction (10%) was able to form filamentous structure. In contrast, nearly all RecA proteins form fine helical nucleoprotein filaments with ssDNA under identical conditions. RecA-mediated recombinase activity is initiated through the nucleation of RecA onto ssDNA to form helical nucleoprotein filaments. Our results support the notion that ScDmc1 becomes catalytically active only when it forms a helical nucleoprotein filament with ssDNA. PMID:15835894

  7. The EGD1 product, a yeast homolog of human BTF3, may be involved in GAL4 DNA binding.

    PubMed Central

    Parthun, M R; Mangus, D A; Jaehning, J A

    1992-01-01

    A variety of techniques, including filter binding, footprinting, and gel retardation, can be used to assay the transcriptional activator GAL4 (Gal4p) through the initial steps of its purification from yeast cells. Following DNA affinity chromatography, Gal4p still bound DNA selectively when assayed by filter binding or footprinting. However, the affinity-purified protein was no longer capable of forming a stable complex with DNA, as assayed by gel retardation. Mixing the purified Gal4p with the flowthrough fraction from the DNA affinity column restored gel retardation complex formation. Gel retardation assays were used to monitor the purification of a heat-stable Gal4p-DNA complex stabilization activity from the affinity column flowthrough. The activity coeluted from the final purification step with polypeptides of 21 and 27 kDa. The yeast gene encoding the 21-kDa protein was cloned on the basis of its N-terminal amino acid sequence. The gene, named EGD1 (enhancer of GAL4 DNA binding), encodes a highly basic protein (21% lysine and arginine) with a predicted molecular mass of 16.5 kDa. The amino acid sequence of the EGD1 product, Egd1p, is highly similar to that of the human protein BTF3 (X. M. Zheng, D. Black, P. Chambon, and J. M. Egly, Nature [London] 344:556-559, 1990). Although an egd1 null mutant was viable and Gal+, induction of the galactose-regulated genes in the egd1 mutant strain was significantly reduced when cells were shifted from glucose to galactose. Images PMID:1448098

  8. A DNA polymerase alpha accessory protein, Mcl1, is required for propagation of centromere structures in fission yeast.

    PubMed

    Natsume, Toyoaki; Tsutsui, Yasuhiro; Sutani, Takashi; Dunleavy, Elaine M; Pidoux, Alison L; Iwasaki, Hiroshi; Shirahige, Katsuhiko; Allshire, Robin C; Yamao, Fumiaki

    2008-01-01

    Specialized chromatin exists at centromeres and must be precisely transmitted during DNA replication. The mechanisms involved in the propagation of these structures remain elusive. Fission yeast centromeres are composed of two chromatin domains: the central CENP-A(Cnp1) kinetochore domain and flanking heterochromatin domains. Here we show that fission yeast Mcl1, a DNA polymerase alpha (Pol alpha) accessory protein, is critical for maintenance of centromeric chromatin. In a screen for mutants that alleviate both central domain and outer repeat silencing, we isolated several cos mutants, of which cos1 is allelic to mcl1. The mcl1-101 mutation causes reduced CENP-A(Cnp1) in the central domain and an aberrant increase in histone acetylation in both domains. These phenotypes are also observed in a mutant of swi7(+), which encodes a catalytic subunit of Pol alpha. Mcl1 forms S-phase-specific nuclear foci, which colocalize with those of PCNA and Pol alpha. These results suggest that Mcl1 and Pol alpha are required for propagation of centromere chromatin structures during DNA replication. PMID:18493607

  9. Proteome-wide Identification of Novel Ceramide-binding Proteins by Yeast Surface cDNA Display and Deep Sequencing.

    PubMed

    Bidlingmaier, Scott; Ha, Kevin; Lee, Nam-Kyung; Su, Yang; Liu, Bin

    2016-04-01

    Although the bioactive sphingolipid ceramide is an important cell signaling molecule, relatively few direct ceramide-interacting proteins are known. We used an approach combining yeast surface cDNA display and deep sequencing technology to identify novel proteins binding directly to ceramide. We identified 234 candidate ceramide-binding protein fragments and validated binding for 20. Most (17) bound selectively to ceramide, although a few (3) bound to other lipids as well. Several novel ceramide-binding domains were discovered, including the EF-hand calcium-binding motif, the heat shock chaperonin-binding motif STI1, the SCP2 sterol-binding domain, and the tetratricopeptide repeat region motif. Interestingly, four of the verified ceramide-binding proteins (HPCA, HPCAL1, NCS1, and VSNL1) and an additional three candidate ceramide-binding proteins (NCALD, HPCAL4, and KCNIP3) belong to the neuronal calcium sensor family of EF hand-containing proteins. We used mutagenesis to map the ceramide-binding site in HPCA and to create a mutant HPCA that does not bind to ceramide. We demonstrated selective binding to ceramide by mammalian cell-produced wild type but not mutant HPCA. Intriguingly, we also identified a fragment from prostaglandin D2synthase that binds preferentially to ceramide 1-phosphate. The wide variety of proteins and domains capable of binding to ceramide suggests that many of the signaling functions of ceramide may be regulated by direct binding to these proteins. Based on the deep sequencing data, we estimate that our yeast surface cDNA display library covers ∼60% of the human proteome and our selection/deep sequencing protocol can identify target-interacting protein fragments that are present at extremely low frequency in the starting library. Thus, the yeast surface cDNA display/deep sequencing approach is a rapid, comprehensive, and flexible method for the analysis of protein-ligand interactions, particularly for the study of non-protein ligands. PMID

  10. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.

    PubMed

    Saparbaev, M; Laval, J

    1994-06-21

    The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested. PMID:8016081

  11. Excision of hypoxanthine from DNA containing dIMP residues by the Escherichia coli, yeast, rat, and human alkylpurine DNA glycosylases.

    PubMed Central

    Saparbaev, M; Laval, J

    1994-01-01

    The deamination of adenine residues in DNA generates hypoxanthine, which is mutagenic since it gives rise to an A.T to G.C transition. Hypoxanthine is removed by hypoxanthine DNA glycosylase activity present in Escherichia coli and mammalian cells. Using polydeoxyribonucleotides or double-stranded synthetic oligonucleotides that contain dIMP residues, we show that this activity in E. coli is associated with the 3-methyladenine DNA glycosylase II coded for by the alkA gene. This conclusion is based on the following facts: (i) the two enzymatic activities have the same chromatographic behavior on various supports and they have the same molecular weight, (ii) both are induced during the adaptive response, (iii) a multicopy plasmid bearing the alkA gene overproduces both activities, (iv) homogeneous preparation of AlkA has both enzymatic activities, (v) the E. coli alkA- mutant does not show any detectable hypoxanthine DNA glycosylase activity. Under the same experimental conditions, but using different substrates, the same amount of AlkA protein liberates 1 pmol of 3-methyladenine from alkylated DNA and 1.2 fmol of hypoxanthine from dIMP-containing DNA. The Km for the latter substrate is 420 x 10(-9) M as compared to 5 x 10(-9) M for alkylated DNA. Hypoxanthine is released as a free base during the reaction. Duplex oligodeoxynucleotides containing hypoxanthine positioned opposite T, G, C, and A were cleaved efficiently. ANPG protein, APDG protein, and MAG protein--the 3-methyladenine DNA glycosylases of human, rat, and yeast origin, respectively--were also able to release hypoxanthine from various DNA substrates containing dIMP residues. The mammalian enzyme is by far the most efficient hypoxanthine DNA glycosylase of all the enzymes tested. Images PMID:8016081

  12. Complete DNA Sequence of Kuraishia capsulata Illustrates Novel Genomic Features among Budding Yeasts (Saccharomycotina)

    PubMed Central

    Morales, Lucia; Noel, Benjamin; Porcel, Betina; Marcet-Houben, Marina; Hullo, Marie-Francoise; Sacerdot, Christine; Tekaia, Fredj; Leh-Louis, Véronique; Despons, Laurence; Khanna, Varun; Aury, Jean-Marc; Barbe, Valérie; Couloux, Arnaud; Labadie, Karen; Pelletier, Eric; Souciet, Jean-Luc; Boekhout, Teun; Gabaldon, Toni; Wincker, Patrick; Dujon, Bernard

    2013-01-01

    The numerous yeast genome sequences presently available provide a rich source of information for functional as well as evolutionary genomics but unequally cover the large phylogenetic diversity of extant yeasts. We present here the complete sequence of the nuclear genome of the haploid-type strain of Kuraishia capsulata (CBS1993T), a nitrate-assimilating Saccharomycetales of uncertain taxonomy, isolated from tunnels of insect larvae underneath coniferous barks and characterized by its copious production of extracellular polysaccharides. The sequence is composed of seven scaffolds, one per chromosome, totaling 11.4 Mb and containing 6,029 protein-coding genes, ∼13.5% of which being interrupted by introns. This GC-rich yeast genome (45.7%) appears phylogenetically related with the few other nitrate-assimilating yeasts sequenced so far, Ogataea polymorpha, O. parapolymorpha, and Dekkera bruxellensis, with which it shares a very reduced number of tRNA genes, a novel tRNA sparing strategy, and a common nitrate assimilation cluster, three specific features to this group of yeasts. Centromeres were recognized in GC-poor troughs of each scaffold. The strain bears MAT alpha genes at a single MAT locus and presents a significant degree of conservation with Saccharomyces cerevisiae genes, suggesting that it can perform sexual cycles in nature, although genes involved in meiosis were not all recognized. The complete absence of conservation of synteny between K. capsulata and any other yeast genome described so far, including the three other nitrate-assimilating species, validates the interest of this species for long-range evolutionary genomic studies among Saccharomycotina yeasts. PMID:24317973

  13. Epidemiologic Study of Malassezia Yeasts in Acne Patients by Analysis of 26S rDNA PCR-RFLP

    PubMed Central

    Song, Young Chan; Hahn, Hyung Jin; Kim, Ji Young; Ko, Jong Hyun; Choe, Yong Beom; Ahn, Kyu Joong

    2011-01-01

    Background Although acne is a common follicular inflammatory dermatosis, studies of the relationship between Malassezia yeasts and acne have rarely been conducted. Objective We sought to identify Malassezia yeasts from acne patients and establish a relationship between specific types of species of Malassezia and acne. Methods Sixty acne patients were enrolled. Each strain obtained was identified as one of eleven species by 26S rDNA PCR-RFLP. We then compared these data with those of age- and sex-matched healthy subjects. Results Growth of Malassezia was evident in fewer patients with acne (50%) in comparison to controls (70.6%). M. restricta was dominant in patients with acne (23.9%), whereas M. globosa was most common (26.7%) in healthy controls. In the patients group, the rate was the highest (71.7%) in the twenties and, in terms of body site, the rate was the highest (60%) in the chest. In the control group, the rate was the highest (75.0%) in the thirties and in the forehead (85.0%). Conclusion The detection rate of Malassezia yeasts was conspicuously low in the acne patients group. Statistically significant differences were observed between the patient and the control groups in the twenties and thirties, and in terms of body site, in the forehead and chest. PMID:21909202

  14. Integrity of chromatin and replicating DNA in nuclei released from fission yeast by semi-automated grinding in liquid nitrogen

    PubMed Central

    2011-01-01

    Background Studies of nuclear function in many organisms, especially those with tough cell walls, are limited by lack of availability of simple, economical methods for large-scale preparation of clean, undamaged nuclei. Findings Here we present a useful method for nuclear isolation from the important model organism, the fission yeast, Schizosaccharomyces pombe. To preserve in vivo molecular configurations, we flash-froze the yeast cells in liquid nitrogen. Then we broke their tough cell walls, without damaging their nuclei, by grinding in a precision-controlled motorized mortar-and-pestle apparatus. The cryo-ground cells were resuspended and thawed in a buffer designed to preserve nuclear morphology, and the nuclei were enriched by differential centrifugation. The washed nuclei were free from contaminating nucleases and have proven well-suited as starting material for genome-wide chromatin analysis and for preparation of fragile DNA replication intermediates. Conclusions We have developed a simple, reproducible, economical procedure for large-scale preparation of endogenous-nuclease-free, morphologically intact nuclei from fission yeast. With appropriate modifications, this procedure may well prove useful for isolation of nuclei from other organisms with, or without, tough cell walls. PMID:22088094

  15. Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA gene.

    PubMed Central

    Kurtzman, C P; Robnett, C J

    1997-01-01

    Clinically important species of Candida and related organisms were compared for extent of nucleotide divergence in the 5' end of the large-subunit (26S) ribosomal DNA (rDNA) gene. This rDNA region is sufficiently variable to allow reliable separation of all known clinically significant yeast species. Of the 204 described species examined, 21 appeared to be synonyms of previously described organisms. Phylogenetic relationships among the species are presented. PMID:9114410

  16. ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei.

    PubMed

    Pasero, P; Braguglia, D; Gasser, S M

    1997-06-15

    We describe an in vitro replication assay from yeast in which the addition of intact nuclei to an S-phase nuclear extract results in the incorporation of deoxynucleotides into genomic DNA at spatially discrete foci. When BrdUTP is substituted for dTTP, part of the newly synthesized DNA shifts to a density on CsCl gradients, indicative of semiconservative replication. Initiation occurs in an origin-specific manner and can be detected in G1- or S-phase nuclei, but not in G2-phase or mitotic nuclei. The S-phase extract contains a heat- and 6-DMAP-sensitive component necessary to promote replication in G1-phase nuclei. Replication of nuclear DNA is blocked at the restrictive temperature in an orc2-1 mutant, and the inactive Orc2p cannot be complemented in trans by an extract containing wild-type ORC. The initiation of DNA replication in cln-deficient nuclei blocked in G1 indicates that the ORC-dependent prereplication complex is formed before Start. This represents the first nonviral and nonembryonic replication system in which DNA replication initiates in an ORC-dependent and origin-specific manner in vitro. PMID:9203578

  17. Reconstitution of a MEC1-independent checkpoint in yeast by expression of a novel human fork head cDNA.

    PubMed Central

    Pati, D; Keller, C; Groudine, M; Plon, S E

    1997-01-01

    A novel human cDNA, CHES1 (checkpoint suppressor 1), has been isolated by suppression of the mec1-1 checkpoint mutation in Saccharomyces cerevisiae. CHES1 suppresses a number of DNA damage-activated checkpoint mutations in S. cerevisiae, including mec1, rad9, rad24, dun1, and rad53. CHES1 suppression of sensitivity to DNA damage is specific for checkpoint-defective strains, in contrast to DNA repair-defective strains. Presence of CHES1 but not a control vector resulted in G2 delay after UV irradiation in checkpoint-defective strains, with kinetics, nuclear morphology, and cycloheximide resistance similar to those of a wild-type strain. CHES1 can also suppress the lethality, UV sensitivity, and G2 checkpoint defect of a mec1 null mutation. In contrast to this activity, CHES1 had no measurable effect on the replication checkpoint as assayed by hydroxyurea sensitivity of a mec1 strain. Sequence analysis demonstrates that CHES1 is a novel member of the fork head/Winged Helix family of transcription factors. Suppression of the checkpoint-defective phenotype requires a 200-amino-acid domain in the carboxy terminus of the protein which is distinct from the DNA binding site. Analysis of CHES1 activity is most consistent with activation of an alternative MEC1-independent checkpoint pathway in budding yeast. PMID:9154802

  18. 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

  19. Activation of budding yeast replication origins and suppression of lethal DNA damage effects on origin function by ectopic expression of the co-chaperone protein Mge1.

    PubMed

    Trabold, Peter A; Weinberger, Martin; Feng, Li; Burhans, William C

    2005-04-01

    Initiation of DNA replication in eukaryotes requires the origin recognition complex (ORC) and other proteins that interact with DNA at origins of replication. In budding yeast, the temperature-sensitive orc2-1 mutation alters these interactions in parallel with defects in initiation of DNA replication and in checkpoints that depend on DNA replication forks. Here we show that DNA-damaging drugs modify protein-DNA interactions at budding yeast replication origins in association with lethal effects that are enhanced by the orc2-1 mutation or suppressed by a different mutation in ORC. A dosage suppressor screen identified the budding yeast co-chaperone protein Mge1p as a high copy suppressor of the orc2-1-specific lethal effects of adozelesin, a DNA-alkylating drug. Ectopic expression of Mge1p also suppressed the temperature sensitivity and initiation defect conferred by the orc2-1 mutation. In wild type cells, ectopic expression of Mge1p also suppressed the lethal effects of adozelesin in parallel with the suppression of adozelesin-induced alterations in protein-DNA interactions at origins, stimulation of initiation of DNA replication, and binding of the precursor form of Mge1p to nuclear chromatin. Mge1p is the budding yeast homologue of the Escherichia coli co-chaperone protein GrpE, which stimulates initiation at bacterial origins of replication by promoting interactions of initiator proteins with origin sequences. Our results reveal a novel, proliferation-dependent cytotoxic mechanism for DNA-damaging drugs that involves alterations in the function of initiation proteins and their interactions with DNA. PMID:15647270

  20. DNA polymerase alpha associated protein P1, a murine homolog of yeast MCM3, changes its intranuclear distribution during the DNA synthetic period.

    PubMed Central

    Kimura, H; Nozaki, N; Sugimoto, K

    1994-01-01

    We isolated a murine gene for the DNA polymerase alpha associated protein P1, which shares high homology with the budding yeast MCM3 protein, which is a member of a protein family involved in the early event of DNA replication having a putative DNA-dependent ATPase motif. Using a polyclonal anti-P1 antibody raised against a beta-galactosidase-P1 fusion protein, we identified at least two forms of P1 protein in the nucleus of a mouse cell line, an underphosphorylated form that was associated with a particular nuclear structure and a hyperphosphorylated form loosely bound to the nucleus. During progression through S phase, P1 disappeared, first from the euchromatic region, then from the heterochromatic region, apparently in parallel with temporally ordered DNA replication. Thus, it is likely that the underphosphorylated P1 is dissociated from the nuclear structure after DNA replication by cell cycle-dependent phosphorylation. This is the first direct observation of a protein whose behavior is consistent with that of a hypothetical factor which restricts the chromatin to replicate once per cell cycle in higher eukaryotes. Images PMID:7925275

  1. Roles of C-Terminal Region of Yeast and Human Rad52 in Rad51-Nucleoprotein Filament Formation and ssDNA Annealing

    PubMed Central

    Khade, Nilesh V.; Sugiyama, Tomohiko

    2016-01-01

    Yeast Rad52 (yRad52) has two important functions at homologous DNA recombination (HR); annealing complementary single-strand DNA (ssDNA) molecules and recruiting Rad51 recombinase onto ssDNA (recombination mediator activity). Its human homolog (hRAD52) has a lesser role in HR, and apparently lacks mediator activity. Here we show that yRad52 can load human Rad51 (hRAD51) onto ssDNA complexed with yeast RPA in vitro. This is biochemically equivalent to mediator activity because it depends on the C-terminal Rad51-binding region of yRad52 and on functional Rad52-RPA interaction. It has been reported that the N-terminal two thirds of both yRad52 and hRAD52 is essential for binding to and annealing ssDNA. Although a second DNA binding region has been found in the C-terminal region of yRad52, its role in ssDNA annealing is not clear. In this paper, we also show that the C-terminal region of yRad52, but not of hRAD52, is involved in ssDNA annealing. This suggests that the second DNA binding site is required for the efficient ssDNA annealing by yRad52. We propose an updated model of Rad52-mediated ssDNA annealing. PMID:27362509

  2. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins

    SciTech Connect

    Matviw, Yu, G.; Young, D. )

    1992-11-01

    The adenylyl cyclases of both Saccharomyces cerevisiae and Schizosaccharomyces pombe are associated with related proteins named CAP. In S. cerevisiae, CAP is required for cellular responses mediated by the RAS/cyclic AMP pathway. Both yeast CAPs appear to be bifunctional proteins: The N-terminal domains are required for the proper function of adenylyl cyclase, while loss of the C-terminal domains results in morphological and nutritional defects that appear to be unrelated to the cAMP pathways. Expression of either yeast CAP in the heterologous yeast suppresses phenotypes associated with loss of the C-terminal domain of the endogenous CAP but does not suppress loss of the N-terminal domain. On the basis of the homology between the two yeast CAP proteins, we have designed degenerate oligonucleotides that we used to detect, by the polymerase chain reaction method, a human cDNA fragment encoding a CAP-related peptide. Using the polymerase chain reaction fragment as a probe, we isolated a human cDNA clone encoding a 475-amino-acid protein that is homologous to the yeast CAP proteins. Expressions of the human CAP protein in S. cerevisiae suppresses the phenotypes associated with loss of the C-terminal domain of CAP but does not suppress phenotypes associated with loss of the N-terminal domain. Thus, CAP proteins have been structurally and, to some extent, functionally conserved in evolution between yeasts and mammals. 42 refs., 5 figs.

  3. Trans-complementation by human apurinic endonuclease (Ape) of hypersensitivity to DNA damage and spontaneous mutator phenotype in apn1-yeast.

    PubMed Central

    Wilson, D M; Bennett, R A; Marquis, J C; Ansari, P; Demple, B

    1995-01-01

    Abasic (AP) sites in DNA are potentially lethal and mutagenic. 'Class II' AP endonucleases initiate the repair of these and other DNA lesions. In yeast, the predominant enzyme of this type is Apn1, and its elimination sensitizes the cells to killing by simple alkylating agents or oxidants, and raises the rate of spontaneous mutation. We investigated the ability of the major human class II AP endonuclease, Ape, which is structurally unrelated to Apn1, to replace the yeast enzyme in vivo. Confocal immunomicroscopy studies indicate that approximately 25% of the Ape expressed in yeast is present in the nucleus. High-level Ape expression corresponding to approximately 7000 molecules per nucleus, equal to the normal Apn1 copy number, restored resistance to methyl methanesulfonate to near wild-type levels in Apn1-deficient (apn1-) yeast. Ape expression in apn1- yeast provided little protection against H2O2 challenges, consistent with the weak 3'-repair diesterase activity of the human enzyme. Ape expression at approximately 2000 molecules per nucleus reduced the spontaneous mutation rate of apn1- yeast to that seen for wild-type cells. Because Ape has a powerful AP endonuclease but weak 3'-diesterase activity, these findings indicate that endogenously generated AP sites can drive spontaneous mutagenesis. Images PMID:8559661

  4. Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins.

    PubMed

    Jay, Kyle A; Smith, Dana L; Blackburn, Elizabeth H

    2016-07-15

    Telomeres cap the ends of chromosomes, protecting them from degradation and inappropriate DNA repair processes that can lead to genomic instability. A short telomere elicits increased telomerase action on itself that replenishes telomere length, thereby stabilizing the telomere. In the prolonged absence of telomerase activity in dividing cells, telomeres eventually become critically short, inducing a permanent cell cycle arrest (senescence). We recently showed that even early after telomerase inactivation (ETI), yeast cells have accelerated mother cell aging and mildly perturbed cell cycles. Here, we show that the complete disruption of DNA damage response (DDR) adaptor proteins in ETI cells causes severe growth defects. This synthetic-lethality phenotype was as pronounced as that caused by extensive DNA damage in wild-type cells but showed genetic dependencies distinct from such damage and was completely alleviated by SML1 deletion, which increases deoxynucleoside triphosphate (dNTP) pools. Our results indicated that these deleterious effects in ETI cells cannot be accounted for solely by the slow erosion of telomeres due to incomplete replication that leads to senescence. We propose that normally occurring telomeric DNA replication stress is resolved by telomerase activity and the DDR in two parallel pathways and that deletion of Sml1 prevents this stress. PMID:27161319

  5. Early Loss of Telomerase Action in Yeast Creates a Dependence on the DNA Damage Response Adaptor Proteins

    PubMed Central

    Jay, Kyle A.; Smith, Dana L.

    2016-01-01

    Telomeres cap the ends of chromosomes, protecting them from degradation and inappropriate DNA repair processes that can lead to genomic instability. A short telomere elicits increased telomerase action on itself that replenishes telomere length, thereby stabilizing the telomere. In the prolonged absence of telomerase activity in dividing cells, telomeres eventually become critically short, inducing a permanent cell cycle arrest (senescence). We recently showed that even early after telomerase inactivation (ETI), yeast cells have accelerated mother cell aging and mildly perturbed cell cycles. Here, we show that the complete disruption of DNA damage response (DDR) adaptor proteins in ETI cells causes severe growth defects. This synthetic-lethality phenotype was as pronounced as that caused by extensive DNA damage in wild-type cells but showed genetic dependencies distinct from such damage and was completely alleviated by SML1 deletion, which increases deoxynucleoside triphosphate (dNTP) pools. Our results indicated that these deleterious effects in ETI cells cannot be accounted for solely by the slow erosion of telomeres due to incomplete replication that leads to senescence. We propose that normally occurring telomeric DNA replication stress is resolved by telomerase activity and the DDR in two parallel pathways and that deletion of Sml1 prevents this stress. PMID:27161319

  6. The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants

    SciTech Connect

    Hipp, Katharina; Rau, Peter; Schäfer, Benjamin; Gronenborn, Bruno; Jeske, Holger

    2014-08-15

    Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. - Highlights: • A potential cyclin interaction motif is conserved in geminivirus Rep proteins. • In ACMV Rep, this motif (RXL) is essential for rereplication of fission yeast DNA. • Mutating RXL abrogated viral infection completely in Nicotiana benthamiana. • Expression of a nanovirus Clink protein in yeast did not induce rereplication. • Plant viruses may have evolved multiple routes to exploit host DNA synthesis.

  7. Widespread compensatory evolution conserves DNA-encoded nucleosome organization in yeast.

    PubMed

    Kenigsberg, Ephraim; Bar, Amir; Segal, Eran; Tanay, Amos

    2010-01-01

    Evolution maintains organismal fitness by preserving genomic information. This is widely assumed to involve conservation of specific genomic loci among species. Many genomic encodings are now recognized to integrate small contributions from multiple genomic positions into quantitative dispersed codes, but the evolutionary dynamics of such codes are still poorly understood. Here we show that in yeast, sequences that quantitatively affect nucleosome occupancy evolve under compensatory dynamics that maintain heterogeneous levels of A+T content through spatially coupled A/T-losing and A/T-gaining substitutions. Evolutionary modeling combined with data on yeast polymorphisms supports the idea that these substitution dynamics are a consequence of weak selection. This shows that compensatory evolution, so far believed to affect specific groups of epistatically linked loci like paired RNA bases, is a widespread phenomenon in the yeast genome, affecting the majority of intergenic sequences in it. The model thus derived suggests that compensation is inevitable when evolution conserves quantitative and dispersed genomic functions. PMID:21203484

  8. Yeast RNA polymerase II initiates transcription in vitro at TATA sequences proximal to potential non-B forms of the DNA template.

    PubMed Central

    Lescure, B; Arcangioli, B

    1984-01-01

    Pure yeast RNA polymerase II selectively initiates an abortive in vitro transcript within a TATA box of the yeast iso-1 cytochrome c gene promoter. Using a series of promoter deletions we show that a DNA sequence located upstream of the TATA box is needed for an efficient in vitro transcription. Supercoiling of the DNA template is an absolute requirement for the specific in vitro transcription. Examination of the DNA structure near several in vitro initiation sites shows that the common features observed are the presence of a TATA sequence in which RNA synthesis is initiated, and which is proximal to a potential non-B form of the DNA (a B to Z transition or a cruciform structure). Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:6376116

  9. The DNA-Binding Domain of Yeast Rap1 Interacts with Double-Stranded DNA in Multiple Binding Modes

    PubMed Central

    2015-01-01

    Saccharomyces cerevisiae repressor-activator protein 1 (Rap1) is an essential protein involved in multiple steps of DNA regulation, as an activator in transcription, as a repressor at silencer elements, and as a major component of the shelterin-like complex at telomeres. All the known functions of Rap1 require the known high-affinity and specific interaction of the DNA-binding domain with its recognition sequences. In this work, we focus on the interaction of the DNA-binding domain of Rap1 (Rap1DBD) with double-stranded DNA substrates. Unexpectedly, we found that while Rap1DBD forms a high-affinity 1:1 complex with its DNA recognition site, it can also form lower-affinity complexes with higher stoichiometries on DNA. These lower-affinity interactions are independent of the presence of the recognition sequence, and we propose they originate from the ability of Rap1DBD to bind to DNA in two different binding modes. In one high-affinity binding mode, Rap1DBD likely binds in the conformation observed in the available crystal structures. In the other alternative lower-affinity binding mode, we propose that a single Myb-like domain of the Rap1DBD makes interactions with DNA, allowing for more than one protein molecule to bind to the DNA substrates. Our findings suggest that the Rap1DBD does not simply target the protein to its recognition sequence but rather it might be a possible point of regulation. PMID:25382181

  10. Tel2p, a regulator of yeast telomeric length in vivo, binds to single-stranded telomeric DNA in vitro.

    PubMed

    Kota, R S; Runge, K W

    1999-09-01

    The telomeres of the yeast Saccharomyces cerevisiae consist of a duplex region of TG(1-3) repeats that acquire a single-stranded 3' extension of the TG(1-3) strand at the end of S-phase. The length of these repeats is kept within a defined range by regulators such as the TEL2-encoded protein (Tel2p). Here we show that Tel2p can specifically bind to single-stranded TG(1-3). Tel2p binding produced several shifted bands; however, only the slowest migrating band contained Tel2p. Methylation protection and interference experiments as well as gel shift experiments using inosine-containing probes indicated that the faster migrating bands resulted from Tel2p-mediated formation of DNA secondary structures held together by G-G interactions. Tel2p bound to single-stranded substrates that were at least 19 bases in length and contained 14 bases of TG(1-3), and also to double-stranded/single-stranded hybrid substrates with a 3' TG(1-3) overhang. Tel2p binding to a hybrid substrate with a 24 base single-stranded TG(1-3) extension also produced a band characteristic of G-G-mediated secondary structures. These data suggest that Tel2p could regulate telomeric length by binding to the 3' single-stranded TG(1-3) extension present at yeast telomeres. PMID:10525964

  11. Role of Inositol Phosphosphingolipid Phospholipase C1, the Yeast Homolog of Neutral Sphingomyelinases in DNA Damage Response and Diseases.

    PubMed

    Tripathi, Kaushlendra

    2015-01-01

    Sphingolipids play a very crucial role in many diseases and are well-known as signaling mediators in many pathways. Sphingolipids are produced during the de novo process in the ER (endoplasmic reticulum) from the nonsphingolipid precursor and comprise both structural and bioactive lipids. Ceramide is the central core of the sphingolipid pathway, and its production has been observed following various treatments that can induce several different cellular effects including growth arrest, DNA damage, apoptosis, differentiation, and senescence. Ceramides are generally produced through the sphingomyelin hydrolysis and catalyzed by the enzyme sphingomyelinase (SMase) in mammals. Presently, there are many known SMases and they are categorized into three groups acid SMases (aSMases), alkaline SMases (alk-SMASES), and neutral SMases (nSMases). The yeast homolog of mammalians neutral SMases is inositol phosphosphingolipid phospholipase C. Yeasts generally have inositol phosphosphingolipids instead of sphingomyelin, which may act as a homolog of mammalian sphingomyelin. In this review, we shall explain the structure and function of inositol phosphosphingolipid phospholipase C1, its localization inside the cells, mechanisms, and its roles in various cell responses during replication stresses and diseases. This review will also give a new basis for our understanding for the mechanisms and nature of the inositol phosphosphingolipid phospholipase C1/nSMase. PMID:26346287

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

    PubMed

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

    2016-09-01

    The yeast strains of the Saccharomyces sensu stricto complex involved in beer production are a heterogeneous group whose genetic and genomic features are not adequately determined. Thus, the aim of the present study was to provide a genetic characterization of selected group of commercially available brewing yeasts both ale top-fermenting and lager bottom-fermenting strains. Molecular karyotyping revealed that the diversity of chromosome patterns and four strains with the most accented genetic variabilities were selected and subjected to genome-wide array-based comparative genomic hybridization (array-CGH) analysis. The differences in the gene copy number were found in five functional gene categories: (1) maltose metabolism and transport, (2) response to toxin, (3) siderophore transport, (4) cellular aldehyde metabolic process, and (5) L-iditol 2-dehydrogenase activity (p < 0.05). In the Saflager W-34/70 strain (Fermentis) with the most affected array-CGH profile, loss of aryl-alcohol dehydrogenase (AAD) gene dosage correlated with an imbalanced redox state, oxidative DNA damage and breaks, lower levels of nucleolar proteins Nop1 and Fob1, and diminished tolerance to fermentation-associated stress stimuli compared to other strains. We suggest that compromised stress response may not only promote oxidant-based changes in the nucleolus state that may affect fermentation performance but also provide novel directions for future strain improvement. PMID:27299603

  13. A 6. 5-Mb yeast artificial chromosome contig incorporating 33 DNA markers on the human X chromosome at Xq22

    SciTech Connect

    Vetrie, D.; Kendall, E.; Coffey, A.; Hassock, S.; Collins, J.; Todd, C.; Bobrow, M.; Bentley, D.R. ); Lehrach, H. ); Harris, A. )

    1994-01-01

    The Xq22 region of the human X chromosome contains genes for a number of inherited disorders. Sixty-nine yeast artificial chromosome clones have been isolated and assembled into a 6.5-Mb contig that contains 33 DNA markers localized to this region. This contig extends distally from DXS366 to beyond DXS87 and includes the genes involved in X-linked agammaglobulinemia (btk), Fabry disease (GLA), and Pelizaeus-Merzbacher disease (PLP). The order of markers in this contig is consistent with the known genetic and physical mapping information of Xq22. This cloned material provides a source from which to isolate other genes located in this part of the X chromosome. 45 refs., 2 figs., 2 tabs.

  14. Biochemical and genetic characterization of Hmi1p, a yeast DNA helicase involved in the maintenance of mitochondrial DNA.

    PubMed

    Monroe, Danny S; Leitzel, Adelaide K; Klein, Hannah L; Matson, Steven W

    2005-12-01

    The HMI1 gene encodes a DNA helicase that localizes to the mitochondria and is required for maintenance of the mitochondrial DNA (mtDNA) genome of Saccharomyces cerevisiae. Identified based on its homology with E. coli uvrD, the HMI1 gene product, Hmi1p, has been presumed to be involved in the replication of the 80 kb linear S. cerevisiae mtDNA genome. Here we report the purification of Hmi1p to apparent homogeneity and provide a characterization of the helicase reaction and the ATPase reaction with regard to NTP preference, divalent cation preference and the stimulatory effects of different nucleic acids on Hmi1p-catalysed ATPase activity. Genetic complementation assays indicate that mitochondrial localization of Hmi1p is essential for its role in mtDNA metabolism. The helicase activity, however, is not essential. Point mutants that lack ATPase/helicase activity partially complement a strain lacking Hmi1p. We suggest several possible roles for Hmi1p in mtDNA metabolism. PMID:16358299

  15. Dissection of the DNA binding domain of yeast Zn-finger protein Rme1p, a repressor of meiotic activator IME1.

    PubMed

    Shimizu, M; Hara, M; Murase, A; Shindo, H; Mitchell, A P

    1997-01-01

    A series of deletion mutants of the yeast Zn-finger protein Rme1p (Repressor of Meiosis) fused with maltose binding protein (MBP) were constructed, purified, and characterized to examine the DNA binding domain. It was shown by gel retardation assay that the DNA binding domain of Rme1p was attributed to C-terminal amino acid residues 171 to 300. All three Zn-fingers are involved in the DNA binding domain, but they are not sufficient for DNA binding ability. Notably, the C-terminal region (residues 285-300) is essential for DNA binding. Provided that the region folds into alpha-helix, the basic amino acid residues may form a ridge on one side of the helix, whereas the hydrophobic residues may form it on the other side. Thus, the DNA binding domain of Rme1p would be dissected two regions. The roles of C-terminal region in DNA recognition will be discussed. PMID:9586056

  16. Primary structure of yeast mitochondrial DNA-coded phenylalanine-tRNA.

    PubMed

    Martin, R P; Sibler, A P; Schneller, J M; Keith, G; Stahl, A J; Dirheimer, G

    1978-12-01

    Mitochondrial tRNAPhe from Saccharomyces cerevisiae isolated by two-dimensional gel electrophoresis was sequenced by fingerprinting uniformly labeled 32 P-tRNA as well as by 5'-end postlabeling techniques. Its sequence was found to be: pG-C-U-U-U-U-A-U-A-G-C-U-U-A-G-D-G-G-D-A-A-A-G-C-m22G-A-U-A-A-A-phi-U-G-A-A-m1G-A-phi-U-U-A-U-U-U-A-C-A-U-G-U-A-G-U-phi-C-G-A-U-U-C-U-C-A-U-U-A-A-G-G-G-C-A-C-C-A. The secondary structure we propose, in order to maximize base pairing in the phiC stem and to allow tertiary interaction between G15 and C46, excludes U50 from base pairing giving a bulge in the phiC stem. No conclusion can be drawn concerning the endosymbiotic theory of mitochondria evolution by comparing the primary structure of mt. tRNAPhe with other sequenced tRNAsPhe. This mt.tRNAPhe lacks some of the structural elements reported to be involved in the yeast cytoplasmic phenylalanyl-tRNA ligase recognition site and cannot be aminoacylated by purified yeast cytoplasmic phenylalanyl-tRNA ligase. PMID:370774

  17. Comparison of the Accuracy of Two Conventional Phenotypic Methods and Two MALDI-TOF MS Systems with That of DNA Sequencing Analysis for Correctly Identifying Clinically Encountered Yeasts

    PubMed Central

    Chao, Qiao-Ting; Lee, Tai-Fen; Teng, Shih-Hua; Peng, Li-Yun; Chen, Ping-Hung; Teng, Lee-Jene; Hsueh, Po-Ren

    2014-01-01

    We assessed the accuracy of species-level identification of two commercially available matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems (Bruker Biotyper and Vitek MS) and two conventional phenotypic methods (Phoenix 100 YBC and Vitek 2 Yeast ID) with that of rDNA gene sequencing analysis among 200 clinical isolates of commonly encountered yeasts. The correct identification rates of the 200 yeast isolates to species or complex (Candida parapsilosis complex, C. guilliermondii complex and C. rugosa complex) levels by the Bruker Biotyper, Vitek MS (using in vitro devices [IVD] database), Phoenix 100 YBC and Vitek 2 Yeast ID (Sabouraud's dextrose agar) systems were 92.5%, 79.5%, 89%, and 74%, respectively. An additional 72 isolates of C. parapsilosis complex and 18 from the above 200 isolates (30 in each of C. parapsilosis, C. metapsilosis, and C. orthopsilosis) were also evaluated separately. Bruker Biotyper system could accurately identify all C. parapsilosis complex to species level. Using Vitek 2 MS (IVD) system, all C. parapsilosis but none of C. metapsilosis, or C. orthopsilosis could be accurately identified. Among the 89 yeasts misidentified by the Vitek 2 MS (IVD) system, 39 (43.8%), including 27 C. orthopsilosis isolates, could be correctly identified Using the Vitek MS Plus SARAMIS database for research use only. This resulted in an increase in the rate of correct identification of all yeast isolates (87.5%) by Vitek 2 MS. The two species in C. guilliermondii complex (C. guilliermondii and C. fermentati) isolates were correctly identified by cluster analysis of spectra generated by the Bruker Biotyper system. Based on the results obtained in the current study, MALDI-TOF MS systems present a promising alternative for the routine identification of yeast species, including clinically commonly and rarely encountered yeast species and several species belonging to C. parapsilosis complex, C. guilliermondii complex

  18. Maximal stimulation of meiotic recombination by a yeast transcription factor requires the transcription activation domain and a DNA-binding domain.

    PubMed Central

    Kirkpatrick, D T; Fan, Q; Petes, T D

    1999-01-01

    The DNA sequences located upstream of the yeast HIS4 represent a very strong meiotic recombination hotspot. Although the activity of this hotspot requires the transcription activator Rap1p, the level of HIS4 transcription is not directly related to the level of recombination. We find that the recombination-stimulating activity of Rap1p requires the transcription activation domain of the protein. We show that a hybrid protein with the Gal4p DNA-binding domain and the Rap1p activation domain can stimulate recombination in a strain in which Gal4p-binding sites are inserted upstream of HIS4. In addition, we find recombination hotspot activity associated with the Gal4p DNA-binding sites that is independent of known transcription factors. We suggest that yeast cells have two types of recombination hotspots, alpha (transcription factor dependent) and beta (transcription factor independent). PMID:10224246

  19. Identification of Novel Protein–Ligand Interactions by Exon Microarray Analysis of Yeast Surface Displayed cDNA Library Selection Outputs

    PubMed Central

    Bidlingmaier, Scott; Liu, Bin

    2016-01-01

    Yeast surface display is widely utilized to screen large libraries for proteins or protein fragments with specific binding properties. We have previously constructed and utilized yeast surface displayed human cDNA libraries to identify protein fragments that bind to various target ligands. Conventional approaches employ monoclonal screening and sequencing of polyclonal outputs that have been enriched for binding to a target molecule by several rounds of affinity-based selection. Frequently, a small number of clones will dominate the selection output, making it difficult to comprehensively identify potentially important interactions due to low representation in the selection output. We have developed a novel method to address this problem. By analyzing selection outputs using high-density human exon microarrays, the full potential of selection output diversity can be revealed in one experiment. FACS-based selection using yeast surface displayed human cDNA libraries combined with exon microarray analysis of the selection outputs is a powerful way of rapidly identifying protein fragments with affinity for any soluble ligand that can be fluorescently detected, including small biological molecules and drugs. In this report we present protocols for exon microarray-based analysis of yeast surface display human cDNA library selection outputs. PMID:26060075

  20. DNA repair defects sensitize cells to anticodon nuclease yeast killer toxins.

    PubMed

    Klassen, Roland; Wemhoff, Sabrina; Krause, Jens; Meinhardt, Friedhelm

    2011-03-01

    Killer toxins from Kluyveromyces lactis (zymocin) and Pichia acaciae (PaT) were found to disable translation in target cells by virtue of anticodon nuclease (ACNase) activities on tRNA(Glu) and tRNA(Gln), respectively. Surprisingly, however, ACNase exposure does not only impair translation, but also affects genome integrity and concomitantly DNA damage occurs. Previously, it was shown that homologous recombination protects cells from ACNase toxicity. Here, we have analyzed whether other DNA repair pathways are functional in conferring ACNase resistance as well. In addition to HR, base excision repair (BER) and postreplication repair (PRR) promote clear resistance to either, PaT and zymocin. Comparative toxin sensitivity analysis of BER mutants revealed that its ACNase protective function is due to the endonucleases acting on apurinic (AP) sites, whereas none of the known DNA glycosylases is involved. Because PaT and zymocin require the presence of the ELP3/TRM9-dependent wobble uridine modification 5-methoxy-carbonyl-methyl (mcm(5)) for tRNA cleavage, we analyzed toxin response in DNA repair mutants additionally lacking such tRNA modifications. ACNase resistance caused by elp3 or trm9 mutations was found to rescue hypersensitivity of DNA repair defects, consistent with DNA damage to occur as a consequence of tRNA cleavage. The obtained genetic evidence promises to reveal new aspects into the mechanism linking translational fidelity and genome surveillance. PMID:21188417

  1. Cernunnos interacts with the XRCC4 x DNA-ligase IV complex and is homologous to the yeast nonhomologous end-joining factor Nej1.

    PubMed

    Callebaut, Isabelle; Malivert, Laurent; Fischer, Alain; Mornon, Jean-Paul; Revy, Patrick; de Villartay, Jean-Pierre

    2006-05-19

    DNA double strand breaks are considered as the most harmful DNA lesions and are repaired by either homologous recombination or nonhomologous end joining (NHEJ). A new NHEJ factor, Cernunnos, has been identified, the defect of which leads to a severe immunodeficiency condition associated with microcephaly and other developmental defects in humans. This presentation is reminiscent to that of DNA-ligase IV deficiency and suggests a possible interplay between Cernunnos and the XRCC4 x DNA-ligase IV complex. We show here that Cernunnos physically interacts with the XRCC4 x DNA-ligase IV complex. Moreover, a combination of sensitive methods of sequence analysis revealed that Cernunnos can be associated with the XRCC4 family of proteins and that it corresponds to the genuine homolog of the yeast Nej1 protein. Altogether these results shed new lights on the last step, the DNA religation, of the NHEJ pathway. PMID:16571728

  2. A new set of rDNA-NTS-based multiple integrative cassettes for the development of antibiotic-marker-free recombinant yeasts.

    PubMed

    Moon, Hye Yun; Lee, Dong Wook; Sim, Gyu Hun; Kim, Hong-Jin; Hwang, Jee Youn; Kwon, Mun-Gyeong; Kang, Bo-Kyu; Kim, Jong Man; Kang, Hyun Ah

    2016-09-10

    The traditional yeast Saccharomyces cerevisiae has been widely used as a host system to produce recombinant proteins and metabolites of great commercial value. To engineer recombinant yeast that stably maintains expression cassettes without an antibiotic resistance gene, we developed new multiple integration cassettes by exploiting the non-transcribed spacer (NTS) of ribosomal DNA (rDNA) in combination with defective selection markers. The 5' and 3'-fragments of rDNA-NTS2 were used as flanking sequences for the expression cassettes carrying a set of URA3, LEU2, HIS3, and TRP1 selection markers with truncated promoters of different lengths. The integration numbers of NTS-based expression cassettes, ranging from one to ∼30 copies, showed a proportional increase with the extent of decreased expression of the auxotrophic markers. The NTS-based cassettes were used to construct yeast strains expressing the capsid protein of red-spotted grouper necrosis virus (RG-NNVCP) in a copy number-dependent manner. Oral administration of the recombinant yeast, harboring ∼30 copies of the integrated RG-NNVCP cassettes, provoked efficient immune responses in mice. In contrast, for the NTS cassettes expressing a truncated 3-hydroxyl-3-methylglutaryl-CoA reductase, the integrant carrying only 4 copies was screened as the highest producer of squalene, showing a 150-fold increase compared to that of the wild-type strain. The multiple integrated cassettes were stably retained under prolonged nonselective conditions. Altogether, our results strongly support that rDNA-NTS integrative cassettes are useful tools to construct recombinant yeasts carrying optimal copies of a desired expression cassette without an antibiotic marker gene, which are suitable as oral vaccines or feed additives for animal and human consumption. PMID:27411901

  3. A role for DNA polymerase α in epigenetic control of transcriptional silencing in fission yeast

    PubMed Central

    Nakayama, Jun-ichi; Allshire, Robin C.; Klar, Amar J.S.; Grewal, Shiv I.S.

    2001-01-01

    In the fission yeast Schizosaccharomyces pombe, transcriptional silencing at the mating-type region, centromeres and telomeres is epigenetically controlled, and results from the assembly of higher order chromatin structures. Chromatin proteins associated with these silenced loci are believed to serve as molecular bookmarks that help promote inheritance of the silenced state during cell division. Specifically, a chromodomain protein Swi6 is believed to be an important determinant of the epigenetic imprint. Here, we show that a mutation in DNA polymerase α (polα) affects Swi6 localization at the mating-type region and causes a 45-fold increase in spontaneous transition from the silenced epigenetic state to the expressed state. We also demonstrate that polα mutant cells are defective in Swi6 localization at centromeres and telomeres. Genetic analysis suggests that Polα and Swi6 are part of the same silencing pathway. Interestingly, we found that Swi6 directly binds to Polα in vitro. Moreover, silencing-defective mutant Polα displays reduced binding to Swi6 protein. This work indicates involvement of a DNA replication protein, Polα, in heterochromatin assembly and inheritance of epigenetic chromatin structures. PMID:11387218

  4. Condensin HEAT Subunits Required for DNA Repair, Kinetochore/Centromere Function and Ploidy Maintenance in Fission Yeast

    PubMed Central

    Xu, Xingya; Nakazawa, Norihiko; Yanagida, Mitsuhiro

    2015-01-01

    Condensin, a central player in eukaryotic chromosomal dynamics, contains five evolutionarily-conserved subunits. Two SMC (structural maintenance of chromosomes) subunits contain ATPase, hinge, and coiled-coil domains. One non-SMC subunit is similar to bacterial kleisin, and two other non-SMC subunits contain HEAT (similar to armadillo) repeats. Here we report isolation and characterization of 21 fission yeast (Schizosaccharomyces pombe) mutants for three non-SMC subunits, created using error-prone mutagenesis that resulted in single-amino acid substitutions. Beside condensation, segregation, and DNA repair defects, similar to those observed in previously isolated SMC and cnd2 mutants, novel phenotypes were observed for mutants of HEAT-repeats containing Cnd1 and Cnd3 subunits. cnd3-L269P is hypersensitive to the microtubule poison, thiabendazole, revealing defects in kinetochore/centromere and spindle assembly checkpoints. Three cnd1 and three cnd3 mutants increased cell size and doubled DNA content, thereby eliminating the haploid state. Five of these mutations reside in helix B of HEAT repeats. Two non-SMC condensin subunits, Cnd1 and Cnd3, are thus implicated in ploidy maintenance. PMID:25764183

  5. Impaired coenzyme A synthesis in fission yeast causes defective mitosis, quiescence-exit failure, histone hypoacetylation and fragile DNA

    PubMed Central

    Nakamura, Takahiro; Pluskal, Tomáš; Nakaseko, Yukinobu; Yanagida, Mitsuhiro

    2012-01-01

    Biosynthesis of coenzyme A (CoA) requires a five-step process using pantothenate and cysteine in the fission yeast Schizosaccharomyces pombe. CoA contains a thiol (SH) group, which reacts with carboxylic acid to form thioesters, giving rise to acyl-activated CoAs such as acetyl-CoA. Acetyl-CoA is essential for energy metabolism and protein acetylation, and, in higher eukaryotes, for the production of neurotransmitters. We isolated a novel S. pombe temperature-sensitive strain ppc1-537 mutated in the catalytic region of phosphopantothenoylcysteine synthetase (designated Ppc1), which is essential for CoA synthesis. The mutant becomes auxotrophic to pantothenate at permissive temperature, displaying greatly decreased levels of CoA, acetyl-CoA and histone acetylation. Moreover, ppc1-537 mutant cells failed to restore proliferation from quiescence. Ppc1 is thus the product of a super-housekeeping gene. The ppc1-537 mutant showed combined synthetic lethal defects with five of six histone deacetylase mutants, whereas sir2 deletion exceptionally rescued the ppc1-537 phenotype. In synchronous cultures, ppc1-537 cells can proceed to the S phase, but lose viability during mitosis failing in sister centromere/kinetochore segregation and nuclear division. Additionally, double-strand break repair is defective in the ppc1-537 mutant, producing fragile broken DNA, probably owing to diminished histone acetylation. The CoA-supported metabolism thus controls the state of chromosome DNA. PMID:23091701

  6. [Expression Of DNA-Encoded Antidote to Organophosphorus Toxins in the Methylotrophic Yeast Pichia Pastoris].

    PubMed

    Terekhov, S S; Bobik, T V; Mokrushina, Yu A; Stepanova, A V; Aleksandrova, N M; Smirnov, I V; Belogurov, A A; Ponomarenko, N A; Gabibov, A G

    2016-01-01

    A platform for the cloning and expression of active human butyrylcholinesterase (BuChE) in the yeast Pichia pastoris is first presented. Genetic constructs for BuChE gene expression, separately and in conjunction with a proline-rich peptide called proline-rich attachment domain (PRAD), are based on the vector pPICZαA. It is shown that the highest level of production is achieved in the expression of a BuChE gene without PRAD pPICZαA. It is found that one can obtain up to 125 mg of active enzyme from 1 L of culture medium at an optimal pH environment (pH 7.6), an optical seed culture density of 3 o.u., and an optimum methanol addition mode of (0.5% methanol in the first day and 0.2% thereafter from the second day). PMID:27266247

  7. Yeast linker histone Hho1p is required for efficient RNA polymerase I processivity and transcriptional silencing at the ribosomal DNA

    PubMed Central

    Levy, Anat; Eyal, Miri; Hershkovits, Gitit; Salmon-Divon, Mali; Klutstein, Michael; Katcoff, Don Jay

    2008-01-01

    Nucleosome core particles in eukaryotes are linked by a stretch of DNA that is usually associated with a linker histone. Here, we show in yeast, that the presence of yeast linker histone Hho1p represses expression of a pol II transcribed gene (MET15) embedded in the rDNA. In vivo deletions of Hho1p sequences showed that the second globular domain is sufficient for that repression, whereas the presence of the N terminus is required for its derepression. In contrast, a run-on assay confirmed by a ChIP experiment showed that Hho1p is required for maximal pol I processivity during rDNA transcription. Psoralen accessibility experiments indicated that Hho1p is necessary for normal rDNA compaction. DNA array expression analysis comparing RNA transcripts in wild-type and hho1 strains before and after a heat-shock showed that Hho1p is necessary to achieve wild-type mRNA levels of transcripts that encode ribosomal components. Taken together, our results suggest that Hho1p is involved in rDNA compaction, and like core histones, is required for efficient rDNA transcription by pol I. PMID:18687885

  8. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation.

    PubMed

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J; Fox, Catherine A

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  9. High Throughput Analyses of Budding Yeast ARSs Reveal New DNA Elements Capable of Conferring Centromere-Independent Plasmid Propagation

    PubMed Central

    Hoggard, Timothy; Liachko, Ivan; Burt, Cassaundra; Meikle, Troy; Jiang, Katherine; Craciun, Gheorghe; Dunham, Maitreya J.; Fox, Catherine A.

    2016-01-01

    The ability of plasmids to propagate in Saccharomyces cerevisiae has been instrumental in defining eukaryotic chromosomal control elements. Stable propagation demands both plasmid replication, which requires a chromosomal replication origin (i.e., an ARS), and plasmid distribution to dividing cells, which requires either a chromosomal centromere for segregation or a plasmid-partitioning element. While our knowledge of yeast ARSs and centromeres is relatively advanced, we know less about chromosomal regions that can function as plasmid partitioning elements. The Rap1 protein-binding site (RAP1) present in transcriptional silencers and telomeres of budding yeast is a known plasmid-partitioning element that functions to anchor a plasmid to the inner nuclear membrane (INM), which in turn facilitates plasmid distribution to daughter cells. This Rap1-dependent INM-anchoring also has an important chromosomal role in higher-order chromosomal structures that enhance transcriptional silencing and telomere stability. Thus, plasmid partitioning can reflect fundamental features of chromosome structure and biology, yet a systematic screen for plasmid partitioning elements has not been reported. Here, we couple deep sequencing with competitive growth experiments of a plasmid library containing thousands of short ARS fragments to identify new plasmid partitioning elements. Competitive growth experiments were performed with libraries that differed only in terms of the presence or absence of a centromere. Comparisons of the behavior of ARS fragments in the two experiments allowed us to identify sequences that were likely to drive plasmid partitioning. In addition to the silencer RAP1 site, we identified 74 new putative plasmid-partitioning motifs predicted to act as binding sites for DNA binding proteins enriched for roles in negative regulation of gene expression and G2/M-phase associated biology. These data expand our knowledge of chromosomal elements that may function in plasmid

  10. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain.

    PubMed

    Morrill, Summer A; Exner, Alexandra E; Babokhov, Michael; Reinfeld, Bradley I; Fuchs, Stephen M

    2016-05-27

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24-26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1 In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  11. DNA Instability Maintains the Repeat Length of the Yeast RNA Polymerase II C-terminal Domain*

    PubMed Central

    Morrill, Summer A.; Exner, Alexandra E.; Babokhov, Michael; Reinfeld, Bradley I.

    2016-01-01

    The C-terminal domain (CTD) of RNA polymerase II in eukaryotes is comprised of tandemly repeating units of a conserved seven-amino acid sequence. The number of repeats is, however, quite variable across different organisms. Furthermore, previous studies have identified evidence of rearrangements within the CTD coding region, suggesting that DNA instability may play a role in regulating or maintaining CTD repeat number. The work described here establishes a clear connection between DNA instability and CTD repeat number in Saccharomyces cerevisiae. First, analysis of 36 diverse S. cerevisiae isolates revealed evidence of numerous past rearrangements within the DNA sequence that encodes the CTD. Interestingly, the total number of CTD repeats was relatively static (24–26 repeats in all strains), suggesting a balancing act between repeat expansion and contraction. In an effort to explore the genetic plasticity within this region, we measured the rates of repeat expansion and contraction using novel reporters and a doxycycline-regulated expression system for RPB1. In efforts to determine the mechanisms leading to CTD repeat variability, we identified the presence of DNA secondary structures, specifically G-quadruplex-like DNA, within the CTD coding region. Furthermore, we demonstrated that mutating PIF1, a G-quadruplex-specific helicase, results in increased CTD repeat length polymorphisms. We also determined that RAD52 is necessary for CTD repeat expansion but not contraction, identifying a role for recombination in repeat expansion. Results from these DNA rearrangements may help explain the CTD copy number variation seen across eukaryotes, as well as support a model of CTD expansion and contraction to maintain CTD integrity and overall length. PMID:27026700

  12. Characterization of vanadate-dependent NADH oxidation activity and isolation of yeast DNA which complements a class 1 vanadate resistance mutation

    SciTech Connect

    Minasi, L.E.

    1989-01-01

    A vanadate-dependent NADH oxidation activity has been characterized in plasma membranes from the yeast S cerevisiae. NADH oxidation activity was maximally stimulated at pH 5.0 in phosphate buffer. NADH oxidation was not dependent on the concentration of plasma membranes. The vanadate-dependent NADH oxidation activity was abolished under anaerobic conditions and the concomitant uptake of oxygen occurred during NADH oxidation. The activity was inhibited by superoxide dismutase and stimulated by the presence of paraquat. These results indicate that the vanadate stimulation of NADH oxidation in yeast plasma membranes occurs as a result of the vanadate-dependent oxidation of NADH by superoxide, generated by a plasma membrane NADH oxidase. {sup 51}V-NMR results indicated that a phosphate-vanadate anhydride was the stimulatory species in pH 5.0 and pH 7.0 phosphate buffer. Yeast DNA has been isolated which complements a class 1 vanadate resistance mutation.

  13. Sen1, the yeast homolog of human senataxin, plays a more direct role than Rad26 in transcription coupled DNA repair.

    PubMed

    Li, Wentao; Selvam, Kathiresan; Rahman, Sheikh A; Li, Shisheng

    2016-08-19

    Rad26, a DNA dependent ATPase that is homologous to human CSB, has been well known to play an important role in transcription coupled DNA repair (TCR) in the yeast Saccharomyces cerevisiae Sen1, a DNA/RNA helicase that is essential for yeast cell viability and homologous to human senataxin, has been known to be required for transcriptional termination of short noncoding RNA genes and for a fail-safe transcriptional termination mechanism of protein-coding genes. Sen1 has also been shown to protect the yeast genome from transcription-associated recombination by resolving RNA:DNA hybrids naturally formed during transcription. Here, we show that the N-terminal non-essential region of Sen1 plays an important role in TCR, whereas the C-terminal nonessential region and the helicase activity of Sen1 are largely dispensable for the repair. Unlike Rad26, which becomes completely dispensable for TCR in cells lacking the TCR repressor Spt4, Sen1 is still required for efficient TCR in the absence of Spt4. Also unlike Rad26, which is important for repair at many but not all damaged sites in the transcribed strand of a gene, Sen1 is required for efficient repair at essentially all the damaged sites. Our results indicate that Sen1 plays a more direct role than Rad26 in TCR. PMID:27179024

  14. In situ hybridization to cytogenetic bands of yeast artificial chromosomes covering 50% of human Xq24-Xq28 DNA

    PubMed Central

    Montanaro, Vittorio; Casamassimi, Amelia; D'Urso, Michele; Yoon, Jae-Young; Freije, Wadiha; Schlessinger, David; Muenke, Maximilian; Nussbaum, Robert L.; Saccone, Salvatore; Maugeri, Silvana; Santoro, Anna Maria; Motta, Salvatore; Della Valle, Giuliano

    1991-01-01

    From the collection described by Abidi et al., 102 yeast artificial chromosomes (YACs) with human DNA inserts more than 300 kb in length were assigned to chromosomal band positions on early metaphase chromosomes by in situ hybridization using the biotin-avidin method. All the YACs hybridized within the Xq24-Xqter region, supporting the origin of the vast majority of the YACs from single human X-chromosomal sites. With assignments precise to ±0.5 bands, YACs were distributed among cytogenetic bands to roughly equal extents. Thus, there is no gross bias in the cloning of DNA from different bands into large YACs. To test band assignments further, hybridizations were carried out blind, and band positions were then compared with (1) probe localizations in cases in which a reported location was present in one of the YACs; (2) cross-hybridization of a labeled YAC with others in the collection; and (3) hybridization to a panel of DNAs from a series of hybrid cells containing Xq DNA truncated at various regions. Of 31 cases in which YACs contained a probe with a previously reported location, 28 in situ assignments were in agreement, and 14 other assignments, including one of the three discordant with probe localization, were confirmed by YAC cross-hybridization studies. Results with a group of nine YACs were further confirmed with a panel of somatic cell hybrid DNAs from that region. Five YACs hybridized both to Xq25 and to a second site (four in Xq27 and one in Xq28), suggestive of some duplication of DNA of the hybrid cell and perhaps in normal X chromosomes. The in situ assignments are thus sufficient to place YACs easily and systematically within bins of about 7–10 Mb and to detect some possible anomalies. Furthermore, on the basis of expectations for random cloning of DNA in YACs, the assigned YACs probably cover more than 50% of the total Xq24-Xq28 region. This provides one way to initiate the assembly of YAC contigs over extended chromosomal regions. Images

  15. Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants.

    PubMed

    Ojini, Irene; Gammie, Alison

    2015-09-01

    Resistance to cancer therapy is a major obstacle in the long-term treatment of cancer. A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring. Here, we exploit the mutator phenotype of mismatch repair defective yeast cells combined with whole genome sequencing to identify drug resistance mutations in key pathways involved in the development of chemoresistance. The utility of this approach was demonstrated via the identification of the known CAN1 and TOP1 resistance targets for two compounds, canavanine and camptothecin, respectively. We have also experimentally validated the plasma membrane transporter HNM1 as the primary drug resistance target of mechlorethamine. Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis. In the case of bactobolin, a promising anticancer drug, the endocytosis pathway was identified as the drug resistance target responsible for conferring resistance. Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance. The rapid and robust nature of these techniques, using Saccharomyces cerevisiae as a model organism, should accelerate the identification of drug resistance targets and guide the development of novel therapeutic combination strategies to prevent the development of chemoresistance in various cancers. PMID:26199284

  16. Rapid Identification of Chemoresistance Mechanisms Using Yeast DNA Mismatch Repair Mutants

    PubMed Central

    Ojini, Irene; Gammie, Alison

    2015-01-01

    Resistance to cancer therapy is a major obstacle in the long-term treatment of cancer. A greater understanding of drug resistance mechanisms will ultimately lead to the development of effective therapeutic strategies to prevent resistance from occurring. Here, we exploit the mutator phenotype of mismatch repair defective yeast cells combined with whole genome sequencing to identify drug resistance mutations in key pathways involved in the development of chemoresistance. The utility of this approach was demonstrated via the identification of the known CAN1 and TOP1 resistance targets for two compounds, canavanine and camptothecin, respectively. We have also experimentally validated the plasma membrane transporter HNM1 as the primary drug resistance target of mechlorethamine. Furthermore, the sequencing of mitoxantrone-resistant strains identified inactivating mutations within IPT1, a gene encoding inositolphosphotransferase, an enzyme involved in sphingolipid biosynthesis. In the case of bactobolin, a promising anticancer drug, the endocytosis pathway was identified as the drug resistance target responsible for conferring resistance. Finally, we show that that rapamycin, an mTOR inhibitor previously shown to alter the fitness of the ipt1 mutant, can effectively prevent the formation of mitoxantrone resistance. The rapid and robust nature of these techniques, using Saccharomyces cerevisiae as a model organism, should accelerate the identification of drug resistance targets and guide the development of novel therapeutic combination strategies to prevent the development of chemoresistance in various cancers. PMID:26199284

  17. Deficiencies in mitochondrial DNA compromise the survival of yeast cells at critically high temperatures.

    PubMed

    Zubko, Elena I; Zubko, Mikhajlo K

    2014-01-01

    To address possible roles of mitochondrial genes in adaptation of eukaryotic cells to critical temperatures, we compared thermotolerance of mitochondrial rho mutants and wild type cells of six rho positive yeast species: Candida glabrata, Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces eubayanus, Saccharomyces paradoxus and Saccharomyces pastorianus. All rho mutants manifested compromised thermotolerance as a common phenotype. Analysis of viabilities at critical temperatures (32-45 °C) showed the reduction of maximum permissive temperatures (MPTs) in all rho mutants in comparison to their wild type counterparts. Degrees of the compromised thermotolerance depended on maximum permissive temperatures for wild type cells: the highest levels of MPT reductions for rho mutants took place in species and strains with highest MPTs for wild types. Short term exposures of S. cerevisiae cells (up to 3.5h) at non-permissive temperatures (45 °C and 50 °C) also lead to more rapid cell death of rho mutants as compared to wild type cells. We conclude that: (1) compromised thermotolerance could be a generic phenotypic property of rho mutants; (2) the enhanced thermotolerance of cells possessing mitochondrial genomes could be one of selective advantages in adaptation to environmental factors, in particular to enhanced temperatures. PMID:23890722

  18. Involvement of budding yeast Rad5 in translesion DNA synthesis through physical interaction with Rev1

    PubMed Central

    Xu, Xin; Lin, Aiyang; Zhou, Cuiyan; Blackwell, Susan R.; Zhang, Yiran; Wang, Zihao; Feng, Qianqian; Guan, Ruifang; Hanna, Michelle D.; Chen, Zhucheng; Xiao, Wei

    2016-01-01

    DNA damage tolerance (DDT) is responsible for genomic stability and cell viability by bypassing the replication block. In Saccharomyces cerevisiae DDT employs two parallel branch pathways to bypass the DNA lesion, namely translesion DNA synthesis (TLS) and error-free lesion bypass, which are mediated by sequential modifications of PCNA. Rad5 has been placed in the error-free branch of DDT because it contains an E3 ligase domain required for PCNA polyubiquitination. Rad5 is a multi-functional protein and may also play a role in TLS, since it interacts with the TLS polymerase Rev1. In this study we mapped the Rev1-interaction domain in Rad5 to the amino acid resolution and demonstrated that Rad5 is indeed involved in TLS possibly through recruitment of Rev1. Genetic analyses show that the dual functions of Rad5 can be separated and reconstituted. Crystal structure analysis of the Rad5–Rev1 interaction reveals a consensus RFF motif in the Rad5 N-terminus that binds to a hydrophobic pocket within the C-terminal domain of Rev1 that is highly conserved in eukaryotes. This study indicates that Rad5 plays a critical role in pathway choice between TLS and error-free DDT. PMID:27001510

  19. Dual requirement for the yeast MMS19 gene in DNA repair and RNA polymerase II transcription.

    PubMed Central

    Lauder, S; Bankmann, M; Guzder, S N; Sung, P; Prakash, L; Prakash, S

    1996-01-01

    Genetic and biochemical studies of Saccharomyces cerevisiae have indicated the involvement of a large number of protein factors in nucleotide excision repair (NER) of UV-damaged DNA. However, how MMS19 affects this process has remained unclear. Here, we report on the isolation of the MMS19 gene and the determination of its role in NER and other cellular processes. Genetic and biochemical evidence indicates that besides its function in NER, MMS19 also affects RNA polymerase II (Pol II) transcription. mms19delta cells do not grow at 37 degrees C, and mutant extract exhibits a thermolabile defect in Pol II transcription. Thus, Mms19 protein resembles TFIIH in that it is required for both transcription and DNA repair. However, addition of purified Mms19 protein does not alleviate the transcriptional defect of the mms19delta extract, nor does it stimulate the incision of UV-damaged DNA reconstituted from purified proteins. Interestingly, addition of purified TFIIH corrects the transcriptional defect of the mms19delta extract. Mms19 is, however, not a component of TFIIH or of Pol II holoenzyme. These and other results suggest that Mms19 affects NER and transcription by influencing the activity of TFIIH as an upstream regulatory element. It is proposed that mutations in the human MMS19 counterpart could result in syndromes in which both NER and transcription are affected. PMID:8943333

  20. Involvement of budding yeast Rad5 in translesion DNA synthesis through physical interaction with Rev1.

    PubMed

    Xu, Xin; Lin, Aiyang; Zhou, Cuiyan; Blackwell, Susan R; Zhang, Yiran; Wang, Zihao; Feng, Qianqian; Guan, Ruifang; Hanna, Michelle D; Chen, Zhucheng; Xiao, Wei

    2016-06-20

    DNA damage tolerance (DDT) is responsible for genomic stability and cell viability by bypassing the replication block. In Saccharomyces cerevisiae DDT employs two parallel branch pathways to bypass the DNA lesion, namely translesion DNA synthesis (TLS) and error-free lesion bypass, which are mediated by sequential modifications of PCNA. Rad5 has been placed in the error-free branch of DDT because it contains an E3 ligase domain required for PCNA polyubiquitination. Rad5 is a multi-functional protein and may also play a role in TLS, since it interacts with the TLS polymerase Rev1. In this study we mapped the Rev1-interaction domain in Rad5 to the amino acid resolution and demonstrated that Rad5 is indeed involved in TLS possibly through recruitment of Rev1. Genetic analyses show that the dual functions of Rad5 can be separated and reconstituted. Crystal structure analysis of the Rad5-Rev1 interaction reveals a consensus RFF motif in the Rad5 N-terminus that binds to a hydrophobic pocket within the C-terminal domain of Rev1 that is highly conserved in eukaryotes. This study indicates that Rad5 plays a critical role in pathway choice between TLS and error-free DDT. PMID:27001510

  1. DNA topoisomerase III localizes to centromeres and affects centromeric CENP-A levels in fission yeast.

    PubMed

    Norman-Axelsson, Ulrika; Durand-Dubief, Mickaël; Prasad, Punit; Ekwall, Karl

    2013-01-01

    Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-A(Cnp1) at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-A(Cnp1) occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-A(Cnp1) at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-A(Cnp1) in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-A(Cnp1) nucleosomes. PMID:23516381

  2. DNA Topoisomerase III Localizes to Centromeres and Affects Centromeric CENP-A Levels in Fission Yeast

    PubMed Central

    Norman-Axelsson, Ulrika; Durand-Dubief, Mickaël; Prasad, Punit; Ekwall, Karl

    2013-01-01

    Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-ACnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-ACnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-ACnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-ACnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-ACnp1 nucleosomes. PMID:23516381

  3. Condensin suppresses recombination and regulates double-strand break processing at the repetitive ribosomal DNA array to ensure proper chromosome segregation during meiosis in budding yeast

    PubMed Central

    Li, Ping; Jin, Hui; Yu, Hong-Guo

    2014-01-01

    During meiosis, homologues are linked by crossover, which is required for bipolar chromosome orientation before chromosome segregation at anaphase I. The repetitive ribosomal DNA (rDNA) array, however, undergoes little or no meiotic recombination. Hyperrecombination can cause chromosome missegregation and rDNA copy number instability. We report here that condensin, a conserved protein complex required for chromosome organization, regulates double-strand break (DSB) formation and repair at the rDNA gene cluster during meiosis in budding yeast. Condensin is highly enriched at the rDNA region during prophase I, released at the prophase I/metaphase I transition, and reassociates with rDNA before anaphase I onset. We show that condensin plays a dual role in maintaining rDNA stability: it suppresses the formation of Spo11-mediated rDNA breaks, and it promotes DSB processing to ensure proper chromosome segregation. Condensin is unnecessary for the export of rDNA breaks outside the nucleolus but required for timely repair of meiotic DSBs. Our work reveals that condensin coordinates meiotic recombination with chromosome segregation at the repetitive rDNA sequence, thereby maintaining genome integrity. PMID:25103240

  4. NMR chemical shift perturbation mapping of DNA binding by a zinc-finger domain from the yeast transcription factor ADR1.

    PubMed Central

    Schmiedeskamp, M.; Rajagopal, P.; Klevit, R. E.

    1997-01-01

    Mutagenesis studies have revealed that the minimal DNA-binding domain of the yeast transcription factor ADR1 consists of two Cys2-His2 zinc fingers plus an additional 20 residues proximal and N-terminal to the fingers. We have assigned NMR 1H, 15N, and 13C chemical shifts for the entire minimal DNA-binding domain of ADR1 both free and bound to specific DNA. 1H chemical shift values suggest little structural difference between the zinc fingers in this construct and in single-finger constructs, and 13C alpha chemical shift index analysis indicates little change in finger structure upon DNA binding. 1H chemical shift perturbations upon DNA binding are observed, however, and these are mapped to define the protein-DNA interface. The two zinc fingers appear to bind DNA with different orientations, as the entire helix of finger 1 is perturbed, while only the extreme N-terminus of the finger 2 helix is affected. Furthermore, residues N-terminal to the first finger undergo large chemical shift changes upon DNA binding suggesting a role at the protein-DNA interface. A striking correspondence is observed between the protein-DNA interface mapped by chemical shift changes and that previously mapped by mutagenesis. PMID:9300483

  5. Structural analysis of the interactions between hsp70 chaperones and the yeast DNA replication protein Orc4p.

    PubMed

    Moreno-del Alamo, María; Sánchez-Gorostiaga, Alicia; Serrano, Ana M; Prieto, Alicia; Cuéllar, Jorge; Martín-Benito, Jaime; Valpuesta, José M; Giraldo, Rafael

    2010-10-15

    Hsp70 chaperones, besides their role in assisting protein folding, are key modulators of protein disaggregation, being consistently found as components of most macromolecular assemblies isolated in proteome-wide affinity purifications. A wealth of structural information has been recently acquired on Hsp70s complexed with Hsp40 and NEF co-factors and with small hydrophobic target peptides. However, knowledge of how Hsp70s recognize large protein substrates is still limited. Earlier, we reported that homologue Hsp70 chaperones (DnaK in Escherichia coli and Ssa1-4p/Ssb1-2p in Saccharomyces cerevisiae) bind strongly, both in vitro and in vivo, to the AAA+ domain in the Orc4p subunit of yeast origin recognition complex (ORC). ScORC is the paradigm for eukaryotic DNA replication initiators and consists of six distinct protein subunits (ScOrc1p-ScOrc 6p). Here, we report that a hydrophobic sequence (IL(4)) in the initiator specific motif (ISM) in Orc4p is the main target for DnaK/Hsp70. The three-dimensional electron microscopy reconstruction of a stable Orc4p(2)-DnaK complex suggests that the C-terminal substrate-binding domain in the chaperone clamps the AAA+ IL(4) motif in one Orc4p molecule, with the substrate-binding domain lid subdomain wedging apart the other Orc4p subunit. Pairwise co-expression in E. coli shows that Orc4p interacts with Orc1/2/5p. Mutation of IL(4) selectively disrupts Orc4p interaction with Orc2p. Allelic substitution of ORC4 by mutants in each residue of IL(4) results in lethal (I184A) or thermosensitive (L185A and L186A) initiation-defective phenotypes in vivo. The interplay between Hsp70 chaperones and the Orc4p-IL(4) motif might have an adaptor role in the sequential, stoichiometric assembly of ScORC subunits. PMID:20732327

  6. Genomic heterogeneity and structural variation in soybean near isogenic lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Near-isogenic lines (NILs) are a critical genetic resource for the soybean research community. The ability to identify and characterize the genes driving the phenotypic differences between NILs is limited by the degree to which differential genetic introgressions can be resolved. Furthermore, the ge...

  7. A yeast-endonuclease-generated DNA break induces antigenic switching in Trypanosoma brucei.

    PubMed

    Boothroyd, Catharine E; Dreesen, Oliver; Leonova, Tatyana; Ly, K Ina; Figueiredo, Luisa M; Cross, George A M; Papavasiliou, F Nina

    2009-05-14

    Trypanosoma brucei is the causative agent of African sleeping sickness in humans and one of the causes of nagana in cattle. This protozoan parasite evades the host immune system by antigenic variation, a periodic switching of its variant surface glycoprotein (VSG) coat. VSG switching is spontaneous and occurs at a rate of about 10(-2)-10(-3) per population doubling in recent isolates from nature, but at a markedly reduced rate (10(-5)-10(-6)) in laboratory-adapted strains. VSG switching is thought to occur predominantly through gene conversion, a form of homologous recombination initiated by a DNA lesion that is used by other pathogens (for example, Candida albicans, Borrelia sp. and Neisseria gonorrhoeae) to generate surface protein diversity, and by B lymphocytes of the vertebrate immune system to generate antibody diversity. Very little is known about the molecular mechanism of VSG switching in T. brucei. Here we demonstrate that the introduction of a DNA double-stranded break (DSB) adjacent to the approximately 70-base-pair (bp) repeats upstream of the transcribed VSG gene increases switching in vitro approximately 250-fold, producing switched clones with a frequency and features similar to those generated early in an infection. We were also able to detect spontaneous DSBs within the 70-bp repeats upstream of the actively transcribed VSG gene, indicating that a DSB is a natural intermediate of VSG gene conversion and that VSG switching is the result of the resolution of this DSB by break-induced replication. PMID:19369939

  8. Telomere-related functions of yeast KU in the repair of bleomycin-induced DNA damage.

    PubMed

    Tam, Angela T Y; Pike, Brietta L; Hammet, Andrew; Heierhorst, Jörg

    2007-06-01

    Bleomycins are small glycopeptide cancer chemotherapeutics that give rise to 3'-modified DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, DSBs are predominantly repaired by RAD52-dependent homologous recombination (HR) with some support by Yku70/Yku80 (KU)-dependent pathways. The main DSB repair function of KU is believed to be as part of the non-homologous end-joining (NHEJ) pathway, but KU also functions in a "chromosome healing" pathway that seals DSBs by de novo telomere addition. We report here that rad52Deltayku70Delta double mutants are considerably more bleomycin hypersensitive than rad52Deltalig4Delta cells that lack the NHEJ-specific DNA ligase 4. Moreover, the telomere-specific KU mutation yku80-135i also dramatically increases rad52Delta bleomycin hypersensitivity, almost to the level of rad52Deltayku80Delta. The results indicate that telomere-specific functions of KU play a more prominent role in the repair of bleomycin-induced damage than its NHEJ functions, which could have important clinical implications for bleomycin-based combination chemotherapies. PMID:17442269

  9. Comparative Analysis of in vivo Interactions Between Rev1 Protein and Other Y-Family DNA Polymerases in Animals and Yeasts

    PubMed Central

    Kosarek, J. Nicole; Woodruff, Rachel V.; Rivera-Begeman, Amanda; Guo, Caixia; D’Souza, Sanjay; Koonin, Eugene V.; Walker, Graham C.; Friedberg, Errol C.

    2008-01-01

    Summary Eukaryotes are endowed with multiple specialized DNA polymerases, some (if not all) of which are believed to play important roles in the tolerance of base damage during DNA replication. Among these DNA polymerases, Rev1 protein (a deoxycytidyl transferase) from vertebrates interacts with several other specialized polymerases via a highly conserved C-terminal region. The present studies assessed whether these interactions are retained in more experimentally tractable model systems, including yeasts, flies, and the nematode C. elegans. We observed a physical interaction between Rev1 protein and other Y-family polymerases in the fruit fly Drosophila melanogaster. However, despite the fact that the C-terminal region of Drosophila and yeast Rev1 are conserved from vertebrates to a similar extent, such interactions were not observed in S. cerevisiae or S. pombe. With respect to regions in specialized DNA polymerases that are required for interaction with Rev1, we find predicted disorder to be an underlying structural commonality. The results of this study suggest that special consideration should be exercised when making mechanistic extrapolations regarding translesion DNA synthesis from one eukaryotic system to another. PMID:18242152

  10. Sequencer-Based Capillary Gel Electrophoresis (SCGE) Targeting the rDNA Internal Transcribed Spacer (ITS) Regions for Accurate Identification of Clinically Important Yeast Species

    PubMed Central

    Chen, Sharon C.-A.; Wang, He; Zhang, Li; Fan, Xin; Xu, Zhi-Peng; Cheng, Jing-Wei; Kong, Fanrong; Zhao, Yu-Pei; Xu, Ying-Chun

    2016-01-01

    Accurate species identification of Candida, Cryptococcus, Trichosporon and other yeast pathogens is important for clinical management. In the present study, we developed and evaluated a yeast species identification scheme by determining the rDNA internal transcribed spacer (ITS) region length types (LTs) using a sequencer-based capillary gel electrophoresis (SCGE) approach. A total of 156 yeast isolates encompassing 32 species were first used to establish a reference SCGE ITS LT database. Evaluation of the ITS LT database was then performed on (i) a separate set of (n = 97) clinical isolates by SCGE, and (ii) 41 isolates of 41 additional yeast species from GenBank by in silico analysis. Of 156 isolates used to build the reference database, 41 ITS LTs were identified, which correctly identified 29 of the 32 (90.6%) species, with the exception of Trichosporon asahii, Trichosporon japonicum and Trichosporon asteroides. In addition, eight of the 32 species revealed different electropherograms and were subtyped into 2–3 different ITS LTs each. Of the 97 test isolates used to evaluate the ITS LT scheme, 96 (99.0%) were correctly identified to species level, with the remaining isolate having a novel ITS LT. Of the additional 41 isolates for in silico analysis, none was misidentified by the ITS LT database except for Trichosporon mucoides whose ITS LT profile was identical to that of Trichosporon dermatis. In conclusion, yeast identification by the present SCGE ITS LT assay is a fast, reproducible and accurate alternative for the identification of clinically important yeasts with the exception of Trichosporon species. PMID:27105313

  11. Surveying expression level polymorphism and single-feature polymorphism in near-isogenic wheat lines differing for the Yr5 stripe rust resistance locus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA polymorphisms are valuable for several applications including genotyping, molecular mapping and marker-assisted selection. The Affymetrix Wheat GeneChip was used to survey expression level polymorphisms (ELPs) and single-feature polymorphisms (SFPs) between two near-isogenic wheat genotypes (BC7...

  12. Surveying expression level polymorphism and single-feature polymorphism in near-isogenic wheat lines differing for the Yr5 stripe rust resistance locus

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA polymorphisms are valuable for several applications including genotyping, molecular mapping and marker-assisted selection. The Affymetrix Wheat GeneChip was used to survey expression level polymorphisms (ELPs) and single-feature polymorphisms (SFPs) between two near-isogenic wheat genotypes (BC...

  13. Microarray-Based Genetic Mapping Using Soybean Near-Isogenic Lines and Generation of SNP Markers in the Rag1 Aphid-Resistance Interval

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A strategy using near-isogenic lines (NILs) and Affymetrix Soybean GeneChip microarrays was employed to identify genetic markers closely linked to the soybean aphid [Aphis glycines Matsumura (Hemiptera: Aphididae)] resistance gene Rag1 in soybean [Glycine max (L.) Merr.]. Genomic DNA from the aphid ...

  14. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes*

    PubMed Central

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-01-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  15. Construction and analysis of the cDNA subtraction library of yeast and mycelial phases of Sporothrix globosa isolated in China: identification of differentially expressed genes.

    PubMed

    Hu, Qing-bi; He, Yu; Zhou, Xun

    2015-12-01

    Species included in the Sporothrix schenckii complex are temperature-dependent with dimorphic growth and cause sporotrichosis that is characterized by chronic and fatal lymphocutaneous lesions. The putative species included in the Sporothrix complex are S. brasiliensis, S. globosa, S. mexicana, S. pallida, S. schenckii, and S. lurei. S. globosa is the causal agent of sporotrichosis in China, and its pathogenicity appears to be closely related to the dimorphic transition, i.e. from the mycelial to the yeast phase, it adapts to changing environmental conditions. To determine the molecular mechanisms of the switching process that mediates the dimorphic transition of S. globosa, suppression subtractive hybridization (SSH) was used to prepare a complementary DNA (cDNA) subtraction library from the yeast and mycelial phases. Bioinformatics analysis was performed to profile the relationship between differently expressed genes and the dimorphic transition. Two genes that were expressed at higher levels by the yeast form were selected, and their differential expression levels were verified using a quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR). It is believed that these differently expressed genes are involved in the pathogenesis of S. globosa infection in China. PMID:26642182

  16. DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and {gamma}-rays

    SciTech Connect

    Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep . E-mail: rakwal-68@aist.go.jp; Iwahashi, Hitoshi

    2006-07-21

    Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma ({gamma})-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and {gamma}-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and {gamma}-rays). Similarly, for X- and {gamma}-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and {gamma}-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-a-vis their energy levels.

  17. Paramagnetic cobalt as a probe of the orientation of an accessory DNA-binding region of the yeast ADR1 zinc-finger protein.

    PubMed

    Schmiedeskamp, M; Klevit, R E

    1997-11-18

    The minimal DNA-binding domain of the yeast ADR1 transcription factor consists of two Cys2-His2 zinc fingers and an additional 20 residues N-terminal and proximal to the fingers. The accessory sequence likely plays a role in contacting DNA. Paramagnetic cobalt was incorporated into the fingers of an ADR1 DNA-binding construct (ADR1z) to serve as a probe of the proximity of the accessory sequence to the zinc fingers. NMR signals from the accessory region are not perturbed by cobalt incorporation. Previous studies showed that this region is random coil in the ADR1z construct in the absence of DNA; it does not adopt a fixed orientation with respect to the cobalt sites. In contrast, many residues of the accessory region are perturbed by cobalt in the DNA-bound form of the protein, suggesting this region becomes constrained. This observation agrees with previous results showing a disorder-to-order transition for the accessory region upon DNA binding. Furthermore, these results indicate that the accessory region lies close to the fingers in the protein-DNA complex. This region thus does not extend along the DNA away from the zinc fingers; it more likely binds the same stretch of DNA contacted by the zinc fingers. Comparison to the behavior of other zinc-finger proteins that utilize an accessory DNA-binding sequence suggested that the region of ADR1 proximal to the zinc fingers might form an alpha-helix. Analysis of sequential NOEs in the accessory region of DNA-bound ADR1z reveals no helical structure. PMID:9369471

  18. Single-step co-integration of multiple expressible heterologous genes into the ribosomal DNA of the methylotrophic yeast Hansenula polymorpha.

    PubMed

    Klabunde, J; Diesel, A; Waschk, D; Gellissen, G; Hollenberg, C P; Suckow, M

    2002-05-01

    We have investigated the methylotrophic yeast Hansenula polymorpha as a host for the co-integration and expression of multiple heterologous genes using an rDNA integration approach. The ribosomal DNA (rDNA) of H. polymorpha was found to consist of a single rDNA cluster of about 50-60 repeats of an 8-kb unit located on chromosome II. A 2.4-kb segment of H. polymorpha rDNA encompassing parts of the 25S, the complete 5S and the non-transcribed spacer region between 25S and 18S rDNA was isolated and inserted into conventional integrative H. polymorpha plasmids harboring the Saccharomyces- cerevisiae-derived URA3 gene for selection. These rDNA plasmids integrated homologously into the rDNA repeats of a H. polymorpha (odc1) host as several independent clusters. Anticipating that this mode of multiple-cluster integration could be used for the simultaneous integration of several distinct rDNA plasmids, the host strain was co-transformed with a mixture of up to three different plasmids, all bearing the same URA3 selection marker. Transformations indeed resulted in mitotically stable strains harboring one, two, or all three plasmids integrated into the rDNA. The overall copy number of the plasmids integrated did not exceed the number of rDNA repeats present in the untransformed host strain, irrespective of the number of different plasmids involved. Strains harboring different plasmids co-expressed the introduced genes, resulting in functional proteins. Thus, this approach provides a new and attractive tool for the rapid generation of recombinant strains that simultaneously co-produce several proteins in desired stoichiometric ratios. PMID:12021801

  19. A Functional Yeast Survival Screen of Tumor-Derived cDNA Libraries Designed to Identify Anti-Apoptotic Mammalian Oncogenes

    PubMed Central

    Melzer, Inga Maria; Moser, Julia; Siele, Dagmar; Köhl, Ulrike; Rieker, Ralf Joachim; Wachter, David Lukas; Agaimy, Abbas; Herpel, Esther; Baumgarten, Peter; Mittelbronn, Michel; Rakel, Stefanie; Kögel, Donat; Böhm, Stefanie; Gutschner, Tony; Diederichs, Sven; Zörnig, Martin

    2013-01-01

    Yeast cells can be killed upon expression of pro-apoptotic mammalian proteins. We have established a functional yeast survival screen that was used to isolate novel human anti-apoptotic genes overexpressed in treatment-resistant tumors. The screening of three different cDNA libraries prepared from metastatic melanoma, glioblastomas and leukemic blasts allowed for the identification of many yeast cell death-repressing cDNAs, including 28% of genes that are already known to inhibit apoptosis, 35% of genes upregulated in at least one tumor entity and 16% of genes described as both anti-apoptotic in function and upregulated in tumors. These results confirm the great potential of this screening tool to identify novel anti-apoptotic and tumor-relevant molecules. Three of the isolated candidate genes were further analyzed regarding their anti-apoptotic function in cell culture and their potential as a therapeutic target for molecular therapy. PAICS, an enzyme required for de novo purine biosynthesis, the long non-coding RNA MALAT1 and the MAST2 kinase are overexpressed in certain tumor entities and capable of suppressing apoptosis in human cells. Using a subcutaneous xenograft mouse model, we also demonstrated that glioblastoma tumor growth requires MAST2 expression. An additional advantage of the yeast survival screen is its universal applicability. By using various inducible pro-apoptotic killer proteins and screening the appropriate cDNA library prepared from normal or pathologic tissue of interest, the survival screen can be used to identify apoptosis inhibitors in many different systems. PMID:23717670

  20. Apn1 AP-endonuclease is essential for the repair of oxidatively damaged DNA bases in yeast frataxin-deficient cells

    PubMed Central

    Lefevre, Sophie; Brossas, Caroline; Auchère, Françoise; Boggetto, Nicole; Camadro, Jean-Michel; Santos, Renata

    2012-01-01

    Frataxin deficiency results in mitochondrial dysfunction and oxidative stress and it is the cause of the hereditary neurodegenerative disease Friedreich ataxia (FA). Here, we present evidence that one of the pleiotropic effects of oxidative stress in frataxin-deficient yeast cells (Δyfh1 mutant) is damage to nuclear DNA and that repair requires the Apn1 AP-endonuclease of the base excision repair pathway. Major phenotypes of Δyfh1 cells are respiratory deficit, disturbed iron homeostasis and sensitivity to oxidants. These phenotypes are weak or absent under anaerobiosis. We show here that exposure of anaerobically grown Δyfh1 cells to oxygen leads to down-regulation of antioxidant defenses, increase in reactive oxygen species, delay in G1- and S-phases of the cell cycle and damage to mitochondrial and nuclear DNA. Nuclear DNA lesions in Δyfh1 cells are primarily caused by oxidized bases and single-strand breaks that can be detected 15–30 min after oxygen exposition. The Apn1 enzyme is essential for the repair of the DNA lesions in Δyfh1 cells. Compared with Δyfh1, the double Δyfh1Δapn1 mutant shows growth impairment, increased mutagenesis and extreme sensitivity to H2O2. On the contrary, overexpression of the APN1 gene in Δyfh1 cells decreases spontaneous and induced mutagenesis. Our results show that frataxin deficiency in yeast cells leads to increased DNA base oxidation and requirement of Apn1 for repair, suggesting that DNA damage and repair could be important features in FA disease progression. PMID:22706278

  1. The RXL motif of the African cassava mosaic virus Rep protein is necessary for rereplication of yeast DNA and viral infection in plants.

    PubMed

    Hipp, Katharina; Rau, Peter; Schäfer, Benjamin; Gronenborn, Bruno; Jeske, Holger

    2014-08-01

    Geminiviruses, single-stranded DNA plant viruses, encode a replication-initiator protein (Rep) that is indispensable for virus replication. A potential cyclin interaction motif (RXL) in the sequence of African cassava mosaic virus Rep may be an alternative link to cell cycle controls to the known interaction with plant homologs of retinoblastoma protein (pRBR). Mutation of this motif abrogated rereplication in fission yeast induced by expression of wildtype Rep suggesting that Rep interacts via its RXL motif with one or several yeast proteins. The RXL motif is essential for viral infection of Nicotiana benthamiana plants, since mutation of this motif in infectious clones prevented any symptomatic infection. The cell-cycle link (Clink) protein of a nanovirus (faba bean necrotic yellows virus) was investigated that activates the cell cycle by binding via its LXCXE motif to pRBR. Expression of wildtype Clink and a Clink mutant deficient in pRBR-binding did not trigger rereplication in fission yeast. PMID:24999043

  2. The Protein Level of Rev1, a TLS Polymerase in Fission Yeast, Is Strictly Regulated during the Cell Cycle and after DNA Damage

    PubMed Central

    Uchiyama, Masashi; Terunuma, Junko; Hanaoka, Fumio

    2015-01-01

    Translesion DNA synthesis provides an alternative DNA replication mechanism when template DNA is damaged. In fission yeast, Eso1 (polη), Kpa1/DinB (polκ), Rev1, and Polζ (a complex of Rev3 and Rev7) have been identified as translesion synthesis polymerases. The enzymatic characteristics and protein-protein interactions of these polymerases have been intensively characterized; however, how these proteins are regulated during the cell cycle remains unclear. Therefore, we examined the cell cycle oscillation of translesion polymerases. Interestingly, the protein levels of Rev1 peaked during G1 phase and then decreased dramatically at the entry of S phase; this regulation was dependent on the proteasome. Temperature-sensitive proteasome mutants, such as mts2-U31 and mts3-U32, stabilized Rev1 protein when the temperature was shifted to the restrictive condition. In addition, deletion of pop1 or pop2, subunits of SCF ubiquitin ligase complexes, upregulated Rev1 protein levels. Besides these effects during the cell cycle, we also observed upregulation of Rev1 protein upon DNA damage. This upregulation was abolished when rad3, a checkpoint protein, was deleted or when the Rev1 promoter was replaced with a constitutive promoter. From these results, we hypothesize that translesion DNA synthesis is strictly controlled through Rev1 protein levels in order to avoid unwanted mutagenesis. PMID:26147350

  3. Determination of fruit origin by using 26S rDNA fingerprinting of yeast communities by PCR-DGGE: preliminary application to Physalis fruits from Egypt.

    PubMed

    El Sheikha, Aly Farag; Condur, Ana; Métayer, Isabelle; Nguyen, Doan Duy Le; Loiseau, Gérard; Montet, Didier

    2009-10-01

    The determination of geographical origin is a demand of the traceability system of import-export food products. One hypothesis for tracing the source of a product is by global analysis of the microbial communities of the food and statistical linkage of this analysis to the geographical origin of the food. For this purpose, a molecular technique employing 26S rDNA profiles generated by PCR-DGGE was used to detect the variation in yeast community structures of three species of Physalis fruit (Physalis ixocarpa Brat, Physalis pubescens L, Physalis pruinosa L) from four Egyptian regions (Qalyoubia, Minufiya, Beheira and Alexandria Governments). When the 26S rDNA profiles were analysed by multivariate analysis, distinct microbial communities were detected. The band profiles of Physalis yeasts from different Governments were specific for each location and could be used as a bar code to discriminate the origin of the fruits. This method is a new traceability tool which provides fruit products with a unique biological bar code and makes it possible to trace back the fruits to their original location. PMID:19784935

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

    PubMed Central

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

    2015-01-01

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

  5. cDNA structure, tissue distribution, and chromosomal localization of rat PC7, a novel mammalian proprotein convertase closest to yeast kexin-like proteinases.

    PubMed Central

    Seidah, N G; Hamelin, J; Mamarbachi, M; Dong, W; Tardos, H; Mbikay, M; Chretien, M; Day, R

    1996-01-01

    By using reverse transcription-coupled PCR on rat anterior pituitary RNA, we isolated a 285-bp cDNA coding for a novel subtilisin/kexin-like protein convertase (PC), called rat (r) PC7. By screening rat spleen and PC12 cell lambda gt11 cDNA libraries, we obtained a composite 3.5-kb full-length cDNA sequence of rPC7. The open reading frame codes for a prepro-PC with a 36-amino acid signal peptide, a 104-amino acid prosegment ending with a cleavable RAKR sequence, and a 747-amino acid type I membrane-bound glycoprotein, representing the mature form of this serine proteinase. Phylogenetic analysis suggests that PC7 represents the most divergent enzyme of the mammalian convertase family and that it is the closest member to the yeast convertases krp and kexin. Northern blot analyses demonstrated a widespread expression with the richest source of rPC7 mRNA being the colon and lymphoid-associated tissues. In situ hybridization revealed a distinctive tissue distribution that sometimes overlaps with that of furin, suggesting that PC7 has widespread proteolytic functions. The gene for PC7 (Pcsk7) was mapped to mouse chromosome 9 by linkage analysis of an interspecific backcross DNA panel. Images Fig. 3 Fig. 4 Fig. 5 PMID:8622945

  6. The budding yeast Centromere DNA Element II wraps a stable Cse4 hemisome in either orientation in vivo

    PubMed Central

    Henikoff, Steven; Ramachandran, Srinivas; Krassovsky, Kristina; Bryson, Terri D; Codomo, Christine A; Brogaard, Kristin; Wang, Ji-Ping; Henikoff, Jorja G

    2014-01-01

    In budding yeast, a single cenH3 (Cse4) nucleosome occupies the ∼120-bp functional centromere, however conflicting structural models for the particle have been proposed. To resolve this controversy, we have applied H4S47C-anchored cleavage mapping, which reveals the precise position of histone H4 in every nucleosome in the genome. We find that cleavage patterns at centromeres are unique within the genome and are incompatible with symmetrical structures, including octameric nucleosomes and (Cse4/H4)2 tetrasomes. Centromere cleavage patterns are compatible with a precisely positioned core structure, one in which each of the 16 yeast centromeres is occupied by oppositely oriented Cse4/H4/H2A/H2B hemisomes in two rotational phases within the population. Centromere-specific hemisomes are also inferred from distances observed between closely-spaced H4 cleavages, as predicted from structural modeling. Our results indicate that the orientation and rotational position of the stable hemisome at each yeast centromere is not specified by the functional centromere sequence. DOI: http://dx.doi.org/10.7554/eLife.01861.001 PMID:24737863

  7. Negative superhelicity promotes ATP-dependent binding of yeast RAD3 protein to ultraviolet-damaged DNA.

    PubMed

    Sung, P; Watkins, J F; Prakash, L; Prakash, S

    1994-03-18

    The RAD3 gene of Saccharomyces cerevisiae is required for excision repair of UV-damaged DNA and is essential for cell viability. Remarkable homology exists between RAD3 and the human excision repair gene XPD, whose mutational inactivation underlies the cancer-prone disorder in xeroderma pigmentosum group D patients. Our previous work demonstrated that RAD3-encoded protein contains a DNA helicase activity. Here, we show that RAD3 binds preferentially to UV-damaged DNA over nondamaged DNA. Removal of pyrimidine dimers from damaged DNA by enzymatic photoreactivation does not affect binding, suggesting an affinity of RAD3 for pyrimidine (6-4) pyrimidone photoproducts. Damage-specific binding by RAD3 is strongly dependent on ATP and on the degree of negative superhelicity in DNA. The requirement of superhelicity in damage binding may target RAD3 to regions of DNA undergoing transcription, resulting in the preferential repair of these regions. The rad3 Arg-48 mutant protein, which lacks the DNA helicase activity, also binds UV-damaged DNA preferentially, indicating that DNA helicase and damage binding are two distinct and separable functional entities in RAD3. PMID:8132553

  8. Epidemiologic Study of Malassezia Yeasts in Seborrheic Dermatitis Patients by the Analysis of 26S rDNA PCR-RFLP

    PubMed Central

    Oh, Byung Ho; Choe, Yong Beom; Ahn, Kyu Joong

    2010-01-01

    Background This case-control study concerns a molecular biological method based on the data gathered from a group of Korean subjects to examine the distribution of Malassezia yeasts in seborrheic dermatitis (SD) patients. Cultures for Malassezia yeasts were taken from the foreheads, cheeks and chests of 60 patients with SD and in 60 healthy controls of equivalent age. Objective The purpose of this study is to identify the relationship between certain species of Malassezia and SD. This was done by analyzing the differences in the distribution of Malassezia species in terms of age and body parts of the host with healthy controls. Methods 26S rDNA PCR-RFLP, a fast and accurate molecular biological method, was used to overcome the limits of morphological and biochemical methods. Results The positive Malassezia culture rate was 51.7% in patients with SD, which was lower than that of healthy adults (63.9%). M. restricta was dominant in patients with SD (19.5%). Likewise, M. restricta was identified as a common species (20.5%) in healthy controls. In the ages 31~40, M. restricta was found to be the most common species (31.6%) among SD patients. Conclusion According to the results of the study, the most frequently isolated species was M. restricta (19.5%) in patients with SD. There was no statistically significant difference in the distribution of Malassezia species between the SD patients and healthy control groups. PMID:20548904

  9. Yeast Infections

    MedlinePlus

    ... antibiotics, it can multiply and cause an infection. Yeast infections affect different parts of the body in different ways: Thrush is a yeast infection that causes white patches in your mouth Candida ...

  10. Identification of a novel alpha-amylase by expression of a newly cloned human amy3 cDNA in yeast.

    PubMed

    Shiosaki, K; Takata, K; Omichi, K; Tomita, N; Horii, A; Ogawa, M; Matsubara, K

    1990-05-14

    A novel amylase gene (amy3) that differs in nucleotide sequence from salivary amylase gene (amy1) and pancreatic amylase gene (amy2) has been described [Tomita et al., Gene 76 (1989) 11-18], but whether this gene can ever code for an active enzyme has not been shown. We prepared cDNA of this gene from an mRNA obtained from lung carcinoid tissue, and expressed it in Saccharomyces cerevisiae under the control of an acid phosphatase promoter. The product was secreted into culture media, and showed enzymatic activity, demonstrating that this novel alpha-amylase gene (amy3) can code for a functional isozyme. We purified this enzyme, and compared its biological properties with those of salivary and pancreatic human amylases similarly expressed in yeast. We observed that the novel amylase isozyme is more heat-sensitive than others, and that its substrate specificity is different from the other two isozymes. PMID:2197187

  11. More than 10% of yeast genes are related to genome stability and influence cellular senescence via rDNA maintenance.

    PubMed

    Saka, Kimiko; Takahashi, Akihiro; Sasaki, Mariko; Kobayashi, Takehiko

    2016-05-19

    Genome instability triggers cellular senescence and is a common cause of cancer. The ribosomal RNA genes (rDNA), due to their repetitive structure, form a fragile site with frequent rearrangements. To identify eukaryotic factors that connect reduced genome stability to senescence we screened 4,876 strains of a Saccharomyces cerevisiae deletion library for aberrant rDNA and found 708 genes that contribute to its upkeep. 28 mutants caused abnormalities in non-rDNA chromosomes and among them 12 mutants have abnormalities both in rDNA and in non-rDNA chromosomes. Many mutated genes have not previously been implicated with genome maintenance nor their homologues with tumorigenesis in mammals. The link between rDNA state and senescence was broken after deletion of factors related with DNA polymerase ϵ. These mutations also suppressed the short lifespan phenotype of a sir2 mutant, suggesting a model in which molecular events at the heart of the replication fork induce abnormal rDNA recombination and are responsible for the emergence of an aging signal. PMID:26912831

  12. A yeast gene, MGS1, encoding a DNA-dependent AAA(+) ATPase is required to maintain genome stability.

    PubMed

    Hishida, T; Iwasaki, H; Ohno, T; Morishita, T; Shinagawa, H

    2001-07-17

    Changes in DNA superhelicity during DNA replication are mediated primarily by the activities of DNA helicases and topoisomerases. If these activities are defective, the progression of the replication fork can be hindered or blocked, which can lead to double-strand breaks, elevated recombination in regions of repeated DNA, and genome instability. Hereditary diseases like Werner's and Bloom's Syndromes are caused by defects in DNA helicases, and these diseases are associated with genome instability and carcinogenesis in humans. Here we report a Saccharomyces cerevisiae gene, MGS1 (Maintenance of Genome Stability 1), which encodes a protein belonging to the AAA(+) class of ATPases, and whose central region is similar to Escherichia coli RuvB, a Holliday junction branch migration motor protein. The Mgs1 orthologues are highly conserved in prokaryotes and eukaryotes. The Mgs1 protein possesses DNA-dependent ATPase and single-strand DNA annealing activities. An mgs1 deletion mutant has an elevated rate of mitotic recombination, which causes genome instability. The mgs1 mutation is synergistic with a mutation in top3 (encoding topoisomerase III), and the double mutant exhibits severe growth defects and markedly increased genome instability. In contrast to the mgs1 mutation, a mutation in the sgs1 gene encoding a DNA helicase homologous to the Werner and Bloom helicases suppresses both the growth defect and the increased genome instability of the top3 mutant. Therefore, evolutionarily conserved Mgs1 may play a role together with RecQ family helicases and DNA topoisomerases in maintaining proper DNA topology, which is essential for genome stability. PMID:11459965

  13. Yeast high mobility group protein HMO1 stabilizes chromatin and is evicted during repair of DNA double strand breaks

    PubMed Central

    Panday, Arvind; Xiao, LiJuan; Grove, Anne

    2015-01-01

    DNA is packaged into condensed chromatin fibers by association with histones and architectural proteins such as high mobility group (HMGB) proteins. However, this DNA packaging reduces accessibility of enzymes that act on DNA, such as proteins that process DNA after double strand breaks (DSBs). Chromatin remodeling overcomes this barrier. We show here that the Saccharomyces cerevisiae HMGB protein HMO1 stabilizes chromatin as evidenced by faster chromatin remodeling in its absence. HMO1 was evicted along with core histones during repair of DSBs, and chromatin remodeling events such as histone H2A phosphorylation and H3 eviction were faster in absence of HMO1. The facilitated chromatin remodeling in turn correlated with more efficient DNA resection and recruitment of repair proteins; for example, inward translocation of the DNA-end-binding protein Ku was faster in absence of HMO1. This chromatin stabilization requires the lysine-rich C-terminal extension of HMO1 as truncation of the HMO1 C-terminal tail phenocopies hmo1 deletion. Since this is reminiscent of the need for the basic C-terminal domain of mammalian histone H1 in chromatin compaction, we speculate that HMO1 promotes chromatin stability by DNA bending and compaction imposed by its lysine-rich domain and that it must be evicted along with core histones for efficient DSB repair. PMID:25979266

  14. A novel cis-acting element required for DNA damage-inducible expression of yeast DIN7

    SciTech Connect

    Yoshitani, Ayako; Yoshida, Minoru; Ling Feng

    2008-01-04

    Din7 is a DNA damage-inducible mitochondrial nuclease that modulates the stability of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae. How DIN7 gene expression is regulated, however, has remained largely unclear. Using promoter sequence alignment, we found a highly conserved 19-bp sequence in the promoter regions of DIN7 and NTG1, which encodes an oxidative stress-inducible base-excision-repair enzyme. Deletion of the 19-bp sequence markedly reduced the hydroxyurea (HU)-enhanced DIN7 promoter activity. In addition, nuclear fractions prepared from HU-treated cells were used in in vitro band shift assays to reveal the presence of currently unidentified trans-acting factor(s) that preferentially bound to the 19-bp region. These results suggest that the 19-bp sequence is a novel cis-acting element that is required for the regulation of DIN7 expression in response to HU-induced DNA damage.

  15. C(m)CGG methylation-independent parent-of-origin effects on genome-wide transcript levels in isogenic reciprocal F1 triploid plants.

    PubMed

    Donoghue, Mark T A; Fort, Antoine; Clifton, Rachel; Zhang, Xu; McKeown, Peter C; Voigt-Zielinksi, M L; Borevitz, Justin O; Spillane, Charles

    2014-01-01

    Triploid F1 hybrids generated via reciprocal interploidy crosses between genetically distinct parental plants can display parent-of-origin effects on gene expression or phenotypes. Reciprocal triploid F1 isogenic plants generated from interploidy crosses in the same genetic background allow investigation on parent-of-origin-specific (parental) genome-dosage effects without confounding effects of hybridity involving heterozygous mutations. Whole-genome transcriptome profiling was conducted on reciprocal F1 isogenic triploid (3x) seedlings of A. thaliana. The genetically identical reciprocal 3x genotypes had either an excess of maternally inherited 3x(m) or paternally inherited 3x(p) genomes. We identify a major parent-of-origin-dependent genome-dosage effect on transcript levels, whereby 602 genes exhibit differential expression between the reciprocal F1 triploids. In addition, using methylation-sensitive DNA tiling arrays, constitutive and polymorphic CG DNA methylation patterns at CCGG sites were analysed, which revealed that paternal-excess F1 triploid seedling C(m)CGG sites are overall hypermethylated. However, no correlation exists between C(m)CGG methylation polymorphisms and transcriptome dysregulation between the isogenic reciprocal F1 triploids. Overall, our study indicates that parental genome-dosage effects on the transcriptome levels occur in paternal-excess triploids, which are independent of C(m)CGG methylation polymorphisms. Such findings have implications for understanding parental effects and genome-dosage effects on gene expression and phenotypes in polyploid plants. PMID:24212467

  16. Functional characterization of a rice de novo DNA methyltransferase, OsDRM2, expressed in Escherichia coli and yeast

    SciTech Connect

    Pang, Jinsong; Dong, Mingyue; Li, Ning; Zhao, Yanli; Liu, Bao

    2013-03-01

    Highlights: ► A rice de novo DNA methyltransferase OsDRM2 was cloned. ► In vitro methylation activity of OsDRM2 was characterized with Escherichia coli. ► Assays of OsDRM2 in vivo methylation were done with Saccharomyces cerevisiae. ► OsDRM2 methylation activity is not preferential to any type of cytosine context. ► The activity of OsDRM2 is independent of RdDM pathway. - Abstract: DNA methylation of cytosine nucleotides is an important epigenetic modification that occurs in most eukaryotic organisms and is established and maintained by various DNA methyltransferases together with their co-factors. There are two major categories of DNA methyltransferases: de novo and maintenance. Here, we report the isolation and functional characterization of a de novo methyltransferase, named OsDRM2, from rice (Oryza sativa L.). The full-length coding region of OsDRM2 was cloned and transformed into Escherichia coli and Saccharomyces cerevisiae. Both of these organisms expressed the OsDRM2 protein, which exhibited stochastic de novo methylation activity in vitro at CG, CHG, and CHH di- and tri-nucleotide patterns. Two lines of evidence demonstrated the de novo activity of OsDRM2: (1) a 5′-CCGG-3′ containing DNA fragment that had been pre-treated with OsDRM2 protein expressed in E. coli was protected from digestion by the CG-methylation-sensitive isoschizomer HpaII; (2) methylation-sensitive amplified polymorphism (MSAP) analysis of S. cerevisiae genomic DNA from transformants that had been introduced with OsDRM2 revealed CG and CHG methylation levels of 3.92–9.12%, and 2.88–6.93%, respectively, whereas the mock control S. cerevisiae DNA did not exhibit cytosine methylation. These results were further supported by bisulfite sequencing of the 18S rRNA and EAF5 genes of the transformed S. cerevisiae, which exhibited different DNA methylation patterns, which were observed in the genomic DNA. Our findings establish that OsDRM2 is an active de novo DNA

  17. A novel DNA binding motif for yeast zinc cluster proteins: the Leu3p and Pdr3p transcriptional activators recognize everted repeats.

    PubMed Central

    Hellauer, K; Rochon, M H; Turcotte, B

    1996-01-01

    The Gal4, Put3, and Ppr1 yeast zinc cluster proteins bind as homodimers to DNA sequences composed of palindromic CGG triplets. Spacing between the triplets specifies the target site for a given zinc cluster protein. In addition, Hap1p, another zinc cluster protein, also recognizes CGG triplets but only when oriented as a direct repeat. Unexpectedly, our results show that Leu3p, another member of this family, also recognizes CGG triplets but oriented in opposite directions and spaced by 4 nucleotides (an everted repeat or inverted palindrome: CCG-N4-CGG). This constitutes a novel DNA motif for zinc cluster proteins. Moreover, the presence of this motif was shown to be essential for in vivo activation by Leu3p of a minimal reporter containing one copy of a target site for this activator. We also provide evidence that another member of this family, Pdr3p, binds to an everted repeat spaced by 0 nucleotides (CCGCGG). Thus, our results show that three CGG motifs are used by members of the zinc cluster family: palindromes, direct repeats, and everted repeats. PMID:8887639

  18. Yeast DNA ligase IV mutations reveal a nonhomologous end joining function of BRCT1 distinct from XRCC4/Lif1 binding

    PubMed Central

    Chiruvella, Kishore K.; Renard, Brian M.; Birkeland, Shanda R.; Sunder, Sham; Liang, Zhuobin; Wilson, Thomas E.

    2014-01-01

    LIG4/Dnl4 is the DNA ligase that (re)joins DNA double-strand breaks (DSBs) via nonhomologous end joining (NHEJ), an activity supported by binding of its tandem BRCT domains to the ligase accessory protein XRCC4/Lif1. We screened a panel of 88 distinct ligase mutants to explore the structure-function relationships of the yeast Dnl4 BRCT domains and inter-BRCT linker in NHEJ. Screen results suggested two distinct classes of BRCT mutations with differential effects on Lif1 interaction as compared to NHEJ completion. Validated constructs confirmed that D800K and GG(868:869)AA mutations, which target the Lif1 binding interface, showed a severely defective Dnl4-Lif1 interaction but a less consistent and often small decrease in NHEJ activity in some assays, as well as nearly normal levels of Dnl4 accumulation at DSBs. In contrast, mutants K742A and KTT(742:744)ATA, which target the β3-α2 region of the first BRCT domain, substantially decreased NHEJ function commensurate with a large defect in Dnl4 recruitment to DSBs, despite a comparatively greater preservation of the Lif1 interaction. Together, these separation-of-function mutants indicate that Dnl4 BRCT1 supports DSB recruitment and NHEJ in a manner distinct from Lif1 binding and reveal a complexity of Dnl4 BRCT domain functions in support of stable DSB association. PMID:25457772

  19. Use of a ring chromosome and pulsed-field gels to study interhomolog recombination, double-strand DNA breaks and sister-chromatid exchange in yeast

    SciTech Connect

    Game, J.C. ); Sitney, K.C.; Cook, V.E.; Mortimer, R.K. )

    1989-12-01

    The authors describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome II (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. They demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints. They present data on the timing of commitment to meiotic recombination scored genetically. They have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-size circles originating in part from sister-chromatid exchange, which they find to be frequent during meiosis.

  20. Use of a Ring Chromosome and Pulsed-Field Gels to Study Interhomolog Recombination, Double-Strand DNA Breaks and Sister-Chromatid Exchange in Yeast

    PubMed Central

    Game, J. C.; Sitney, K. C.; Cook, V. E.; Mortimer, R. K.

    1989-01-01

    We describe a system that uses pulsed-field gels for the physical detection of recombinant DNA molecules, double-strand DNA breaks (DSB) and sister-chromatid exchange in the yeast Saccharomyces cerevisiae. The system makes use of a circular variant of chromosome III (Chr. III). Meiotic recombination between this ring chromosome and a linear homolog produces new molecules of sizes distinguishable on gels from either parental molecule. We demonstrate that these recombinant molecules are not present either in strains with two linear Chr. III molecules or in rad50 mutants, which are defective in meiotic recombination. In conjunction with the molecular endpoints, we present data on the timing of commitment to meiotic recombination scored genetically. We have used x-rays to linearize circular Chr. III, both to develop a sensitive method for measuring frequency of DSB and as a means of detecting double-sized circles originating in part from sister-chromatid exchange, which we find to be frequent during meiosis. PMID:2693206

  1. A unique nucleosome arrangement, maintained actively by chromatin remodelers facilitates transcription of yeast tRNA genes

    PubMed Central

    2013-01-01

    Background RNA polymerase (pol) III transcribes a unique class of genes with intra-genic promoters and high transcriptional activity. The major contributors to the pol III transcriptome, tRNAs genes are found scattered on all chromosomes of yeast. A prototype tDNA of <150 bp length, is generally considered nucleosome-free while some pol III-transcribed genes have been shown to have nucleosome-positioning properties. Results Using high resolution ChIP-chip and ChIP-seq methods, we found several unique features associated with nucleosome profiles on all tRNA genes of budding yeast, not seen on nucleosome-dense counterparts in fission yeast and resting human CD4+ T cells. The nucleosome-free region (NFR) on all but three yeast tDNAs is found bordered by an upstream (US) nucleosome strongly positioned at −140 bp position and a downstream (DS) nucleosome at variable positions with respect to the gene terminator. Perturbation in this nucleosomal arrangement interferes with the tRNA production. Three different chromatin remodelers generate and maintain the NFR by targeting different gene regions. Isw1 localizes to the gene body and makes it nucleosome-depleted, Isw2 maintains periodicity in the upstream nucleosomal array, while RSC targets the downstream nucleosome. Direct communication of pol III with RSC serves as a stress-sensory mechanism for these genes. In its absence, the downstream nucleosome moves towards the gene terminator. Levels of tRNAs from different families are found to vary considerably as different pol III levels are seen even on isogenes within a family. Pol III levels show negative correlation with the nucleosome occupancies on different genes. Conclusions Budding yeast tRNA genes maintain an open chromatin structure, which is not due to sequence-directed nucleosome positioning or high transcription activity of genes. Unlike 5′ NFR on pol II-transcribed genes, the tDNA NFR, which facilitates tDNA transcription, results from action of chromatin

  2. A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3

    PubMed Central

    Chaurasiya, Kathy R.; Geertsema, Hylkje; Cristofari, Gaël; Darlix, Jean-Luc; Williams, Mark C.

    2012-01-01

    Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which drive the rearrangement of nucleic acid conformation. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein have been extensively studied, and nucleic acid aggregation, duplex destabilization and rapid binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. In addition, it is unclear whether a single zinc finger is sufficient to optimize the properties characteristic of HIV-1 NC. We used single-molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. We found that wild type Ty3 NC aggregates single- and double-stranded DNA, weakly stabilizes dsDNA, and exhibits rapid binding kinetics. Single-molecule studies in the presence of Ty3 NC mutants show that the N-terminal basic residues and the unique zinc finger at the C-terminus are required for optimum chaperone activity in this system. While the single zinc finger is capable of optimizing Ty3 NC's DNA interaction kinetics, two zinc fingers may be necessary in order to facilitate the DNA destabilization exhibited by HIV-1 NC. PMID:21917850

  3. A single zinc finger optimizes the DNA interactions of the nucleocapsid protein of the yeast retrotransposon Ty3.

    PubMed

    Chaurasiya, Kathy R; Geertsema, Hylkje; Cristofari, Gaël; Darlix, Jean-Luc; Williams, Mark C

    2012-01-01

    Reverse transcription in retroviruses and retrotransposons requires nucleic acid chaperones, which drive the rearrangement of nucleic acid conformation. The nucleic acid chaperone properties of the human immunodeficiency virus type-1 (HIV-1) nucleocapsid (NC) protein have been extensively studied, and nucleic acid aggregation, duplex destabilization and rapid binding kinetics have been identified as major components of its activity. However, the properties of other nucleic acid chaperone proteins, such as retrotransposon Ty3 NC, a likely ancestor of HIV-1 NC, are not well understood. In addition, it is unclear whether a single zinc finger is sufficient to optimize the properties characteristic of HIV-1 NC. We used single-molecule DNA stretching as a method for detailed characterization of Ty3 NC chaperone activity. We found that wild type Ty3 NC aggregates single- and double-stranded DNA, weakly stabilizes dsDNA, and exhibits rapid binding kinetics. Single-molecule studies in the presence of Ty3 NC mutants show that the N-terminal basic residues and the unique zinc finger at the C-terminus are required for optimum chaperone activity in this system. While the single zinc finger is capable of optimizing Ty3 NC's DNA interaction kinetics, two zinc fingers may be necessary in order to facilitate the DNA destabilization exhibited by HIV-1 NC. PMID:21917850

  4. Genomic Heterogeneity and Structural Variation in Soybean Near Isogenic Lines

    PubMed Central

    Stec, Adrian O.; Bhaskar, Pudota B.; Bolon, Yung-Tsi; Nolan, Rebecca; Shoemaker, Randy C.; Vance, Carroll P.; Stupar, Robert M.

    2013-01-01

    Near isogenic lines (NILs) are a critical genetic resource for the soybean research community. The ability to identify and characterize the genes driving the phenotypic differences between NILs is limited by the degree to which differential genetic introgressions can be resolved. Furthermore, the genetic heterogeneity extant among NIL sub-lines is an unaddressed research topic that might have implications for how genomic and phenotypic data from NILs are utilized. In this study, a recently developed high-resolution comparative genomic hybridization (CGH) platform was used to investigate the structure and diversity of genetic introgressions in two classical soybean NIL populations, respectively varying in protein content and iron deficiency chlorosis (IDC) susceptibility. There were three objectives: assess the capacity for CGH to resolve genomic introgressions, identify introgressions that are heterogeneous among NIL sub-lines, and associate heterogeneous introgressions with susceptibility to IDC. Using the CGH approach, introgression boundaries were refined and previously unknown introgressions were revealed. Furthermore, heterogeneous introgressions were identified within seven sub-lines of the IDC NIL “IsoClark.” This included three distinct introgression haplotypes linked to the major iron susceptible locus on chromosome 03. A phenotypic assessment of the seven sub-lines did not reveal any differences in IDC susceptibility, indicating that the genetic heterogeneity among the lines does not have a significant impact on the primary NIL phenotype. PMID:23630538

  5. Differential suppression of DNA repair deficiencies of Yeast rad50, mre11 and xrs2 mutants by EXO1 and TLC1 (the RNA component of telomerase).

    PubMed Central

    Lewis, L Kevin; Karthikeyan, G; Westmoreland, James W; Resnick, Michael A

    2002-01-01

    Rad50, Mre11, and Xrs2 form a nuclease complex that functions in both nonhomologous end-joining (NHEJ) and recombinational repair of DNA double-strand breaks (DSBs). A search for highly expressed cDNAs that suppress the DNA repair deficiency of rad50 mutants yielded multiple isolates of two genes: EXO1 and TLC1. Overexpression of EXO1 or TLC1 increased the resistance of rad50, mre11, and xrs2 mutants to ionizing radiation and MMS, but did not increase resistance in strains defective in recombination (rad51, rad52, rad54, rad59) or NHEJ only (yku70, sir4). Increased Exo1 or TLC1 RNA did not alter checkpoint responses or restore NHEJ proficiency, but DNA repair defects of yku70 and rad27 (fen) mutants were differentially suppressed by the two genes. Overexpression of Exo1, but not mutant proteins containing substitutions in the conserved nuclease domain, increased recombination and suppressed HO and EcoRI endonuclease-induced killing of rad50 strains. exo1 rad50 mutants lacking both nuclease activities exhibited a high proportion of enlarged, G2-arrested cells and displayed a synergistic decrease in DSB-induced plasmid:chromosome recombination. These results support a model in which the nuclease activity of the Rad50/Mre11/Xrs2 complex is required for recombinational repair, but not NHEJ. We suggest that the 5'-3' exo activity of Exo1 is able to substitute for Rad50/Mre11/Xrs2 in rescission of specific classes of DSB end structures. Gene-specific suppression by TLC1, which encodes the RNA subunit of the yeast telomerase complex, demonstrates that components of telomerase can also impact on DSB repair pathways. PMID:11805044

  6. Genomic Editing Tools to Model Human Diseases with Isogenic Pluripotent Stem Cells

    PubMed Central

    Kim, Huen Suk; Bernitz, Jeffrey M.; Lee, Dung-Fang

    2014-01-01

    Patient-specific induced pluripotent stem cells (iPSCs) are considered a versatile resource in the field of biomedicine. As iPSCs are generated on an individual basis, iPSCs may be the optimal cellular material to use for disease modeling, drug discovery, and the development of patient-specific cellular therapies. Recently, to gain an in-depth understanding of human pathologies, patient-specific iPSCs have been used to model human diseases with some iPSC-derived cells recapitulating pathological phenotypes in vitro. However, complex multigenic diseases generally have not resulted in concise conclusions regarding the underlying mechanisms of disease, in large part due to genetic variations between disease-state and control iPSCs. To circumvent this, the use of genomic editing tools to generate perfect isogenic controls is gaining momentum. To date, DNA binding domain-based zinc finger nucleases and transcription activator-like effector nucleases have been utilized to create genetically defined conditions in patient-specific iPSCs, with some examples leading to the successful identification of novel mechanisms of disease. As the feasibility and utility of genomic editing tools in iPSCs improve, along with the introduction of the clustered regularly interspaced short palindromic repeat system, understanding the features and limitations of genomic editing tools and their applications to iPSC technology is critical to expending the field of human disease modeling. PMID:25075441

  7. Dpb11 Controls the Association between DNA Polymerases α and ɛ and the Autonomously Replicating Sequence Region of Budding Yeast

    PubMed Central

    Masumoto, Hiroshi; Sugino, Akio; Araki, Hiroyuki

    2000-01-01

    Dpb11 is required for chromosomal DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Here, we report detection of a physical complex containing Dpb11 and DNA polymerase ɛ (Dpb11-Polɛ complex). During the S phase of the cell cycle, Dpb11 associated preferentially with DNA fragments containing autonomously replicating sequences (ARSs), at the same time as Polɛ associated with these fragments. Association of Dpb11 and Polɛ with these fragments was mutually dependent, suggesting that the Dpb11-Polɛ complex associates with the ARS. Moreover, Dpb11 was required for the association of Polα-primase with the fragments. Thus, it seems likely that association of the Dpb11-Polɛ complex with the ARS fragments is required for the association of the Polα-primase complex. Hydroxyurea inhibits late-origin firing in S. cerevisiae, and the checkpoint genes, RAD53 and MEC1, are involved in this inhibition. In the presence of hydroxyurea at temperatures permissive for cell growth, Polɛ in dpb11-1 cells associated with early- and late-origin fragments. In wild-type cells, however, it associated only with early-origin fragments. This indicates that Dpb11 may also be involved in the regulation of late-origin firing. Overall, these results suggest that Dpb11 controls the association between DNA polymerases α and ɛ and the ARS. PMID:10733584

  8. The versatile thymine DNA-glycosylase: a comparative characterization of the human, Drosophila and fission yeast orthologs.

    PubMed

    Hardeland, Ulrike; Bentele, Marc; Jiricny, Josef; Schär, Primo

    2003-05-01

    Human thymine-DNA glycosylase (TDG) is well known to excise thymine and uracil from G.T and G.U mismatches, respectively, and was therefore proposed to play a central role in the cellular defense against genetic mutation through spontaneous deamination of 5-methylcytosine and cytosine. In this study, we characterized two newly discovered orthologs of TDG, the Drosophila melanogaster Thd1p and the Schizosaccharomyces pombe Thp1p proteins, with an objective to address the function of this subfamily of uracil-DNA glycosylases from an evolutionary perspective. A systematic biochemical comparison of both enzymes with human TDG revealed a number of biologically significant facts. (i) All eukaryotic TDG orthologs have broad and species-specific substrate spectra that include a variety of damaged pyrimidine and purine bases; (ii) the common most efficiently processed substrates of all are uracil and 3,N4- ethenocytosine opposite guanine and 5-fluorouracil in any double-stranded DNA context; (iii) 5-methylcytosine and thymine derivatives are processed with an appreciable efficiency only by the human and the Drosophila enzymes; (iv) none of the proteins is able to hydrolyze a non-damaged 5'-methylcytosine opposite G; and (v) the double strand and mismatch dependency of the enzymes varies with the substrate and is not a stringent feature of this subfamily of DNA glycosylases. These findings advance our current view on the role of TDG proteins and document that they have evolved with high structural flexibility to counter a broad range of DNA base damage in accordance with the specific needs of individual species. PMID:12711670

  9. Uniparental Inheritance of Mitochondrial Genes in Yeast: Dependence on Input Bias of Mitochondrial DNA and Preliminary Investigations of the Mechanism

    PubMed Central

    Birky, C. William; Demko, Catherine A.; Perlman, Philip S.; Strausberg, Robert

    1978-01-01

    In Saccharomyces cerevisiae, previous studies on the inheritance of mitochondrial genes controlling antibiotic resistance have shown that some crosses produce a substantial number of uniparental zygotes , which transmit to their diploid progeny mitochondrial alleles from only one parent. In this paper, we show that uniparental zygotes are formed especially when one parent (majority parent) contributes substantially more mitochondrial DNA molecules to the zygote than does the other (minority) parent. Cellular contents of mitochondrial DNA (mtDNA) are increased in these experiments by treatment with cycloheximide, alpha-factor, or the uvsρ5 nuclear mutation. In such a biased cross, some zygotes are uniparental for mitochondrial alleles from the majority parent, and the frequency of such zygotes increases with increasing bias. In two- and three-factor crosses, the cap1, ery1, and oli1 loci behave coordinately, rather than independently; minority markers tend to be transmitted or lost as a unit, suggesting that the uniparental mechanism acts on entire mtDNA molecules rather than on individual loci. This rules out the possibility that uniparental inheritance can be explained by the conversion of minority markers to the majority alleles during recombination. Exceptions to the coordinate behavior of different loci can be explained by marker rescue via recombination. Uniparental inheritance is largely independent of the position of buds on the zygote. We conclude that it is due to the failure of minority markers to replicate in some zygotes, possibly involving the rapid enzymatic destruction of such markers. We have considered two general classes of mechanisms: (1) random selection of molecules for replication, as for example by competition for replicating sites on a membrane; and (2) differential marking of mtDNA molecules in the two parents, possibly by modification enzymes, followed by a mechanism that "counts" molecules and replicates only the majority type. These

  10. Detection of leucine-independent DNA site occupancy of the yeast Leu3p transcriptional activator in vivo.

    PubMed Central

    Kirkpatrick, C R; Schimmel, P

    1995-01-01

    The product of the Saccharomyces cerevisiae LEU3 gene, Leu3p, is a transcriptional activator which regulates leucine biosynthesis in response to intracellular levels of leucine through the biosynthetic intermediate alpha-isopropylmalate. We devised a novel assay to examine the DNA site occupancy of Leu3p under different growth conditions, using a reporter gene with internal Leu3p-binding sites. Expression of the reporter is inhibited by binding of nuclear Leu3p to these sites; inhibition is dependent on the presence of the sites in the reporter, on the integrity of the Leu3p DNA-binding domain, and, surprisingly, on the presence of a transcriptional activation domain in the inhibiting protein. By this assay, Leu3p was found to occupy its binding site under all conditions tested, including high and low levels of leucine and in the presence and absence of alpha-isopropylmalate. The localization of Leu3p to the nucleus was confirmed by immunofluorescence staining of cells expressing epitope-tagged Leu3p derivatives. We conclude that Leu3p regulates transcription in vivo without changing its intracellular localization and DNA site occupancy. PMID:7623798

  11. The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome

    PubMed Central

    Pevala, Vladimír; Truban, Dominika; Bauer, Jacob A.; Košťan, Július; Kunová, Nina; Bellová, Jana; Brandstetter, Marlene; Marini, Victoria; Krejčí, Lumír; Tomáška, Ľubomír; Nosek, Jozef; Kutejová, Eva

    2016-01-01

    To study the mechanisms involved in the maintenance of a linear mitochondrial genome we investigated the biochemical properties of the recombination protein Mgm101 from Candida parapsilosis. We show that CpMgm101 complements defects associated with the Saccharomyces cerevisiae mgm101–1ts mutation and that it is present in both the nucleus and mitochondrial nucleoids of C. parapsilosis. Unlike its S. cerevisiae counterpart, CpMgm101 is associated with the entire nucleoid population and is able to bind to a broad range of DNA substrates in a non-sequence specific manner. CpMgm101 is also able to catalyze strand annealing and D-loop formation. CpMgm101 forms a roughly C-shaped trimer in solution according to SAXS. Electron microscopy of a complex of CpMgm101 with a model mitochondrial telomere revealed homogeneous, ring-shaped structures at the telomeric single-stranded overhangs. The DNA-binding properties of CpMgm101, together with its DNA recombination properties, suggest that it can play a number of possible roles in the replication of the mitochondrial genome and the maintenance of its telomeres. PMID:26743001

  12. Yeast gene CMR1/YDL156W is consistently co-expressed with genes participating in DNA-metabolic processes in a variety of stringent clustering experiments

    PubMed Central

    Abu-Jamous, Basel; Fa, Rui; Roberts, David J.; Nandi, Asoke K.

    2013-01-01

    The binarization of consensus partition matrices (Bi-CoPaM) method has, among its unique features, the ability to perform ensemble clustering over the same set of genes from multiple microarray datasets by using various clustering methods in order to generate tunable tight clusters. Therefore, we have used the Bi-CoPaM method to the most synchronized 500 cell-cycle-regulated yeast genes from different microarray datasets to produce four tight, specific and exclusive clusters of co-expressed genes. We found 19 genes formed the tightest of the four clusters and this included the gene CMR1/YDL156W, which was an uncharacterized gene at the time of our investigations. Two very recent proteomic and biochemical studies have independently revealed many facets of CMR1 protein, although the precise functions of the protein remain to be elucidated. Our computational results complement these biological results and add more evidence to their recent findings of CMR1 as potentially participating in many of the DNA-metabolism processes such as replication, repair and transcription. Interestingly, our results demonstrate the close co-expressions of CMR1 and the replication protein A (RPA), the cohesion complex and the DNA polymerases α, δ and ɛ, as well as suggest functional relationships between CMR1 and the respective proteins. In addition, the analysis provides further substantial evidence that the expression of the CMR1 gene could be regulated by the MBF complex. In summary, the application of a novel analytic technique in large biological datasets has provided supporting evidence for a gene of previously unknown function, further hypotheses to test, and a more general demonstration of the value of sophisticated methods to explore new large datasets now so readily generated in biological experiments. PMID:23349438

  13. Phylogenetic position of the yeast-like symbiotes of Tagosodes orizicolus (Homoptera: Delphacidae) based on 18S ribosomal DNA partial sequences.

    PubMed

    Xet-Mull, Ana M; Quesada, Tania; Espinoza, Ana M

    2004-09-01

    Tagosodes orizicolus Muir (Homoptera: Delphacidae), the endemic delphacid species of tropical America carries yeast-like symbiotes (YLS) in the abdominal fat bodies and the ovarial tissues, like other rice planthoppers of Asia. These YLS are obligate symbiotes, which are transmitted transovarially, and maintain a mutualistic relationship with the insect host. This characteristic has made in vitro culture and classification of YLS rather difficult using conventional methods. Nevertheless, microorganisms of similar characteristics have been successfully classified by using molecular taxonomy. In the present work, the YLS of Tagosodes orizicolus (YLSTo) were purified on Percoll gradients, and specific segments of 18S rDNA were amplified by PCR, cloned and sequenced. Sequences were aligned by means of the CLUSTAL V (DNASTAR) program; phylogenetic trees were constructed with the Phylogeny Inference Package (PHYLIP), showing that YLSTo belong to the fungi class Pyrenomycetes, phylum Ascomycota. Similarities between 98% and 100% were observed among YLS of the rice delphacids Tagosodes orizicolus, Nilaparvata lugens, Laodelphax striatellus and Sogatella fur cifera, and between 89.8% and 90.8% when comparing the above to YLS of the aphid Hamiltonaphis styraci. These comparisons revealed that delphacid YLS are a highly conserved monophyletic group within the Pyrenomycetes and are closely related to Hypomyces chrysospermus. PMID:17361570

  14. Improved production of fatty acids by Saccharomyces cerevisiae through screening a cDNA library from the oleaginous yeast Yarrowia lipolytica.

    PubMed

    Shi, Shuobo; Ji, Haichuan; Siewers, Verena; Nielsen, Jens

    2016-02-01

    Biological production of fatty acid (FA)-derived products has gained increasing attention to replace petroleum-based fuels and chemicals. FA biosynthesis is highly regulated, and usually it is challenging to design rational engineering strategies. In addition, the conventional 'one sample at a time' method for lipid determination is time consuming and laborious, and it is difficult to screen large numbers of samples. Here, a method for detecting free FAs in viable cells using Nile red staining was developed for use in large-scale screening. Following optimization of the method, it was used for screening a cDNA library from the oleaginous yeast Yarrowia lipolytica for identification of genes/enzymes that were able to enhance free FA accumulation in Saccharomyces cerevisiae. Several novel enzymes resulting in increasing FA accumulation were discovered. These targets include a GPI anchor protein, malate dehydrogenase, glyceraldehyde 3-phosphate dehydrogenase, FA hydroxylase, farnesyltransferase, anoctamin, dihydrolipoamide dehydrogenase and phosphatidylethanolamine-binding protein. The best enzyme resulted in a 2.5-fold improvement in production of free FAs. Our findings not only provide a novel method for high-throughput evaluation of the content of free FAs, but also give new insight into how enzymes from Y. lipolytica may increase the production of fatty acids in S. cerevisiae. PMID:26658002

  15. Structural bases of dimerization of yeast telomere protein Cdc13 and its interaction with the catalytic subunit of DNA polymerase [alpha

    SciTech Connect

    Sun, Jia; Yang, Yuting; Wan, Ke; Mao, Ninghui; Yu, Tai-Yuan; Lin, Yi-Chien; DeZwaan, Diane C.; Freeman, Brian C.; Lin, Jing-Jer; Lue, Neal F.; Lei, Ming

    2011-08-24

    Budding yeast Cdc13-Stn1-Ten1 (CST) complex plays an essential role in telomere protection and maintenance, and has been proposed to be a telomere-specific replication protein A (RPA)-like complex. Previous genetic and structural studies revealed a close resemblance between Stn1-Ten1 and RPA32-RPA14. However, the relationship between Cdc13 and RPA70, the largest subunit of RPA, has remained unclear. Here, we report the crystal structure of the N-terminal OB (oligonucleotide/oligosaccharide binding) fold of Cdc13. Although Cdc13 has an RPA70-like domain organization, the structures of Cdc13 OB folds are significantly different from their counterparts in RPA70, suggesting that they have distinct evolutionary origins. Furthermore, our structural and biochemical analyses revealed unexpected dimerization by the N-terminal OB fold and showed that homodimerization is probably a conserved feature of all Cdc13 proteins. We also uncovered the structural basis of the interaction between the Cdc13 N-terminal OB fold and the catalytic subunit of DNA polymerase {alpha} (Pol1), and demonstrated a role for Cdc13 dimerization in Pol1 binding. Analysis of the phenotypes of mutants defective in Cdc13 dimerization and Cdc13-Pol1 interaction revealed multiple mechanisms by which dimerization regulates telomere lengths in vivo. Collectively, our findings provide novel insights into the mechanisms and evolution of Cdc13.

  16. Brief Report: Isogenic Induced Pluripotent Stem Cell Lines From an Adult With Mosaic Down Syndrome Model Accelerated Neuronal Ageing and Neurodegeneration

    PubMed Central

    Murray, Aoife; Letourneau, Audrey; Canzonetta, Claudia; Stathaki, Elisavet; Gimelli, Stefania; Sloan‐Bena, Frederique; Abrehart, Robert; Goh, Pollyanna; Lim, Shuhui; Baldo, Chiara; Dagna‐Bricarelli, Franca; Hannan, Saad; Mortensen, Martin; Ballard, David; Syndercombe Court, Denise; Fusaki, Noemi; Hasegawa, Mamoru; Smart, Trevor G.; Bishop, Cleo; Antonarakis, Stylianos E.

    2015-01-01

    Abstract Trisomy 21 (T21), Down Syndrome (DS) is the most common genetic cause of dementia and intellectual disability. Modeling DS is beginning to yield pharmaceutical therapeutic interventions for amelioration of intellectual disability, which are currently being tested in clinical trials. DS is also a unique genetic system for investigation of pathological and protective mechanisms for accelerated ageing, neurodegeneration, dementia, cancer, and other important common diseases. New drugs could be identified and disease mechanisms better understood by establishment of well‐controlled cell model systems. We have developed a first nonintegration‐reprogrammed isogenic human induced pluripotent stem cell (iPSC) model of DS by reprogramming the skin fibroblasts from an adult individual with constitutional mosaicism for DS and separately cloning multiple isogenic T21 and euploid (D21) iPSC lines. Our model shows a very low number of reprogramming rearrangements as assessed by a high‐resolution whole genome CGH‐array hybridization, and it reproduces several cellular pathologies seen in primary human DS cells, as assessed by automated high‐content microscopic analysis. Early differentiation shows an imbalance of the lineage‐specific stem/progenitor cell compartments: T21 causes slower proliferation of neural and faster expansion of hematopoietic lineage. T21 iPSC‐derived neurons show increased production of amyloid peptide‐containing material, a decrease in mitochondrial membrane potential, and an increased number and abnormal appearance of mitochondria. Finally, T21‐derived neurons show significantly higher number of DNA double‐strand breaks than isogenic D21 controls. Our fully isogenic system therefore opens possibilities for modeling mechanisms of developmental, accelerated ageing, and neurodegenerative pathologies caused by T21. Stem Cells 2015;33:2077–2084 PMID:25694335

  17. Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro.

    PubMed Central

    Arndt, K M; Ricupero, S L; Eisenmann, D M; Winston, F

    1992-01-01

    A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition. Images PMID:1569955

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

    PubMed Central

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

    1994-01-01

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

  19. Substitution of conserved residues within the active site alters the cleavage religation equilibrium of DNA topoisomerase I.

    PubMed

    Colley, William C; van der Merwe, Marie; Vance, John R; Burgin, Alex B; Bjornsti, Mary-Ann

    2004-12-24

    Eukaryotic DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA and constitutes the cellular target of camptothecin (CPT). Mutation of conserved residues in close proximity to the active site tyrosine (Tyr(727) of yeast Top1p) alters the DNA cleavage religation equilibrium, inducing drug-independent cell lethality. Previous studies indicates that yeast Top1T722Ap and Top1N726Hp cytotoxicity results from elevated levels of covalent enzyme-DNA intermediates. Here we show that Top1T722Ap acts as a CPT mimetic by exhibiting reduced rates of DNA religation, whereas increased Top1N726Hp.DNA complexes result from elevated DNA binding and cleavage. We also report that the combination of the T722A and N726H mutations in a single protein potentiates the cytotoxic action of the enzyme beyond that induced by co-expression of the single mutants. Moreover, the addition of CPT to cells expressing the double top1T722A/N726H mutant did not enhance cell lethality. Thus, independent alterations in DNA cleavage and religation contribute to the lethal phenotype. The formation of distinct cytotoxic lesions was also evidenced by the different responses induced by low levels of these self-poisoning enzymes in isogenic strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. Substitution of Asn(726) with Phe or Tyr also produces self-poisoning enzymes, implicating stacking interactions in the increased kinetics of DNA cleavage by Top1N726Hp and Top1N726Fp. In contrast, replacing the amide side chain of Asn(726) with Gln renders Top1N726Qp resistant to CPT, suggesting that the orientation of the amide within the active site is critical for effective CPT binding. PMID:15489506

  20. Counting Yeast.

    ERIC Educational Resources Information Center

    Bealer, Jonathan; Welton, Briana

    1998-01-01

    Describes changes to a traditional study of population in yeast colonies. Changes to the procedures include: (1) only one culture per student team; (2) cultures are inoculated only once; and (3) the same tube is sampled daily. (DDR)

  1. Epidemiologic Study of Malassezia Yeasts in Patients with Malassezia Folliculitis by 26S rDNA PCR-RFLP Analysis

    PubMed Central

    Ko, Jong Hyun; Choe, Yong Beom; Ahn, Kyu Joong

    2011-01-01

    Background So far, studies on the inter-relationship between Malassezia and Malassezia folliculitis have been rather scarce. Objective We sought to analyze the differences in body sites, gender and age groups, and to determine whether there is a relationship between certain types of Malassezia species and Malassezia folliculitis. Methods Specimens were taken from the forehead, cheek and chest of 60 patients with Malassezia folliculitis and from the normal skin of 60 age- and gender-matched healthy controls by 26S rDNA PCR-RFLP. Results M. restricta was dominant in the patients with Malassezia folliculitis (20.6%), while M. globosa was the most common species (26.7%) in the controls. The rate of identification was the highest in the teens for the patient group, whereas it was the highest in the thirties for the control group. M. globosa was the most predominant species on the chest with 13 cases (21.7%), and M. restricta was the most commonly identified species, with 17 (28.3%) and 12 (20%) cases on the forehead and cheek, respectively, for the patient group. Conclusion Statistically significant differences were observed between the patient and control groups for the people in their teens and twenties, and in terms of the body site, on the forehead only. PMID:21747616

  2. Asynchronous spore germination in isogenic natural isolates of Saccharomyces paradoxus.

    PubMed

    Stelkens, Rike B; Miller, Eric L; Greig, Duncan

    2016-05-01

    Spores from wild yeast isolates often show great variation in the size of colonies they produce, for largely unknown reasons. Here we measure the colonies produced from single spores from six different wild Saccharomyces paradoxus strains. We found remarkable variation in spore colony sizes, even among spores that were genetically identical. Different strains had different amounts of variation in spore colony sizes, and variation was not affected by the number of preceding meioses, or by spore maturation time. We used time-lapse photography to show that wild strains also have high variation in spore germination timing, providing a likely mechanism for the variation in spore colony sizes. When some spores from a laboratory strain make small colonies, or no colonies, it usually indicates a genetic or meiotic fault. Here, we demonstrate that in wild strains spore colony size variation is normal. We discuss and assess potential adaptive and non-adaptive explanations for this variation. PMID:26880797

  3. An AIF orthologue regulates apoptosis in yeast

    PubMed Central

    Wissing, Silke; Ludovico, Paula; Herker, Eva; Büttner, Sabrina; Engelhardt, Silvia M.; Decker, Thorsten; Link, Alexander; Proksch, Astrid; Rodrigues, Fernando; Corte-Real, Manuela; Fröhlich, Kai-Uwe; Manns, Joachim; Candé, Céline; Sigrist, Stephan J.; Kroemer, Guido; Madeo, Frank

    2004-01-01

    Apoptosis-inducing factor (AIF), a key regulator of cell death, is essential for normal mammalian development and participates in pathological apoptosis. The proapoptotic nature of AIF and its mode of action are controversial. Here, we show that the yeast AIF homologue Ynr074cp controls yeast apoptosis. Similar to mammalian AIF, Ynr074cp is located in mitochondria and translocates to the nucleus of yeast cells in response to apoptotic stimuli. Purified Ynr074cp degrades yeast nuclei and plasmid DNA. YNR074C disruption rescues yeast cells from oxygen stress and delays age-induced apoptosis. Conversely, overexpression of Ynr074cp strongly stimulates apoptotic cell death induced by hydrogen peroxide and this effect is attenuated by disruption of cyclophilin A or the yeast caspase YCA1. We conclude that Ynr074cp is a cell death effector in yeast and rename it AIF-1 (Aif1p, gene AIF1). PMID:15381687

  4. Evolution of p53 transactivation specificity through the lens of a yeast-based functional assay.

    PubMed

    Lion, Mattia; Raimondi, Ivan; Donati, Stefano; Jousson, Olivier; Ciribilli, Yari; Inga, Alberto

    2015-01-01

    Co-evolution of transcription factors (TFs) with their respective cis-regulatory network enhances functional diversity in the course of evolution. We present a new approach to investigate transactivation capacity of sequence-specific TFs in evolutionary studies. Saccharomyces cerevisiae was used as an in vivo test tube and p53 proteins derived from human and five commonly used animal models were chosen as proof of concept. p53 is a highly conserved master regulator of environmental stress responses. Previous reports indicated conserved p53 DNA binding specificity in vitro, even for evolutionary distant species. We used isogenic yeast strains where p53-dependent transactivation was measured towards chromosomally integrated p53 response elements (REs). Ten REs were chosen to sample a wide range of DNA binding affinity and transactivation capacity for human p53 and proteins were expressed at two levels using an inducible expression system. We showed that the assay is amenable to study thermo-sensitivity of frog p53, and that chimeric constructs containing an ectopic transactivation domain could be rapidly developed to enhance the activity of proteins, such as fruit fly p53, that are poorly effective in engaging the yeast transcriptional machinery. Changes in the profile of relative transactivation towards the ten REs were measured for each p53 protein and compared to the profile obtained with human p53. These results, which are largely independent from relative p53 protein levels, revealed widespread evolutionary divergence of p53 transactivation specificity, even between human and mouse p53. Fruit fly and human p53 exhibited the largest discrimination among REs while zebrafish p53 was the least selective. PMID:25668429

  5. Suppressive-subtractive cDNA analysis indicates that elevated yeast antioxidant gene expression is associated with increased stress tolerance and biocontrol efficacy in the antagonistic yeast, Candida Oleophila

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Several yeast species have been used as biocontrol agents against postharvest diseases of fruits and vegetables. The objectives of the current study are to develop a better understanding of the role of reactive oxygen species (ROS) in relation to viability and biocontrol activity of Candida oleophi...

  6. Yeast Identification Algorithm Based on Use of the Vitek MS System Selectively Supplemented with Ribosomal DNA Sequencing: Proposal of a Reference Assay for Invasive Fungal Surveillance Programs in China

    PubMed Central

    Zhang, Li; Xiao, Meng; Wang, He; Gao, Ran; Fan, Xin; Brown, Mitchell; Gray, Timothy J.; Kong, Fanrong

    2014-01-01

    Sequence analysis of the internal transcribed spacer (ITS) region was employed as the gold standard method for yeast identification in the China Hospital Invasive Fungal Surveillance Net (CHIF-NET). It has subsequently been found that matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) is potentially a more practical approach for this purpose. In the present study, the performance of the Vitek MS v2.0 system for the identification of yeast isolates collected from patients with invasive fungal infections in the 2011 CHIF-NET was evaluated. A total of 1,243 isolates representing 31 yeast species were analyzed, and the identification results by the Vitek MS v2.0 system were compared to those obtained by ITS sequence analysis. By the Vitek MS v2.0 system, 96.7% (n = 1,202) of the isolates were correctly assigned to the species level and 0.2% (n = 2) of the isolates were identified to the genus level, while 2.4% (n = 30) and 0.7% (n = 9) of the isolates were unidentified and misidentified, respectively. After retesting of the unidentified and misidentified strains, 97.3% (n = 1,209) of the isolates were correctly identified to the species level. Based on these results, a testing algorithm that combines the use of the Vitek MS system with selected supplementary ribosomal DNA (rDNA) sequencing was developed and validated for yeast identification purposes. By employing this algorithm, 99.7% (1,240/1,243) of the study isolates were accurately identified with the exception of two isolates of Candida fermentati and one isolate of Cryptococcus gattii. In conclusion, the proposed identification algorithm could be practically implemented in strategic programs of fungal infection surveillance. PMID:24478490

  7. Lager Yeast Comes of Age

    PubMed Central

    2014-01-01

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

  8. Yeast Droplets

    NASA Astrophysics Data System (ADS)

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

    2002-11-01

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

  9. Genotoxicity of Cytolethal Distending Toxin (CDT) on Isogenic Human Colorectal Cell Lines: Potential Promoting Effects for Colorectal Carcinogenesis

    PubMed Central

    Graillot, Vanessa; Dormoy, Inge; Dupuy, Jacques; Shay, Jerry W.; Huc, Laurence; Mirey, Gladys; Vignard, Julien

    2016-01-01

    The composition of the human microbiota influences tumorigenesis, notably in colorectal cancer (CRC). Pathogenic Escherichia coli possesses a variety of virulent factors, among them the Cytolethal Distending Toxin (CDT). CDT displays dual DNase and phosphatase activities and induces DNA double strand breaks, cell cycle arrest and apoptosis in a broad range of mammalian cells. As CDT could promote malignant transformation, we investigated the cellular outcomes induced by acute and chronic exposures to E. coli CDT in normal human colon epithelial cells (HCECs). Moreover, we conducted a comparative study between isogenic derivatives cell lines of the normal HCECs in order to mimic the mutation of three major genes found in CRC genetic models: APC, KRAS, and TP53. Our results demonstrate that APC and p53 deficient cells showed impaired DNA damage response after CDT exposure, whereas HCECs expressing oncogenic KRASV12 were more resistant to CDT. Compared to normal HCECs, the precancerous derivatives exhibit hallmarks of malignant transformation after a chronic exposure to CDT. HCECs defective in APC and p53 showed enhanced anchorage independent growth and genetic instability, assessed by the micronucleus formation assay. In contrast, the ability to grow independently of anchorage was not impacted by CDT chronic exposure in KRASV12 HCECs, but micronucleus formation is dramatically increased. Thus, CDT does not initiate CRC by itself, but may have promoting effects in premalignant HCECs, involving different mechanisms in function of the genetic alterations associated to CRC. PMID:27047802

  10. A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

    PubMed Central

    Vizeacoumar, Franco J; Arnold, Roland; Vizeacoumar, Frederick S; Chandrashekhar, Megha; Buzina, Alla; Young, Jordan T F; Kwan, Julian H M; Sayad, Azin; Mero, Patricia; Lawo, Steffen; Tanaka, Hiromasa; Brown, Kevin R; Baryshnikova, Anastasia; Mak, Anthony B; Fedyshyn, Yaroslav; Wang, Yadong; Brito, Glauber C; Kasimer, Dahlia; Makhnevych, Taras; Ketela, Troy; Datti, Alessandro; Babu, Mohan; Emili, Andrew; Pelletier, Laurence; Wrana, Jeff; Wainberg, Zev; Kim, Philip M; Rottapel, Robert; O'Brien, Catherine A; Andrews, Brenda; Boone, Charles; Moffat, Jason

    2013-01-01

    Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large-scale sequencing efforts. Using genome-scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co-culture competition assays to generate a high-confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non-isogenic cancer cell lines. For example, the PTEN−/− DiE genes reveal a signature that can preferentially classify PTEN-dependent genotypes across a series of non-isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model. PMID:24104479

  11. NOTICE OF RELEASE OF HARD KERNEL PUROINDOLINE ALLELE NEAR-ISOGENIC LINE HEXAPLOID WHEAT GENETIC STOCKS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Agricultural Research Service, U.S. Department of Agriculture announces the release of seven hard kernel puroindoline allele near-isogenic line (NIL) hexaploid wheat (Triticum aestivum L.) genetic stocks (PI xxxxxx – PI xxxxxx) developed by Dr. Craig F. Morris at the USDA-ARS Western Wheat Quali...

  12. Joint Analysis of Near Isogenic and Recombinant Inbred Line Populations Yields Precise Positional Estimates for QTL

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Near isogenic lines (NILs) are typically constructed to fine-map quantitative trait loci (QTL). The data generated for the initial QTL mapping are usually ignored for fine-mapping purposes. However, combining already-available data from initial recombinant inbred line (RIL) studies with new data fro...

  13. Registration of isogenic HMW-GS Ax1 protein deficient lines of an elite wheat variety

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A series of isogenic wheat lines deficient in high-molecular weight subunit (HMW-GS) proteins encoded on the A-genome of an elite variety of bread wheat (Triticum aestivum L.) was developed by USDA-ARS at the Western Regional Research Center in Albany, CA. This set of A-genome encoded HMW-GS defi...

  14. Genomic heterogeneity and structural variation in soybean near-isogenic lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Near-isogenic lines (NILs) are a critical genetic resource for the soybean research community. The ability to identify and characterize the genes driving the phenotypic differences between NILs is limited by the degree to which differential genetic introgressions can be resolved. Furthermore, the ...

  15. Protein and quality characterization of complete and partial near isogenic lines of waxy wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to evaluate protein composition and its effects on flour quality and physical dough test parameters using waxy wheat near-isogenic lines. Partial waxy (single and double nulls) and waxy (null at all three waxy loci, Wx-A1, Wx-B1, and Wx-D1) lines of N11 set (bread whe...

  16. Microarray analysis of iron deficiency chlorosis in near-isogenic soybean lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    RNA isolated from the roots of two near isogenic lines, which differ in iron efficiency, PI548533 (Clark; iron efficient) and PI547430 (IsoClark; iron inefficient), were compared on a spotted microarray slide containing 9,728 cDNAs from root specific EST libraries. A comparison of RNA transcripts i...

  17. Inhibitory activity of avibactam against selected β-lactamases expressed in an isogenic Escherichia coli strain.

    PubMed

    Giani, Tommaso; Cannatelli, Antonio; Di Pilato, Vincenzo; Testa, Raymond; Nichols, Wright W; Rossolini, Gian Maria

    2016-09-01

    Avibactam restored the in-vitro antibacterial activity of ceftazidime, ceftaroline, and aztreonam against isogenic Escherichia coli expressing class A, class C, and class D β-lactamases. The enzymes included TEM and CTX-M extended spectrum β-lactamases, ACT, CMY and FOX AmpC-type enzymes, and carbapenemases including rarer KPC variants and OXA-139. PMID:27394638

  18. Two glycerol 3-phosphate dehydrogenase isogenes from Candida versatilis SN-18 play an important role in glycerol biosynthesis under osmotic stress.

    PubMed

    Mizushima, Daiki; Iwata, Hisashi; Ishimaki, Yuki; Ogihara, Jun; Kato, Jun; Kasumi, Takafumi

    2016-05-01

    Two isogenes of glycerol 3-phosphate dehydrogenase (GPD) from Candida versatilis SN-18 were cloned and sequenced. These intronless genes (Cagpd1 and Cagpd2) were both predicted to encode a 378 amino acid polypeptide, and the deduced amino acid sequences mutually showed 76% identity. Interestingly, Cagpd1 and Cagpd2 were located tandemly in a locus of genomic DNA within a 262 bp interval. To our knowledge, this represents a novel instance of isogenic genes relating to glucose metabolism. The stress response element (STRE) was found respectively at -93 to -89 bp upstream of the 5'end of Cagpd1 and -707 to -703 bp upstream of Cagpd2, indicating that these genes are involved in osmotic stress response. In heterologous expression using a gpd1Δgpd2Δ double deletion mutant of Saccharomyces cerevisiae, Cagpd1 and Cagpd2 transformants complemented the function of GPD, with Cagpd2 being much more effective than Cagpd1 in promoting growth and glycerol synthesis. Phylogenetic analysis of the amino acid sequences suggested that Cagpd1p and Cagpd2p are NADP(+)-dependent GPDs (EC 1.1.1.94). However, crude enzyme extract from Cagpd1 and Cagpd2 transformants showed GPD activity with only NAD(+) as cofactor. Hence, both Cagpd1p and Cagpd2p are likely NAD(+)-dependent GPDs (EC 1.1.1.8), similar to GPDs from S. cerevisiae and Candida magnoliae. PMID:26906228

  19. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  20. Expressed sequence tag analysis and development of gene associated markers in a near-isogenic plant system of Eragrostis curvula.

    PubMed

    Cervigni, Gerardo D L; Paniego, Norma; Díaz, Marina; Selva, Juan P; Zappacosta, Diego; Zanazzi, Darío; Landerreche, Iñaki; Martelotto, Luciano; Felitti, Silvina; Pessino, Silvina; Spangenberg, Germán; Echenique, Viviana

    2008-05-01

    Eragrostis curvula (Schrad.) Nees is a forage grass native to the semiarid regions of Southern Africa, which reproduces mainly by pseudogamous diplosporous apomixis. A collection of ESTs was generated from four cDNA libraries, three of them obtained from panicles of near-isogenic lines with different ploidy levels and reproductive modes, and one obtained from 12 days-old plant leaves. A total of 12,295 high-quality ESTs were clustered and assembled, rendering 8,864 unigenes, including 1,490 contigs and 7,394 singletons, with a genome coverage of 22%. A total of 7,029 (79.11%) unigenes were functionally categorized by BLASTX analysis against sequences deposited in public databases, but only 37.80% could be classified according to Gene Ontology. Sequence comparison against the cereals genes indexes (GI) revealed 50% significant hits. A total of 254 EST-SSRs were detected from 219 singletons and 35 from contigs. Di- and tri- motifs were similarly represented with percentages of 38.95 and 40.16%, respectively. In addition, 190 SNPs and Indels were detected in 18 contigs generated from 3 to 4 libraries. The ESTs and the molecular markers obtained in this study will provide valuable resources for a wide range of applications including gene identification, genetic mapping, cultivar identification, analysis of genetic diversity, phenotype mapping and marker assisted selection. PMID:18196464

  1. Determining the Contribution of Epidermal Cell Shape to Petal Wettability Using Isogenic Antirrhinum Lines

    PubMed Central

    Whitney, Heather M.; Poetes, Rosa; Steiner, Ullrich; Chittka, Lars; Glover, Beverley J.

    2011-01-01

    The petal epidermis acts not only as a barrier to the outside world but also as a point of interaction between the flower and potential pollinators. The presence of conical petal epidermal cells has previously been shown to influence the attractiveness of the flower to pollinating insects. Using Antirrhinum isogenic lines differing only in the presence of a single epidermal structure, conical cells, we were able to investigate how the structure of the epidermis influences petal wettability by measuring the surface contact angle of water drops. Conical cells have a significant impact on how water is retained on the flower surface, which may have indirect consequences for pollinator behaviour. We discuss how the petal epidermis is a highly multifunctional one and how a battery of methods, including the use of isogenic lines, is required to untangle the impacts of specific epidermal properties in an ecological context. PMID:21423738

  2. A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA

    PubMed Central

    Ling, Feng; Morioka, Hiroshi; Ohtsuka, Eiko; Shibata, Takehiko

    2000-01-01

    A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30°C and shows extensive vegetative petite induction by UV irradiation at 30°C or when cultivated at a higher temperature (37°C). It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration. Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay. mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells. Soon after a temperature shift from 30 to 37°C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells. Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37°C. Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level. These observations are consistent with MHR1 having a possible role in mtDNA repair. PMID:11121487

  3. Functional isogenic modeling of BRCA1 alleles reveals distinct carrier phenotypes

    PubMed Central

    Cochran, Rory L.; Cidado, Justin; Kim, Minsoo; Zabransky, Daniel J.; Croessmann, Sarah; Chu, David; Wong, Hong Yuen; Beaver, Julia A.; Cravero, Karen; Erlanger, Bracha; Parsons, Heather; Heaphy, Christopher M.; Meeker, Alan K.; Lauring, Josh; Park, Ben Ho

    2015-01-01

    Clinical genetic testing of BRCA1 and BRCA2 is commonly performed to identify specific individuals at risk for breast and ovarian cancers who may benefit from prophylactic therapeutic interventions. Unfortunately, it is evident that deleterious BRCA1 alleles demonstrate variable penetrance and that many BRCA1 variants of unknown significance (VUS) exist. In order to further refine hereditary risks that may be associated with specific BRCA1 alleles, we performed gene targeting to establish an isogenic panel of immortalized human breast epithelial cells harboring eight clinically relevant BRCA1 alleles. Interestingly, BRCA1 mutations and VUS had distinct, quantifiable phenotypes relative to isogenic parental BRCA1 wild type cells and controls. Heterozygous cells with known deleterious BRCA1 mutations (185delAG, C61G and R71G) demonstrated consistent phenotypes in radiation sensitivity and genomic instability assays, but showed variability in other assays. Heterozygous BRCA1 VUS cells also demonstrated assay variability, with some VUS demonstrating phenotypes more consistent with deleterious alleles. Taken together, our data suggest that BRCA1 deleterious mutations and VUS can differ in their range of tested phenotypes, suggesting they might impart varying degrees of risk. These results demonstrate that functional isogenic modeling of BRCA1 alleles could aid in classifying BRCA1 mutations and VUS, and determining BRCA allele cancer risk. PMID:26246475

  4. Functional isogenic modeling of BRCA1 alleles reveals distinct carrier phenotypes.

    PubMed

    Cochran, Rory L; Cidado, Justin; Kim, Minsoo; Zabransky, Daniel J; Croessmann, Sarah; Chu, David; Wong, Hong Yuen; Beaver, Julia A; Cravero, Karen; Erlanger, Bracha; Parsons, Heather; Heaphy, Christopher M; Meeker, Alan K; Lauring, Josh; Park, Ben Ho

    2015-09-22

    Clinical genetic testing of BRCA1 and BRCA2 is commonly performed to identify specific individuals at risk for breast and ovarian cancers who may benefit from prophylactic therapeutic interventions. Unfortunately, it is evident that deleterious BRCA1 alleles demonstrate variable penetrance and that many BRCA1 variants of unknown significance (VUS) exist. In order to further refine hereditary risks that may be associated with specific BRCA1 alleles, we performed gene targeting to establish an isogenic panel of immortalized human breast epithelial cells harboring eight clinically relevant BRCA1 alleles. Interestingly, BRCA1 mutations and VUS had distinct, quantifiable phenotypes relative to isogenic parental BRCA1 wild type cells and controls. Heterozygous cells with known deleterious BRCA1 mutations (185delAG, C61G and R71G) demonstrated consistent phenotypes in radiation sensitivity and genomic instability assays, but showed variability in other assays. Heterozygous BRCA1 VUS cells also demonstrated assay variability, with some VUS demonstrating phenotypes more consistent with deleterious alleles. Taken together, our data suggest that BRCA1 deleterious mutations and VUS can differ in their range of tested phenotypes, suggesting they might impart varying degrees of risk. These results demonstrate that functional isogenic modeling of BRCA1 alleles could aid in classifying BRCA1 mutations and VUS, and determining BRCA allele cancer risk. PMID:26246475

  5. Distribution of a limited Sir2 protein pool regulates the strength of yeast rDNA silencing and is modulated by Sir4p.

    PubMed Central

    Smith, J S; Brachmann, C B; Pillus, L; Boeke, J D

    1998-01-01

    Transcriptional silencing in Saccharomyces cerevisiae occurs at the silent mating-type loci HML and HMR, at telomeres, and at the ribosomal DNA (rDNA) locus RDN1. Silencing in the rDNA occurs by a novel mechanism that depends on a single Silent Information Regulator (SIR) gene, SIR2. SIR4, essential for other silenced loci, paradoxically inhibits rDNA silencing. In this study, we elucidate a regulatory mechanism for rDNA silencing based on the finding that rDNA silencing strength directly correlates with cellular Sir2 protein levels. The endogenous level of Sir2p was shown to be limiting for rDNA silencing. Furthermore, small changes in Sir2p levels altered rDNA silencing strength. In rDNA silencing phenotypes, sir2 mutations were shown to be epistatic to sir4 mutations, indicating that SIR4 inhibition of rDNA silencing is mediated through SIR2. Furthermore, rDNA silencing is insensitive to SIR3 overexpression, but is severely reduced by overexpression of full-length Sir4p or a fragment of Sir4p that interacts with Sir2p. This negative effect of SIR4 overexpression was overridden by co-overexpression of SIR2, suggesting that SIR4 directly inhibits the rDNA silencing function of SIR2. Finally, genetic manipulations of SIR4 previously shown to promote extended life span also resulted in enhanced rDNA silencing. We propose a simple model in which telomeres act as regulators of rDNA silencing by competing for limiting amounts of Sir2 protein. PMID:9649515

  6. Enzyme induction, mutagen activation and carcinogen testing in yeast

    SciTech Connect

    Wiseman, A.

    1987-01-01

    This book documents the scientific basis for using yeasts to detect mutagenic chemicals likely to cause cancer in humans, a phenomenon explained by the presence of the enzyme cytochrome P-450 in some tissues. Explains the nature and roles of this enzyme in detail, and explores a range of related topics, including the genetic features of yeast, the mitochondrial DNA system and petite mutants, the molecular biology of transcription of genes in yeast, and enzyme induction. Also examined is DNA repair and how mutagenesis in yeast and other microorganisms relates to the practical detection of mutagens.

  7. Impairment of the DNA binding activity of the TATA-binding protein renders the transcriptional function of Rvb2p/Tih2p, the yeast RuvB-like protein, essential for cell growth.

    PubMed

    Ohdate, Hidezumi; Lim, Chun Ren; Kokubo, Tetsuro; Matsubara, Kenichi; Kimata, Yukio; Kohno, Kenji

    2003-04-25

    In Saccharomyces cerevisiae, two highly conserved proteins, Rvb1p/Tih1p and Rvb2p/Tih2p, have been demonstrated to be major components of the chromatin-remodeling INO80 complex. The mammalian orthologues of these two proteins have been shown to physically associate with the TATA-binding protein (TBP) in vitro but not clearly in vivo. Here we show that yeast proteins interact with TBP under both conditions. To assess the functional importance of these interactions, we examined the effect of mutating both TIH2/RVB2 and SPT15, which encodes TBP, on yeast cell growth. Intriguingly, only those spt15 mutations that affected the ability of TBP to bind to the TATA box caused synthetic growth defects in a tih2-ts160 background. This suggests that Tih2p might be important in recruiting TBP to the promoter. A DNA microarray technique was used to identify genes differentially expressed in the tih2-ts160 strain grown at the restrictive temperature. Only 34 genes were significantly and reproducibly affected; some up-regulated and others down-regulated. We compared the transcription of several of these Tih2p target genes in both wild type and various mutant backgrounds. We found that the transcription of some genes depends on functions possessed by both Tih2p and TBP and that these functions are substantially impaired in the spt15/tih2-ts160 double mutants that confer synthetic growth defects. PMID:12576485

  8. Yeast K1 killer toxin forms ion channels in sensitive yeast spheroplasts and in artificial liposomes.

    PubMed Central

    Martinac, B; Zhu, H; Kubalski, A; Zhou, X L; Culbertson, M; Bussey, H; Kung, C

    1990-01-01

    The patch-clamp technique was used to examine the plasma membranes of sensitive yeast spheroplasts exposed to partially purified killer toxin preparations. Asolectin liposomes in which the toxin was incorporated were also examined. Excised inside-out patches from these preparations often revealed at 118 pS conductance appearing in pairs. The current through this conductance flickered rapidly among three states: dwelling mostly at the unit-open state, less frequently at the two-unit-open state, and more rarely at the closed state. Membrane voltages from -80 to 80 mV had little influence on the opening probability. The current reversed near the equilibrium potential of K+ in asymmetric KCl solutions and also reversed near O mV at symmetric NaCl vs. KCl solutions. The two levels of the conductance were likely due to the toxin protein, as treatment of spheroplasts or liposomes with extracellular protein preparations from isogenic yeasts deleted for the toxin gene gave no such conductance levels. These results show that in vivo the killer-toxin fraction can form a cation channel that seldom closes regardless of membrane voltage. We suggest that this channel causes the death of sensitive yeast cells. Images PMID:1696721

  9. Mutational analysis of the D1/E1 core helices and the conserved N-terminal region of yeast transcription factor IIB (TFIIB): identification of an N-terminal mutant that stabilizes TATA-binding protein-TFIIB-DNA complexes.

    PubMed Central

    Bangur, C S; Pardee, T S; Ponticelli, A S

    1997-01-01

    The general transcription factor IIB (TFIIB) plays an essential role in transcription of protein-coding genes by RNA polymerase II. We have used site-directed mutagenesis to assess the role of conserved amino acids in several important regions of yeast TFIIB. These include residues in the highly conserved amino-terminal region and basic residues in the D1 and E1 core domain alpha-helices. Acidic substitutions of residues K190 (D1) and K201 (E1) resulted in growth impairments in vivo, reduced basal transcriptional activity in vitro, and an inability to form stable TFIIB-TATA-binding protein-DNA (DB) complexes. Significantly, these mutants retained the ability to respond to acidic activators in vivo and to the Gal4-VP16 activator in vitro, supporting the view that these basic residues play a role in basal transcription. In addition, 14 single-amino-acid substitutions were introduced in the conserved amino-terminal region. Three of these mutants, the L50D, R64E, and R78L mutants, displayed altered growth properties in vivo and were compromised for supporting transcription in vitro. The L50D mutant was impaired for RNA polymerase II interaction, while the R64E mutant exhibited altered transcription start site selection both in vitro and in vivo and, surprisingly, was more active than the wild type in the formation of stable DB complexes. These results support the view that the amino-terminal domain is involved in the direct interaction between yeast TFIIB and RNA polymerase II and suggest that this domain may interact with DNA and/or modulate the formation of a DB complex. PMID:9372909

  10. UV light-induced DNA lesions cause dissociation of yeast RNA polymerases-I and establishment of a specialized chromatin structure at rRNA genes

    PubMed Central

    Tremblay, Maxime; Charton, Romain; Wittner, Manuel; Levasseur, Geneviève; Griesenbeck, Joachim; Conconi, Antonio

    2014-01-01

    The cytotoxicity of UV light-induced DNA lesions results from their interference with transcription and replication. DNA lesions arrest elongating RNA polymerases, an event that triggers transcription-coupled nucleotide excision repair. Since arrested RNA polymerases reduce the accessibility of repair factors to DNA lesions, they might be displaced. The fate of arrested RNA polymerases-II at DNA lesions has been extensively studied, yielding partially contradictory results. Considerably less is known about RNA polymerases-I that transcribe nucleosomes-depleted rRNA genes at very high rate. To investigate the fate of arrested RNA polymerases-I at DNA lesions, chromatin-immunoprecipitation, electron microscopy, transcription run-on, psoralen-cross-linking and chromatin-endogenous cleavage were employed. We found that RNA polymerases-I density increased at the 5′-end of the gene, likely due to continued transcription initiation followed by elongation and pausing/release at the first DNA lesion. Most RNA polymerases-I dissociated downstream of the first DNA lesion, concomitant with chromatin closing that resulted from deposition of nucleosomes. Although nucleosomes were deposited, the high mobility group-box Hmo1 (component of actively transcribed rRNA genes) remained associated. After repair of DNA lesions, Hmo1 containing chromatin might help to restore transcription elongation and reopening of rRNA genes chromatin. PMID:24097442

  11. The DNA binding specificity of the basic region of the yeast transcriptional activator GCN4 can be changed by substitution of a single amino acid.

    PubMed Central

    Suckow, M; von Wilcken-Bergmann, B; Müller-Hill, B

    1993-01-01

    The X-ray structure of a GCN4 DNA complex (1) shows, that specific DNA binding of the GCN4 basic region is mediated by a complicated network of base pair and DNA backbone contacts. According to the X-ray structure, alanine -14 of the basic region of GCN4 (we define the first leucine of the leucine zipper as +1) makes a hydrophobic contact to the methyl group of the thymine next to the center of the GCN4 binding site 5' ATGACTCAT 3'. We tested the DNA binding properties of the nineteen derivatives of GCN4, which carry all possible amino acids in position -14 of the basic region. Substitution of alanine -14 of GCN4 by either asparagine or cysteine changes the DNA binding specificity. Serine in this position broadens the specificity for position 1 of the target, whereas other amino acids either retain or decrease GCN4 specificity. Images PMID:8502548

  12. The activity of the histone chaperone yeast Asf1 in the assembly and disassembly of histone H3/H4–DNA complexes

    PubMed Central

    Donham, Douglas C.; Scorgie, Jean K.; Churchill, Mair E. A.

    2011-01-01

    The deposition of the histones H3/H4 onto DNA to give the tetrasome intermediate and the displacement of H3/H4 from DNA are thought to be the first and the last steps in nucleosome assembly and disassembly, respectively. Anti-silencing function 1 (Asf1) is a chaperone of the H3/H4 dimer that functions in both of these processes. However, little is known about the thermodynamics of chaperone–histone interactions or the direct role of Asf1 in the formation or disassembly of histone–DNA complexes. Here, we show that Saccharomyces cerevisiae Asf1 shields H3/H4 from unfavorable DNA interactions and aids the formation of favorable histone–DNA interactions through the formation of disomes. However, Asf1 was unable to disengage histones from DNA for tetrasomes formed with H3/H4 and strong nucleosome positioning DNA sequences or tetrasomes weakened by mutant (H3K56Q/H4) histones or non-positioning DNA sequences. Furthermore, Asf1 did not associate with preformed tetrasomes. These results are consistent with the measured affinity of Asf1 for H3/H4 dimers of 2.5 nM, which is weaker than the association of H3/H4 for DNA. These studies support a mechanism by which Asf1 aids H3/H4 deposition onto DNA but suggest that additional factors or post-translational modifications are required for Asf1 to remove H3/H4 from tetrasome intermediates in chromatin. PMID:21447559

  13. Transcriptional activators in yeast

    PubMed Central

    2006-01-01

    Eukaryotic transcription activation domains (ADs) are not well defined on the proteome scale. We systematicallly tested ∼6000 yeast proteins for transcriptional activity using a yeast one-hybrid system and identified 451 transcriptional activators. We then determined their transcription activation strength using fusions to the Gal4 DNA-binding domain and a His3 reporter gene which contained a promoter with a Gal4-binding site. Among the 132 strongest activators 32 are known transcription factors while another 35 have no known function. Although zinc fingers, helix–loop–helix domains and several other domains are highly overrepresented among the activators, only few contain characterized ADs. We also found some striking correlations: the stronger the activation activity, the more acidic, glutamine-rich, proline-rich or asparagine-rich the activators were. About 29% of the activators have been found previously to specifically interact with the transcription machinery, while 10% are known to be components of transcription regulatory complexes. Based on their transcriptional activity, localization and interaction patterns, at least six previously uncharacterized proteins are suggested to be bona fide transcriptional regulators (namely YFL049W, YJR070C, YDR520C, YGL066W/Sgf73, YKR064W and YCR082W/Ahc2). PMID:16464826

  14. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell.

    PubMed

    Luo, Yumei; Chen, Zikai; Zhu, Detu; Tu, Haitao; Pan, Shen Quan

    2015-01-01

    The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells. PMID:26425545

  15. Yeast Actin-Related Protein ARP6 Negatively Regulates Agrobacterium-Mediated Transformation of Yeast Cell

    PubMed Central

    Luo, Yumei; Chen, Zikai; Zhu, Detu; Tu, Haitao; Pan, Shen Quan

    2015-01-01

    The yeasts, including Saccharomyces cerevisiae and Pichia pastoris, are single-cell eukaryotic organisms that can serve as models for human genetic diseases and hosts for large scale production of recombinant proteins in current biopharmaceutical industry. Thus, efficient genetic engineering tools for yeasts are of great research and economic values. Agrobacterium tumefaciens-mediated transformation (AMT) can transfer T-DNA into yeast cells as a method for genetic engineering. However, how the T-DNA is transferred into the yeast cells is not well established yet. Here our genetic screening of yeast knockout mutants identified a yeast actin-related protein ARP6 as a negative regulator of AMT. ARP6 is a critical member of the SWR1 chromatin remodeling complex (SWR-C); knocking out some other components of the complex also increased the transformation efficiency, suggesting that ARP6 might regulate AMT via SWR-C. Moreover, knockout of ARP6 led to disruption of microtubule integrity, higher uptake and degradation of virulence proteins, and increased DNA stability inside the cells, all of which resulted in enhanced transformation efficiency. Our findings have identified molecular and cellular mechanisms regulating AMT and a potential target for enhancing the transformation efficiency in yeast cells. PMID:26425545

  16. DNA

    ERIC Educational Resources Information Center

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  17. Cloning of a human cDNA encoding a CDC2-related kinase by complementation of a budding yeast cdc28 mutation

    SciTech Connect

    Ninomiya-Tsuji, Jun ); Nomoto, Satoshi; Matsumoto, Kunihiro ); Yasuda, Hideyo ); Reed, S.I. )

    1991-10-15

    The authors have cloned two different human cDNAs that can complement cdc28 mutations of budding yeast Saccharomyces cerevisiae. One corresponds to a gene encoding human p34{sup CDC2} kinase, and the other to a gene (CDK2; cell division kinase) that has not been characterized previously. The CDK2 protein is highly homologous to p34{sup CDC2} kinase and more significantly is homologous to Xenopus Eg1 kinase, suggesting that CDK2 is the human homolog of Eg1. The human CDC2 and CDK2 genes were both able to complement the inviability of a null allele of S. cerevisiae CDC28. This result indicates that the CDK2 protein has a biological activity closely related to the CDC28 and p34{sup CDC2} kinases. However, CDK2 was unable to complement cdc2 mutants in fission yeast Schizosaccharomyces pombe under the condition where the human CDC2 gene could complement them. CDK2 mRNA appeared late in G{sub 1} or in early S phase, slightly before CDC2 mRNA, after growth stimulation in normal human fibroblast cells. These results suggest that in human cells, two different CDC2-like kinases may regulate the cell cycle at distinct stages.

  18. DNA secondary structures are associated with recombination in major Plasmodium falciparum variable surface antigen gene families

    PubMed Central

    Sander, Adam F.; Lavstsen, Thomas; Rask, Thomas S.; Lisby, Michael; Salanti, Ali; Fordyce, Sarah L.; Jespersen, Jakob S.; Carter, Richard; Deitsch, Kirk W.; Theander, Thor G.; Pedersen, Anders Gorm; Arnot, David E.

    2014-01-01

    Many bacterial, viral and parasitic pathogens undergo antigenic variation to counter host immune defense mechanisms. In Plasmodium falciparum, the most lethal of human malaria parasites, switching of var gene expression results in alternating expression of the adhesion proteins of the Plasmodium falciparum-erythrocyte membrane protein 1 class on the infected erythrocyte surface. Recombination clearly generates var diversity, but the nature and control of the genetic exchanges involved remain unclear. By experimental and bioinformatic identification of recombination events and genome-wide recombination hotspots in var genes, we show that during the parasite’s sexual stages, ectopic recombination between isogenous var paralogs occurs near low folding free energy DNA 50-mers and that these sequences are heavily concentrated at the boundaries of regions encoding individual Plasmodium falciparum-erythrocyte membrane protein 1 structural domains. The recombinogenic potential of these 50-mers is not parasite-specific because these sequences also induce recombination when transferred to the yeast Saccharomyces cerevisiae. Genetic cross data suggest that DNA secondary structures (DSS) act as inducers of recombination during DNA replication in P. falciparum sexual stages, and that these DSS-regulated genetic exchanges generate functional and diverse P. falciparum adhesion antigens. DSS-induced recombination may represent a common mechanism for optimizing the evolvability of virulence gene families in pathogens. PMID:24253306

  19. Development of two isogenic sweet corn hybrids differing for glycinebetaine content.

    PubMed

    Rhodes, D; Rich, P J; Brunk, D G; Ju, G C; Rhodes, J C; Pauly, M H; Hansen, L A

    1989-11-01

    A hybrid of sweet corn, Zea mays L. (;1720'; Rogers Brothers Seed Co.), was found to be comprised of glycinebetaine-positive and glycinebetaine-deficient individuals in a 1:1 mixture. This phenomenon was traced to segregation for a single, nuclear, dominant gene determining leaf glycinebetaine content within the female inbred parent of this hybrid. Selection for homozygous recessive (glycinebetaine-deficient) and homozygous dominant (glycinebetaine-positive) genotypes of the female inbred parent enabled production of two isogenic versions of hybrid ;1720' differing with respect to a single copy of the dominant allele, by mating these female parent selections with the common homozygous recessive (glycinebetaine-deficient) male parent. These two isogenic hybrids are shown to differ by a factor of 300- to 400-fold in glycinebetaine titer of young expanding leaves of salinized plants, but exhibit no striking differences in the levels of free amino acids or the level of N-methylnicotinic acid (nicotinic acid betaine; trigonelline). The only significant difference between the two hybrids in terms of amino acid composition was found to be in the level of alanine under nonsalinized conditions. The betaine-deficient hybrid exhibited a 14% lower alanine level than the betaine-positive hybrid. Betaine deficiency was not associated with altered stress-induced accumulation of amino acids such as proline, serine, and asparagine plus aspartate, attesting to the high specificity of the genetic difference between these isogenic hybrids with respect to betaine accumulation. This germplasm offers unique opportunities to test whether a single dominant allele determining stress-induced betaine accumulation capacity influences stress resistance in maize. PMID:16667120

  20. Development of Two Isogenic Sweet Corn Hybrids Differing for Glycinebetaine Content 1

    PubMed Central

    Rhodes, David; Rich, Patrick J.; Brunk, Dennis G.; Ju, Grace C.; Rhodes, Judith C.; Pauly, Michael H.; Hansen, Leon A.

    1989-01-01

    A hybrid of sweet corn, Zea mays L. (`1720'; Rogers Brothers Seed Co.), was found to be comprised of glycinebetaine-positive and glycinebetaine-deficient individuals in a 1:1 mixture. This phenomenon was traced to segregation for a single, nuclear, dominant gene determining leaf glycinebetaine content within the female inbred parent of this hybrid. Selection for homozygous recessive (glycinebetaine-deficient) and homozygous dominant (glycinebetaine-positive) genotypes of the female inbred parent enabled production of two isogenic versions of hybrid `1720' differing with respect to a single copy of the dominant allele, by mating these female parent selections with the common homozygous recessive (glycinebetaine-deficient) male parent. These two isogenic hybrids are shown to differ by a factor of 300- to 400-fold in glycinebetaine titer of young expanding leaves of salinized plants, but exhibit no striking differences in the levels of free amino acids or the level of N-methylnicotinic acid (nicotinic acid betaine; trigonelline). The only significant difference between the two hybrids in terms of amino acid composition was found to be in the level of alanine under nonsalinized conditions. The betaine-deficient hybrid exhibited a 14% lower alanine level than the betaine-positive hybrid. Betaine deficiency was not associated with altered stress-induced accumulation of amino acids such as proline, serine, and asparagine plus aspartate, attesting to the high specificity of the genetic difference between these isogenic hybrids with respect to betaine accumulation. This germplasm offers unique opportunities to test whether a single dominant allele determining stress-induced betaine accumulation capacity influences stress resistance in maize. PMID:16667120

  1. In vivo genomic footprint of a yeast centromere.

    PubMed Central

    Densmore, L; Payne, W E; Fitzgerald-Hayes, M

    1991-01-01

    We have used in vivo genomic footprinting to investigate the protein-DNA interactions within the conserved DNA elements (CDEI, CDEII, and CDEIII) in the centromere from chromosome III of the yeast Saccharomyces cerevisiae. The in vivo footprint pattern obtained from wild-type cells shows that some guanines within the centromere DNA are protected from methylation by dimethyl sulfate. These results are consistent with studies demonstrating that yeast cells contain sequence-specific centromere DNA-binding proteins. Our in vivo experiments on chromosomes with mutant centromeres show that some mutations which affect chromosome segregation also alter the footprint pattern caused by proteins bound to the centromere DNA. The results of this study provide the first fine-structure map of proteins bound to centromere DNA in living yeast cells and suggest a direct correlation between these protein-DNA interactions and centromere function. Images PMID:1986217

  2. Comparison of barley malt alpha-amylase isozymes 1 and 2: construction of cDNA hybrids by in vivo recombination and their expression in yeast.

    PubMed

    Juge, N; Søgaard, M; Chaix, J C; Martin-Eauclaire, M F; Svensson, B; Marchis-Mouren, G; Guo, X J

    1993-08-25

    Germinating barley produces two alpha-amylase isozymes, AMY1 and AMY2, having 80% amino acid (aa) sequence identity and differing with respect to a number of functional properties. Recombinant AMY1 (re-AMY1) and AMY2 (re-AMY2) are produced in yeast, but whereas all re-AMY1 is secreted, re-AMY2 accumulates within the cell and only traces are secreted. Expression of AMY1::AMY2 hybrid cDNAs may provide a means of understanding the difference in secretion efficiency between the two isozymes. Here, the efficient homologous recombination system of the yeast, Saccharomyces cerevisiae, was used to generate hybrids of barley AMY with the N-terminal portion derived from AMY1, including the signal peptide (SP), and the C-terminal portion from AMY2. Hybrid cDNAs were thus generated that encode either the SP alone, or the SP followed by the N-terminal 21, 26, 53, 67 or 90 aa from AMY1 and the complementary C-terminal sequences from AMY2. Larger amounts of re-AMY are secreted by hybrids containing, in addition to the SP, 53 or more aa of AMY1. In contrast, only traces of re-AMY are secreted for hybrids having 26 or fewer aa of AMY1. In this case, re-AMY hybrid accumulates intracellularly. Transformants secreting hybrid enzymes also accumulated some re-AMY within the cell. The AMY1 SP, therefore, does not ensure re-AMY2 secretion and a certain portion of the N-terminal sequence of AMY1 is required for secretion of a re-AMY1::AMY2 hybrid. PMID:8359683

  3. Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes.

    PubMed

    Burch, Tanya C; Rhim, Johng S; Nyalwidhe, Julius O

    2016-01-01

    Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells) and RC77T/E (malignant cells) were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mitochondria and the Mitochondrial Energy Metabolism RT(2) PCR arrays. Forty-seven genes were differentially regulated between the two cell lines. The interaction and regulatory networks of these genes were generated by Ingenuity Pathway Analysis. UCP2 was the most significantly upregulated gene in primary adenocarcinoma cells in the current study. The overexpression of UCP2 upon malignant transformation was further validated using human prostatectomy clinical specimens. Conclusions. This study demonstrates the overexpression of multiple genes that are involved in mitochondria biogenesis, bioenergetics, and modulation of apoptosis. These genes may play a role in malignant transformation and disease progression. The upregulation of some of these genes in clinical samples indicates that some of the differentially transcribed genes could be the potential targets for therapeutic interventions. PMID:27478826

  4. Microevolutionary distribution of isogenicity in a self-fertilizing fish (Kryptolebias marmoratus) in the Florida Keys.

    PubMed

    Tatarenkov, Andrey; Earley, Ryan L; Taylor, D Scott; Avise, John C

    2012-12-01

    The mangrove rivulus Kryptolebias marmoratus and a closely related species are the world's only vertebrates that routinely self-fertilize. Such uniqueness presents a model for understanding why this reproductive mode, common in plants and invertebrates, is so rare in vertebrates. A survey of 32 highly polymorphic loci in >200 specimens of mangrove rivulus from multiple locales in the Florida Keys, USA, revealed extensive population-genetic structure on microspatial and micro-temporal scales. Observed heterozygosities were severely constrained, as expected for a hermaphroditic species with a mixed-mating system and low rates of outcrossing. Despite the pronounced population structure and the implied restrictions on effective gene flow, isogenicity (genetic identity across individuals) within and among local inbred populations was surprisingly low even after factoring out probable de novo mutations. Results indicate that neither frequent bottlenecks nor directional genetic adaptation to local environmental conditions were the primary driving forces impacting multilocus population-genetic architecture in this self-fertilizing vertebrate species. On the other hand, a high diversity of isogenic lineages within relatively small and isolated local populations is consistent with the action of diversifying selection driven by the extreme spatio-temporal environmental variability that is characteristic of mangrove habitats. PMID:22593558

  5. Mitochondria Biogenesis and Bioenergetics Gene Profiles in Isogenic Prostate Cells with Different Malignant Phenotypes

    PubMed Central

    Burch, Tanya C.; Rhim, Johng S.

    2016-01-01

    Background. The most significant hallmarks of cancer are directly or indirectly linked to deregulated mitochondria. In this study, we sought to profile mitochondria associated genes in isogenic prostate cell lines with different tumorigenic phenotypes from the same patient. Results. Two isogenic human prostate cell lines RC77N/E (nonmalignant cells) and RC77T/E (malignant cells) were profiled for expression of mitochondrial biogenesis and energy metabolism genes by qRT-PCR using the Human Mitochondria and the Mitochondrial Energy Metabolism RT2 PCR arrays. Forty-seven genes were differentially regulated between the two cell lines. The interaction and regulatory networks of these genes were generated by Ingenuity Pathway Analysis. UCP2 was the most significantly upregulated gene in primary adenocarcinoma cells in the current study. The overexpression of UCP2 upon malignant transformation was further validated using human prostatectomy clinical specimens. Conclusions. This study demonstrates the overexpression of multiple genes that are involved in mitochondria biogenesis, bioenergetics, and modulation of apoptosis. These genes may play a role in malignant transformation and disease progression. The upregulation of some of these genes in clinical samples indicates that some of the differentially transcribed genes could be the potential targets for therapeutic interventions. PMID:27478826

  6. Microevolutionary Distribution of Isogenicity in a Self-fertilizing Fish (Kryptolebias marmoratus) in the Florida Keys

    PubMed Central

    Tatarenkov, Andrey; Earley, Ryan L.; Taylor, D. Scott; Avise, John C.

    2012-01-01

    The mangrove rivulus Kryptolebias marmoratus and a closely related species are the world’s only vertebrates that routinely self-fertilize. Such uniqueness presents a model for understanding why this reproductive mode, common in plants and invertebrates, is so rare in vertebrates. A survey of 32 highly polymorphic loci in >200 specimens of mangrove rivulus from multiple locales in the Florida Keys, USA, revealed extensive population-genetic structure on microspatial and micro-temporal scales. Observed heterozygosities were severely constrained, as expected for a hermaphroditic species with a mixed-mating system and low rates of outcrossing. Despite the pronounced population structure and the implied restrictions on effective gene flow, isogenicity (genetic identity across individuals) within and among local inbred populations was surprisingly low even after factoring out probable de novo mutations. Results indicate that neither frequent bottlenecks nor directional genetic adaptation to local environmental conditions were the primary driving forces impacting multilocus population-genetic architecture in this self-fertilizing vertebrate species. On the other hand, a high diversity of isogenic lineages within relatively small and isolated local populations is consistent with the action of diversifying selection driven by the extreme spatio-temporal environmental variability that is characteristic of mangrove habitats. PMID:22593558

  7. Vaginal Yeast Infections (For Parents)

    MedlinePlus

    ... Can I Help a Friend Who Cuts? Vaginal Yeast Infections KidsHealth > For Teens > Vaginal Yeast Infections Print ... side effect of taking antibiotics. What Is a Yeast Infection? A yeast infection is a common infection ...

  8. Vaginal Yeast Infection

    MedlinePlus

    ... t diagnose this condition by a person’s medical history and physical examination. They usually diagnose yeast infection by examining vaginal secretions under a microscope for evidence of yeast. Treatment Various antifungal vaginal ...

  9. Vaginal yeast infection

    MedlinePlus

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

  10. Vaginal yeast infection

    MedlinePlus

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

  11. Transcriptional and cellular responses to defective mitochondrial proteolysis in fission yeast.

    PubMed

    Guha, Suranjana; López-Maury, Luis; Shaw, Michael; Bähler, Jürg; Norbury, Chris J; Agashe, Vishwas R

    2011-04-29

    Lon and m-AAA are the principal, regulated proteases required for protein maturation and turnover in the mitochondrial matrix of diverse species. To understand their roles in fission yeast (Schizosaccharomyces pombe) mitochondria, we generated deletion strains lacking Lon and m-AAA, individually (Δlon1 and Δm-AAA) or together, Δlon1Δm-AAA (Δ/Δ). All three strains were viable but incapable of respiratory growth on a non-fermentable carbon source due to mitochondrial dysfunction. Confocal and electron microscopy revealed a decrease in membrane potential and ultrastructural changes in Δlon1, Δm-AAA and Δ/Δ mitochondria, consistent with a respiratory defect and aggregation of proteins in the mitochondrial matrix. To understand the global adaptations required for cell survival in the absence of Lon and m-AAA proteases, we compared genome-wide gene expression signatures of the deletion strains with the isogenic wild-type strain. Deletion of lon1 caused a distinctive transcriptional footprint of just 12 differentially expressed genes, 9 of which were up-regulated genes located on the proximal mitochondrial genome (mitochondrial DNA). In contrast, m-AAA deletion caused a much larger transcriptional response involving 268 almost exclusively nuclear genes. Genes ameliorating stress and iron assimilation were up-regulated, while diverse mitochondrial genes and other metabolic enzymes were down-regulated. The connection with iron dysregulation was further explored using biochemical, chemical and cellular assays. Although Δm-AAA and Δ/Δ contained more cellular iron than the wild-type strain, their transcriptomes strongly resembled a signature normally evoked by iron insufficiency or disrupted assembly of iron-sulfur clusters in mitochondria. Based on these findings, we posit that excess iron accumulation could contribute to the pathology of human neurodegenerative disorders arising from defects in m-AAA function. PMID:21354177

  12. Casein Kinase 1γ Ensures Monopolar Growth Polarity under Incomplete DNA Replication Downstream of Cds1 and Calcineurin in Fission Yeast

    PubMed Central

    Koyano, Takayuki; Konishi, Manabu; Martin, Sophie G.; Ohya, Yoshikazu; Hirata, Dai

    2015-01-01

    Cell polarity is essential for various cellular functions during both proliferative and developmental stages, and it displays dynamic alterations in response to intracellular and extracellular cues. However, the molecular mechanisms underlying spatiotemporal control of polarity transition are poorly understood. Here, we show that fission yeast Cki3 (a casein kinase 1γ homolog) is a critical regulator to ensure persistent monopolar growth during S phase. Unlike the wild type, cki3 mutant cells undergo bipolar growth when S phase is blocked, a condition known to delay transition from monopolar to bipolar growth (termed NETO [new end takeoff]). Consistent with this role, Cki3 kinase activity is substantially increased, and cells lose their viability in the absence of Cki3 upon an S-phase block. Cki3 acts downstream of the checkpoint kinase Cds1/Chk2 and calcineurin, and the latter physically interacts with Cki3. Autophosphorylation in the C terminus is inhibitory toward Cki3 kinase activity, and calcineurin is responsible for its dephosphorylation. Cki3 localizes to the plasma membrane, and this localization requires the palmitoyltransferase complex Erf2-Erf4. Membrane localization is needed not only for proper NETO timing but also for Cki3 kinase activity. We propose that Cki3 acts as a critical inhibitor of cell polarity transition under S-phase arrest. PMID:25691662

  13. Analysis of two benzo[a]pyrene-resistant mutants of the mouse hepatoma Hepa-1 P(1)450 gene via cDNA expression in yeast.

    PubMed Central

    Kimura, S; Smith, H H; Hankinson, O; Nebert, D W

    1987-01-01

    Two benzo[a]pyrene-resistant mutant clones (c1 and c37) of the mouse hepatoma Hepa-1 wild-type (wt) cell line were examined for their lack of P(1)450 [aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH)] activity. From lambda gt11 cDNA libraries, the nearly full-length P(1)450 cDNAs of wt, c1 and c37 were isolated and sequenced. The c1 cDNA was found to have a single mutation leading to premature termination of the protein after Asn-414; a rapidly migrating band corresponding to this truncated protein was found on Western immunoblots. The c37 cDNA was found to have two point mutations, leading to Leu-118----Arg-118 and Arg-245----Pro-245, but otherwise to encode the normal (524-residue) protein; the mature protein was confirmed by Western blot analysis. P(1)450 cDNA from wt, c1 and c37 and chimeric cDNAs between wt and c37 were inserted into the expression vector pAAH5 and expressed in Saccharomyces cerevisiae strain 50.L4. The Leu-118----Arg-118 mutation alone was found to have negligible effect on AHH activity, while the Arg-245----Pro-245 mutation alone leads to a 2- to 3-fold decrease in enzyme activity. The two mutations together totally abrogate AHH activity. The biologic mutant c37 provides the first evidence for the importance of Arg-245, and the complementary function of Leu-118, in normal P(1)450 enzymic function. This alteration in a single amino acid from arginine to proline might block electron flow directly, or change secondary structure of the protein, such that normal monooxygenation of benzo[a]pyrene cannot occur. Images Fig. 1. Fig. 2. Fig. 5. PMID:3308449

  14. Fast and sensitive detection of genetically modified yeasts in wine.

    PubMed

    León, Carlos; García-Cañas, Virginia; González, Ramón; Morales, Pilar; Cifuentes, Alejandro

    2011-10-21

    In this work, a novel screening methodology based on the combined use of multiplex polymerase chain reaction (PCR) and capillary gel electrophoresis with laser induced fluorescence (CGE-LIF) is developed for the fast and sensitive detection of genetically modified yeasts in wine. As model, a recombinant EKD-13 Saccaromyces cerevisiae strain was selected and different wines were prepared using either recombinant or conventional yeasts. Special emphasis is put on the yeast DNA extraction step, exploring different commercial and non-commercial methods, in order to overcome the important difficulty of obtaining amplifiable DNA from wine samples. To unequivocally detect the transgenic yeast, two specific segments of the transgenic construction were amplified. In addition, a third primer pair was used as amplification control to confirm the quality of the yeast DNA obtained from the extraction step. CGE-LIF provides high sensitivity, good analysis speed and impressive resolution of DNA fragments, making this technique very convenient to optimize multiplex PCR parameters and to analyze the amplified DNA fragments. Thus, the CGE-LIF method provided %RSD values for DNA migration times lower than 0.82% (n=10) with the same capillary and lower than 1.92% (n=15) with three different capillaries, allowing the adequate size determination of the PCR products with an error lower than 4% compared to the theoretically expected. The whole method developed in this work requires less than one working day and grants the sensitive detection of transgenic yeasts in wine samples. PMID:21296357

  15. The Analysis Of The Correlations And Regressions Between Some Characters On A Wheat Isogenic Varities Assortment

    NASA Astrophysics Data System (ADS)

    Păniţă, Ovidiu

    2015-09-01

    In the years 2012-2014 on Banu-Maracine DRS there were tested an assortment of 25 isogenic lines of wheat (Triticum aestivum ssp.vulgare), the analyzed characters being the number of seeds/spike, seeds weight/spike (g), no. of spikes/m2, weight of a thousand seeds (WTS) (g) and no. of emerged plants/m2. Based on recorded data and statistical processing of those, they were identified a numbers of links between these characters. Also available regression models were identified between some of the studied characters. Based on component analysis, no. of seeds/spike and seeds weight/spike are components that influence in excess of 88% variance analysis, a total of seven genotypes with positive scores for both factors.

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

    NASA Astrophysics Data System (ADS)

    Sakharkar, Kishore R.; Sakharkar, Meena K.

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

  17. Production of alpha-amylase by yeast

    SciTech Connect

    Thomse, K.K.

    1987-01-01

    The enzyme alpha-amylase confers to an organism the enzymatic activity for the degradation of polyglucosides with alpha-1,4 glycosidic bonds such as starch and glycogen which are among the major storage compounds in plants and animals. Most alpha-amylases are single polypeptides of molecular weights around 50,000 dalton. They are generally found in the digestive tract of animals and in germinating seeds. Among the products released upon enzymatic degradation of polyglucosides maltose, a sugar that can be utilized as carbon source by yeast, is a major constituent. A cDNA segment complementary to mouse salivary amylase messenger RNA has been inserted into the yeast expression vector pMA56 behind the promoter of the gene encoding alcohol dehydrogenase I of yeast. Yeast transformants harboring plasmids with the normal orientation of the promoter and the mouse amylase cDNA gene produce amylase and release the enzyme in free form into the culture medium. Approximately 90% of the amylase activity is found in the medium. Yeast strains carrying MAL allele and transformed with a plasmid which directed the synthesis of mouse alpha-amylase were tested on plates containing starch and in batch fermentations using different high molecular weight sugars and oligosaccharides as carbon source. The results of these experiments will be discussed. (Refs. 21).

  18. Identification of soybean proteins and genes differentially regulated in near isogenic lines differing in resistance to aphid infestation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The soybean aphid, a plant sap sucking insect, is an important soybean pest in the USA causing significant yield losses. The Rag2 gene of soybean provides resistance to soybean aphid biotypes 1 and 2. Transcriptomic and proteomic analyses were performed on near isogenic lines (NILs) with the Rag2 al...

  19. Fusarium spp. Associated with Field-Grown Grain of Near-Isogenic Low Lignin and Wild-Type Sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fusarium spp. associated with field-grown grain of near-isogenic low lignin and wild-type sorghum. Deanna Funnell-Harris and Jeff Pedersen, USDA-ARS, Lincoln, NE Previous studies indicated that low lignin brown midrib (bmr) sorghum may be more resistant to Fusarium spp. than wild-type and that phen...

  20. Evaluation of two southern leaf blight resistance QTL for their effect on yield and disease resistance in isogenic maize hybrids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plants need to balance resources between yield and defense. This phenomenon has rarely been investigated in the context of naturally-occurring quantitative resistance alleles in an agricultural production environment. B73-3B and B73-6A are two near-isogenic lines (NILs) in the background of the mai...

  1. Whole genome expression analysis of near isogenic lines for the soybean E1 gene under short and long day conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Control of soybean flowering time is important for geographic adaptation, and maximizing yield and has been shown in soybean be to be controlled by a series of genes (E genes) that condition time to flowering. The E genes are available as near isogenic lines (NILs) making them an excellent model sys...

  2. Activation of an Alternative, Rec12 (Spo11)-Independent Pathway of Fission Yeast Meiotic Recombination in the Absence of a DNA Flap Endonuclease

    PubMed Central

    Farah, Joseph A.; Cromie, Gareth; Davis, Luther; Steiner, Walter W.; Smith, Gerald R.

    2005-01-01

    Spo11 or a homologous protein appears to be essential for meiotic DNA double-strand break (DSB) formation and recombination in all organisms tested. We report here the first example of an alternative, mutationally activated pathway for meiotic recombination in the absence of Rec12, the Spo11 homolog of Schizosaccharomyces pombe. Rad2, a FEN-1 flap endonuclease homolog, is involved in processing Okazaki fragments. In its absence, meiotic recombination and proper segregation of chromosomes were restored in rec12Δ mutants to nearly wild-type levels. Although readily detectable in wild-type strains, meiosis-specific DSBs were undetectable in recombination-proficient rad2Δ rec12Δ strains. On the basis of the biochemical properties of Rad2, we propose that meiotic recombination by this alternative (Rec*) pathway can be initiated by non-DSB lesions, such as nicks and gaps, which accumulate during premeiotic DNA replication in the absence of Okazaki fragment processing. We compare the Rec* pathway to alternative pathways of homologous recombination in other organisms. PMID:16118186

  3. Methylations of histone H3 lysine 9 and lysine 36 are functionally linked to DNA replication checkpoint control in fission yeast

    SciTech Connect

    Kim, Hyun Soo; Rhee, Dong Keun; Jang, Yeun Kyu

    2008-04-04

    Recently, histone H4 lysine 20 and H3 lysine 79 methylations were functionally linked to DNA damage checkpoint. The crosstalk between histone methylation and the S-M checkpoint, however, has remained unclear. Here, we show that H3 lysine 9 (K9) and lysine 36 (K36) methylations catalyzed by two histone methyltransferases Clr4 and Set2 are involved in hydroxyurea (HU)-induced replication checkpoint. The clr4-set2 double mutants besides histone H3-K9 and K36 double mutants exhibited HU-sensitivity, a defective HU-induced S-M checkpoint, and a significant reduction of HU-induced phosphorylation of Cdc2. Intriguingly, the clr4-set2 double mutations impaired the HU-induced accumulation of a mitotic inhibitor Mik1. Double mutants in Alp13 and Swi6, which can specifically bind to H3-K36 and K9 methylations, exhibited phenotypes similar to those of the clr4-set2 mutants. Together, these findings suggest that methylations of histone H3-K9 and K36 by Clr4 and Set2 are functionally linked to DNA replication checkpoint via accumulation of Mik1.

  4. Subcellular localization of the yeast proteome

    PubMed Central

    Kumar, Anuj; Agarwal, Seema; Heyman, John A.; Matson, Sandra; Heidtman, Matthew; Piccirillo, Stacy; Umansky, Lara; Drawid, Amar; Jansen, Ronald; Liu, Yang; Cheung, Kei-Hoi; Miller, Perry; Gerstein, Mark; Roeder, G. Shirleen; Snyder, Michael

    2002-01-01

    Protein localization data are a valuable information resource helpful in elucidating eukaryotic protein function. Here, we report the first proteome-scale analysis of protein localization within any eukaryote. Using directed topoisomerase I-mediated cloning strategies and genome-wide transposon mutagenesis, we have epitope-tagged 60% of the Saccharomyces cerevisiae proteome. By high-throughput immunolocalization of tagged gene products, we have determined the subcellular localization of 2744 yeast proteins. Extrapolating these data through a computational algorithm employing Bayesian formalism, we define the yeast localizome (the subcellular distribution of all 6100 yeast proteins). We estimate the yeast proteome to encompass ∼5100 soluble proteins and >1000 transmembrane proteins. Our results indicate that 47% of yeast proteins are cytoplasmic, 13% mitochondrial, 13% exocytic (including proteins of the endoplasmic reticulum and secretory vesicles), and 27% nuclear/nucleolar. A subset of nuclear proteins was further analyzed by immunolocalization using surface-spread preparations of meiotic chromosomes. Of these proteins, 38% were found associated with chromosomal DNA. As determined from phenotypic analyses of nuclear proteins, 34% are essential for spore viability—a percentage nearly twice as great as that observed for the proteome as a whole. In total, this study presents experimentally derived localization data for 955 proteins of previously unknown function: nearly half of all functionally uncharacterized proteins in yeast. To facilitate access to these data, we provide a searchable database featuring 2900 fluorescent micrographs at http://ygac.med.yale.edu. PMID:11914276

  5. Biocavity laser spectroscopy of genetically altered yeast cells and isolated yeast mitochondria

    NASA Astrophysics Data System (ADS)

    Gourley, Paul L.; Hendricks, Judy K.; McDonald, Anthony E.; Copeland, R. Guild; Naviaux, Robert K.; Yaffe, Michael P.

    2006-02-01

    We report an analysis of 2 yeast cell mutants using biocavity laser spectroscopy. The two yeast strains differed only by the presence or absence of mitochondrial DNA. Strain 104 is a wild-type (ρ +) strain of the baker's yeast, Saccharomyces cerevisiae. Strain 110 was derived from strain 104 by removal of its mitochondrial DNA (mtDNA). Removal of mtDNA causes strain 110 to grow as a "petite" (ρ -), named because it forms small colonies (of fewer cells because it grows more slowly) on agar plates supplemented with a variety of different carbon sources. The absence of mitochondrial DNA results in the complete loss of all the mtDNA-encoded proteins and RNAs, and loss of the pigmented, heme-containing cytochromes a and b. These cells have mitochondria, but the mitochondria lack the normal respiratory chain complexes I, III, IV, and V. Complex II is preserved because its subunits are encoded by genes located in nuclear DNA. The frequency distributions of the peak shifts produced by wild-type and petite cells and mitochondria show striking differences in the symmetry and patterns of the distributions. Wild-type ρ + cells (104) and mitochondria produced nearly symmetric, Gaussian distributions. The ρ - cells (110) and mitochondria showed striking asymmetry and skew that appeared to follow a Poisson distribution.

  6. Pexophagy in yeasts.

    PubMed

    Oku, Masahide; Sakai, Yasuyoshi

    2016-05-01

    Pexophagy, selective degradation of peroxisomes via autophagy, is the main system for reducing organelle abundance. Elucidation of the molecular machinery of pexophagy has been pioneered in studies of the budding yeast Saccharomyces cerevisiae and the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha. Recent analyses using these yeasts have elucidated the molecular machineries of pexophagy, especially in terms of the interactions and modifications of the so-called adaptor proteins required for guiding autophagic membrane biogenesis on the organelle surface. Based on the recent findings, functional relevance of pexophagy and another autophagic pathway, mitophagy (selective autophagy of mitochondria), is discussed. We also discuss the physiological importance of pexophagy in these yeast systems. PMID:26409485

  7. A method to capture large DNA fragments from genomic DNA.

    PubMed

    Ball, Geneviève; Filloux, Alain; Voulhoux, Romé

    2014-01-01

    The gene capture technique is a powerful tool that allows the cloning of large DNA regions (up to 80 kb), such as entire genomic islands, without using restriction enzymes or DNA amplification. This technique takes advantage of the high recombinant capacity of the yeast. A "capture" vector containing both ends of the target DNA region must first be constructed. The target region is then captured by co-transformation and recombination in yeast between the "capture" vector and appropriate genomic DNA. The selected recombinant plasmid can be verified by sequencing and transferred in the bacteria for multiple applications. This chapter describes a protocol specifically adapted for Pseudomonas aeruginosa genomic DNA capture. PMID:24818928

  8. Binding of the Fkh1 Forkhead Associated Domain to a Phosphopeptide within the Mph1 DNA Helicase Regulates Mating-Type Switching in Budding Yeast

    PubMed Central

    Su, Zhangli; Cherney, Rachel; Choi, Koyi; Denu, John; Zhao, Xiaolan; Fox, Catherine A.

    2016-01-01

    The Saccharomyces cerevisiae Fkh1 protein has roles in cell-cycle regulated transcription as well as a transcription-independent role in recombination donor preference during mating-type switching. The conserved FHA domain of Fkh1 regulates donor preference by juxtaposing two distant regions on chromosome III to promote their recombination. A model posits that this Fkh1-mediated long-range chromosomal juxtaposition requires an interaction between the FHA domain and a partner protein(s), but to date no relevant partner has been described. In this study, we used structural modeling, 2-hybrid assays, and mutational analyses to show that the predicted phosphothreonine-binding FHA domain of Fkh1 interacted with multiple partner proteins. The Fkh1 FHA domain was important for its role in cell-cycle regulation, but no single interaction partner could account for this role. In contrast, Fkh1’s interaction with the Mph1 DNA repair helicase regulated donor preference during mating-type switching. Using 2-hybrid assays, co-immunoprecipitation, and fluorescence anisotropy, we mapped a discrete peptide within the regulatory Mph1 C-terminus required for this interaction and identified two threonines that were particularly important. In vitro binding experiments indicated that at least one of these threonines had to be phosphorylated for efficient Fkh1 binding. Substitution of these two threonines with alanines (mph1-2TA) specifically abolished the Fkh1-Mph1 interaction in vivo and altered donor preference during mating-type switching to the same degree as mph1Δ. Notably, the mph1-2TA allele maintained other functions of Mph1 in genome stability. Deletion of a second Fkh1-interacting protein encoded by YMR144W also resulted in a change in Fkh1-FHA-dependent donor preference. We have named this gene FDO1 for Forkhead one interacting protein involved in donor preference. We conclude that a phosphothreonine-mediated protein-protein interface between Fkh1-FHA and Mph1 contributes

  9. Transcriptomic and Proteomic Research To Explore Bruchid-Resistant Genes in Mungbean Isogenic Lines.

    PubMed

    Lin, Wu-Jui; Ko, Chia-Yun; Liu, Mao-Sen; Kuo, Chien-Yen; Wu, Dung-Chi; Chen, Chien-Yu; Schafleitner, Roland; Chen, Long-Fang O; Lo, Hsiao-Feng

    2016-08-31

    Mungbean (Vigna radiata (L.) Wilczek) is an important rotation legume crop for human nutrition in Asia. Bruchids (Callosobruchus spp.) currently cause heavy damage as pests of grain legumes during storage. We used omics-related technologies to study the mechanisms of bruchid resistance in seeds of the nearly isogenic lines VC1973A (bruchid-susceptible) and VC6089A (bruchid-resistant). A total of 399 differentially expressed genes (DEGs) were identified between the two lines by transcriptome sequencing. Among these DEGs, 251 exhibited high expression levels and 148 expressed low expression levels in seeds of VC6089A. Forty-five differential proteins (DPs) were identified by isobaric tags for relative and absolute quantification (iTRAQ); 21 DPs had higher abundances in VC6089A, and 24 DPs had higher abundances in VC1973A. According to transcriptome and proteome data, only three DEGs/DPs, including resistant-specific protein (g39185), gag/pol polyprotein (g34458), and aspartic proteinase (g5551), were identified and located on chromosomes 5, 1, and 7, respectively. Both g39185 and g34458 genes encode a protein containing a BURP domain. In previous research on bruchid molecular markers, the g39185 gene located close to the molecular markers of major bruchid-resistant locus may be a bruchid-resistant gene. PMID:27508985

  10. Gene expression patterns in near isogenic lines for wheat rust resistance gene lr34/yr18.

    PubMed

    Hulbert, S H; Bai, J; Fellers, J P; Pacheco, M G; Bowden, R L

    2007-09-01

    ABSTRACT The Lr34/Yr18 resistance gene provides durable, adult-plant, slow rusting resistance to leaf rust, yellow rust, and several other diseases of wheat. Flag leaves may exhibit spontaneous leaf tip necrosis and tips are more resistant than leaf bases. Despite the importance of this gene, the mechanism of resistance is unknown. Patterns of expression for 55,052 transcripts were examined by microarray analysis in mock-inoculated flag leaves of two pairs of wheat near isogenic lines for Lr34/Yr18 (Jupateco 73S/Jupateco 73R and Thatcher/Thatcher-Lr34). The Thatcher isolines were also examined for patterns of expression after inoculation with leaf rust. Mock-inoculated leaf tips of resistant plants showed up-regulation of 57 transcripts generally associated with ABA inducibility, osmotic stress, cold stress, and/or seed maturation. Several transcripts may be useful as expression markers for Lr34/Yr18. Five transcripts were also up-regulated in resistant leaf bases. The possible role of these transcripts in resistance is discussed. In mock-inoculated plants, pathogenesis-related (PR) proteins were not up-regulated in resistant flag leaves compared with that in susceptible flag leaves. In inoculated plants, the same set of PR proteins was up-regulated in both resistant and susceptible flag leaves. However, expression was often higher in resistant plants, suggesting a possible role for Lr34/Yr18 in priming of defense responses. PMID:18944173

  11. Individuality and Transgenerational Inheritance of Social Dominance and Sex Pheromones in Isogenic Male Mice.

    PubMed

    Fang, Qi; Zhang, Yao-Hua; Shi, Yao-Long; Zhang, Jin-Hua; Zhang, Jian-Xu

    2016-06-01

    Phenotypic variation and its epigenetic regulations within the inbred isogenic mice have long intrigued biologists. Here, we used inbred C57BL/6 mice to examine the individual differences and the inheritance of social dominance and male pheromones, expecting to create a model for studying the underlying epigenetic mechanisms for the evolution of these traits. We used a repeated male-male contest paradigm to form stable dominance-submission relationships between paired males and make superior or inferior quality manifest. Females showed olfactory preferences for the urine of dominant males to that of subordinate opponents. Gas chromatography-mass spectrometer analysis revealed that dominance-related or superior quality related pheromones were actually exaggerated male pheromone components (e.g., E-β-farnesene, hexadecanol, and 1-hexadecanol acetate) of preputial gland origin. Although the socially naïve sons of both dominant and subordinate males elicited the same female attraction when reaching adulthood, the former could dominated over the latter during undergoing the male-male competition and then gained more attraction of females. Our results demonstrated that social dominance or superior quality and the related pheromones were heritable and could be expressed through the interaction between aggression-related epigenotypes and male-male contests. It suggested that the evolution of sexually selected traits could be epigenetically determined and promoted through female mate choice. The epigenetic mechanisms driving the individual differences in behavior and male pheromones deserve further studies. PMID:27283352

  12. Targeted isolation of cloned genomic regions by recombineering for haplotype phasing and isogenic targeting.

    PubMed

    Nedelkova, Marta; Maresca, Marcello; Fu, Jun; Rostovskaya, Maria; Chenna, Ramu; Thiede, Christian; Anastassiadis, Konstantinos; Sarov, Mihail; Stewart, A Francis

    2011-11-01

    Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (∼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery. PMID:21852329

  13. Targeted isolation of cloned genomic regions by recombineering for haplotype phasing and isogenic targeting

    PubMed Central

    Nedelkova, Marta; Maresca, Marcello; Fu, Jun; Rostovskaya, Maria; Chenna, Ramu; Thiede, Christian; Anastassiadis, Konstantinos; Sarov, Mihail; Stewart, A. Francis

    2011-01-01

    Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (∼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery. PMID:21852329

  14. Comparative Proteomics of Leaves from Phytase-Transgenic Maize and Its Non-transgenic Isogenic Variety.

    PubMed

    Tan, Yanhua; Yi, Xiaoping; Wang, Limin; Peng, Cunzhi; Sun, Yong; Wang, Dan; Zhang, Jiaming; Guo, Anping; Wang, Xuchu

    2016-01-01

    To investigate unintended effects in genetically modified crops (GMCs), a comparative proteomic analysis between the leaves of the phytase-transgenic maize and the non-transgenic plants was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 57 differentially expressed proteins (DEPs) were successfully identified, which represents 44 unique proteins. Functional classification of the identified proteins showed that these DEPs were predominantly involved in carbohydrate transport and metabolism category, followed by post-translational modification. KEGG pathway analysis revealed that most of the DEPs participated in carbon fixation in photosynthesis. Among them, 15 proteins were found to show protein-protein interactions with each other, and these proteins were mainly participated in glycolysis and carbon fixation. Comparison of the changes in the protein and tanscript levels of the identified proteins showed that most proteins had a similar pattern of changes between proteins and transcripts. Our results suggested that although some significant differences were observed, the proteomic patterns were not substantially different between the leaves of the phytase-transgenic maize and the non-transgenic isogenic type. Moreover, none of the DEPs was identified as a new toxic protein or an allergenic protein. The differences between the leaf proteome might be attributed to both genetic modification and hybrid influence. PMID:27582747

  15. Two transaldolase isogenes from Moniliella megachiliensis behave in a different way depending on the stress class.

    PubMed

    Iwata, Hisashi; Mizushima, Daiki; Kobayashi, Yosuke; Ookura, Tetsuya; Ogihara, Jun; Kato, Jun; Kasumi, Takafumi

    2015-02-01

    We cloned and sequenced two transaldolase genes from Moniliella megachiliensis, a microorganism known to produce a significant amount of erythritol under hyper-osmotic stress. The amino acid sequences encoded by these two genes (MmTAL1, MmTAL2) showed 72% homology to each other. An AP-1 (ap response element) associated with oxidative stress was found in the promoter region of MmTAL1, while four STREs (stress response element) associated with osmotic stress were found in the promoter region of MmTAL2. In early-stage cultivation (up to 2 h), MmTAL1 was specifically expressed in response to oxidative stress generated by the presence of 0.15 mM menadione; expression level 3-fold higher than before stress loading. MmTAL2 was expressed in response to osmotic stress caused by 1.2 M NaCl; expression level was 21-fold higher than stress-free control. Erythritol accumulated intracellularly under osmotic and oxidative stress, approximately 30-fold and 35-fold, respectively. We therefore concluded that M. megachiliensis selectively uses two isogenes and produces erythritol during early-stage response to stress, depending on the type of environmental stress. PMID:25127884

  16. Proteomic Analysis of Isogenic Rice Reveals Proteins Correlated with Aroma Compound Biosynthesis at Different Developmental Stages.

    PubMed

    Wongpia, Aphinya; Roytrakul, Sittiruk; Nomura, Mika; Tajima, Shigeyuki; Lomthaisong, Khemika; Mahatheeranont, Sugunya; Niamsup, Hataichanoke

    2016-02-01

    Fragrant rice has a potent flavor compound, 2-acetyl-1-pyrroline (2AP). A better understanding of the 2AP biosynthetic pathway is gained by proteomic analysis of two isogenic lines of Thai jasmine rice, Oryza sativa L. cv. Khao Dawk Mali 105, which differ only in the aromatic gene Os2AP. The protein profiles of two lines, from six growth stages, seedling to grain filling, had 41 identifiable protein spots. Four of these spots were betaine aldehyde dehydrogenase, a key enzyme responsible for 2AP production. This enzyme occurred in every growth stage of the non-aromatic rice line except smaller amount detected in the hard grain-filling stage of the aromatic line. Glyceraldehyde 3-phosphate dehydrogenase and aspartate aminotransferase, observed in the aromatic line, may involve in the metabolism of precursors for 2AP biosynthesis. In addition, glutamine synthetase and 1-cys peroxiredoxin A which function in ammonia reassimilation and hydrogen peroxide detoxification were unique in the aromatic line. However, proteins that correspond to photosynthesis and the nutrient reservoir were only detected in lower abundances. This possibly explains why the aroma rice grain weight is low. Our study proposed the possible role of these remarkable proteins which involved in 2AP biosynthesis in jasmine rice. PMID:26646386

  17. Transcriptome analysis of near-isogenic lines provides molecular insights into starch biosynthesis in maize kernel.

    PubMed

    Xiao, Yingni; Thatcher, Shawn; Wang, Min; Wang, Tingting; Beatty, Mary; Zastrow-Hayes, Gina; Li, Lin; Li, Jiansheng; Li, Bailin; Yang, Xiaohong

    2016-08-01

    Starch is the major component in maize kernels, providing a stable carbohydrate source for humans and livestock as well as raw material for the biofuel industry. Increasing maize kernel starch content will help meet industry demands and has the potential to increase overall yields. We developed a pair of maize near-isogenic lines (NILs) with different alleles for a starch quantitative trait locus on chromosome 3 (qHS3), resulting in different kernel starch content. To investigate the candidate genes for qHS3 and elucidate their effects on starch metabolism, RNA-Seq was performed for the developing kernels of the NILs at 14 and 21 d after pollination (DAP). Analysis of genomic and transcriptomic data identified 76 genes with nonsynonymous single nucleotide polymorphisms and 384 differentially expressed genes (DEGs) in the introgressed fragment, including a hexokinase gene, ZmHXK3a, which catalyzes the conversion of glucose to glucose-6-phosphate and may play a key role in starch metabolism. The expression pattern of all DEGs in starch metabolism shows that altered expression of the candidate genes for qHS3 promoted starch synthesis, with positive consequences for kernel starch content. These results expand the current understanding of starch biosynthesis and accumulation in maize kernels and provide potential candidate genes to increase starch content. PMID:26676690

  18. Inheritance of Propoxur Resistance in a Near-Isogenic Line of Musca domestica (Diptera: Muscidae).

    PubMed

    Shan, Chao; Zhang, Yi; Ma, Zhuo; Gao, Xiwu

    2016-04-01

    Propoxur, a carbamate insecticide, has been used worldwide for the control of house flies (Musca domestica L.) for many decades. Resistance levels to propoxur have been detected in field populations of house flies in many parts of the world, including China. In this study, a near-isogenic house fly line (N-PRR) resistant to propoxur was used to determine the mode of inheritance. Bioassay results showed no significant differences in LD50 values or in the slope of log dose-probit lines between the reciprocal F1 and F1’ progenies, and the degree of dominance (D) was more than −1 and less than 0. Chi-square analysis of the responses of self-bred (F2, F2’) and backcross progenies (BC1, BC2, BC1’, and BC2’) indicated that a single gene was responsible for resistance. Propoxur resistance in the N-PRR strain of house fly was inherited as a single, major, autosomal, and incompletely recessive factor. These results should be useful to reveal the mode of inheritance and the development trend of propoxur resistance and develop a systematic strategy for the resistance management in house flies. PMID:26921225

  19. Comparative Proteomics of Leaves from Phytase-Transgenic Maize and Its Non-transgenic Isogenic Variety

    PubMed Central

    Tan, Yanhua; Yi, Xiaoping; Wang, Limin; Peng, Cunzhi; Sun, Yong; Wang, Dan; Zhang, Jiaming; Guo, Anping; Wang, Xuchu

    2016-01-01

    To investigate unintended effects in genetically modified crops (GMCs), a comparative proteomic analysis between the leaves of the phytase-transgenic maize and the non-transgenic plants was performed using two-dimensional gel electrophoresis and mass spectrometry. A total of 57 differentially expressed proteins (DEPs) were successfully identified, which represents 44 unique proteins. Functional classification of the identified proteins showed that these DEPs were predominantly involved in carbohydrate transport and metabolism category, followed by post-translational modification. KEGG pathway analysis revealed that most of the DEPs participated in carbon fixation in photosynthesis. Among them, 15 proteins were found to show protein-protein interactions with each other, and these proteins were mainly participated in glycolysis and carbon fixation. Comparison of the changes in the protein and tanscript levels of the identified proteins showed that most proteins had a similar pattern of changes between proteins and transcripts. Our results suggested that although some significant differences were observed, the proteomic patterns were not substantially different between the leaves of the phytase-transgenic maize and the non-transgenic isogenic type. Moreover, none of the DEPs was identified as a new toxic protein or an allergenic protein. The differences between the leaf proteome might be attributed to both genetic modification and hybrid influence. PMID:27582747

  20. Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains

    PubMed Central

    Lengronne, Armelle; Pasero, Philippe; Bensimon, Aaron; Schwob, Etienne

    2001-01-01

    Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK+ yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8–9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 ‘early’ origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants. PMID:11266543

  1. Analysis of chromosomal integration and deletions of yeast plasmids.

    PubMed Central

    Cameron, J R; Philippsen, P; Davis, R W

    1977-01-01

    Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA. Images PMID:331256

  2. Induction of transpositions of MGE Dm412 by {gamma}-radiation in an isogenic line of Drosophila melanogaster

    SciTech Connect

    Zabanov, S.A.; Vasil`eva, L.A.; Ratner, V.A. |

    1995-06-01

    In an isogenic line of Drosophila, transpositions of mobile genetic elements (MGE) Dm412 were induced by {gamma}-radiation at doses of 300, 800, and 1300 R. The rates of induced transpositions were (for each dose, respectively) 3.9 x 10{sup {minus}3}, 1.0 x 10{sup {minus}2}, and 1.87 x 10{sup {minus}2} events per occupied site per haploid genome of the isogenic line per generation. Thus, the transposition rate increased linearly with the radiation dose. The specific rate of {gamma}-radiation-induced transpositions was (1.3 {+-} 0.6) x 10{sup {minus}5} per occupied site per haploid genome of the isogenic line per Roentgen per generation. {gamma}-Radiation-induced hot transposition sites and haplotypes, very similar to those induced by heat shock, were found. It was suggested that the mechanism of induction by {gamma}-radiation involves the heat shock system. Thus, it is more similar to the mechanism of temperature induction than to the direct mutational effect of {gamma}-radiation. Estimates of induced transposition rates per genome for each dose were calculated as 1.1, 3.0, and 5.6 events, respectively, per genome per generation. This level probably corresponds to the subthreshold level of genomes near the {open_quotes}catastrophic border of transpositional losses.{close_quotes} 21 refs., 1 fig., 4 tabs.

  3. Effects of xenogeneic, allogeneic and isogeneic thymus grafts on lymphocyte populations in peripheral lymphoid organs of the nude rat.

    PubMed

    Hougen, H P; Klausen, B; Stenvang, J P; Kraemmer, J; Rygaard, J

    1987-04-01

    In order to gain information about the effect of xenografted, allografted and isografted thymic tissue on peripheral lymphoid organs of immune-deficient rats, athymic nude LEW rats of ninth backcross-intercross were grafted with fetal calf and neonatal BDIX and LEW thymus. Adrenalectomy was also performed in some animals in order to obtain a possible enhancement of the immunological reconstitution. Both groups of isogeneic-thymus-grafted animals had more T helper cells than the nude controls. Furthermore, they had more densely populated paracortical areas in the inguinal lymph nodes and higher lymphocyte counts in the thoracic duct lymph. Finally, the inguinal lymph nodes contained germinal centres. Xenogeneic and allogeneic thymus transplants did not induce constant changes in the parameters observed compared with the untreated nudes. No clear difference was observed between the adrenalectomized and non-adrenalectomized thymic-isografted animals. We therefore conclude that of all the experimental animals examined the isografted nude rats show by far the best response and that adrenalectomy seems unnecessary for the success of neonatal isogeneic thymus grafts. We also conclude that the isogeneic-thymus-grafted nude rat is a suitable tool for immunological reconstitution studies. PMID:3496487

  4. Recombinant DNA means and method

    SciTech Connect

    Alford, B.L.; Mao, J.I.; Moir, D.T.; Taunton-Rigby, A.; Vovis, G.F.

    1987-05-19

    This patent describes a transformed living cell selected from the group consisting of fungi, yeast and bacteria, and containing genetic material derived from recombinant DNA material and coding for bovine rennin.

  5. Fitness of Isogenic Colony Morphology Variants of Pseudomonas aeruginosa in Murine Airway Infection

    PubMed Central

    Rakhimova, Elza; Munder, Antje; Wiehlmann, Lutz; Bredenbruch, Florian; Tümmler, Burkhard

    2008-01-01

    Chronic lung infections with Pseudomonas aeruginosa are associated with the diversification of the persisting clone into niche specialists and morphotypes, a phenomenon called ‘dissociative behaviour’. To explore the potential of P. aeruginosa to change its morphotype by single step loss-of–function mutagenesis, a signature-tagged mini-Tn5 plasposon library of the cystic fibrosis airway isolate TBCF10839 was screened for colony morphology variants under nine different conditions in vitro. Transposon insertion into 1% of the genome changed colony morphology into eight discernable morphotypes. Half of the 55 targets encode features of primary or secondary metabolism whereby quinolone production was frequently affected. In the other half the transposon had inserted into genes of the functional categories transport, regulation or motility/chemotaxis. To mimic dissociative behaviour of isogenic strains in lungs, pools of 25 colony morphology variants were tested for competitive fitness in an acute murine airway infection model. Six of the 55 mutants either grew better or worse in vivo than in vitro, respectively. Metabolic proficiency of the colony morphology variant was a key determinant for survival in murine airways. The most common morphotype of self-destructive autolysis did unexpectedly not impair fitness. Transposon insertions into homologous genes of strain PAO1 did not reproduce the TBCF10839 mutant morphotypes for 16 of 19 examined loci pointing to an important role of the genetic background on colony morphology. Depending on the chosen P. aeruginosa strain, functional genome scans will explore other areas of the evolutionary landscape. Based on our discordant findings of mutant phenotypes in P. aeruginosa strains PAO1, PA14 and TBCF10839, we conclude that the current focus on few reference strains may miss modes of niche adaptation and dissociative behaviour that are relevant for the microevolution of complex traits in the wild. PMID:18301762

  6. Nitrile Metabolizing Yeasts

    NASA Astrophysics Data System (ADS)

    Bhalla, Tek Chand; Sharma, Monica; Sharma, Nitya Nand

    Nitriles and amides are widely distributed in the biotic and abiotic components of our ecosystem. Nitrile form an important group of organic compounds which find their applications in the synthesis of a large number of compounds used as/in pharmaceutical, cosmetics, plastics, dyes, etc>. Nitriles are mainly hydro-lyzed to corresponding amide/acid in organic chemistry. Industrial and agricultural activities have also lead to release of nitriles and amides into the environment and some of them pose threat to human health. Biocatalysis and biotransformations are increasingly replacing chemical routes of synthesis in organic chemistry as a part of ‘green chemistry’. Nitrile metabolizing organisms or enzymes thus has assumed greater significance in all these years to convert nitriles to amides/ acids. The nitrile metabolizing enzymes are widely present in bacteria, fungi and yeasts. Yeasts metabolize nitriles through nitrilase and/or nitrile hydratase and amidase enzymes. Only few yeasts have been reported to possess aldoxime dehydratase. More than sixty nitrile metabolizing yeast strains have been hither to isolated from cyanide treatment bioreactor, fermented foods and soil. Most of the yeasts contain nitrile hydratase-amidase system for metabolizing nitriles. Transformations of nitriles to amides/acids have been carried out with free and immobilized yeast cells. The nitrilases of Torulopsis candida>and Exophiala oligosperma>R1 are enantioselec-tive and regiospecific respectively. Geotrichum>sp. JR1 grows in the presence of 2M acetonitrile and may have potential for application in bioremediation of nitrile contaminated soil/water. The nitrilase of E. oligosperma>R1 being active at low pH (3-6) has shown promise for the hydroxy acids. Immobilized yeast cells hydrolyze some additional nitriles in comparison to free cells. It is expected that more focus in future will be on purification, characterization, cloning, expression and immobilization of nitrile metabolizing

  7. Forces in yeast flocculation

    PubMed Central

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

    2014-01-01

    In the baker's yeast Saccharomyces cerevisiae, cell-cell adhesion (“flocculation”) is conferred by a family of lectin-like proteins known as the flocculin (Flo) proteins. Knowledge of the adhesive and mechanical properties of flocculins is important for understanding the mechanisms of yeast adhesion, and may help controlling yeast behaviour in biotechnology. We use single-molecule and single-cell atomic force microscopy (AFM) to explore the nanoscale forces engaged in yeast flocculation, focusing on the role of Flo1 as a prototype of flocculins. Using AFM tips labelled with mannose, we detect single flocculins on Flo1-expressing cells, showing they are widely exposed on the cell surface. When subjected to force, individual Flo1 proteins display two distinct force responses, i.e. weak lectin binding forces and strong unfolding forces reflecting the force-induced extension of hydrophobic tandem repeats. We demonstrate that cell-cell adhesion bonds also involve multiple weak lectin interactions together with strong unfolding forces, both associated with Flo1 molecules. Single-molecule and single-cell data correlate with microscale cell adhesion behaviour, suggesting strongly that Flo1 mechanics is critical for yeast flocculation. These results favour a model in which not only weak lectin-sugar interactions are involved in yeast flocculation but also strong hydrophobic interactions resulting from protein unfolding. PMID:25515338

  8. Forces in yeast flocculation

    NASA Astrophysics Data System (ADS)

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

    2015-01-01

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

  9. Altered Transcriptional Control Networks with Trans-Differentiation of Isogenic Mutant-KRas NSCLC Models

    PubMed Central

    Haley, John A.; Haughney, Elizabeth; Ullman, Erica; Bean, James; Haley, John D.; Fink, Marc Y.

    2014-01-01

    Background: The capacity of cancer cells to undergo epithelial mesenchymal trans-differentiation has been implicated as a factor driving metastasis, through the acquisition of enhanced migratory/invasive cell programs and the engagement of anti-apoptotic mechanisms promoting drug and radiation resistance. Our aim was to define molecular signaling changes associated with mesenchymal trans-differentiation in two KRas mutant NSCLC models. We focused on central transcription and epigenetic regulators predicted to be important for mesenchymal cell survival. Experimental design: We have modeled trans-differentiation and cancer stemness in inducible isogenic mutant-KRas H358 and A549 non-small cell lung cell backgrounds. As expected, our models show mesenchymal-like tumor cells acquire novel mechanisms of cellular signaling not apparent in their epithelial counterparts. We employed large-scale quantitative phosphoproteomic, proteomic, protein–protein interaction, RNA-Seq, and network function prediction approaches to dissect the molecular events associated with the establishment and maintenance of the mesenchymal state. Results: Gene-set enrichment and pathway prediction indicated BMI1, KDM5B, RUNX2, MYC/MAX, NFκB, LEF1, and HIF1 target networks were significantly enriched in the trans-differentiation of H358 and A549 NSCLC models. Physical overlaps between multiple networks implicate NR4A1 as an overlapping control between TCF and NFκB pathways. Enrichment correlations also indicated marked decrease in cell cycling, which occurred early in the EMT process. RNA abundance time course studies also indicated early expression of epigenetic and chromatin regulators within 8–24 h, including CITED4, RUNX3, CMBX1, and SIRT4. Conclusion: Multiple transcription and epigenetic pathways where altered between epithelial and mesenchymal tumor cell states, notably the polycomb repressive complex-1, HP1γ, and BAF/Swi-Snf. Network analysis suggests redundancy in the activation

  10. Cloning of the cDNA of the heme-regulated eukaryotic initiation factor 2. alpha. (eIF-2. alpha. ) kinase of rabbit reticulocytes: Homology to yeast GCN2 protein kinase and human double-stranded-RNA-dependent eIF-2. alpha. kinase

    SciTech Connect

    Chen, J.J.; Throop, M.S.; Kuo, I.; Pal, J.K.; Brodsky, M.; London, I.M. ); Gehrke, L. Harvard Medical School, Boston, MA )

    1991-09-01

    The authors have cloned the cDNA of the heme-regulated eIF-2{alpha} kinase (HRI) of rabbit reticulocytes. In vitro translation of mRNA transcribed from the HRI cDNA yields a 90-kDa polypeptide that exhibits eIF-2{alpha} kinase activity and is recognized by a monoclonal antibody directed against authentic HRI. The open reading frame sequence of the HRI cDNA contains all 11 catalytic domains of protein kinases with consensus sequences of protein-serine/threonine kinases in conserved catalytic domains VI and VIII. The HRI cDNA also contains an insert of {approx} 140 amino acids between catalytic domains V and VI. The HRI cDNA coding sequence has extensive homology to GCN2 protein kinase of Saccharomyces cerevisiae and to human double-stranded-RNA-dependent eIF-2{alpha} kinase. This observation suggests that GCN2 protein kinase may be an eIF-2{alpha} kinase in yeast. In addition, HRI has an unusually high degree of homology to three protein kinases (NimA, Wee1, and CDC2) that are involved in the regulation of the cell cycle.

  11. Isolation of MECP2-null Rett Syndrome patient hiPS cells and isogenic controls through X-chromosome inactivation

    PubMed Central

    Cheung, Aaron Y.L.; Horvath, Lindsay M.; Grafodatskaya, Daria; Pasceri, Peter; Weksberg, Rosanna; Hotta, Akitsu; Carrel, Laura; Ellis, James

    2011-01-01

    Rett syndrome (RTT) is a neurodevelopmental autism spectrum disorder that affects girls due primarily to mutations in the gene encoding methyl-CpG binding protein 2 (MECP2). The majority of RTT patients carry missense and nonsense mutations leading to a hypomorphic MECP2, while null mutations leading to the complete absence of a functional protein are rare. MECP2 is an X-linked gene subject to random X-chromosome inactivation resulting in mosaic expression of mutant MECP2. The lack of human brain tissue motivates the need for alternative human cellular models to study RTT. Here we report the characterization of a MECP2 mutation in a classic female RTT patient involving rearrangements that remove exons 3 and 4 creating a functionally null mutation. To generate human neuron models of RTT, we isolated human induced pluripotent stem (hiPS) cells from RTT patient fibroblasts. RTT-hiPS cells retained the MECP2 mutation, are pluripotent and fully reprogrammed, and retained an inactive X-chromosome in a nonrandom pattern. Taking advantage of the latter characteristic, we obtained a pair of isogenic wild-type and mutant MECP2 expressing RTT-hiPS cell lines that retained this MECP2 expression pattern upon differentiation into neurons. Phenotypic analysis of mutant RTT-hiPS cell-derived neurons demonstrated a reduction in soma size compared with the isogenic control RTT-hiPS cell-derived neurons from the same RTT patient. Analysis of isogenic control and mutant hiPS cell-derived neurons represents a promising source for understanding the pathogenesis of RTT and the role of MECP2 in human neurons. PMID:21372149

  12. Genomic and Phenotypic Characterization of a Wild Medaka Population: Towards the Establishment of an Isogenic Population Genetic Resource in Fish

    PubMed Central

    Spivakov, Mikhail; Auer, Thomas O.; Peravali, Ravindra; Dunham, Ian; Dolle, Dirk; Fujiyama, Asao; Toyoda, Atsushi; Aizu, Tomoyuki; Minakuchi, Yohei; Loosli, Felix; Naruse, Kiyoshi; Birney, Ewan; Wittbrodt, Joachim

    2014-01-01

    Oryzias latipes (medaka) has been established as a vertebrate genetic model for more than a century and recently has been rediscovered outside its native Japan. The power of new sequencing methods now makes it possible to reinvigorate medaka genetics, in particular by establishing a near-isogenic panel derived from a single wild population. Here we characterize the genomes of wild medaka catches obtained from a single Southern Japanese population in Kiyosu as a precursor for the establishment of a near-isogenic panel of wild lines. The population is free of significant detrimental population structure and has advantageous linkage disequilibrium properties suitable for the establishment of the proposed panel. Analysis of morphometric traits in five representative inbred strains suggests phenotypic mapping will be feasible in the panel. In addition, high-throughput genome sequencing of these medaka strains confirms their evolutionary relationships on lines of geographic separation and provides further evidence that there has been little significant interbreeding between the Southern and Northern medaka population since the Southern/Northern population split. The sequence data suggest that the Southern Japanese medaka existed as a larger older population that went through a relatively recent bottleneck approximately 10,000 years ago. In addition, we detect patterns of recent positive selection in the Southern population. These data indicate that the genetic structure of the Kiyosu medaka samples is suitable for the establishment of a vertebrate near-isogenic panel and therefore inbreeding of 200 lines based on this population has commenced. Progress of this project can be tracked at http://www.ebi.ac.uk/birney-srv/medaka-ref-panel. PMID:24408034

  13. Mapping Yeast Transcriptional Networks

    PubMed Central

    Hughes, Timothy R.; de Boer, Carl G.

    2013-01-01

    The term “transcriptional network” refers to the mechanism(s) that underlies coordinated expression of genes, typically involving transcription factors (TFs) binding to the promoters of multiple genes, and individual genes controlled by multiple TFs. A multitude of studies in the last two decades have aimed to map and characterize transcriptional networks in the yeast Saccharomyces cerevisiae. We review the methodologies and accomplishments of these studies, as well as challenges we now face. For most yeast TFs, data have been collected on their sequence preferences, in vivo promoter occupancy, and gene expression profiles in deletion mutants. These systematic studies have led to the identification of new regulators of numerous cellular functions and shed light on the overall organization of yeast gene regulation. However, many yeast TFs appear to be inactive under standard laboratory growth conditions, and many of the available data were collected using techniques that have since been improved. Perhaps as a consequence, comprehensive and accurate mapping among TF sequence preferences, promoter binding, and gene expression remains an open challenge. We propose that the time is ripe for renewed systematic efforts toward a complete mapping of yeast transcriptional regulatory mechanisms. PMID:24018767

  14. Yeasts in spa establishments.

    PubMed

    Svorcová, L

    1982-05-01

    It was investigated occurrence of yeasts on bathsurfaces, in sauna rooms, in swimming and therapeutic pool water. The number of yeasts decreased depending on patients age, if the rooms were furnished with bath. The lowest contamination was found after bath of 40-60 years-old women. In the saunas were yeasts not found on the upper benches with temperature above 55 degrees C. Much higher counts on lower benches and wood mats with temperature 35-40 degrees C, on basin walls and bottom-up to 10(4)-10(6)/100 cm2. It was isolated 172 yeast strains. The occurrence of some selected strains is given in Table 7, with the toxic effect of disinfectants. The most strains were resistant to Peracetic acid and Chloramin B. Since most of the isolated and determinated strains were found in contaminated environment or during various diseases, the yeasts of the genus Cryptococcus, Candida, Rhodotorula, Torulopsis and Metschnikowia should not occur in bath establishment, and should be classified among indicators of contamination of environment including water. PMID:7124167

  15. Oxygen requirements of yeasts.

    PubMed Central

    Visser, W; Scheffers, W A; Batenburg-van der Vegte, W H; van Dijken, J P

    1990-01-01

    Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth. Images PMID:2082825

  16. Breeding research on sake yeasts in Japan: history, recent technological advances, and future perspectives.

    PubMed

    Kitagaki, Hiroshi; Kitamoto, Katsuhiko

    2013-01-01

    Sake is an alcoholic beverage of Japan, with a tradition lasting more than 1,300 years; it is produced from rice and water by fermenting with the koji mold Aspergillus oryzae and sake yeast Saccharomyces cerevisiae. Breeding research on sake yeasts was originally developed in Japan by incorporating microbiological and genetic research methodologies adopted in other scientific areas. Since the advent of a genetic paradigm, isolation of yeast mutants has been a dominant approach for the breeding of favorable sake yeasts. These sake yeasts include (a) those that do not form foams (produced by isolating a mutant that does not stick to foams, thus decreasing the cost of sake production); (b) those that do not produce urea, which leads to the formation of ethyl carbamate, a possible carcinogen (isolated by positive selection in a canavanine-, arginine-, and ornithine-containing medium); (c) those that produce an increased amount of ethyl caproate, an apple-like flavor (produced by isolating a mutant resistant to cerulenin, an inhibitor of fatty-acid synthesis); and (d) those that produce a decreased amount of pyruvate (produced by isolating a mutant resistant to an inhibitor of mitochondrial transport, thus decreasing the amount of diacetyl). Given that sake yeasts perform sexual reproduction, sporulation and mating are potent approaches for their breeding. Recently, the genome sequences of sake yeasts have been determined and made publicly accessible. By utilizing this information, the quantitative trait loci (QTLs) for the brewing characteristics of sake yeasts have been identified, which paves a way to DNA marker-assisted selection of the mated strains. Genetic engineering technologies for experimental yeast strains have recently been established by academic groups, and these technologies have also been applied to the breeding of sake yeasts. Sake yeasts whose genomes have been modified with these technologies correspond to genetically modified organisms (GMOs

  17. Design and construction of two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast.

    PubMed

    Abdemami, Baharak; Shokrgozar, Mohammad Ali; Shahreza, Hossein Khanahmad; Ghavami, Mehdi

    2011-01-01

    Collagens are the most abundant proteins in the human body. Their main function is to provide structural and mechanical support for the tissues, but they are also involved in a number of other biological functions including cell attachment, migration and differentiation. Collagens and gelatins are widely used in pharmaceutical and medical applications. Every year, more than 50,000 tons of collagen and gelatin are used in medical applications. These materials may have some viral and prion impurity and/or stimulate allergic response in human body. Therefore, scientists have produced human collagen in recombinant systems. In this study we have constructed two yeast shuttle vectors containing human procollagen genes expression cassette for expression in yeast. Total RNA was extracted from human skin fibroblast cell line, and cDNA synthesis was done by oligo dt. Then gene fragments were amplified from the cDNA with the necessary changes by Polymerase Chain Reaction (PCR). Finally they were cloned in yeast vector pPICZαA containing regulatory sequences for expressing and secreting the polypeptide product. Two yeast shuttle vectors containing human COL1A1 and COL1A2 expression cassettes were created. Final constructs were confirmed by enzymatic digestion, PCR of desired fragment and sequencing. The yeast shuttle vectors containing human COL1A1 and COL1A2 can be transferred into the yeast in the later stages to determine the scale of expression. PMID:23407617

  18. Global Analysis of Transcription Factor-Binding Sites in Yeast Using ChIP-Seq

    PubMed Central

    Lefrançois, Philippe; Gallagher, Jennifer E. G.; Snyder, Michael

    2016-01-01

    Transcription factors influence gene expression through their ability to bind DNA at specific regulatory elements. Specific DNA-protein interactions can be isolated through the chromatin immunoprecipitation (ChIP) procedure, in which DNA fragments bound by the protein of interest are recovered. ChIP is followed by high-throughput DNA sequencing (Seq) to determine the genomic provenance of ChIP DNA fragments and their relative abundance in the sample. This chapter describes a ChIP-Seq strategy adapted for budding yeast to enable the genome-wide characterization of binding sites of transcription factors (TFs) and other DNA-binding proteins in an efficient and cost-effective way. Yeast strains with epitope-tagged TFs are most commonly used for ChIP-Seq, along with their matching untagged control strains. The initial step of ChIP involves the cross-linking of DNA and proteins. Next, yeast cells are lysed and sonicated to shear chromatin into smaller fragments. An antibody against an epitope-tagged TF is used to pull down chromatin complexes containing DNA and the TF of interest. DNA is then purified and proteins degraded. Specific barcoded adapters for multiplex DNA sequencing are ligated to ChIP DNA. Short DNA sequence reads (28–36 base pairs) are parsed according to the barcode and aligned against the yeast reference genome, thus generating a nucleotide-resolution map of transcription factor-binding sites and their occupancy. PMID:25213249

  19. [Overexpression of FKS1 to improve yeast autolysis-stress].

    PubMed

    Li, Jia; Wang, Jinjing; Li, Qi

    2015-09-01

    With the development of high gravity brewing, yeast cells are exposed to multiple brewing-associated stresses, such as increased osmotic pressure, enhanced alcohol concentration and nutritional imbalance. These will speed up yeast autolysis, which seriously influence beer flavor and quality. To increase yeast anti-autolytic ability, FKS1 overexpression strain was constructed by 18S rDNA. The concentration of β-1,3-glucan of overexpression strain was 62% higher than that of wild type strain. Meantime, FKS1 overexpression strain increased anti-stress ability at 8% ethanol, 0.4 mol/L NaCl and starvation stress. Under simulated autolysis, FKS1 showed good anti-autolytic ability by slower autolysis. These results confirms the potential of FKS1 overexpression to tackle yeast autolysis in high-gravity brewing. PMID:26955712

  20. Yeast killer systems.

    PubMed Central

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

    1997-01-01

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

  1. [Fructose transporter in yeasts].

    PubMed

    Lazar, Zbigniew; Dobrowolski, Adam; Robak, Małgorzata

    2014-01-01

    Study of hexoses transporter started with discovery of galactose permease in Saccharomyces cerevisiae. Glucose, fructose and mannose assimilation is assumed by numerous proteins encoded by different genes. To date over 20 hexoses transporters, belonging to Sugar Porter family and to Major Facilitator Superfamily, were known. Genome sequence analysis of Candida glabrata, Kluyveromyces lactis, Yarrowia lipolytica, S. cerevisaie and Debaryomyces hansenii reveled potential presence of 17-48 sugar porter proteins. Glucose transporters in S. cerevisiae have been already characterized. In this paper, hexoses transporters, responsible for assimilation of fructose by cells, are presented and compared. Fructose specific transporter are described for yeasts: Zygosaccharomyces rouxii, Zygosaccharomyces bailli, K. lactis, Saccharomyces pastorianus, S. cerevisiae winemaking strain and for fungus Botritys cinerea and human (Glut5p). Among six yeasts transporters, five are fructose specific, acting by facilitated diffusion or proton symport. Yeasts monosaccharides transporter studies allow understanding of sugars uptake and metabolism important aspects, even in higher eukaryotes cells. PMID:25033548

  2. Evolutionary history of Ascomyceteous Yeasts

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Yeasts are important for many industrial and biotechnological processes and show remarkable diversity despite morphological similarities. We have sequenced the genomes of 20 ascomyceteous yeasts of taxonomic and industrial importance including members of Saccharomycotina and Taphrinomycotina. A comp...

  3. Genetic profiling of yeast industrial strains using in situ comparative genomic hybridization (CGH).

    PubMed

    Wnuk, Maciej; Panek, Anita; Golec, Ewelina; Magda, Michal; Deregowska, Anna; Adamczyk, Jagoda; Lewinska, Anna

    2015-09-20

    The genetic differences and changes in genomic stability may affect fermentation processes involving baker's, brewer's and wine yeast strains. Thus, it seems worthwhile to monitor the changes in genomic DNA copy number of industrial strains. In the present study, we developed an in situ comparative genomic hybridization (CGH) to investigate the ploidy and genetic differences between selected industrial yeast strains. The CGH-based system was validated using the laboratory Saccharomyces cerevisiae yeast strains (haploid BY4741 and diploid BY4743). DNA isolated from BY4743 cells was considered a reference DNA. The ploidy and DNA gains and losses of baker's, brewer's and wine strains were revealed. Taken together, the in situ CGH was shown a helpful molecular tool to identify genomic differences between yeast industrial strains. Moreover, the in situ CGH-based system may be used at the single-cell level of analysis to supplement array-based techniques and high-throughput analyses at the population scale. PMID:26116136

  4. Genetics of Yeasts

    NASA Astrophysics Data System (ADS)

    Querol, Amparo; Fernández-Espinar, M. Teresa; Belloch, Carmela

    The use of yeasts in biotechnology processes dates back to ancient days. Before 7000 BC, beer was produced in Sumeria. Wine was made in Assyria in 3500 BC, and ancient Rome had over 250 bakeries, which were making leavened bread by 100 BC. And milk has been made into Kefyr and Koumiss in Asia for many centuries (Demain, Phaff, & Kurtzman, 1999). However, the importance of yeast in the food and beverage industries was only realized about 1860, when their role in food manufacturing became evident.

  5. L-arabinose fermenting yeast

    DOEpatents

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

    2010-12-07

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

  6. Profile and relative concentrations of fatty acids in corn and soybean seeds from transgenic and isogenic crops.

    PubMed

    Jiménez, J J; Bernal, J L; Nozal, M J; Toribio, L; Bernal, J

    2009-10-23

    In this work 44 fatty acids, which were analyzed as methyl esters by GC/MS in scan mode, have been determined in genetically modified corn and soybean seeds. Their relative concentrations have been compared with those of isogenic lines grown in the same conditions. Studied compounds comprised saturated and unsaturated fatty acids, including cis/trans isomers and minor fatty acids. A classical soxhlet extraction and an accelerated solvent extraction have been assayed to extract the fatty compounds from seeds and the GC separation has been carried out on a biscyanopropylpolysiloxane chromatographic column. Soxhlet extraction was selected as the most convenient and applied to compare the samples. Specific compounds, which could denote the origin of the crop have not been observed, but for some sample pairs, significant differences have been found in relation to the percentage of certain acids; the highest differences for major acids were 4.1% in corn and 4.8% in soybean. The concentrations of long chain acids such as 24:0, 26:0 and 28:0 were higher in some isogenic lines whereas the concentrations of short chain acids such as 6:0, 8:0, 9:0, 10:0 and 12:0 were higher in their transgenic counterparts. PMID:19716136

  7. An Isogenic Human ESC Platform for Functional Evaluation of Genome-wide-Association-Study-Identified Diabetes Genes and Drug Discovery.

    PubMed

    Zeng, Hui; Guo, Min; Zhou, Ting; Tan, Lei; Chong, Chi Nok; Zhang, Tuo; Dong, Xue; Xiang, Jenny Zhaoying; Yu, Albert S; Yue, Lixia; Qi, Qibin; Evans, Todd; Graumann, Johannes; Chen, Shuibing

    2016-09-01

    Genome-wide association studies (GWASs) have increased our knowledge of loci associated with a range of human diseases. However, applying such findings to elucidate pathophysiology and promote drug discovery remains challenging. Here, we created isogenic human ESCs (hESCs) with mutations in GWAS-identified susceptibility genes for type 2 diabetes. In pancreatic beta-like cells differentiated from these lines, we found that mutations in CDKAL1, KCNQ1, and KCNJ11 led to impaired glucose secretion in vitro and in vivo, coinciding with defective glucose homeostasis. CDKAL1 mutant insulin+ cells were also hypersensitive to glucolipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-specific defects in vitro and in vivo by inhibiting the FOS/JUN pathway. Our approach of a proof-of-principle platform, which uses isogenic hESCs for functional evaluation of GWAS-identified loci and identification of a drug candidate that rescues gene-specific defects, paves the way for precision therapy of metabolic diseases. PMID:27524441

  8. Surface Spreading and Immunostaining of Yeast Chromosomes.

    PubMed

    Grubb, Jennifer; Brown, M Scott; Bishop, Douglas K

    2015-01-01

    The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described. PMID:26325523

  9. Conversion of pentoses by yeasts

    SciTech Connect

    Gong, C.S.; Claypool, T.A.; Maun, C.M.; Mccracken, L.D.; Tsao, G.T.; Ueng, P.P.

    1983-01-01

    The utilization and conversion of D-xylose, D-xyulose, L-arabinose, and xylitol by yeast strains have been investigated with the following results: 1) The majority of yeasts tested utilize D-xylose and produce polyols, ethanol, and organic acids. The type and amount of products formed varies with the yeast strains used. The most commonly detected product is xylitol. 2) The majority of yeasts tested utilize D-xylulose aerobically and fermentatively to produce ethanol, xylitol D-arabitol, and organic acids. The type and amount of products varies depending upon the yeast strains used. 3) Xylitol is a poor carbon and energy source for most yeasts tested. Some yeast strains produce small amounts of ethanol from xylitol. 4) Most yeast strains utilize L-arabinose, and L-arabitol is the common product. Small amounts of ethanol are also produced by some yeast strains. 5) Of the four substrates examined, D-xylulose was the preferred substrate, followed by D-xylose, L-arabinose, and xylitol. 6) Mutant yeast strains that exhibit different metabolic product patterns can be induced and isolated from Candida sp. Saccharomyces cerevisiae, and other yeasts. These mutant strains can be used for ethanol production from D-xylose as well as for the study of metabolic regulation of pentose utilization in yeasts.

  10. Delimination of brewing yeast strains using different molecular techniques.

    PubMed

    Tornai-Lehoczki, J; Dlauchy, D

    2000-12-01

    In general, the genetic characteristics, the phenotype and the microbial purity of the production brewing yeast strains are among the most important factors in maintaining a consistently good quality of products. Analysis of restriction fragment length polymorphism (RFLP) patterns of 18S rRNA-coding DNA was investigated to group ale and lager strains. All production brewing yeast strains showed the same RFLP pattern as the type strain and synonym type strains of S. cerevisiae, and were quite different from the type and synonym type strains of S. pastorianus. Based on these data, all production brewing yeast strains investigated in this study appeared to belong to S. cerevisiae. Electrophoretic karyotyping and random amplified polymorphic DNA (RAPD) analysis appeared to be suitable methods for distinguishing not only the type and synonym type strain of S. cerevisiae and S. pastorianus, but also the ale and the lager strains. PMID:11139020

  11. Horizontal Transfer of Genetic Material among Saccharomyces Yeasts

    PubMed Central

    Marinoni, Gaelle; Manuel, Martine; Petersen, Randi Føns; Hvidtfeldt, Jeanne; Sulo, Pavol; Piškur, Jure

    1999-01-01

    The genus Saccharomyces consists of several species divided into the sensu stricto and the sensu lato groups. The genomes of these species differ in the number and organization of nuclear chromosomes and in the size and organization of mitochondrial DNA (mtDNA). In the present experiments we examined whether these yeasts can exchange DNA and thereby create novel combinations of genetic material. Several putative haploid, heterothallic yeast strains were isolated from different Saccharomyces species. All of these strains secreted an a- or α-like pheromone recognized by S. cerevisiae tester strains. When interspecific crosses were performed by mass mating between these strains, hybrid zygotes were often detected. In general, the less related the two parental species were, the fewer hybrids they gave. For some crosses, viable hybrids could be obtained by selection on minimal medium and their nuclear chromosomes and mtDNA were examined. Often the frequency of viable hybrids was very low. Sometimes putative hybrids could not be propagated at all. In the case of sensu stricto yeasts, stable viable hybrids were obtained. These contained both parental sets of chromosomes but mtDNA from only one parent. In the case of sensu lato hybrids, during genetic stabilization one set of the parental chromosomes was partially or completely lost and the stable mtDNA originated from the same parent as the majority of the nuclear chromosomes. Apparently, the interspecific hybrid genome was genetically more or less stable when the genetic material originated from phylogenetically relatively closely related parents; both sets of nuclear genetic material could be transmitted and preserved in the progeny. In the case of more distantly related parents, only one parental set, and perhaps some fragments of the other one, could be found in genetically stabilized hybrid lines. The results obtained indicate that Saccharomyces yeasts have a potential to exchange genetic material. If Saccharomyces

  12. Multipurpose Transposon-Insertion Libraries in Yeast.

    PubMed

    Kumar, Anuj

    2016-01-01

    Libraries of transposon-insertion alleles constitute powerful and versatile tools for large-scale analysis of yeast gene function. Transposon-insertion libraries are constructed most simply through mutagenesis of a plasmid-based genomic DNA library; modification of the mutagenizing transposon by incorporation of yeast selectable markers, recombination sites, and an epitope tag enables the application of insertion alleles for phenotypic screening and protein localization. In particular, yeast genomic DNA libraries have been mutagenized with modified bacterial transposons carrying the URA3 marker, lox recombination sites, and sequence encoding multiple copies of the hemagglutinin (HA) epitope. Mutagenesis with these transposons has yielded a large resource of insertion alleles affecting nearly 4000 yeast genes in total. Through well-established protocols, these insertion libraries can be introduced into the desired strain backgrounds and the resulting insertional mutants can be screened or systematically analyzed. Relative to alternative methods of UV irradiation or chemical mutagenesis, transposon-insertion alleles can be easily identified by PCR-based approaches or high-throughput sequencing. Transposon-insertion libraries also provide a cost-effective alternative to targeted deletion approaches, although, in contrast to start-codon to stop-codon deletions, insertion alleles might not represent true null-mutants. For protein-localization studies, transposon-insertion alleles can provide encoded epitope tags in-frame with internal codons; in many cases, these transposon-encoded epitope tags can provide a more accurate localization for proteins in which terminal sequences are crucial for intracellular targeting. Thus, overall, transposon-insertion libraries can be used quickly and economically and have a particular utility in screening for desired phenotypes and localization patterns in nonstandard genetic backgrounds. PMID:27250950

  13. Molecular characterization of Atlas 66 derived wheat near-isogenic lines contrasting in Aluminum (Al) tolerance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aluminum (Al) toxicity is the major limiting factors for wheat growth in acidic soils. Genetic improvement of Al tolerance is one of the most cost-effective solutions to improve wheat productivity. The objective of this study was to identify DNA markers associated with the Al-tolerance in wheat usin...

  14. Opportunistic Pathogenic Yeasts

    NASA Astrophysics Data System (ADS)

    Banerjee, Uma

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

  15. Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis.

    PubMed

    Chatterjee, Gautam; Sankaranarayanan, Sundar Ram; Guin, Krishnendu; Thattikota, Yogitha; Padmanabhan, Sreedevi; Siddharthan, Rahul; Sanyal, Kaustuv

    2016-02-01

    The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species--Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast. PMID:26845548

  16. Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis

    PubMed Central

    Chatterjee, Gautam; Sankaranarayanan, Sundar Ram; Guin, Krishnendu; Thattikota, Yogitha; Padmanabhan, Sreedevi; Siddharthan, Rahul; Sanyal, Kaustuv

    2016-01-01

    The centromere, on which kinetochore proteins assemble, ensures precise chromosome segregation. Centromeres are largely specified by the histone H3 variant CENP-A (also known as Cse4 in yeasts). Structurally, centromere DNA sequences are highly diverse in nature. However, the evolutionary consequence of these structural diversities on de novo CENP-A chromatin formation remains elusive. Here, we report the identification of centromeres, as the binding sites of four evolutionarily conserved kinetochore proteins, in the human pathogenic budding yeast Candida tropicalis. Each of the seven centromeres comprises a 2 to 5 kb non-repetitive mid core flanked by 2 to 5 kb inverted repeats. The repeat-associated centromeres of C. tropicalis all share a high degree of sequence conservation with each other and are strikingly diverged from the unique and mostly non-repetitive centromeres of related Candida species—Candida albicans, Candida dubliniensis, and Candida lusitaniae. Using a plasmid-based assay, we further demonstrate that pericentric inverted repeats and the underlying DNA sequence provide a structural determinant in CENP-A recruitment in C. tropicalis, as opposed to epigenetically regulated CENP-A loading at centromeres in C. albicans. Thus, the centromere structure and its influence on de novo CENP-A recruitment has been significantly rewired in closely related Candida species. Strikingly, the centromere structural properties along with role of pericentric repeats in de novo CENP-A loading in C. tropicalis are more reminiscent to those of the distantly related fission yeast Schizosaccharomyces pombe. Taken together, we demonstrate, for the first time, fission yeast-like repeat-associated centromeres in an ascomycetous budding yeast. PMID:26845548

  17. Cloning of genes that complement yeast hexokinase and glucokinase mutants.

    PubMed Central

    Walsh, R B; Kawasaki, G; Fraenkel, D G

    1983-01-01

    Genes complementing the glucose-negative fructose-negative Saccharomyces cerevisiae triple mutant strain (hxkl hxk2 glk1), which lacks hexokinase PI, hexokinase PII, and glucokinase, were obtained from a pool of yeast DNA in the multicopy plasmid YEp13. Images PMID:6341351

  18. Affected chromosome homeostasis and genomic instability of clonal yeast cultures.

    PubMed

    Adamczyk, Jagoda; Deregowska, Anna; Panek, Anita; Golec, Ewelina; Lewinska, Anna; Wnuk, Maciej

    2016-05-01

    Yeast cells originating from one single colony are considered genotypically and phenotypically identical. However, taking into account the cellular heterogeneity, it seems also important to monitor cell-to-cell variations within a clone population. In the present study, a comprehensive yeast karyotype screening was conducted using single chromosome comet assay. Chromosome-dependent and mutation-dependent changes in DNA (DNA with breaks or with abnormal replication intermediates) were studied using both single-gene deletion haploid mutants (bub1, bub2, mad1, tel1, rad1 and tor1) and diploid cells lacking one active gene of interest, namely BUB1/bub1, BUB2/bub2, MAD1/mad1, TEL1/tel1, RAD1/rad1 and TOR1/tor1 involved in the control of cell cycle progression, DNA repair and the regulation of longevity. Increased chromosome fragility and replication stress-mediated chromosome abnormalities were correlated with elevated incidence of genomic instability, namely aneuploid events-disomies, monosomies and to a lesser extent trisomies as judged by in situ comparative genomic hybridization (CGH). The tor1 longevity mutant with relatively balanced chromosome homeostasis was found the most genomically stable among analyzed mutants. During clonal yeast culture, spontaneously formed abnormal chromosome structures may stimulate changes in the ploidy state and, in turn, promote genomic heterogeneity. These alterations may be more accented in selected mutated genetic backgrounds, namely in yeast cells deficient in proper cell cycle regulation and DNA repair. PMID:26581629

  19. L-arabinose fermenting yeast

    SciTech Connect

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

    2014-09-23

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

  20. L-arabinose fermenting yeast

    SciTech Connect

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

    2013-02-12

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

  1. Molecular polymorphism distribution in phenotypically distinct populations of wine yeast strains.

    PubMed Central

    Nadal, D; Colomer, B; Piña, B

    1996-01-01

    Electrophoretic karyotyping and mitochondrial DNA restriction analysis were used to analyze natural yeast populations from fermenting musts in El Penedès, Spain. Both analyses revealed a considerable degree of polymorphism, indicating heterogeneous natural populations. By specifically designed genetic selection protocols, strains showing potentially interesting phenotypes, such as high tolerance to ethanol and temperature or the ability to grow and to ferment in wine-water-sugar mixtures, were isolated from these natural populations. Genetic analysis showed a strong correlation between the selected phenotypes and mitochondrial DNA polymorphisms. Karyotype analysis revealed several genetically similar yeast lineages in the natural yeast microflora, which we interpret as genetically isolated subpopulations of yeast strains with distinct genetic traits, which may correspond to specific microenvironments. Thus, molecular polymorphism analysis may be useful not only to study the geographical distribution of natural yeast strains but also to identify strains with specific phenotypic properties. PMID:8787392

  2. Biosynthesis of the Torpedo californica Acetylcholine Receptor α Subunit in Yeast

    NASA Astrophysics Data System (ADS)

    Fujita, Norihisa; Nelson, Nathan; Fox, Thomas D.; Claudio, Toni; Lindstrom, Jon; Riezman, Howard; Hess, George P.

    1986-03-01

    Yeast cells were transformed with a plasmid containing complementary DNA encoding the α subunit of the Torpedo californica acetylcholine receptor. These cells synthesized a protein that had the expected molecular weight, antigenic specificity, and ligand-binding properties of the α subunit. The subunit was inserted into the yeast plasma membrane, demonstrating that yeast has the apparatus to express a membrane-bound receptor protein and to insert such a foreign protein into its plasma membrane. The α subunit constituted approximately 1 percent of the total yeast membrane proteins, and its density was about the same in the plasma membrane of yeast and in the receptor-rich electric organ of Electrophorus electricus. In view of the available technology for obtaining large quantities of yeast proteins, it may now be possible to obtain amplified amounts of interesting membrane-bound proteins for physical and biochemical studies.

  3. Studying Functions of All Yeast Genes Simultaneously

    NASA Technical Reports Server (NTRS)

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

    2006-01-01

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

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

    PubMed

    Brysch-Herzberg, Michael; Seidel, Martin

    2015-12-01

    The yeast diversity on wine grapes in Germany, one of the most northern wine growing regions of the world, was investigated by means of a culture dependent approach. All yeast isolates were identified by sequence analysis of the D1/D2 domain of the 26S rDNA and the ITS region. Besides Hanseniaspora uvarum and Metschnikowia pulcherrima, which are well known to be abundant on grapes, Metschnikowia viticola, Rhodosporidium babjevae, and Curvibasidium pallidicorallinum, as well as two potentially new species related to Sporidiobolus pararoseus and Filobasidium floriforme, turned out to be typical members of the grape yeast community. We found M. viticola in about half of the grape samples in high abundance. Our data strongly suggest that M. viticola is one of the most important fermenting yeast species on grapes in the temperate climate of Germany. The frequent occurrence of Cu. pallidicorallinum and strains related to F. floriforme is a new finding. The current investigation provides information on the distribution of recently described yeast species, some of which are known from a very few strains up to now. Interestingly yeasts known for their role in the wine making process, such as Saccharomyces cerevisiae, Saccharomyces bayanus ssp. uvarum, Torulaspora delbrueckii, and Zygosaccharomyces bailii, were not found in the grape samples. PMID:26292165

  5. Metabolite Profiling of Soybean Seed Extracts from Near-Isogenic Low and Normal Phytate Lines Using Orthogonal Separation Strategies.

    PubMed

    Jervis, Judith; Kastl, Christin; Hildreth, Sherry B; Biyashev, Ruslan; Grabau, Elizabeth A; Saghai-Maroof, Mohammad A; Helm, Richard F

    2015-11-11

    Untargeted metabolomic profiling using liquid chromatography-mass spectrometry (LC-MS) was applied to lipid-depleted methanolic extracts of soybean seeds utilizing orthogonal chromatographic separations (reversed-phase and hydrophilic interaction) in both positive and negative ionization modes. Four near-isogenic lines (NILs) differing in mutations for two genes encoding highly homologous multidrug resistant proteins (MRPs) were evaluated. The double mutant exhibited a low phytate phenotype, whereas the other three NILs, the two single mutants and the wild type, did not. Principal component analysis (PCA) of the four LC-MS data sets fully separated the low phytate line from the other three. While the levels of neutral oligosaccharides were the same for all lines, there were significant metabolite differences residing in the levels of malonyl isoflavones, soyasaponins, and arginine. Two methanol-soluble polypeptides were also found as differing in abundance levels, one of which was identified as the allergen Gly m 1. PMID:26487475

  6. Production of Double-stranded DNA Ministrings

    PubMed Central

    Wong, Shirley; Lam, Peggy; Nafissi, Nafiseh; Denniss, Steven; Slavcev, Roderick

    2016-01-01

    We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis. Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of 'minicircle' DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells. We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable. PMID:26967586

  7. Production of Double-stranded DNA Ministrings.

    PubMed

    Wong, Shirley; Lam, Peggy; Nafissi, Nafiseh; Denniss, Steven; Slavcev, Roderick

    2016-01-01

    We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis. Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of 'minicircle' DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells. We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable. PMID:26967586

  8. Phylogeny of yeasts and related filamentous fungi within Pucciniomycotina determined from multigene sequence analyses

    PubMed Central

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

    2015-01-01

    In addition to rusts, the subphylum Pucciniomycotina (Basidiomycota) includes a large number of unicellular or dimorphic fungi which are usually studied as yeasts. Ribosomal DNA sequence analyses have shown that the current taxonomic system of the pucciniomycetous yeasts which is based on phenotypic criteria is not concordant with the molecular phylogeny and many genera are polyphyletic. Here we inferred the molecular phylogeny of 184 pucciniomycetous yeast species and related filamentous fungi using maximum likelihood, maximum parsimony and Bayesian inference analyses based on the sequences of seven genes, including the small subunit ribosomal DNA (rDNA), the large subunit rDNA D1/D2 domains, the internal transcribed spacer regions (ITS 1 and 2) of rDNA including the 5.8S rDNA gene; the nuclear protein-coding genes of the two subunits of DNA polymerase II (RPB1 and RPB2) and the translation elongation factor 1-α (TEF1); and the mitochondrial gene cytochrome b (CYTB). A total of 33 monophyletic clades and 18 single species lineages were recognised among the pucciniomycetous yeasts employed, which belonged to four major lineages corresponding to Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes and Mixiomycetes. These lineages remained independent from the classes Atractiellomycetes, Classiculomycetes, Pucciniomycetes and Tritirachiomycetes formed by filamentous taxa in Pucciniomycotina. An updated taxonomic system of pucciniomycetous yeasts implementing the ‘One fungus = One name’ principle will be proposed based on the phylogenetic framework presented here. PMID:26955197

  9. Amylase mRNA transcripts in normal tissues and neoplasms: the implication of different expressions of amylase isogenes.

    PubMed

    Seyama, K; Nukiwa, T; Takahashi, K; Takahashi, H; Kira, S

    1994-01-01

    To understand the cellular origin and mechanism of gene expression in amylase-producing cancers, the phenotyping of amylase isogenes by the polymerase chain reaction and restriction-fragment-length polymorphism using restriction endonucleases TaqI, DdeI, HinfI, and AfaI were performed for 3 amylase-producing lung adenocarcinomas, 16 lung cancers without hyperamylasemia, other human malignant neoplasms, cultured cell lines, and normal tissues. In addition, amylase mRNA transcripts were semi-quantified by the limited polymerase chain reaction. Amylase mRNA transcripts were detected in all of the tissues examined. The AMY1 gene (salivary type) was exclusively and highly expressed in the salivary glands and the amylase-producing lung adenocarcinomas. Coexpression of the AMY1 gene and AMY2 gene (pancreatic type) was observed in most of the lung cancers without hyperamylasemia, lung tissue, and cells scraped from the tracheal epithelium, thyroid, and female genital tract (ovary, fallopian tube, and uterus cervix), while minimal levels of mRNA transcripts of the AMY2 gene were detected in other malignant neoplasms, various normal tissues, and the cultured cell lines. All mRNA transcripts identified as being those of the AMY2 gene were further identified as being from the AMY2B gene except for the transcripts from the pancreas, in which the AMY2A gene and AMY2B gene were coexpressed. On the basis of these results, the clinical occurrence of amylase-producing cancer likely relates to the tissues expressing the AMY1 gene, while the AMY2B gene, which evolutionarily is the oldest gene among human amylase isogenes, is constitutively expressed in various tissues. PMID:7507116

  10. Vascular Analysis as a Proxy for Mechanostransduction Response in an Isogenic, Irradiated Murine Model of Mandibular Distraction Osteogenesis

    PubMed Central

    Deshpande, Sagar S.; Donneys, Alexis; Kang, Steven Y.; Page, Erin E.; Felice, Peter A.; Kiryakoza, Lauren; Nelson, Noah S.; Rodriguez, Jose L; Deshpande, Samir S.; Buchman, Steven R

    2014-01-01

    Introduction Head and neck cancer is a debilitating and disfiguring disease. Although numerous treatment options exist, an array of debilitating side effects accompany them, causing physiological and social problems. Distraction osteogenesis (DO) can avoid many of the pathologies of current reconstructive strategies; however, due to the deleterious effects of radiation on bone vascularity, DO is generally ineffective. This makes investigating the effects of radiation on neovasculature during DO and creating quantifiable metrics to gauge the success of future therapies vital. The purpose of this study was to develop a novel isogenic rat model of impaired vasculogenesis of the regenerate mandible in order to determine quantifiable metrics of vascular injury and associated damage. Methods Male Lewis Rats were divided into two groups: DO only (n=5) AND Radiation Therapy (XRT) + DO (n=7). Afterwards, a distraction device was surgically implanted into the mandible. Finally, they were distracted a total of 5.1mm. Animals were perfused with a radiopaque casting agent concomitant with euthanasia, and subsequently demineralization, microcomputed tomography, and vascular analysis were performed. Results Vessel Volume Fraction, Vessel Thickness, Vessel Number, and Degree of Anisotropy were diminished by radiation. Vessel Separation was increased by radiation. Conclusion The DO group experienced vigorous vessel formation during distraction and neovascularization with a clear, directional progression, while the XRT/DO group saw weak vessel formation during distraction and neovascularization. Further studies are warranted to more deeply examine the impairments in osteogenic mechanotransductive pathways following radiation in the murine mandible. This isogenic model provides quantifiable metrics for future studies requiring a controlled approach to immunogenicity. PMID:25173587

  11. Extracellular Polysaccharides Produced by Yeasts and Yeast-Like Fungi

    NASA Astrophysics Data System (ADS)

    van Bogaert, Inge N. A.; de Maeseneire, Sofie L.; Vandamme, Erick J.

    Several yeasts and yeast-like fungi are known to produce extracellular polysaccharides. Most of these contain D-mannose, either alone or in combination with other sugars or phosphate. A large chemical and structural variability is found between yeast species and even among different strains. The types of polymers that are synthesized can be chemically characterized as mannans, glucans, phosphoman-nans, galactomannans, glucomannans and glucuronoxylomannans. Despite these differences, almost all of the yeast exopolysaccharides display some sort of biological activity. Some of them have already applications in chemistry, pharmacy, cosmetics or as probiotic. Furthermore, some yeast exopolysaccharides, such as pullulan, exhibit specific physico-chemical and rheological properties, making them useful in a wide range of technical applications. A survey is given here of the production, the characteristics and the application potential of currently well studied yeast extracellular polysaccharides.

  12. Iron toxicity in yeast.

    PubMed

    Wiśnicka, R; Krzepiłko, A; Wawryn, J; Biliński, T

    1997-01-01

    It has been found that yeast cells are sensitive to iron overload only when grown on glucose as a carbon source. Effective concentration of ferrous iron is much higher than that found in natural environments. Effects of ferrous iron are strictly oxygen dependent, what suggest that the formation of hydroxyl radicals in the Fenton reaction is a cause of the toxicity. Respiratory deficiency and pretreatment of cells with antimycin A prevent toxic effects in the late exponential phase of growth, whereas uncouplers and 2mM magnesium salts completely protect even the most vulnerable exponential cells. Generally, toxic effects correlate with the ability of cells to take up this metal. The results presented suggest that during ferrous iron overload iron is transported through the unspecific divalent cation uptake system which is known in fungi. The data suggest that recently described high and low affinity systems of iron uptake in yeast are the only source of iron in natural environments. PMID:9516981

  13. Water Transport in Yeasts.

    PubMed

    Sabir, Farzana; Prista, Catarina; Madeira, Ana; Moura, Teresa; Loureiro-Dias, Maria C; Soveral, Graça

    2016-01-01

    Water moves across membranes through the lipid bilayer and through aquaporins, in this case in a regulated manner. Aquaporins belong to the MIP superfamily and two subfamilies are represented in yeasts: orthodox aquaporins considered to be specific water channels and aquaglyceroporins (heterodox aquaporins). In Saccharomyces cerevisiae genome, four aquaporin isoforms were identified, two of which are genetically close to orthodox aquaporins (ScAqy1 and ScAqy2) and the other two are more closely related to the aquaglyceroporins (ScFps1 and ScAqy3). Advances in the establishment of water channels structure are reviewed in this chapter in relation with the mechanisms of selectivity, conductance and gating. Aquaporins are important for key aspects of yeast physiology. They have been shown to be involved in sporulation, rapid freeze-thaw tolerance, osmo-sensitivity, and modulation of cell surface properties and colony morphology, although the underlying exact mechanisms are still unknown. PMID:26721272

  14. A multivariate analysis using physiology and behavior to characterize robustness in two isogenic lines of rainbow trout exposed to a confinement stress.

    PubMed

    Sadoul, Bastien; Leguen, Isabelle; Colson, Violaine; Friggens, Nicolas C; Prunet, Patrick

    2015-03-01

    Robustness is a complex trait difficult to characterize and phenotype. In the present study, two features of robustness in rainbow trout were investigated: sensitivity and resilience to an acute stressor. For that purpose, oxygen consumption, cortisol release, group dispersion and group activity of two isogenic lines of juvenile rainbow trout were followed before and after an environmental challenge. The effect of a 4h confinement protocol (~140kg/m(3)), which is generally considered as a highly stressful challenge, was investigated. Temporal patterns produced by this experiment were analyzed using multivariate statistics on curve characteristics to describe physiological and behavioral adaptive systems for each isogenic line. The two isogenic lines were found to be highly divergent in their corticosteroid reactivity. However, no correlation between physiological and behavioral sensitivity or resilience was observed. Furthermore, the multivariate analysis results indicated two separate and independent fish group coping strategies, i.e. by favoring either behavioral or physiological responses. In addition, considerable intra-line variabilities were observed, suggesting the importance of micro-environment effects on perturbation sensitivities. In this context, cortisol release rate variability was found to be related to the pre-stress social environment, with a strong correlation between pre-stress aggressiveness and cortisol release rate amplitude. Overall, this approach allowed us to extract important characteristics from dynamic data in physiology and behavior to describe components of robustness in two isogenic lines of rainbow trout. PMID:25481357

  15. Low Lignin (Brown Mid-rib) Sorghum Genotypes Restrict Growth of Fusarium Spp. as Compared with Near-Isogenic Wild-Type Sorghum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    To increase usability of sorghum for bioenergy and forages, two different brown midrib (bmr) genes, bmr-6 and bmr-12, were backcrossed into five elite backgrounds, resulting in reduced lignin near-isogenic genotypes. Field-grown grain from bmr-6 and bmr-12 plants had significantly reduced colonizati...

  16. Comparative analysis of genetic background in eight near-isogenic wheat lines with different H genes conferring resistance to Hessian fly

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Near-isogenic lines (NILs) are useful tools for investigating gene expression, detecting closely linked markers, or cloning the genes. However, the reliability of using NILs for genetic and genomic analysis relies on the homogeneity of the genetic background. In this study, a set of eight NILs (Car...

  17. Characterization of cottonseed nutrients composition in near isogenic cotton (Gossypium hirsutum L.) mutant lines for fuzzless seed trait under well-watered and water stress conditions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cotton mutant near isogenic lines (NILs) for fuzzless seed trait has been used to investigate cell biology, genetic, and molecular processes of fiber initiation, development, fiber yield and quality. However, there is no information available on the effect of fuzzless seed trait on cottonseed nutrie...

  18. Responses of Maize (Zea mays L.) near isogenic lines carrying Wsm1, Wsm2 and Wsm3 to three viruses in the Potyviridae

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Three independent genes controlling resistance to Wheat streak mosaic virus (WSMV; family Potyviridae; genus Tritimovirus) were previously identified on chromosomes six (Wsm1), three (Wsm2) and ten (Wsm3) in the resistant maize (Zea mays L.) inbred line Pa405. Near isogenic lines (NIL) carrying eac...

  19. Impact of Diuraphis noxia (Mordvilko) and Rhopalosiphum padi (L.) (Hemiptera: Aphididae) on primary physiology of four near-isogenic wheat lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The impact of feeding injury by the Russian wheat aphid, Diuraphis noxia (Mordvilko), and bird cherry oat aphid, Rhopalosiphum padi (L.), (Hemiptera: Aphididae) on susceptible and resistant wheat, Triticum aestivum L., near-isogenic lines ‘Tugela’ (susceptible), Tugela-Dn1 (antibiotic), -Dn2 (tolera...

  20. Glossy and Nonglossy Near-isogenic Lines USVL115-GL, USVL115-NG, USVL188-GL and USVL188-NG of Broccoli Derived from Doubled Haploids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two pairs of near-isogenic lines of green-sprouting broccoli (Brassica oleracea L. var. italica), designated USVL115-GL and USVL115-NG, and USVL188-GL and USVL188-NG, were released by the Agricultural Research Service of the U.S. Department of Agriculture in 2008. Each of the released pairs (double...

  1. Yield and fiber quality of five pairs of near-isogenic cotton (Gossypium hirsutum L.) lines expressing the fuzzless/linted and fuzzy/linted seed phenotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fuzzless cotton often has traits desirable to the cotton industry, including longer fibers, reduced short fiber content, fewer neps, and improved ginning efficiency. This two-year field study described yield and fiber properties of five pairs of fuzzy and fuzzless near-isogenic lines, developed from...

  2. Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations.

    PubMed

    Helfrich, Stefan; Pfeifer, Eugen; Krämer, Christina; Sachs, Christian Carsten; Wiechert, Wolfgang; Kohlheyer, Dietrich; Nöh, Katharina; Frunzke, Julia

    2015-11-01

    Almost all bacterial genomes contain DNA of viral origin, including functional prophages or degenerated phage elements. A frequent but often unnoted phenomenon is the spontaneous induction of prophage elements (SPI) even in the absence of an external stimulus. In this study, we have analyzed SPI of the large, degenerated prophage CGP3 (187 kbp), which is integrated into the genome of the Gram-positive Corynebacterium glutamicum ATCC 13032. Time-lapse fluorescence microscopy of fluorescent reporter strains grown in microfluidic chips revealed the sporadic induction of the SOS response as a prominent trigger of CGP3 SPI but also displayed a considerable fraction (∼30%) of RecA-independent SPI. Whereas approx. 20% of SOS-induced cells recovered from this stress and resumed growth, the spontaneous induction of CGP3 always led to a stop of growth and likely cell death. A carbon source starvation experiment clearly emphasized that SPI only occurs in actively proliferating cells, whereas sporadic SOS induction was still observed in resting cells. These data highlight the impact of sporadic DNA damage on the activity of prophage elements and provide a time-resolved, quantitative description of SPI as general phenomenon of bacterial populations. PMID:26235130

  3. Utilization of hexamethylenetetramine (urotropine) by bacteria and yeasts.

    PubMed

    Middelhoven, Wouter J; van Doesburg, Wim

    2007-02-01

    A slow growing bacterial population able to utilize hexamethylelenetetramine (urotropine) as sole source of carbon, nitrogen and energy was isolated from soil. From this crude enrichment culture two bacteria were isolated and identified as Brevundimonas diminuta and a Phyllobacterium sp. by sequencing of 16S ribosomal DNA. These bacteria also grew on urotropine but at a lower rate than the enrichment culture. Addition of glucose to the latter resulted in growth of some yeasts that overgrew the bacteria. Assimilation of urotropine as sole nitrogen source is very common among yeasts, 46 out of 60 species tested showed this characteristic. PMID:17043911

  4. Effect of endogenous Hsp104 chaperone in yeast models of sporadic and familial Parkinson's disease.

    PubMed

    Gade, Vamshidhar R; Kardani, Jay; Roy, Ipsita

    2014-10-01

    Molecular chaperones constitute a major component of the cellular stress response machinery in neurodegenerative diseases, many of which are characterized by the misfolding and aggregation of endogenous cellular proteins into generic amyloid macrostructures. Heterologous expression of the yeast protein remodelling factor Hsp104 has been proposed as a possible therapeutic approach in such disease conditions. Hsp104 is unique in its ability to act as a protein 'disaggregase' by removing smaller units from amyloid fibrils and has no homologue in metazoa. The effect of Hsp104 is strongly modulated by its expression level. We show that at endogenous levels, the presence of Hsp104 has a deleterious effect on protein aggregation in two different strains of yeast. Overexpression of wild-type and mutant human α-synuclein in a well-validated yeast model of Parkinson's disease and in an isogenic Hsp104-deleted strain resulted in lower oxidative stress and reduced damage to cellular proteins in the latter case. This translated to lower cytotoxicity and increased cell viability. Endocytotic defect caused due to aggregation of α-syuclein was also rescued in cells lacking Hsp104. Our results show that the effect of overexpression of a chaperone on protein misfolding/aggregation cannot be predicted from its function in the host expression platform. PMID:25161148

  5. Effect of auxotrophies on yeast performance in aerated fed-batch reactor

    SciTech Connect

    Landi, Carmine; Paciello, Lucia; Alteriis, Elisabetta de; Brambilla, Luca; Parascandola, Palma

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer The paper contributes to fill the gap existing between the basic and applied research. Black-Right-Pointing-Pointer Mathematical model sheds light on the physiology of auxotrophic yeast strains. Black-Right-Pointing-Pointer Yeast behavior in fed-batch is influenced by biological and environmental determinants. Black-Right-Pointing-Pointer Process optimization would make possible the production of heterologous proteins which are not yet on the market. -- Abstract: A systematic investigation on the effects of auxotrophies on the performance of yeast in aerated fed-batch reactor was carried out. Six isogenic strains from the CEN.PK family of Saccharomyces cerevisiae, one prototroph and five auxotrophs, were grown in aerated fed-batch reactor using the same operative conditions and a proper nutritional supplementation. The performance of the strains, in terms of final biomass decreased with increasing the number of auxotrophies. Auxotrophy for leucine exerted a profound negative effect on the performance of the strains. Accumulation of reactive oxygen species (ROS) in the cells of the strain carrying four auxotrophies and its significant viability loss, were indicative of an oxidative stress response induced by exposure of cells to the environmental conditions. The mathematical model was fundamental to highlight how the carbon flux, depending on the number and type of auxotrophies, was diverted towards the production of increasingly large quantities of energy for maintenance.

  6. Isolation of yeast artificial chromosomes free of endogenous yeast chromosomes: construction of alternate hosts with defined karyotypic alterations.

    PubMed Central

    Hamer, L; Johnston, M; Green, E D

    1995-01-01

    An intrinsic feature of yeast artificial chromosomes (YACs) is that the cloned DNA is generally in the same size range (i.e., approximately 200-2000 kb) as the endogenous yeast chromosomes. As a result, the isolation of YAC DNA, which typically involves separation by pulsed-field gel electrophoresis, is frequently confounded by the presence of a comigrating or closely migrating endogenous yeast chromosome(s). We have developed a strategy that reliably allows the isolation of any YAC free of endogenous yeast chromosomes. Using recombination-mediated chromosome fragmentation, a set of Saccharomyces cerevisiae host strains was systematically constructed. Each strain contains defined alterations in its electrophoretic karyotype, which provide a large-size interval devoid of endogenous chromosomes (i.e., a karyotypic "window"). All of the constructed strains contain the kar1-delta 15 mutation, thereby allowing the efficient transfer of a YAC from its original host into an appropriately selected window strain using the kar1-transfer procedure. This approach provides a robust and efficient means to obtain relatively pure YAC DNA regardless of YAC size. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8524833

  7. A Millifluidic Study of Cell-to-Cell Heterogeneity in Growth-Rate and Cell-Division Capability in Populations of Isogenic Cells of Chlamydomonas reinhardtii

    PubMed Central

    Damodaran, Shima P.; Eberhard, Stephan; Boitard, Laurent; Rodriguez, Jairo Garnica; Wang, Yuxing; Bremond, Nicolas; Baudry, Jean; Bibette, Jérôme; Wollman, Francis-André

    2015-01-01

    To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes. PMID:25760649

  8. A millifluidic study of cell-to-cell heterogeneity in growth-rate and cell-division capability in populations of isogenic cells of Chlamydomonas reinhardtii.

    PubMed

    Damodaran, Shima P; Eberhard, Stephan; Boitard, Laurent; Rodriguez, Jairo Garnica; Wang, Yuxing; Bremond, Nicolas; Baudry, Jean; Bibette, Jérôme; Wollman, Francis-André

    2015-01-01

    To address possible cell-to-cell heterogeneity in growth dynamics of isogenic cell populations of Chlamydomonas reinhardtii, we developed a millifluidic drop-based device that not only allows the analysis of populations grown from single cells over periods of a week, but is also able to sort and collect drops of interest, containing viable and healthy cells, which can be used for further experimentation. In this study, we used isogenic algal cells that were first synchronized in mixotrophic growth conditions. We show that these synchronized cells, when placed in droplets and kept in mixotrophic growth conditions, exhibit mostly homogeneous growth statistics, but with two distinct subpopulations: a major population with a short doubling-time (fast-growers) and a significant subpopulation of slowly dividing cells (slow-growers). These observations suggest that algal cells from an isogenic population may be present in either of two states, a state of restricted division and a state of active division. When isogenic cells were allowed to propagate for about 1000 generations on solid agar plates, they displayed an increased heterogeneity in their growth dynamics. Although we could still identify the original populations of slow- and fast-growers, drops inoculated with a single progenitor cell now displayed a wider diversity of doubling-times. Moreover, populations dividing with the same growth-rate often reached different cell numbers in stationary phase, suggesting that the progenitor cells differed in the number of cell divisions they could undertake. We discuss possible explanations for these cell-to-cell heterogeneities in growth dynamics, such as mutations, differential aging or stochastic variations in metabolites and macromolecules yielding molecular switches, in the light of single-cell heterogeneities that have been reported among isogenic populations of other eu- and prokaryotes. PMID:25760649

  9. Phenotypic and genotypic diversity of wine yeasts used for acidic musts.

    PubMed

    Kunicka-Styczyńska, Alina; Rajkowska, Katarzyna

    2012-05-01

    The aim of this study was to examine the physiological and genetic stability of the industrial wine yeasts Saccharomyces cerevisiae and Saccharomyces bayanus var. uvarum under acidic stress during fermentation. The yeasts were sub-cultured in aerobic or fermentative conditions in media with or without l-malic acid. Changes in the biochemical profiles, karyotypes, and mitochondrial DNA profiles were assessed after minimum 50 generations. All yeast segregates showed a tendency to increase the range of compounds used as sole carbon sources. The wild strains and their segregates were aneuploidal or diploidal. One of the four strains of S. cerevisiae did not reveal any changes in the electrophoretic profiles of chromosomal and mitochondrial DNA, irrespective of culture conditions. The extent of genomic changes in the other yeasts was strain-dependent. In the karyotypes of the segregates, the loss of up to 2 and the appearance up to 3 bands was noted. The changes in their mtDNA patterns were much broader, reaching 5 missing and 10 additional bands. The only exception was S. bayanus var. uvarum Y.00779, characterized by significantly greater genome plasticity only under fermentative stress. Changes in karyotypes and mtDNA profiles prove that fermentative stress is the main driving force of the adaptive evolution of the yeasts. l-malic acid does not influence the extent of genomic changes and the resistance of wine yeasts exhibiting increased demalication activity to acidic stress is rather related to their ability to decompose this acid. The phenotypic changes in segregates, which were found even in yeasts that did not reveal deviations in their DNA profiles, show that phenotypic characterization may be misleading in wine yeast identification. Because of yeast gross genomic diversity, karyotyping even though it does not seem to be a good discriminative tool, can be useful in determining the stability of wine yeasts. Restriction analysis of mitochondrial DNA appears to be

  10. Genomics and the making of yeast biodiversity

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  11. A survey of yeasts in traditional sausages of southern Italy.

    PubMed

    Gardini, F; Suzzi, G; Lombardi, A; Galgano, F; Crudele, M A; Andrighetto, C; Schirone, M; Tofalo, R

    2001-07-01

    The evolution of the yeast population during manufacturing and ripening of 'salsiccia sotto sugna', a typical salami of the Lucania region (southern Italy), was investigated. Four different batches, produced in four farms in Lucania, were studied. Each batch showed a specific yeast population, and the most frequently isolated yeasts belonged to Debaryomyces hansenii and its anamorph Candida famata, and Rhodotorula mucilaginosa. Yarrowia lipolytica was isolated from three sausage batches. The Y. lipolytica isolates were further characterised, in particular for their lipolytic activity on pork fat. Lipolytic activity was maximal at pH 5.5, with oleic and palmitic acids as major free fatty acids produced. The use of randomly amplified polymorphic DNA-polymerase chain reaction allowed the detection of a high genetic heterogeneity among the isolates phenotypically assigned to the species Y. lipolytica. PMID:12702361

  12. Whole Genome Analysis of a Wine Yeast Strain

    PubMed Central

    Hauser, Nicole C.; Fellenberg, Kurt; Gil, Rosario; Bastuck, Sonja; Hoheisel, Jörg D.

    2001-01-01

    Saccharomyces cerevisiae strains frequently exhibit rather specific phenotypic features needed for adaptation to a special environment. Wine yeast strains are able to ferment musts, for example, while other industrial or laboratory strains fail to do so. The genetic differences that characterize wine yeast strains are poorly understood, however. As a first search of genetic differences between wine and laboratory strains, we performed DNA-array analyses on the typical wine yeast strain T73 and the standard laboratory background in S288c. Our analysis shows that even under normal conditions, logarithmic growth in YPD medium, the two strains have expression patterns that differ significantly in more than 40 genes. Subsequent studies indicated that these differences correlate with small changes in promoter regions or variations in gene copy number. Blotting copy numbers vs. transcript levels produced patterns, which were specific for the individual strains and could be used for a characterization of unknown samples. PMID:18628902

  13. Recent Taxonomic Developments with Candida and Other Opportunistic Yeasts

    PubMed Central

    Lockhart, Shawn R.

    2015-01-01

    Increases in susceptible patient populations and advances in identification methods have resulted in the continued recognition of novel yeasts as agents of human infection. Most of these agents are members of the well-recognized genera Candida, Cryptococcus, Trichosporon, and Rhodotorula. Some of these agents are “cryptic species,” members of species complexes, and may not be detectable using classical carbohydrate assimilation-based methods of yeast identification. Such species require DNA- or MALDI-based methods for correct identification, although sporadic isolates may not routinely require delineation to the individual species level. The coming end of the fungal taxonomy rules requiring separate names for sexual and asexual forms of the same fungus will hopefully allow greater clarity, as names for medically important yeast can now be based on the needs of the medical mycology community and the common goal of better communication between laboratory and clinician. PMID:26526658

  14. Molecular Phylogeny of the Yeasts: Impact on Classification and Prediction of Biotechnological Properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA sequence analysis and other DNA-based methodologies have transformed the way in which yeasts are identified and classified. Development of species-specific gene sequence databases has provided a barcode system for rapid identification of known species and the recognition of undescribed species. ...

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

    PubMed

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

    2015-02-01

    Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3-4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. PMID:25499885

  16. Phosphorylation and Cellular Function of the Human Rpa2 N-Terminus in the Budding Yeast Saccharomyces cerevisiae

    PubMed Central

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

    2015-01-01

    Maintenance of genome integrity is critical for proper cell growth. This occurs through accurate DNA replication and repair of DNA lesions. A key factor involved in both DNA replication and the DNA damage response is the heterotrimeric single-stranded DNA (ssDNA) binding complex Replication Protein A (RPA). Although the RPA complex appears to be structurally conserved throughout eukaryotes, the primary amino acid sequence of each subunit can vary considerably. Examination of sequence differences along with the functional interchangeability of orthologous RPA subunits or regions could provide insight into important regions and their functions. This might also allow for study in simpler systems. We determined that substitution of yeast Replication Factor A (RFA) with human RPA does not support yeast cell viability. Exchange of a single yeast RFA subunit with the corresponding human RPA subunit does not function due to lack of inter-species subunit interactions. Substitution of yeast Rfa2 with domains/regions of human Rpa2 important for Rpa2 function (i.e., the N-terminus and the loop 3–4 region) supports viability in yeast cells, and hybrid proteins containing human Rpa2 N-terminal phospho-mutations result in similar DNA damage phenotypes to analogous yeast Rfa2 N-terminal phospho-mutants. Finally, the human Rpa2 N-terminus (NT) fused to yeast Rfa2 is phosphorylated in a manner similar to human Rpa2 in human cells, indicating that conserved kinases recognize the human domain in yeast. The implication is that budding yeast represents a potential model system for studying not only human Rpa2 N-terminal phosphorylation, but also phosphorylation of Rpa2 N-termini from other eukaryotic organisms. PMID:25499885

  17. Yeast identification in floral nectar of Mimulus aurantiacus (Invited)

    NASA Astrophysics Data System (ADS)

    Kyauk, C.; Belisle, M.; Fukami, T.

    2009-12-01

    Nectar is such a sugar-rich resource that serves as a natural habitat in which microbes thrive. As a result, yeasts arrive to nectar on the bodies of pollinators such as hummingbirds and bees. Yeasts use the sugar in nectar for their own needs when introduced. This research focuses on the identification of different types of yeast that are found in the nectar of Mimulus aurantiacus (commonly known as sticky monkey-flower). Unopened Mimulus aurantiacus flower buds were tagged at Jasper Ridge and bagged three days later. Floral nectar was then extracted and plated on potato dextrose agar. Colonies on the plates were isolated and DNA was extracted from each sample using QIAGEN DNeasy Plant Mini Kit. The DNA was amplified through PCR and ran through gel electrophoresis. The PCR product was used to clone the nectar samples into an E.coli vector. Finally, a phylogenetic tree was created by BLAST searching sequences in GenBank using the Internal Transcribed Space (ITS) locus. It was found that 18 of the 50 identified species were Candida magnifica, 14 was Candida rancensis, 6 were Crytococcus albidus and there were 3 or less of the following: Starmella bombicola, Candida floricola, Aureobasidium pullulans, Pichia kluyvera, Metschnikowa cibodaserisis, Rhodotorua colostri, and Malassezia globosa. The low diversity of the yeast could have been due to several factors: time of collection, demographics of Jasper Ridge, low variety of pollinators, and sugar concentration of the nectar. The results of this study serve as a necessary first step for a recently started research project on ecological interactions between plants, pollinators, and nectar-living yeast. More generally, this research studies the use of the nectar-living yeast community as a natural microcosm for addressing basic questions about the role of dispersal and competitive and facilitative interactions in ecological succession.

  18. Binding of HIV-1 Vpr Protein to the Human Homolog of the Yeast DNA Repair Protein RAD23 (hHR23A) Requires Its Xeroderma Pigmentosum Complementation Group C Binding (XPCB) Domain as Well as the Ubiquitin-associated 2 (UBA2) Domain*

    PubMed Central

    Jung, Jinwon; Byeon, In-Ja L.; DeLucia, Maria; Koharudin, Leonardus M. I.; Ahn, Jinwoo; Gronenborn, Angela M.

    2014-01-01

    The human homolog of the yeast DNA repair protein RAD23, hHR23A, has been found previously to interact with the human immunodeficiency virus, type 1 accessory protein Vpr. hHR23A is a modular protein containing an N-terminal ubiquitin-like (UBL) domain and two ubiquitin-associated domains (UBA1 and UBA2) separated by a xeroderma pigmentosum complementation group C binding (XPCB) domain. All domains are connected by flexible linkers. hHR23A binds ubiquitinated proteins and acts as a shuttling factor to the proteasome. Here, we show that hHR23A utilizes both the UBA2 and XPCB domains to form a stable complex with Vpr, linking Vpr directly to cellular DNA repair pathways and their probable exploitation by the virus. Detailed structural mapping of the Vpr contacts on hHR23A, by NMR, revealed substantial contact surfaces on the UBA2 and XPCB domains. In addition, Vpr binding disrupts an intramolecular UBL-UBA2 interaction. We also show that Lys-48-linked di-ubiquitin, when binding to UBA1, does not release the bound Vpr from the hHR23A-Vpr complex. Instead, a ternary hHR23A·Vpr·di-UbK48 complex is formed, indicating that Vpr does not necessarily abolish hHR23A-mediated shuttling to the proteasome. PMID:24318982

  19. New and emerging yeast pathogens.

    PubMed Central

    Hazen, K C

    1995-01-01

    The most common yeast species that act as agents of human disease are Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, and Cryptococcus neoformans. The incidence of infections by other yeasts has increased during the past decade. The most evident emerging pathogens are Malassezia furfur, Trichosporon beigelii, Rhodotorula species, Hansenula anomala, Candida lusitaniae, and Candida krusei. Organisms once considered environmental contaminants or only industrially important, such as Candida utilis and Candida lipolytica, have now been implicated as agents of fungemia, onychomycosis, and systemic disease. The unusual yeasts primarily infect immunocompromised patients, newborns, and the elderly. The role of central venous catheter removal and antifungal therapy in patient management is controversial. The antibiograms of the unusual yeasts range from resistant to the most recent azoles and amphotericin B to highly susceptible to all antifungal agents. Current routine methods for yeast identification may be insufficient to identify the unusual yeasts within 2 days after isolation. The recognition of unusual yeasts as agents of sometimes life-threatening infection and their unpredictable antifungal susceptibilities increase the burden on the clinical mycology laboratory to pursue complete species identification and MIC determinations. Given the current and evolving medical practices for management of seriously ill patients, further evaluations of the clinically important data about these yeasts are needed. PMID:8665465

  20. Chronic hemodynamic unloading regulates the morphologic development of newborn mouse hearts transplanted into the ear of isogeneic adult mice.

    PubMed Central

    Rossi, M. A.

    1992-01-01

    The morphologic development of newborn mouse hearts transplanted into the pinna of the ears of isogeneic adult mice was assessed in comparison to in situ ventricular myocardium of recipients. The grafted hearts became vascularized from the auricular artery at the base of the ear, and although these preparations appeared not to be intrinsically innervated, most of them showed grossly visible pulsatile activity. Since they were not subjected to hemodynamic load due to working against a pressure gradient, this technique provided an interesting experimental model for studies on the growth of chronically unloaded tissue. The ultrastructure of the myocardium from neonatal mouse hearts, which were fixed immediately after dissection, revealed no differences in comparison to previously published observations. By 2 months, there was virtually no change in the myocardial cell size as compared with newborn mouse cardiac tissue. The heterotopic hearts showed a mature ultrastructural appearance, with parallel bands of myofibrils alternating with rows of mitochondria and differentiated intercalated discs comparable to in situ myocardium. The interstitial space was widened due to fibrous tissue, with activated fibroblasts and a few mononuclear cells. In contrast, by 6 months after transplantation, the heterotopic myocardium showed a dispersion of the measured cell diameter of myocytes, with atrophy of a certain population of cells and hypertrophy in others; nevertheless, the mean cell diameter was similar to that observed in 2-month grafts. The myocytes showed significant dissociation from each other in fibrous tissue and a cellular infiltrate composed predominantly of mononuclear cells, and greater variability of the parallel arrangement of cells. They often contained myofibrils coursing in different directions rather than in parallel. Normal-sized or predominantly atrophic degenerated myocytes, characterized by a wide variety of ultrastructural alterations, were present. By 12

  1. Phage and Yeast Display.

    PubMed

    Sheehan, Jared; Marasco, Wayne A

    2015-02-01

    Despite the availability of antimicrobial drugs, the continued development of microbial resistance--established through escape mutations and the emergence of resistant strains--limits their clinical utility. The discovery of novel, therapeutic, monoclonal antibodies (mAbs) offers viable clinical alternatives in the treatment and prophylaxis of infectious diseases. Human mAb-based therapies are typically nontoxic in patients and demonstrate high specificity for the intended microbial target. This specificity prevents negative impacts on the patient microbiome and avoids driving the resistance of nontarget species. The in vitro selection of human antibody fragment libraries displayed on phage or yeast surfaces represents a group of well-established technologies capable of generating human mAbs. The advantage of these forms of microbial display is the large repertoire of human antibody fragments present during a single selection campaign. Furthermore, the in vitro selection environments of microbial surface display allow for the rapid isolation of antibodies--and their encoding genes--against infectious pathogens and their toxins that are impractical within in vivo systems, such as murine hybridomas. This article focuses on the technologies of phage display and yeast display, as these strategies relate to the discovery of human mAbs for the treatment and vaccine development of infectious diseases. PMID:26104550

  2. Eighteen new oleaginous yeast species.

    PubMed

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

    2016-07-01

    Of 1600 known species of yeasts, about 70 are known to be oleaginous, defined as being able to accumulate over 20 % intracellular lipids. These yeasts have value for fundamental and applied research. A survey of yeasts from the Phaff Yeast Culture Collection, University of California Davis was performed to identify additional oleaginous species within the Basidiomycota phylum. Fifty-nine strains belonging to 34 species were grown in lipid inducing media, and total cell mass, lipid yield and triacylglycerol profiles were determined. Thirty-two species accumulated at least 20 % lipid and 25 species accumulated over 40 % lipid by dry weight. Eighteen of these species were not previously reported to be oleaginous. Triacylglycerol profiles were suitable for biodiesel production. These results greatly expand the number of known oleaginous yeast species, and reveal the wealth of natural diversity of triacylglycerol profiles within wild-type oleaginous Basidiomycetes. PMID:27072563

  3. A Functional Genomic Yeast Screen to Identify Pathogenic Bacterial Proteins

    PubMed Central

    Slagowski, Naomi L; Kramer, Roger W; Morrison, Monica F; LaBaer, Joshua; Lesser, Cammie F

    2008-01-01

    Many bacterial pathogens promote infection and cause disease by directly injecting into host cells proteins that manipulate eukaryotic cellular processes. Identification of these translocated proteins is essential to understanding pathogenesis. Yet, their identification remains limited. This, in part, is due to their general sequence uniqueness, which confounds homology-based identification by comparative genomic methods. In addition, their absence often does not result in phenotypes in virulence assays limiting functional genetic screens. Translocated proteins have been observed to confer toxic phenotypes when expressed in the yeast Saccharomyces cerevisiae. This observation suggests that yeast growth inhibition can be used as an indicator of protein translocation in functional genomic screens. However, limited information is available regarding the behavior of non-translocated proteins in yeast. We developed a semi-automated quantitative assay to monitor the growth of hundreds of yeast strains in parallel. We observed that expression of half of the 19 Shigella translocated proteins tested but almost none of the 20 non-translocated Shigella proteins nor ∼1,000 Francisella tularensis proteins significantly inhibited yeast growth. Not only does this study establish that yeast growth inhibition is a sensitive and specific indicator of translocated proteins, but we also identified a new substrate of the Shigella type III secretion system (TTSS), IpaJ, previously missed by other experimental approaches. In those cases where the mechanisms of action of the translocated proteins are known, significant yeast growth inhibition correlated with the targeting of conserved cellular processes. By providing positive rather than negative indication of activity our assay complements existing approaches for identification of translocated proteins. In addition, because this assay only requires genomic DNA it is particularly valuable for studying pathogens that are difficult to

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

    PubMed

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

    2009-04-01

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

  5. Effect of Bt corn on broiler growth performance and fate of feed-derived DNA in the digestive tract.

    PubMed

    Rossi, F; Morlacchini, M; Fusconi, G; Pietri, A; Mazza, R; Piva, G

    2005-07-01

    The aim of the study was to evaluate the effect on broiler performance of transgenic Bacillus thuringiensis (Bt) corn containing the Cry1A(b) protein compared with the corresponding near isogenic corn and to analyze the degradation of the Cry1A(b) gene in the digestive tract. Ross male broilers (432) were fed for 42 consecutive days with diets containing Bt or isogenic corn. Diet, Bt corn, and the isogenic form of the Bt corn were analyzed for composition and aflatoxin B1, fumonisin B1, and deoxynivalenol contents. Broiler body weight and feed intake were recorded at regular intervals (d 0, 21, and 42). The presence of the Cry1A(b) gene and plant-specific genes Zein and Sh-2 in gut contents of crop, gizzard, jejunum, cecum, and samples of blood was determined in 10 animals per treatment at the end of the trial using a PCR technique. Chemical composition was not different between Bt and its isogenic form, whereas the fumonisin B1 content for Bt was lower than for isogenic corn (2,039 vs. 1,1034 ppb; P < 0.05). The results of the growth study showed no difference for average daily weight gain (129.4 vs. 126.0 g/d), feed intake (63.4 vs. 61.8 g/d), and feed conversion ratio (1.95 vs. 2.02) among the groups. No significant relationship was observed between mycotoxins content and growth performances. Feed-derived DNA is progressively degraded along the digestive tract. Detection frequency of short fragments of maize-specific high copy number Zein gene was high but significantly decreased in distal sectors. An 1,800-bp fragment of the Cry1A(b) gene, corresponding to the minimal functional unit, was detected only in crop and gizzard of birds fed Bt corn. Sh-2 showed the same detection frequency of Cry1A(b) and was also found in birds fed isogenic corn. Blood samples were positive with low frequency only for the Zein gene fragment. No significant difference in DNA detection was observed between birds fed Bt and isogenic corn, indicating that DNA derived from transgenic

  6. [Study of animal viruses in yeast].

    PubMed

    Morikawa, Yuko

    2006-06-01

    Yeast is often considered to be a model eukaryotic organism, in a manner analogous to E. coli as a model prokaryotic organism. Yeast has been extensively characterized and the genomes completely sequenced. Despite the small genome size, yeast displays most of features of higher eukaryotes. The facts that most of cellular machinery is conserved among different eukaryotes and that the powerful technologies of genetics and molecular biology are available have made yeast model eukaryotic cells in biological and biomedical sciences including virology. Cumulative data indicate that yeast can be a host for animal viruses. I briefly describe yeast gene expression and review viral replication in yeast. Great discovery include complete replication of animal viruses and production of virus-like particle vaccines in yeast. Current studies on yeast focus on identification of host factors and machinery used for viral replication. The studies are based on traditional yeast genetics and genome-wide identification using a complete set of yeast deletion strains. PMID:17038807

  7. Spatial organization and dynamics of interphase yeast chromosomes

    NASA Astrophysics Data System (ADS)

    Avsaroglu, Baris; Gordon-Messer, Susannah; Fritsche, Miriam; Ham, Jungoh; Heermann, Dieter W.; Haber, James E.; Kondev, Jane

    2012-02-01

    Understanding how the genome is spatially organized is an important problem in cell biology, due to its key roles in gene expression and DNA recombination. Here we report on a combined experimental and theoretical study of the organization and dynamics of yeast chromosome III which has a functional role in the yeast life cycle, in particular, it is responsible for mating type switching. By imaging two fluorescent markers, one at the spindle pole body (SPB) and the other proximal to the HML locus that is involved in DNA recombination during mating type switching, we measured the cell to cell distribution of distances and the mean square displacement between the markers as a function of time. We compared our experimental results with a random-walk polymer model that takes into account tethering and confinement of chromosomes in the nucleus, and found that the model recapitulates the observed spatial and temporal organization of chromosome III in yeast in quantitative detail. The polymer model makes specific predictions for mating-type switching in yeast, and suggests new experiments to test them.

  8. The Spontaneous Mutation Rate in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Farlow, Ashley; Long, Hongan; Arnoux, Stéphanie; Sung, Way; Doak, Thomas G; Nordborg, Magnus; Lynch, Michael

    2015-10-01

    The rate at which new mutations arise in the genome is a key factor in the evolution and adaptation of species. Here we describe the rate and spectrum of spontaneous mutations for the fission yeast Schizosaccharomyces pombe, a key model organism with many similarities to higher eukaryotes. We undertook an ∼1700-generation mutation accumulation (MA) experiment with a haploid S. pombe, generating 422 single-base substitutions and 119 insertion-deletion mutations (indels) across the 96 replicates. This equates to a base-substitution mutation rate of 2.00 × 10(-10) mutations per site per generation, similar to that reported for the distantly related budding yeast Saccharomyces cerevisiae. However, these two yeast species differ dramatically in their spectrum of base substitutions, the types of indels (S. pombe is more prone to insertions), and the pattern of selection required to counteract a strong AT-biased mutation rate. Overall, our results indicate that GC-biased gene conversion does not play a major role in shaping the nucleotide composition of the S. pombe genome and suggest that the mechanisms of DNA maintenance may have diverged significantly between fission and budding yeasts. Unexpectedly, CpG sites appear to be excessively liable to mutation in both species despite the likely absence of DNA methylation. PMID:26265703

  9. Fine-Structure Mapping of Meiosis-Specific Double-Strand DNA Breaks at a Recombination Hotspot Associated with an Insertion of Telomeric Sequences Upstream of the His4 Locus in Yeast

    PubMed Central

    Xu, F.; Petes, T. D.

    1996-01-01

    Meiotic recombination in Saccharomyces cerevisiae is initiated by double-strand DNA breaks (DSBs). Using two approaches, we mapped the position of DSBs associated with a recombination hotspot created by insertion of telomeric sequences into the region upstream of HIS4. We found that the breaks have no obvious sequence specificity and localize to a region of ~50 bp adjacent to the telomeric insertion. By mapping the breaks and by studies of the exonuclease III sensitivity of the broken ends, we conclude that most of the broken DNA molecules have blunt ends with 3'-hydroxyl groups. PMID:8807286

  10. Spontaneous ATM Gene Reversion in A-T iPSC to Produce an Isogenic Cell Line

    PubMed Central

    Lin, Lucy; Swerdel, Mavis R.; Lazaropoulos, Michael P.; Hoffman, Gary S.; Toro-Ramos, Alana J.; Wright, Jennifer; Lederman, Howard; Chen, Jianmin; Moore, Jennifer C.; Hart, Ronald P.

    2015-01-01

    Summary A spontaneously reverted iPSC line was identified from an A-T subject with heterozygous ATM truncation mutations. The reverted iPSC line expressed ATM protein and was capable of radiation-induced phosphorylation of CHK2 and H2A.X. Genome-wide SNP analysis confirmed a match to source T cells and also to a distinct, non-reverted iPSC line from the same subject. Rearranged T cell receptor sequences predict that the iPSC culture originated as several independently reprogrammed cells that resolved into a single major clone, suggesting that gene correction likely occurred early in the reprogramming process. Gene expression analysis comparing ATM−/− iPSC lines to unrelated ATM+/− cells identifies a large number of differences, but comparing only the isogenic pair of A-T iPSC lines reveals that the primary pathway affected by loss of ATM is a diminished expression of p53-related mRNAs. Gene reversion in culture, although likely a rare event, provided a novel, reverted cell line for studying ATM function. PMID:26677768

  11. Genetic architecture and mechanism of seed number per pod in rapeseed: elucidated through linkage and near-isogenic line analysis

    PubMed Central

    Yang, Yuhua; Shi, Jiaqin; Wang, Xinfa; Liu, Guihua; Wang, Hanzhong

    2016-01-01

    Seed number per pod (SNPP) is one of the major yield components and breeding targets in rapeseed that shows great variation and is invaluable for genetic improvement. To elucidate the genetic architecture and uncover the mechanism of SNPP, we identified five quantitative trait loci (QTLs) using the BnaZNRIL population, which were integrated with those of previous studies by physical map to demonstrate a complex and relatively complete genetic architecture of SNPP. A major QTL, qSN.A6, was successfully fine-mapped from 1910 to 267 kb using near-isogenic line (NIL). In addition, qSN.A6 exhibited an antagonistic pleiotropy on seed weight (SW), which is caused by a physiological interaction in which SNPP acts “upstream” of SW. Because the negative effect of qSN.A6 on SW cannot fully counteract its positive effect on SNPP, it also enhanced the final yield (17.4%), indicating its great potential for utilization in breeding. The following genetic and cytological experiments further confirmed that the different rate of ovule abortion was responsible for the ~5 seed difference between Zhongshuang11 and NIL-qSN.A6. This systematic approach to dissecting the comprehensive genetic architecture of SNPP and characterizing the underlying mechanism has advanced the understanding of SNPP and will facilitate the development of high-yield cultivars. PMID:27067010

  12. Transcriptome Profile of Near-Isogenic Soybean Lines for β-Conglycinin α-Subunit Deficiency during Seed Maturation

    PubMed Central

    Song, Bo; An, Lixin; Han, Yanjing; Gao, Hongxiu; Ren, Hongbo; Zhao, Xue; Wei, Xiaoshuang; Krishnan, Hari B.

    2016-01-01

    Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max) near-isogenic line (cgy-2-NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of β-conglycinin. To identify α-null-related transcriptional changes, the gene expressions of cgy-2-NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF). Seeds at 18 DAF served as the control. Comparison of the transcript profiles identified 3,543 differentially expressed genes (DEGs) between the two genotypes, with 2,193 genes downregulated and 1,350 genes upregulated. The largest numbers of DEGs were identified at 55 DAF. The DEGs identified at 25 DAF represented a unique pattern of GO category distributions. KEGG pathway analyses identified 541 altered metabolic pathways in cgy-2-NIL. At 18DAF, 12 DEGs were involved in arginine and proline metabolism. The cgy-2 allele in the homozygous form modified the expression of several Cupin allergen genes. The cgy-2 allele is an alteration of a functional allele that is closely related to soybean protein amino acid quality, and is useful for hypoallergenic soybean breeding programs that aim to improve seed protein quality. PMID:27532666

  13. Transcriptome Profile of Near-Isogenic Soybean Lines for β-Conglycinin α-Subunit Deficiency during Seed Maturation.

    PubMed

    Song, Bo; An, Lixin; Han, Yanjing; Gao, Hongxiu; Ren, Hongbo; Zhao, Xue; Wei, Xiaoshuang; Krishnan, Hari B; Liu, Shanshan

    2016-01-01

    Crossing, backcrossing, and molecular marker-assisted background selection produced a soybean (Glycine max) near-isogenic line (cgy-2-NIL) containing the cgy-2 allele, which is responsible for the absence of the allergenic α-subunit of β-conglycinin. To identify α-null-related transcriptional changes, the gene expressions of cgy-2-NIL and its recurrent parent DN47 were compared using Illumina high-throughput RNA-sequencing of samples at 25, 35, 50, and 55 days after flowering (DAF). Seeds at 18 DAF served as the control. Comparison of the transcript profiles identified 3,543 differentially expressed genes (DEGs) between the two genotypes, with 2,193 genes downregulated and 1,350 genes upregulated. The largest numbers of DEGs were identified at 55 DAF. The DEGs identified at 25 DAF represented a unique pattern of GO category distributions. KEGG pathway analyses identified 541 altered metabolic pathways in cgy-2-NIL. At 18DAF, 12 DEGs were involved in arginine and proline metabolism. The cgy-2 allele in the homozygous form modified the expression of several Cupin allergen genes. The cgy-2 allele is an alteration of a functional allele that is closely related to soybean protein amino acid quality, and is useful for hypoallergenic soybean breeding programs that aim to improve seed protein quality. PMID:27532666

  14. Identification of Soybean Proteins and Genes Differentially Regulated in Near Isogenic Lines Differing in Resistance to Aphid Infestation.

    PubMed

    Brechenmacher, Laurent; Nguyen, Tran Hong Nha; Zhang, Ning; Jun, Tae-Hwan; Xu, Dong; Mian, M A Rouf; Stacey, Gary

    2015-10-01

    Soybean aphid is an important pest causing significant yield losses. The Rag2 locus confers resistance to soybean aphid biotypes 1 and 2. Transcriptomic and proteomic analyses were done over a 48 h period after aphid infestation using near isogenic lines (NILs) differing at the Rag2 locus. Comparing the Rag2 and/or rag2 lines identified 3445 proteins, of which 396 were differentially regulated between the two lines, including proteins involved in cell wall metabolism, carbohydrate metabolism, and stress response. RNA-seq transcriptomic analysis identified 2361 genes significantly regulated between the resistant and susceptible lines. Genes upregulated in the Rag2 line were annotated as being involved in cell wall, secondary, and hormone metabolism as well as in stress, signaling, and transcriptional responses. Genes downregulated in the Rag2 line were annotated as being involved in photosynthesis and carbon metabolism. Interestingly, two genes (unknown and mitochondrial protease) located within the defined Rag2 locus were expressed significantly higher in the resistant genotype. The expression of a putative NBS-LRR resistant gene within the Rag2 locus was not different between the two soybean lines, but a second NBL-LRR gene located just at the border of the defined Rag2 locus was. Therefore, this gene may be a candidate R gene controlling aphid resistance. PMID:26350764

  15. Functional analysis of a chromosomal deletion associated with myelodysplastic syndromes using isogenic human induced pluripotent stem cells

    PubMed Central

    Kotini, Andriana G; Chang, Chan-Jung; Boussaad, Ibrahim; Delrow, Jeffrey J; Dolezal, Emily K; Nagulapally, Abhinav B; Perna, Fabiana; Fishbein, Gregory A; Klimek, Virginia M; Hawkins, R David; Huangfu, Danwei; Murry, Charles E; Graubert, Timothy; Nimer, Stephen D; Papapetrou, Eirini P

    2015-01-01

    Chromosomal deletions associated with human diseases, such as cancer are common, but synteny issues complicate modeling of these deletions in mice. We use cellular reprogramming and genome engineering to functionally dissect the loss of chromosome 7q [del(7q)], a somatic cytogenetic abnormality present in myelodysplastic syndromes (MDS). We derive del(7q)- and isogenic karyotypically normal induced pluripotent stem cells (iPSCs) from hematopoietic cells of MDS patients and show that the del(7q) iPSCs recapitulate disease-associated phenotypes, including impaired hematopoietic differentiation. These disease phenotypes are rescued by spontaneous dosage correction and can be reproduced in karyotypically normal cells by engineering hemizygosity of defined chr7q segments, in a 20 Mb region. We use a phenotype-rescue screen to identify candidate haploinsufficient genes that might mediate the del(7q)- hematopoietic defect. Our approach highlights the utility of human iPSCs both for functional mapping of disease-associated large-scale chromosomal deletions and for discovery of haploinsufficient genes. PMID:25798938

  16. Genetic architecture and mechanism of seed number per pod in rapeseed: elucidated through linkage and near-isogenic line analysis.

    PubMed

    Yang, Yuhua; Shi, Jiaqin; Wang, Xinfa; Liu, Guihua; Wang, Hanzhong

    2016-01-01

    Seed number per pod (SNPP) is one of the major yield components and breeding targets in rapeseed that shows great variation and is invaluable for genetic improvement. To elucidate the genetic architecture and uncover the mechanism of SNPP, we identified five quantitative trait loci (QTLs) using the BnaZNRIL population, which were integrated with those of previous studies by physical map to demonstrate a complex and relatively complete genetic architecture of SNPP. A major QTL, qSN.A6, was successfully fine-mapped from 1910 to 267 kb using near-isogenic line (NIL). In addition, qSN.A6 exhibited an antagonistic pleiotropy on seed weight (SW), which is caused by a physiological interaction in which SNPP acts "upstream" of SW. Because the negative effect of qSN.A6 on SW cannot fully counteract its positive effect on SNPP, it also enhanced the final yield (17.4%), indicating its great potential for utilization in breeding. The following genetic and cytological experiments further confirmed that the different rate of ovule abortion was responsible for the ~5 seed difference between Zhongshuang11 and NIL-qSN.A6. This systematic approach to dissecting the comprehensive genetic architecture of SNPP and characterizing the underlying mechanism has advanced the understanding of SNPP and will facilitate the development of high-yield cultivars. PMID:27067010

  17. Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome

    PubMed Central

    Bellin, Milena; Casini, Simona; Davis, Richard P; D'Aniello, Cristina; Haas, Jessica; Ward-van Oostwaard, Dorien; Tertoolen, Leon G J; Jung, Christian B; Elliott, David A; Welling, Andrea; Laugwitz, Karl-Ludwig; Moretti, Alessandra; Mummery, Christine L

    2013-01-01

    Patient-specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long-QT syndromes (LQTS). To study the LQT2-associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC-derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC-derived CMs. Further characterization of N996I-HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype. PMID:24213244

  18. Comparative metabolomics profiling of isogenic KRAS wild type and mutant NSCLC cells in vitro and in vivo

    PubMed Central

    Brunelli, Laura; Caiola, Elisa; Marabese, Mirko; Broggini, Massimo; Pastorelli, Roberta

    2016-01-01

    Oncogenes induce metabolic reprogramming on cancer cells. Recently, G12C KRAS mutation in isogenic NSCLC cell line has been shown to be a key player in promoting metabolic rewiring mainly through the regulation of glutamine metabolism to fuel growth and proliferation. Even though cell lines possessing many of the genetic backgrounds of the primary cancer they derive from could be a valuable pre-clinical model, they do not have the additional complexity present in the whole tumor that impact metabolism. This preliminary study is aimed to explore how cancer cell metabolism in culture might recapitulate the metabolic alterations present in vivo. Our result highlighted that the gross metabolic changes observed in G12C KRAS mutant cells growing in culture were also maintained in the derived xenograft model, suggesting that a simple in vitro cell model can give important insights into the metabolic alterations induced by cancer. This is of relevance for guiding effective targeting of those metabolic traits that underlie tumor progression and anticancer treatment responses. PMID:27329432

  19. Impact of Diuraphis noxia and Rhopalosiphum padi (Hemiptera: Aphididae) on primary physiology of four near-isogenic wheat lines.

    PubMed

    Macedo, Tulio B; Peterson, Robert K D; Weaver, David K; Ni, Xinzhi

    2009-02-01

    The impact of feeding injury by the Russian wheat aphid, Diuraphis noxia (Mordvilko) (Hemiptera: Aphididae), and bird cherry-oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae) on susceptible and resistant wheat, Triticum aestivum L., near-isogenic lines 'Tugela' (susceptible), Tugela-Dn1 (antibiotic), Tugela-Dn2 (tolerant), and Tugela-Dn5 (antixenotic) was evaluated by assessing photosynthetic parameters. Photosynthesis and closely related parameters, pigment composition, and nonstructural carbohydrates were measured at 1, 3, and 9 d after aphids were introduced on plants maintained under greenhouse conditions. Overall, R. padi had a higher reproductive capacity within a period of 9 d compared with D. noxia on all lines except Tugela-Dn2. Although the visible injury symptoms associated with aphid injury can be highly species specific, the data indicate that photosynthetic reduction is a common physiological pattern of wheat response to aphid feeding, irrespective of chlorosis elicitation. Although both aphids negatively affected net photosynthesis, D. noxia had a greater impact than R. padi, even when aphid numbers were considerably fewer for D. noxia (100-150 aphids per plant) compared with R. padi (> 200 aphids per plant). The photosynthetic pigment and carbohydrate data suggest that the initial net photosynthesis reduction elicited by aphid feeding may not be directly related to the light reaction portion of the photosynthetic pathway via pigment losses. It is also unlikely that source-sink manipulation is the primary cause for the observed short-term inhibition of photosynthesis. PMID:19253663

  20. Near-isogenic lines of Triticum aestivum with distinct modes of resistance exhibit dissimilar transcriptional regulation during Diuraphis noxia feeding

    PubMed Central

    Botha, Anna-Maria; van Eck, Leon; Burger, N. Francois V.; Swanevelder, Zacharias H.

    2014-01-01

    ABSTRACT Russian wheat aphid (Diuraphis noxia, Kurdjumov) feeding on susceptible Triticum aestivum L. leads to leaf rolling, chlorosis and plant death – symptoms not present in resistant lines. Although the effects of several D. noxia (Dn) resistance genes are known, none have been isolated or characterized. Wheat varieties expressing different Dn genes exhibit distinct modes of D. noxia resistance, such as antibiosis (Dn1), tolerance (Dn2), and antixenosis (Dn5). However, the mechanism whereby feeding aphids are perceived, and how subsequent transcriptional responses are partitioned into resistance categories, remains unclear. Here we report on downstream events in near-isogenic wheat lines containing different Dn genes after D. noxia biotype SA1 feeding. Transcripts involved in stress, signal transduction, photosynthesis, metabolism and gene regulation were differentially regulated during D. noxia feeding. Expression analyses using RT-qPCR and RNA hybridization, as well as enzyme activity profiling, provide evidence that the timing and intensity of pathways induced are critical in the development of particular modes of resistance. Pathways involved include the generation of kinase signalling cascades that lead to a sustained oxidative burst, and a hypersensitive response that is active during antibiosis. Tolerance is a passive resistance mechanism that acts through repair or de novo synthesis of photosystem proteins. Results further suggest that ethylene-mediated pathways are possibly involved in generating volatile compounds and cell wall fortification during the antixenosic response. PMID:25361582

  1. Canopy Photosynthesis and Its Relationship to Plant Productivity in Near-Isogenic Cotton Lines Differing in Leaf Morphology

    PubMed Central

    Wells, Randy; Meredith, William R.; Williford, J. Ray

    1986-01-01

    A 2-year study was conducted to determine the relationships between plant canopy photosynthesis, canopy light interception, and plant productivity of cotton (Gossypium hirsutum L.) exhibiting differing leaf morphologies. The near-isogenic lines were from a single background (MD 65-11) and represented the leaf shapes Normal (small leaf lobing), Sub-Okra (intermediate leaf lobing), Okra (large leaf lobing), and Super Okra (severe leaf lobing). The F1 of a cross Normal × Okra (intermediate leaf lobing) and the F2 (segregating 1:2:1 for Normal Sub-Okra, and Okra, respectively) were also grown. Reduced plant canopies were produced by Okra and Super Okra lines, which translated into increased light penetration to the ground, and hence, in reduced canopy photosynthesis. Integrated canopy photosynthesis (ICAP) was significantly associated with light interception by the plant canopy. Part of the remaining variability in ICAP was associated with confounding factors associated with plant maturity and other unmeasured genotypic factors. Intermediate (F1 and Sub-Okra) and normal leaf types displayed the largest ICAP values in both years. Lint production was positively related to ICAP (R2 = 0.53). The combination of high ICAP values and competitive lint yields indicate that intermediate lobed leaf morphologies offer promise as productive sources of physiological variation for cotton germplasm development. PMID:16665085

  2. The Analysis Of The Strain Influence On Some Yield Elemnts On An Assortment Of Isogenic Wheat Varieties

    NASA Astrophysics Data System (ADS)

    Păniţă, Ovidiu

    2015-09-01

    In the years 2012-2014 on Banu-Maracine DRS were tested an assortment consists of 25 isogenic lines of common wheat (Triticum aestivum ssp.vulgare), with the combination of Rht1&rht8 genes, the analyzed characters being the hight of the strain (cm), the number of seeds/spike, seeds weight/spike (g), no. of spikes/m2, weight of a thousand seeds (WTS) (g) and no. of emerged plants/m2. Based on recorded data and statistical processing of those, they were identified a numbers of links between these characters, links quantified by using the correlation. Regarding the classification based on the average high of the strain, it was found that that there is a strong link between some characters too. Based on component analysis, the no. of seeds/spike and the seeds weight/spikes are components that influence in excess of 83% variance analysis, a total of five genotypes having positive scores for both factors.

  3. Comparative metabolomics profiling of isogenic KRAS wild type and mutant NSCLC cells in vitro and in vivo.

    PubMed

    Brunelli, Laura; Caiola, Elisa; Marabese, Mirko; Broggini, Massimo; Pastorelli, Roberta

    2016-01-01

    Oncogenes induce metabolic reprogramming on cancer cells. Recently, G12C KRAS mutation in isogenic NSCLC cell line has been shown to be a key player in promoting metabolic rewiring mainly through the regulation of glutamine metabolism to fuel growth and proliferation. Even though cell lines possessing many of the genetic backgrounds of the primary cancer they derive from could be a valuable pre-clinical model, they do not have the additional complexity present in the whole tumor that impact metabolism. This preliminary study is aimed to explore how cancer cell metabolism in culture might recapitulate the metabolic alterations present in vivo. Our result highlighted that the gross metabolic changes observed in G12C KRAS mutant cells growing in culture were also maintained in the derived xenograft model, suggesting that a simple in vitro cell model can give important insights into the metabolic alterations induced by cancer. This is of relevance for guiding effective targeting of those metabolic traits that underlie tumor progression and anticancer treatment responses. PMID:27329432

  4. Data supporting the identification of anti-metastatic drug and natural compound targets in isogenic colorectal cancer cells.

    PubMed

    Lee, Jin-Gyun; McKinney, Kimberly Q; Pavlopoulos, Antonis J; Park, Jeong-Hill; Hwang, Sunil

    2014-12-01

    To investigate molecular therapeutic targets in cancer metastasis, comparative proteomic analysis was performed using the isogenic colorectal cancer cell lines SW480 and SW620. Two potential metastasis related molecular targets were identified: fatty acid synthase and histone H4. Subsequently, metastatic SW620 cells were treated with six anti-cancerous components and suppressive effects were observed in target protein expression. Through comprehensive proteomic analysis, three of the tested compounds, oxaliplatin, ginsenoside 20(S)-Rg3 and curcumin, were determined to have a suppressive effect on fatty acid synthase and histone H4 expression [1]. The current article contains one table exhibiting a list of proteins differentially expressed in metastatic SW620 cell lines compared to the primary SW480 cell line (Supplementary Table 1). Additionally, six tables demonstrate proteome changes in SW620 resulting from the treatment of three chemotherapeutics and three natural components (Supplementary Tables 1-7). The anti-metastatic components revealed by the current proteomic analysis represent promising chemotherapeutic candidates for the treatment of colorectal adenocarcinoma. PMID:26217691

  5. Yeast Pol4 Promotes Tel1-Regulated Chromosomal Translocations

    PubMed Central

    Sastre-Moreno, Guillermo; Aguilera, Andrés; Blanco, Luis

    2013-01-01

    DNA double-strand breaks (DSBs) are one of the most dangerous DNA lesions, since their erroneous repair by nonhomologous end-joining (NHEJ) can generate harmful chromosomal rearrangements. PolX DNA polymerases are well suited to extend DSB ends that cannot be directly ligated due to their particular ability to bind to and insert nucleotides at the imperfect template-primer structures formed during NHEJ. Herein, we have devised genetic assays in yeast to induce simultaneous DSBs in different chromosomes in vivo. The repair of these breaks in trans could result in reciprocal chromosomal translocations that were dependent on classical Ku-dependent NHEJ. End-joining events leading to translocations were mainly based on the formation of short base pairing between 3′-overhanging DNA ends coupled to gap-filling DNA synthesis. A major proportion of these events were specifically dependent on yeast DNA polymerase Pol4 activity. In addition, we have discovered that Pol4-Thr540 amino acid residue can be phosphorylated by Tel1/ATM kinase, which could modulate Pol4 activity during NHEJ. Our data suggest that the role of Tel1 in preventing break-induced chromosomal translocations can, to some extent, be due to its stimulating effect on gap-filling activity of Pol4 to repair DSBs in cis. Overall, this work provides further insight to the molecular mechanisms of DSB repair by NHEJ and presents a new perspective to the understanding of how chromosomal translocations are formed in eukaryotic cells. PMID:23874240

  6. Debaryomyces mycophilus sp. nov., a siderophore-dependent yeast isolated from woodlice.

    PubMed

    Thanh, Vu Nguyen; Van Dyk, Martha S; Wingfield, Michael J

    2002-08-01

    Four strains of an ascogenous yeast were isolated from the guts of the woodlice species Armadillidium vulgare (Latreille). This yeast differed from all known yeasts by its inability to grow in culture without the presence of a metabolite produced by some common soil fungi such as Cladosporium cladosporioides, Aspergillus alliaceus, and Penicillium spp. Phylogenetic analysis based on 18S rDNA and 26S rDNA (domain D1/D2) sequences indicated that the yeast represents a new taxon in the genus Debaryomyces. The new species Debaryomyces mycophilus is thus proposed. It was, furthermore, shown that the fungal metabolite necessary for growth of D. mycophilus did not provide the yeast with carbon, nitrogen or vitamins. The active compound was partially purified and it was shown that it is a siderophore used by the yeast as a source of iron. The addition of ferrichrome or high concentrations of FeCl(3) to growth media replaced the obligate dependence on a fungal metabolite. Symbiosis among fungi, based on the availability and utilization of iron, is an aspect of mycology that has not previously been recognized. The addition of chelated iron to isolation media could lead to the discovery of many unknown yeasts and fungi. PMID:12702293

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

    PubMed Central

    van Keulen, H; Thomas, D Y

    1982-01-01

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

  8. Interaction Between Yeasts and Zinc

    NASA Astrophysics Data System (ADS)

    Nicola, Raffaele De; Walker, Graeme

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

  9. Yeasts: From genetics to biotechnology

    SciTech Connect

    Russo, S.; Poli, G.; Siman-Tov, R.B.

    1995-12-31

    Yeasts have been known and used in food and alcoholic fermentations ever since the Neolithic Age. In more recent times, on the basis of their peculiar features and history, yeasts have become very important experimental models in both microbiological and genetic research, as well as the main characters in many fermentative production processes. In the last 40 years, advances in molecular biology and genetic engineering have made possible not only the genetic selection of organisms, but also the genetic modification of some of them, especially the simplest of them, such as bacteria and yeasts. These discoveries have led to the availability of new yeast strains fit to fulfill requests of industrial production and fermentation. Moreover, genetically modified and transformed yeasts have been constructed that are able to produce large amounts of biologically active proteins and enzymes. Thus, recombinant yeasts make it easier to produce drugs, biologically active products, diagnostics, and vaccines, by inexpensive and relatively simple techniques. Yeasts are going to become more and more important in the {open_quotes}biotechnological revolution{close_quotes} by virtue of both their features and their very long and safe use in human nutrition and industry. 175 refs., 4 figs., 6 tabs.

  10. Fission yeast septation

    PubMed Central

    Cortés, Juan C. G.; Ramos, Mariona; Osumi, Masako; Pérez, Pilar; Ribas, Juan Carlos

    2016-01-01

    ABSTRACT In animal cells cytokinesis relies on the contraction of an actomyosin ring that pulls the plasma membrane to create a cleavage furrow, whose ingression finally divides the mother cell into two daughter cells. Fungal cells are surrounded by a tough and flexible structure called cell wall, which is considered to be the functional equivalent of the extracellular matrix in animal cells. Therefore, in addition to cleavage furrow ingression, fungal cytokinesis also requires the centripetal formation of a septum wall structure that develops between the dividing cells, whose genesis must be strictly coordinated with both the actomyosin ring closure and plasma membrane ingression. Here we briefly review what is known about the septum structure and composition in the fission yeast Schizosaccharomyces pombe, the recent progress about the relationship between septum biosynthesis and actomyosin ring constriction, and the importance of the septum and ring in the steady progression of the cleavage furrow. PMID:27574536

  11. Fission yeast septation.

    PubMed

    Cortés, Juan C G; Ramos, Mariona; Osumi, Masako; Pérez, Pilar; Ribas, Juan Carlos

    2016-01-01

    In animal cells cytokinesis relies on the contraction of an actomyosin ring that pulls the plasma membrane to create a cleavage furrow, whose ingression finally divides the mother cell into two daughter cells. Fungal cells are surrounded by a tough and flexible structure called cell wall, which is considered to be the functional equivalent of the extracellular matrix in animal cells. Therefore, in addition to cleavage furrow ingression, fungal cytokinesis also requires the centripetal formation of a septum wall structure that develops between the dividing cells, whose genesis must be strictly coordinated with both the actomyosin ring closure and plasma membrane ingression. Here we briefly review what is known about the septum structure and composition in the fission yeast Schizosaccharomyces pombe, the recent progress about the relationship between septum biosynthesis and actomyosin ring constriction, and the importance of the septum and ring in the steady progression of the cleavage furrow. PMID:27574536

  12. Molecular composition of leaves and stems of genetically modified Bt and near-isogenic non-Bt maize--characterization of lignin patterns.

    PubMed

    Poerschmann, Juergen; Gathmann, Achim; Augustin, Juergen; Langer, Uwe; Górecki, Tadeusz

    2005-01-01

    Transformation of crops, including maize (Zea mays L.), with the cry1Ab gene from Bacillus thuringiensis to combat lepidopteran pests results in pleiotropic effects regarding lignin biosynthesis. Lignin patterns in stems and leaves of two genetically modified Bt-maize varieties (Novelis T and Valmont T) were studied along with their non-Bt near-isolines (Nobilis and Prelude, respectively). Molecular-level based thermochemolysis using tetramethylammonium hydroxide (TMAH) in combination with gas chromatography-mass spectrometry (GC-MS) was used to quantitate the total lignin contents and to identify monomeric lignin subunits including p-hydroxyphenyl (P), guaiacyl (G), and syringyl (S) moieties. The results were supplemented and confirmed by cupric oxide oxidation. The stems of the transgenic lines had higher concentrations of total lignin than the respective isogenic lines: Valmont T/Prelude by 18% and Novelis T/Nobilis by 28%. In contrast, differences in the total lignin concentration of leaves between the transgenic and the respective near-isogenic lines were marginal. There were significant modifications in the ratio of p-hydroxyphenyl/guaiacyl/syringyl molecular marker units of stem lignin between transgenic and isogenic lines. The guaiacyl units (in particular the G18 marker) accounted chiefly for the higher total lignin contents in the transgenic lines. The leaf lignin patterns did not show significant differences in molecular markers between isogenic and transgenic lines. TMAH-induced thermochemolysis--conducted in both the on-line and off-line modes--provided detailed information on the molecular composition of lignin, thus proving superior to the established "wet chemistry" methods of lignin determination. PMID:16091603

  13. Fatty acid patterns of genetically modified Cry3Bb1 expressing Bt-maize MON88017 and its near-isogenic line.

    PubMed

    Poerschmann, Juergen; Rauschen, Stefan; Langer, Uwe; Augustin, Juergen; Górecki, Tadeusz

    2009-01-14

    Fatty acid (FA) profiles of the Bt-maize line MON88017 expressing the Cry3Bb1 protein and its near-isogenic line DKC5143 were examined. Plant compartments under study included leaves taken from different internodes and roots. Sample preparation involved pressurized liquid extraction (PLE) of the biomass, transmethylation of the extracted lipids to give fatty acid methyl esters (FAMEs), and finally GC-MS analysis. The essential quality parameters for the FA profiles included total FA and sum of saturated FA, as well as double-bond index (DBI). FA profiles of the roots--characterized by high concentrations of homomorphic FA including palmitic and stearic acid, along with low concentrations of polyunsaturated surrogates--revealed high similarity between the genetically modified and the near-isogenic line. In contrast, FA profiles of the leaves showed significant differences: higher total FA concentrations and higher DBI were observed for the near-isogenic line. This was overwhelmingly associated with lower concentrations of alpha-linolenic acid (18:3omega3,6,9ccc) in the genetically modified leaf samples. These differences were particularly pronounced for leaves taken from the fourth elongated, above-ground internode. Given the large reported variability in the population of maize lines, MON88017 and its near-isogenic line can be regarded as equivalent with regard to their fatty acid profiles, despite the differences observed for the leaves. Further experiments are needed to assess whether the genetic modification of Bt-maize plants might induce unintended effects with regard to FA profiles. PMID:19067518

  14. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

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

  15. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

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

  16. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

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

  17. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

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

  18. 21 CFR 172.896 - Dried yeasts.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

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

  19. Navigating yeast genome maintenance with functional genomics.

    PubMed

    Measday, Vivien; Stirling, Peter C

    2016-03-01

    Maintenance of genome integrity is a fundamental requirement of all organisms. To address this, organisms have evolved extremely faithful modes of replication, DNA repair and chromosome segregation to combat the deleterious effects of an unstable genome. Nonetheless, a small amount of genome instability is the driver of evolutionary change and adaptation, and thus a low level of instability is permitted in populations. While defects in genome maintenance almost invariably reduce fitness in the short term, they can create an environment where beneficial mutations are more likely to occur. The importance of this fact is clearest in the development of human cancer, where genome instability is a well-established enabling characteristic of carcinogenesis. This raises the crucial question: what are the cellular pathways that promote genome maintenance and what are their mechanisms? Work in model organisms, in particular the yeast Saccharomyces cerevisiae, has provided the global foundations of genome maintenance mechanisms in eukaryotes. The development of pioneering genomic tools inS. cerevisiae, such as the systematic creation of mutants in all nonessential and essential genes, has enabled whole-genome approaches to identifying genes with roles in genome maintenance. Here, we review the extensive whole-genome approaches taken in yeast, with an emphasis on functional genomic screens, to understand the genetic basis of genome instability, highlighting a range of genetic and cytological screening modalities. By revealing the biological pathways and processes regulating genome integrity, these analyses contribute to the systems-level map of the yeast cell and inform studies of human disease, especially cancer. PMID:26323482

  20. Ctf3p, the Mis6 budding yeast homolog, interacts with Mcm22p and Mcm16p at the yeast outer kinetochore.

    PubMed

    Measday, Vivien; Hailey, Dale W; Pot, Isabelle; Givan, Scott A; Hyland, Katherine M; Cagney, Gerard; Fields, Stan; Davis, Trisha N; Hieter, Philip

    2002-01-01

    The budding yeast kinetochore is composed of an inner and outer protein complex, which binds to centromere (CEN) DNA and attaches to microtubules. We performed a genetic synthetic dosage lethality screen to identify novel kinetochore proteins in a collection of chromosome transmission fidelity mutants. Our screen identified several new kinetochore-related proteins including YLR381Wp/Ctf3p, which is a member of a conserved family of centromere-binding proteins. Ctf3p interacts with Mcm22p, Mcm16p, and the outer kinetochore protein Ctf19p. We used chromatin immunoprecipitation to demonstrate that Ctf3p, Mcm22p, and Mcm16p bind to CEN DNA in a Ctf19p-dependent manner. In addition, Ctf3p, Mcm22p, and Mcm16p have a localization pattern similar to other kinetochore proteins. The fission yeast Ctf3p homolog, Mis6, is required for loading of a CENP-A centromere specific histone, Cnp1, onto centromere DNA. We find however that Ctf3p is not required for loading of the budding yeast CENP-A homolog, Cse4p, onto CEN DNA. In contrast, Ctf3p and Ctf19p fail to bind properly to the centromere in a cse4-1 mutant strain. We conclude that the requirements for CENP-A loading onto centromere DNA differ in fission versus budding yeast. PMID:11782448

  1. Marine yeast isolation and industrial application

    PubMed Central

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

    2014-01-01

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

  2. Free-Floating DNA: A New Strand of Technology from the Old Spool of Thought.

    ERIC Educational Resources Information Center

    Wegmann, Larry

    1989-01-01

    A procedure for extracting DNA from yeast using common reagents and equipment is given. Suggestions for variations are provided. A second activity for building a model of DNA from toothpicks is included. (CW)

  3. Enumeration and rapid identification of yeasts during extraction processes of extra virgin olive oil in Tuscany.

    PubMed

    Mari, Eleonora; Guerrini, Simona; Granchi, Lisa; Vincenzini, Massimo

    2016-06-01

    The aim of this study was to evaluate the occurrence of yeast populations during different olive oil extraction processes, carried out in three consecutive years in Tuscany (Italy), by analysing crushed pastes, kneaded pastes, oil from decanter and pomaces. The results showed yeast concentrations ranging between 10(3) and 10(5) CFU/g or per mL. Seventeen dominant yeast species were identified by random amplified polymorphic DNA with primer M13 and their identification was confirmed by restriction fragments length polymorphism of ribosomal internal transcribed spacer and sequencing rRNA genes. The isolation frequencies of each species in the collected samples pointed out that the occurrence of the various yeast species in olive oil extraction process was dependent not only on the yeasts contaminating the olives but also on the yeasts colonizing the plant for oil extraction. In fact, eleven dominant yeast species were detected from the washed olives, but only three of them were also found in oil samples at significant isolation frequency. On the contrary, the most abundant species in oil samples, Yamadazyma terventina, did not occur in washed olive samples. These findings suggest a phenomenon of contamination of the plant for oil extraction that selects some yeast species that could affect the quality of olive oil. PMID:27116959

  4. Polyphenolic substrates and dyes degradation by yeasts from 25 de Mayo/King George Island (Antarctica).

    PubMed

    Rovati, José I; Pajot, Hipólito F; Ruberto, Lucas; Mac Cormack, Walter; Figueroa, Lucía I C

    2013-11-01

    Antarctica offers a range of extreme climatic conditions, such as low temperatures, high solar radiation and low nutrient availability, and constitutes one of the harshest environments on Earth. Despite that, it has been successfully colonized by ’cold-loving’ fungi, which play a key role in decomposition cycles in cold ecosystems. However, knowledge about the ecological role of yeasts in nutrient or organic matter recycling/mineralization remains highly fragmentary. The aim of this work was to study the yeast microbiota in samples collected on 25 de Mayo/King George Island regarding the scope of their ability to degrade polyphenolic substrates such as lignin and azo dyes. Sixty-one yeast isolates were obtained from 37 samples, including soil, rocks, wood and bones. Molecular analyses based on rDNA sequences revealed that 35 yeasts could be identified at the species level and could be classified in the genera Leucosporidiella, Rhodotorula, Cryptococcus, Bullera and Candida. Cryptococcus victoriae was by far the most ubiquitous species. In total, 33% of the yeast isolates examined showed significant activity for dye decolorization, 25% for laccase activity and 38% for ligninolytic activity. Eleven yeasts did not show positive activity in any of the assays performed and no isolates showed positive activity across all tested substrates. A high diversity of yeasts were isolated in this work, possibly including undescribed species and conspicuous Antarctic yeasts, most of them belonging to oligotrophic, slow-growing and metabolically diverse basidiomycetous genera. PMID:24298603

  5. Studying the Replicative Life Span of Yeast Cells

    PubMed Central

    Sinclair, David A.

    2016-01-01

    The budding yeast Saccharomyces cerevisiae is a useful model for elucidating the pathways that control life span and the influence of environmental factors, such as calorie restriction (CR). For 75 years, CR has been studied for its ability to delay diseases of aging in mammals, from cancer to cardiovascular disease (McCay et al., Nutr Rev 33:241–243, 1975). In many other species, reducing calorie intake extends life span, including unicellular organisms (Jiang et al., FASEB J 14:2135–2137, 2000; Lin et al., Science 289:2126–2128, 2000), invertebrates (Rogina and Helfand, Proc Natl Acad Sci U S A 101:15998–16003, 2004), and rodents (Martín-Montalvo et al., Oncogene 30:505–520, 2011). Here we describe how to calorically restrict yeast cells, the methods used to determine the replicative life span (RLS) of budding yeast cells, how to selectively kill daughter cells using the mother enrichment program (MEP), how to measure recombination frequency at the rDNA locus, how to isolate large quantities of old cells, and how to analyze the circular forms of DNA known as extrachromosomal rDNA circles (ERCs), a cause of aging in S. cerevisiae (Petes, Cell 19:765–774, 1980; Sinclair and Guarente, Cell 91:1033–1042, 1997; Defossez et al., Mol Cell 3:447–455, 1999). PMID:23929097

  6. Microscopy of Fission Yeast Sexual Lifecycle.

    PubMed

    Vjestica, Aleksandar; Merlini, Laura; Dudin, Omaya; Bendezu, Felipe O; Martin, Sophie G

    2016-01-01

    The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores. PMID:27022830

  7. A Genetic Incompatibility Accelerates Adaptation in Yeast.

    PubMed

    Bui, Duyen T; Dine, Elliot; Anderson, James B; Aquadro, Charles F; Alani, Eric E

    2015-07-01

    During mismatch repair (MMR) MSH proteins bind to mismatches that form as the result of DNA replication errors and recruit MLH factors such as Mlh1-Pms1 to initiate excision and repair steps. Previously, we identified a negative epistatic interaction involving naturally occurring polymorphisms in the MLH1 and PMS1 genes of baker's yeast. Here we hypothesize that a mutagenic state resulting from this negative epistatic interaction increases the likelihood of obtaining beneficial mutations that can promote adaptation to stress conditions. We tested this by stressing yeast strains bearing mutagenic (incompatible) and non-mutagenic (compatible) mismatch repair genotypes. Our data show that incompatible populations adapted more rapidly and without an apparent fitness cost to high salt stress. The fitness advantage of incompatible populations was rapid but disappeared over time. The fitness gains in both compatible and incompatible strains were due primarily to mutations in PMR1 that appeared earlier in incompatible evolving populations. These data demonstrate a rapid and reversible role (by mating) for genetic incompatibilities in accelerating adaptation in eukaryotes. They also provide an approach to link experimental studies to observational population genomics. PMID:26230253

  8. Anhidrotic ectodermal dysplasia gene region cloned in yeast artificial chromosomes

    SciTech Connect

    Kere, J. |; Grzeschik, K.H.; Limon, J.; Gremaud, M.; Schlessinger, D.; De La Chapelle, A.

    1993-05-01

    Anhidrotic ectodermal dysplasia (EDA), an X-chromosomal recessive disorder, is expressed in a few females with chromosomal translocations involving bands Xq12-q13. Using available DNA markers from the region and somatic cell hybrids the authors mapped the X-chromosomal breakpoints in two such translocations. The breakpoints were further mapped within a yeast artificial chromosome contig constructed by chromosome walking techniques. Genomic DNA markers that map between the two translocation breakpoints were recovered representing putative portions of the EDA gene. 32 refs., 3 figs., 1 tab.

  9. Structure of the yeast Bre1 RING domain

    PubMed Central

    Kumar, Pankaj; Wolberger, Cynthia

    2015-01-01

    Monoubiquitination of histone H2B at Lys123 in yeast plays a critical role in regulating transcription, mRNA export, DNA replication and the DNA damage response. The RING E3 ligase, Bre1, catalyzes monoubiquitination of H2B in concert with the E2 ubiquitin conjugating enzyme, Rad6. The crystal structure of a C-terminal fragment of Bre1 shows that the catalytic RING domain is preceded by an N-terminal helix that mediates coiled-coil interactions with a crystallographically related monomer. Homology modeling suggests that the human homologue of Bre1, RNF20/RNF40, heterodimerizes through similar coiled coil interactions. PMID:25864391

  10. Homing endonuclease target determination using SELEX adapted for yeast surface display.

    PubMed

    Jacoby, Kyle; Scharenberg, Andrew M

    2014-01-01

    Knowing the target sequence of a DNA-binding protein is vital in obtaining fundamental characteristics of the protein and evaluating properties of the protein-DNA interaction. For example, novel homing endonucleases cannot be proven to be functional until a predicted target site is tested. Unfortunately, target site prediction is not always easy, or even possible, depending on the amount of sequence data available. Here we describe a modification of SELEX using yeast surface display that can quickly and inexpensively resolve DNA-binding targets in high throughput for proteins without any prior assumptions or knowledge regarding the target site. This protocol is easily integrated into the yeast surface display pipeline and is leveraged by the expansive number of existing tools for both SELEX and yeast surface display. PMID:24510268

  11. The Yeast Sphingolipid Signaling Landscape

    PubMed Central

    Montefusco, David J.; Matmati, Nabil

    2014-01-01

    Sphingolipids are recognized as signaling mediators in a growing number of pathways, and represent potential targets to address many diseases. The study of sphingolipid signaling in yeast has created a number of breakthroughs in the field, and has the potential to lead future advances. The aim of this article is to provide an inclusive view of two major frontiers in yeast sphingolipid signaling. In the first section, several key studies in the field of sphingolipidomics are consolidated to create a yeast sphingolipidome that ranks nearly all known sphingolipid species by their level in a resting yeast cell. The second section presents an overview of most known phenotypes identified for sphingolipid gene mutants, presented with the intention of illuminating not yet discovered connections outside and inside of the field. PMID:24220500

  12. Growth and manipulation of yeast.

    PubMed

    Treco, D A; Reynolds, A; Lundblad, V

    2001-05-01

    This unit describes preparation of selected media for growing yeast and also discusses strain storage and revival. Protocols are provided for the assay of beta-galactosidase in liquid culture and for transformation using lithium acetate. PMID:18429086

  13. RNA-Seq Analysis of a Soybean Near-Isogenic Line Carrying Bacterial Leaf Pustule-Resistant and -Susceptible Alleles

    PubMed Central

    Kim, Kil Hyun; Kang, Yang Jae; Kim, Dong Hyun; Yoon, Min Young; Moon, Jung-Kyung; Kim, Moon Young; Van, Kyujung; Lee, Suk-Ha

    2011-01-01

    Bacterial leaf pustule (BLP) disease is caused by Xanthomonas axonopodis pv. glycines (Xag). To investigate the plant basal defence mechanisms induced in response to Xag, differential gene expression in near-isogenic lines (NILs) of BLP-susceptible and BLP-resistant soybean was analysed by RNA-Seq. Of a total of 46 367 genes that were mapped to soybean genome reference sequences, 1978 and 783 genes were found to be up- and down-regulated, respectively, in the BLP-resistant NIL relative to the BLP-susceptible NIL at 0, 6, and 12h after inoculation (hai). Clustering analysis revealed that these genes could be grouped into 10 clusters with different expression patterns. Functional annotation based on gene ontology (GO) categories was carried out. Among the putative soybean defence response genes identified (GO:0006952), 134 exhibited significant differences in expression between the BLP-resistant and -susceptible NILs. In particular, pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors and the genes induced by these receptors were highly expressed at 0 hai in the BLP-resistant NIL. Additionally, pathogenesis-related (PR)-1 and -14 were highly expressed at 0 hai, and PR-3, -6, and -12 were highly expressed at 12 hai. There were also significant differences in the expression of the core JA-signalling components MYC2 and JASMONATE ZIM-motif. These results indicate that powerful basal defence mechanisms involved in the recognition of PAMPs or DAMPs and a high level of accumulation of defence-related gene products may contribute to BLP resistance in soybean. PMID:21987089

  14. Compositional differences between near-isogenic GM and conventional maize hybrids are associated with backcrossing practices in conventional breeding.

    PubMed

    Venkatesh, Tyamagondlu V; Cook, Kevin; Liu, Bing; Perez, Timothy; Willse, Alan; Tichich, Ryan; Feng, Ping; Harrigan, George G

    2015-02-01

    Here, we show that differences between genetically modified (GM) and non-GM comparators cannot be attributed unequivocally to the GM trait, but arise because of minor genomic differences in near-isogenic lines. Specifically, this study contrasted the effect of three GM traits (drought tolerance, MON 87460; herbicide resistance, NK603; insect protection, MON 89034) on maize grain composition relative to the effects of residual genetic variation from backcrossing. Important features of the study included (i) marker-assisted backcrossing to generate genetically similar inbred variants for each GM line, (ii) high-resolution genotyping to evaluate the genetic similarity of GM lines to the corresponding recurrent parents and (iii) introgression of the different GM traits separately into a wide range of genetically distinct conventional inbred lines. The F1 hybrids of all lines were grown concurrently at three replicated field sites in the United States during the 2012 growing season, and harvested grain was subjected to compositional analysis. Proximates (protein, starch and oil), amino acids, fatty acids, tocopherols and minerals were measured. The number of statistically significant differences (α = 0.05), as well as magnitudes of difference, in mean levels of these components between corresponding GM variants was essentially identical to that between GM and non-GM controls. The largest sources of compositional variation were the genetic background of the different conventional inbred lines (males and females) used to generate the maize hybrids and location. The lack of any compositional effect attributable to GM suggests the development of modern agricultural biotechnology has been accompanied by a lack of any safety or nutritional concerns. PMID:25196222

  15. Molecular response to the pathogen Phytophthora sojae among ten soybean near isogenic lines revealed by comparative transcriptomics

    PubMed Central

    2014-01-01

    Background Phytophthora root and stem rot (PRR) of soybean, caused by Phytophthora sojae, is controlled by Rps genes. However, little is known regarding the Rps-induced molecular responses to P. sojae and how they actually overlap. We thus sequenced, analyzed, and compared the transcriptomes of 10 near isogenic lines (NILs), each with a unique Rps gene/allele, and the susceptible parent Williams, pre- and post-inoculation with the pathogen. Results A total of 4,330 differentially expressed genes (DEGs) were identified in Williams versus 2,014 to 5,499 DEGs in individual NILs upon inoculation with the pathogen. Comparisons of the DEGs between the NILs and Williams identified incompatible interaction genes (IIGs) and compatible interaction genes (CIGs). Hierarchical cluster and heatmap analyses consistently grouped the NILs into three clusters: Cluster I (Rps1-a), Cluster II (Rps1-b, 1-c and 1-k) and Cluster III (Rps3-a, 3-b, 3-c, 4, 5, and 6), suggesting an overlap in Rps-induced defense signaling among certain NILs. Gene ontology (GO) analysis revealed associations between members of the WRKY family and incompatible reactions and between a number of phytohormone signaling pathways and incompatible/compatible interactions. These associations appear to be distinguished according to the NIL clusters. Conclusions This study characterized genes and multiple branches of putative regulatory networks associated with resistance to P. sojae in ten soybean NILs, and depicted functional “fingerprints” of individual Rps-mediated resistance responses through comparative transcriptomic analysis. Of particular interest are dramatic variations of detected DEGs, putatively involved in ethylene (ET)-, jasmonic acid (JA)-, (reactive oxygen species) ROS-, and (MAP-kinase) MAPK- signaling, among these soybean NILs, implicating their important roles of these signaling in differentiating molecular defense responses. We hypothesize that different timing and robustness in defense

  16. The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats.

    PubMed

    Garcia, J P; Beingesser, J; Fisher, D J; Sayeed, S; McClane, B A; Posthaus, H; Uzal, F A

    2012-06-15

    Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch's postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis. PMID:22296994

  17. The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats

    PubMed Central

    Garcia, J. P.; Beingesser, J.; Fisher, D. J.; Sayeed, S.; McClane, B. A.; Posthaus, H.; Uzal, F. A.

    2012-01-01

    Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch’s postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24 h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24 h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis. PMID:22296994

  18. Use of near-isogenic wheat lines to determine the glutenin composition and functionality requirements for flour tortillas.

    PubMed

    Mondal, Suchismita; Tilley, Michael; Alviola, Juma Novi; Waniska, Ralph D; Bean, Scott R; Glover, Karl D; Hays, Dirk B

    2008-01-01

    In wheat ( Triticum aestivum L), the synthesis of high molecular weight (HMW) glutenins (GS) is controlled by three heterologous genetic loci present on the long arms of group 1 wheat chromosomes. The loci Glu-A1, Glu-B1, and Glu-D1 and their allelic variants play important roles in the functional properties of wheat flour. This study focused on understanding the functionality of these protein subunits on tortilla quality. Near-isogenic wheat lines in which one or more of these loci were absent or deleted were used. Tortillas were prepared from each deletion line and the parent lines. The elimination of certain HMW-GS alleles alter distinct but critical aspects of tortilla quality such as diameter, shelf stability, and overall quality. Two deletion lines possessing HMW-GS 17 + 18 at Glu-B1 and deletions in Glu-A1 and Glu-D1 had significantly larger tortilla diameters, yet tortilla shelf life was compromised or unchanged from the parent lines used to develop the deletion lines or the commercial tortilla flour used as a control. Alternatively, a deletion line possessing Glu-A1 and Glu-D1 (HMW-GS 1, 5 + 10) and a deletion in Glu-B1 also significantly improved tortilla diameters. Whereas the increase in diameter was less than the line possessing only HMW-GS 17 + 18 at Glu-B1, the stability of the tortillas were, however, maintained and improved as compared to the parent lines containing a full compliment of HMW-GS. Thus, the presence of subunits 5 + 10 at Glu-D1 alone or in combination with subunit 1 at Glu-A1 appears to provide a compromise of improvement in dough extensibility for improved tortilla diameters while also providing sufficient gluten strength to maintain ideal shelf stability. PMID:18072743

  19. RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and -susceptible alleles.

    PubMed

    Kim, Kil Hyun; Kang, Yang Jae; Kim, Dong Hyun; Yoon, Min Young; Moon, Jung-Kyung; Kim, Moon Young; Van, Kyujung; Lee, Suk-Ha

    2011-12-01

    Bacterial leaf pustule (BLP) disease is caused by Xanthomonas axonopodis pv. glycines (Xag). To investigate the plant basal defence mechanisms induced in response to Xag, differential gene expression in near-isogenic lines (NILs) of BLP-susceptible and BLP-resistant soybean was analysed by RNA-Seq. Of a total of 46 367 genes that were mapped to soybean genome reference sequences, 1978 and 783 genes were found to be up- and down-regulated, respectively, in the BLP-resistant NIL relative to the BLP-susceptible NIL at 0, 6, and 12h after inoculation (hai). Clustering analysis revealed that these genes could be grouped into 10 clusters with different expression patterns. Functional annotation based on gene ontology (GO) categories was carried out. Among the putative soybean defence response genes identified (GO:0006952), 134 exhibited significant differences in expression between the BLP-resistant and -susceptible NILs. In particular, pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors and the genes induced by these receptors were highly expressed at 0 hai in the BLP-resistant NIL. Additionally, pathogenesis-related (PR)-1 and -14 were highly expressed at 0 hai, and PR-3, -6, and -12 were highly expressed at 12 hai. There were also significant differences in the expression of the core JA-signalling components MYC2 and JASMONATE ZIM-motif. These results indicate that powerful basal defence mechanisms involved in the recognition of PAMPs or DAMPs and a high level of accumulation of defence-related gene products may contribute to BLP resistance in soybean. PMID:21987089

  20. Highly-efficient colony PCR method for red yeasts and its application to identify mutations within two leucine auxotroph mutants.

    PubMed

    Lin, Xinping; Yang, Fan; Zhou, Yongjin; Zhu, Zhiwei; Jin, Guojie; Zhang, Sufang; Zhao, Zongbao Kent

    2012-11-01

    Red yeasts hold great promise in the production of microbial lipids and carotenoids. Genetic study of red yeasts has attracted much attention; however, rapid amplification of genes from red yeast samples remains technically challenging. Here a highly efficient method for the preparation of genomic DNA (gDNA) template, which could be directly used for PCR, was developed. Cells from colonies or liquid cultures were collected and sequentially treated by microwave, plMAN5C, proteinase K and boiling (MMPB) in a single tube to give cell lysates that were qualified as PCR templates. Single-copied gDNA fragments o up to 2.8 kb were successfully amplified. We also demonstrated successful application of this method for species in the Ascomycetes and Basidiomycetes and identification of two leucine auxotroph mutants of Rhodotorula glutinis. This method could be widely employed for the screening and genetic engineering of various yeasts. PMID:23065821

  1. Mechanism of gene amplification via yeast autonomously replicating sequences.

    PubMed

    Sehgal, Shelly; Kaul, Sanjana; Dhar, M K

    2015-01-01

    The present investigation was aimed at understanding the molecular mechanism of gene amplification. Interplay of fragile sites in promoting gene amplification was also elucidated. The amplification promoting sequences were chosen from the Saccharomyces cerevisiae ARS, 5S rRNA regions of Plantago ovata and P. lagopus, proposed sites of replication pausing at Ste20 gene locus of S. cerevisiae, and the bend DNA sequences within fragile site FRA11A in humans. The gene amplification assays showed that plasmid bearing APS from yeast and human beings led to enhanced protein concentration as compared to the wild type. Both the in silico and in vitro analyses were pointed out at the strong bending potential of these APS. In addition, high mitotic stability and presence of TTTT repeats and SAR amongst these sequences encourage gene amplification. Phylogenetic analysis of S. cerevisiae ARS was also conducted. The combinatorial power of different aspects of APS analyzed in the present investigation was harnessed to reach a consensus about the factors which stimulate gene expression, in presence of these sequences. It was concluded that the mechanism of gene amplification was that AT rich tracts present in fragile sites of yeast serve as binding sites for MAR/SAR and DNA unwinding elements. The DNA protein interactions necessary for ORC activation are facilitated by DNA bending. These specific bindings at ORC promote repeated rounds of DNA replication leading to gene amplification. PMID:25685838

  2. Mechanism of Gene Amplification via Yeast Autonomously Replicating Sequences

    PubMed Central

    Dhar, M. K.

    2015-01-01

    The present investigation was aimed at understanding the molecular mechanism of gene amplification. Interplay of fragile sites in promoting gene amplification was also elucidated. The amplification promoting sequences were chosen from the Saccharomyces cerevisiae ARS, 5S rRNA regions of Plantago ovata and P. lagopus, proposed sites of replication pausing at Ste20 gene locus of S. cerevisiae, and the bend DNA sequences within fragile site FRA11A in humans. The gene amplification assays showed that plasmid bearing APS from yeast and human beings led to enhanced protein concentration as compared to the wild type. Both the in silico and in vitro analyses were pointed out at the strong bending potential of these APS. In addition, high mitotic stability and presence of TTTT repeats and SAR amongst these sequences encourage gene amplification. Phylogenetic analysis of S. cerevisiae ARS was also conducted. The combinatorial power of different aspects of APS analyzed in the present investigation was harnessed to reach a consensus about the factors which stimulate gene expression, in presence of these sequences. It was concluded that the mechanism of gene amplification was that AT rich tracts present in fragile sites of yeast serve as binding sites for MAR/SAR and DNA unwinding elements. The DNA protein interactions necessary for ORC activation are facilitated by DNA bending. These specific bindings at ORC promote repeated rounds of DNA replication leading to gene amplification. PMID:25685838

  3. A Three-Dimensional Model of the Yeast Genome

    NASA Astrophysics Data System (ADS)

    Noble, William; Duan, Zhi-Jun; Andronescu, Mirela; Schutz, Kevin; McIlwain, Sean; Kim, Yoo Jung; Lee, Choli; Shendure, Jay; Fields, Stanley; Blau, C. Anthony

    Layered on top of information conveyed by DNA sequence and chromatin are higher order structures that encompass portions of chromosomes, entire chromosomes, and even whole genomes. Interphase chromosomes are not positioned randomly within the nucleus, but instead adopt preferred conformations. Disparate DNA elements co-localize into functionally defined aggregates or factories for transcription and DNA replication. In budding yeast, Drosophila and many other eukaryotes, chromosomes adopt a Rabl configuration, with arms extending from centromeres adjacent to the spindle pole body to telomeres that abut the nuclear envelope. Nonetheless, the topologies and spatial relationships of chromosomes remain poorly understood. Here we developed a method to globally capture intra- and inter-chromosomal interactions, and applied it to generate a map at kilobase resolution of the haploid genome of Saccharomyces cerevisiae. The map recapitulates known features of genome organization, thereby validating the method, and identifies new features. Extensive regional and higher order folding of individual chromosomes is observed. Chromosome XII exhibits a striking conformation that implicates the nucleolus as a formidable barrier to interaction between DNA sequences at either end. Inter-chromosomal contacts are anchored by centromeres and include interactions among transfer RNA genes, among origins of early DNA replication and among sites where chromosomal breakpoints occur. Finally, we constructed a three-dimensional model of the yeast genome. Our findings provide a glimpse of the interface between the form and function of a eukaryotic genome.

  4. Replication-Associated Recombinational Repair: Lessons from Budding Yeast

    PubMed Central

    Bonner, Jaclyn N.; Zhao, Xiaolan

    2016-01-01

    Recombinational repair processes multiple types of DNA lesions. Though best understood in the repair of DNA breaks, recombinational repair is intimately linked to other situations encountered during replication. As DNA strands are decorated with many types of blocks that impede the replication machinery, a great number of genomic regions cannot be duplicated without the help of recombinational repair. This replication-associated recombinational repair employs both the core recombination proteins used for DNA break repair and the specialized factors that couple replication with repair. Studies from multiple organisms have provided insights into the roles of these specialized factors, with the findings in budding yeast being advanced through use of powerful genetics and methods for detecting DNA replication and repair intermediates. In this review, we summarize recent progress made in this organism, ranging from our understanding of the classical template switch mechanisms to gap filling and replication fork regression pathways. As many of the protein factors and biological principles uncovered in budding yeast are conserved in higher eukaryotes, these findings are crucial for stimulating studies in more complex organisms. PMID:27548223

  5. Calling Card Analysis in Budding Yeast.

    PubMed

    Mayhew, David; Mitra, Robi D

    2016-02-01

    Calling card analysis is a high-throughput method for identifying the genomic binding sites of multiple transcription factors in a single experiment in budding yeast. By tagging a DNA-binding protein with a targeting domain that directs the insertion of the Ty5 retrotransposon, the genomic binding sites for that transcription factor are marked. The transposition locations are then identified en masse by Illumina sequencing. The calling card protocol allows for simultaneous analysis of multiple transcription factors. By cloning barcodes into the Ty5 transposon, it is possible to pair a unique barcode with every transcription factor in the experiment. The method presented here uses expression of transcription factors from their native loci; however, it can also be altered to measure binding sites of transcription factors overexpressed from a plasmid. PMID:26832687

  6. Transformation of yeast with synthetic oligonucleotides.

    PubMed Central

    Moerschell, R P; Tsunasawa, S; Sherman, F

    1988-01-01

    Genomic DNA of the yeast, Saccharomyces cerevisiae, can be conveniently and specifically altered by transforming spheroplasts or lithium acetate-treated cells directly with synthetic oligonucleotides. Altered forms of iso-1-cytochrome c were generated by transforming a cyc1 mutant with oligonucleotides and selecting for at least partially functional revertants; the oligonucleotides contained a sequence that corrected the cyc1 mutation and produced additional alterations at nearby sites. Transformation has been accomplished with oligonucleotides as short as 20 nucleotides and with amounts as low as 100 micrograms. This method of site-directed mutagenesis in vivo has been used to produce alterations in the NH2-terminal region of iso-1-cytochrome c in which the NH2-terminal methionine is excised and the penultimate residue is acetylated. PMID:2829192

  7. A yeast model of FUS/TLS-dependent cytotoxicity.

    PubMed

    Ju, Shulin; Tardiff, Daniel F; Han, Haesun; Divya, Kanneganti; Zhong, Quan; Maquat, Lynne E; Bosco, Daryl A; Hayward, Lawrence J; Brown, Robert H; Lindquist, Susan; Ringe, Dagmar; Petsko, Gregory A

    2011-04-01

    FUS/TLS is a nucleic acid binding protein that, when mutated, can cause a subset of familial amyotrophic lateral sclerosis (fALS). Although FUS/TLS is normally located predominantly in the nucleus, the pathogenic mutant forms of FUS/TLS traffic to, and form inclusions in, the cytoplasm of affected spinal motor neurons or glia. Here we report a yeast model of human FUS/TLS expression that recapitulates multiple salient features of the pathology of the disease-causing mutant proteins, including nuclear to cytoplasmic translocation, inclusion formation, and cytotoxicity. Protein domain analysis indicates that the carboxyl-terminus of FUS/TLS, where most of the ALS-associated mutations are clustered, is required but not sufficient for the toxicity of the protein. A genome-wide genetic screen using a yeast over-expression library identified five yeast DNA/RNA binding proteins, encoded by the yeast genes ECM32, NAM8, SBP1, SKO1, and VHR1, that rescue the toxicity of human FUS/TLS without changing its expression level, cytoplasmic translocation, or inclusion formation. Furthermore, hUPF1, a human homologue of ECM32, also rescues the toxicity of FUS/TLS in this model, validating the yeast model and implicating a possible insufficiency in RNA processing or the RNA quality control machinery in the mechanism of FUS/TLS mediated toxicity. Examination of the effect of FUS/TLS expression on the decay of selected mRNAs in yeast indicates that the nonsense-mediated decay pathway is probably not the major determinant of either toxicity or suppression. PMID:21541368

  8. Biotechnological Applications of Dimorphic Yeasts

    NASA Astrophysics Data System (ADS)

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

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

  9. Rapid multiple-level coevolution in experimental populations of yeast killer and nonkiller strains.

    PubMed

    Pieczynska, Magdalena D; Wloch-Salamon, Dominika; Korona, Ryszard; de Visser, J Arjan G M

    2016-06-01

    Coevolution between different biological entities is considered an important evolutionary mechanism at all levels of biological organization. Here, we provide evidence for coevolution of a yeast killer strain (K) carrying cytoplasmic dsRNA viruses coding for anti-competitor toxins and an isogenic toxin-sensitive strain (S) during 500 generations of laboratory propagation. Signatures of coevolution developed at two levels. One of them was coadaptation of K and S. Killing ability of K first increased quickly and was followed by the rapid invasion of toxin-resistant mutants derived from S, after which killing ability declined. High killing ability was shown to be advantageous when sensitive cells were present but costly when they were absent. Toxin resistance evolved via a two-step process, presumably involving the fitness-enhancing loss of one chromosome followed by selection of a recessive resistant mutation on the haploid chromosome. The other level of coevolution occurred between cell and killer virus. By swapping the killer viruses between ancestral and evolved strains, we could demonstrate that changes observed in both host and virus were beneficial only when combined, suggesting that they involved reciprocal changes. Together, our results show that the yeast killer system shows a remarkable potential for rapid multiple-level coevolution. PMID:27168531

  10. Organization of spacer DNA in chromatin.

    PubMed Central

    Lohr, D; Van Holde, K E

    1979-01-01

    Detailed analysis of the DNA fragment patterns produced by DNase I digestion of yeast, HeLa, and chicken erythrocyte nuclei reveals surprising features of nucleosome phasing. First, the spacer regions in phased yeast chromatin must be of lengths (10m + 5) base pairs, where m = 0, 1, 2,....This feature is not seen in parallel studies of chicken erythrocyte chromatin. The 5-base pair increment in the yeast spacer imposes interesting restraints on the higher order structure of yeast chromatin. Second, we have been able to simulate the DNase I cutting patterns and get good agreement with the observed yeast patterns. Third, three different chromatins show a long range periodicity in the DNase I digest pattern, with a period half that of the staphylococcal nuclease repeat. These results suggest that the amount of chromatin observed in discrete extended-ladder bands is a minimum estimate of phasing and in fact phasing may be a more general feature. Images PMID:392519

  11. Cell-Cycle Analyses Using Thymidine Analogues in Fission Yeast

    PubMed Central

    Anda, Silje; Boye, Erik; Grallert, Beata

    2014-01-01

    Thymidine analogues are powerful tools when studying DNA synthesis including DNA replication, repair and recombination. However, these analogues have been reported to have severe effects on cell-cycle progression and growth, the very processes being investigated in most of these studies. Here, we have analyzed the effects of 5-ethynyl-2′-deoxyuridine (EdU) and 5-Chloro-2′-deoxyuridine (CldU) using fission yeast cells and optimized the labelling procedure. We find that both analogues affect the cell cycle, but that the effects can be mitigated by using the appropriate analogue, short pulses of labelling and low concentrations. In addition, we report sequential labelling of two consecutive S phases using EdU and 5-bromo-2′-deoxyuridine (BrdU). Furthermore, we show that detection of replicative DNA synthesis is much more sensitive than DNA-measurements by flow cytometry. PMID:24551125

  12. Unsuspected pyocyanin effect in yeast under anaerobiosis.

    PubMed

    Barakat, Rana; Goubet, Isabelle; Manon, Stephen; Berges, Thierry; Rosenfeld, Eric

    2014-02-01

    The blue-green phenazine, Pyocyanin (PYO), is a well-known virulence factor produced by Pseudomonas aeruginosa, notably during cystic fibrosis lung infections. It is toxic to both eukaryotic and bacterial cells and several mechanisms, including the induction of oxidative stress, have been postulated. However, the mechanism of PYO toxicity under the physiological conditions of oxygen limitation that are encountered by P. aeruginosa and by target organisms in vivo remains unclear. In this study, wild-type and mutant strains of the yeast Saccharomyces cerevisiae were used as an effective eukaryotic model to determine the toxicity of PYO (100-500 μmol/L) under key growth conditions. Under respiro-fermentative conditions (with glucose as substrate), WT strains and certain H2 O2 -hypersensitive strains showed a low-toxic response to PYO. Under respiratory conditions (with glycerol as substrate) all the strains tested were significantly more sensitive to PYO. Four antioxidants were tested but only N-acetylcysteine was capable of partially counteracting PYO toxicity. PYO did not appear to affect short-term respiratory O2 uptake, but it did seem to interfere with cyanide-poisoned mitochondria through a complex III-dependent mechanism. Therefore, a combination of oxidative stress and respiration disturbance could partly explain aerobic PYO toxicity. Surprisingly, the toxic effects of PYO were more significant under anaerobic conditions. More pronounced effects were observed in several strains including a 'petite' strain lacking mitochondrial DNA, strains with increased or decreased levels of ABC transporters, and strains deficient in DNA damage repair. Therefore, even though PYO is toxic for actively respiring cells, O2 may indirectly protect the cells from the higher anaerobic-linked toxicity of PYO. The increased sensitivity to PYO under anaerobic conditions is not unique to S. cerevisiae and was also observed in another yeast, Candida albicans. PMID:24307284

  13. Riboneogenesis in yeast

    PubMed Central

    Clasquin, Michelle F.; Melamud, Eugene; Singer, Alexander; Gooding, Jessica R.; Xu, Xiaohui; Dong, Aiping; Cui, Hong; Campagna, Shawn R.; Savchenko, Alexei; Yakunin, Alexander F.; Rabinowitz, Joshua D.; Caudy, Amy A.

    2011-01-01

    Summary Gluconeogenesis converts three carbon units into glucose. Here we identify an analogous pathway in Saccharomyces cerevisiae for converting three carbon units into ribose, a component of nucleic acids and nucleotides. This riboneogenic pathway involves the enzyme sedoheptulose-1,7-bisphosphatase (SHB17), whose activity was identified based on accumulation of sedoheptulose-1,7-bisphosphate in the corresponding knockout strain. We determined the crystal structure of Shb17 in complex with sedoheptulose-1,7-bisphosphate, and found that the sugar is bound in the closed furan form in the active site. Like fructose-1,6-bisphosphate, sedoheptulose-1,7-bisphosphate is produced by aldolase, in this case from erythrose 4-phosphate and dihydroxyacetone phosphate. Hydrolysis of sedoheptulose-1,7-bisphosphate by SHB17 provides an energetically favorable input to the non-oxidative pentose phosphate pathway to drive ribose production. Flux through SHB17 is enhanced under conditions when ribose demand is high relative to demand for NADPH, including during ribosome biogenesis in metabolically synchronized yeast cells. Thus, riboneogenesis provides a thermodynamically-driven route of ribose production uncoupled from formation of NADPH. PMID:21663798

  14. Yeast Mitochondrial Transcriptomics

    PubMed Central

    Garcia, Mathilde; Darzacq, Xavier; Devaux, Frederic; Singer, Robert H.; Jacq, Claude

    2016-01-01

    Although 30 years ago it was strongly suggested that some cytoplasmic ribosomes are bound to the surface of yeast mitochondria, the mechanisms and the raison d’ětre of this process are not understood. For instance, it is not perfectly known which of the several hundred nuclearly encoded genes have to be translated to the mitochondrial vicinity to guide the import of the corresponding proteins. One can take advantage of several modern methods to address a number of aspects of the site-specific translation process of messenger ribonucleic acid (mRNA) coding for proteins imported into mitochondria. Three complementary approaches are presented to analyze the spatial distribution of mRNAs coding for proteins imported into mitochondria. Starting from biochemical purifications of mitochondria-bound polysomes, we describe a genomewide approach to classify all the cellular mRNAs according to their physical proximity with mitochondria; we also present real-time quantitative reverse transcription polymerase chain reaction monitoring of mRNA distribution to provide a quantified description of this localization. Finally, a fluorescence microscopy approach on a single living cell is described to visualize the in vivo localization of mRNAs involved in mitochondria biogenesis. PMID:18314748

  15. Synthetic Yeast Cooperation

    NASA Astrophysics Data System (ADS)

    Shou, Wenying; Burton, Justin

    2010-03-01

    Cooperation is wide-spread and has been postulated to drive major transitions in evolution. However, Darwinian selection favors ``cheaters'' that consume benefits without paying a fair cost. How did cooperation evolve against the threat of cheaters? To investigate the evolutionary trajectories of cooperation, we created a genetically tractable system that can be observed as it evolves from inception. The system consists of two engineered yeast strains -- a red-fluorescent strain that requires adenine and releases lysine and a yellow-fluorescent strain that requires lysine and releases adenine. Cells that consume but not supply metabolites would be cheaters. From the properties of two cooperating strains, we calculated and experimentally verified the minimal initial cell densities required for the viability of the cooperative system in the absence of exogenously added adenine and lysine. Strikingly, evolved cooperative systems were viable at 100-fold lower initial cell densities than their ancestors. We are investigating the nature and diversity of pro-cooperation changes, the dynamics of cooperator-cheater cocultures, and the effects of spatial environment on cooperation and cheating.

  16. Metabolic regulation of yeast

    NASA Astrophysics Data System (ADS)

    Fiechter, A.

    1982-12-01

    Metabolic regulation which is based on endogeneous and exogeneous process variables which may act constantly or time dependently on the living cell is discussed. The observed phenomena of the regulation are the result of physical, chemical, and biological parameters. These parameters are identified. Ethanol is accumulated as an intermediate product and the synthesis of biomass is reduced. This regulatory effect of glucose is used for the aerobic production of ethanol. Very high production rates are thereby obtained. Understanding of the regulation mechanism of the glucose effect has improved. In addition to catabolite repression, several other mechanisms of enzyme regulation have been described, that are mostly governed by exogeneous factors. Glucose also affects the control of respiration in a third class of yeasts which are unable to make use of ethanol as a substrate for growth. This is due to the lack of any anaplerotic activity. As a consequence, diauxic growth behavior is reduced to a one-stage growth with a drastically reduced cell yield. The pulse chemostat technique, a systematic approach for medium design is developed and medium supplements that are essential for metabolic control are identified.

  17. Gene conversion plays the major role in controlling the stability of large tandem repeats in yeast.

    PubMed Central

    Gangloff, S; Zou, H; Rothstein, R

    1996-01-01

    The genomic stability of the rDNA tandem array in yeast is tightly controlled to allow sequence homogenization and at the same time prevent deleterious rearrangements. In our study, we show that gene conversion, and not unequal sister chromatid exchange, is the predominant recombination mechanism regulating the expansion and contraction of the rDNA array. Furthermore, we found that RAD52, which is essential for gene conversion, is required for marker duplication stimulated in the absence of the two yeast type I topoisomerases. Our results have implications for the mechanisms regulating genomic stability of repetitive sequence families found in all eukaryotes. Images PMID:8612596

  18. [Finding of dairy yeasts Kluyveromyces lactis var. lactis in natural habitats].

    PubMed

    Naumov, G I; Naumova, E S; Glushakova, A M; Kachalkin, A V; Chernov, I Y

    2014-01-01

    Well-known yeasts Kluyveromyces lactis var. lactis, which are usually associated with dairy prod- ucts, were discovered in nature (in woodland park soil under Impatiens glandulifera Royle plants). Reliable identification of the yeasts was carried out using physiological criteria (lactose and maltose utilization) and molecular markers (nucleotide sequence of the 5.8S-ITS rDNA fragment, pulsed-field electrophoresis, and Southern hybridization of chromosomal DNA with the LAC4 probe). Ecology of KI. lactis var. lactis is discussed. PMID:25941717

  19. Genetic Instability of Heterozygous, Hybrid, Natural Wine Yeasts

    PubMed Central

    Ramírez, Manuel; Vinagre, Antonia; Ambrona, Jesús; Molina, Felipe; Maqueda, Matilde; Rebollo, JoséE.

    2004-01-01

    We describe a genetic instability found in natural wine yeasts but not in the common laboratory strains of Saccharomyces cerevisiae. Spontaneous cyh2R/cyh2R mutants resistant to high levels of cycloheximide can be directly isolated from cyh2S/cyh2S wine yeasts. Heterozygous cyh2R/cyh2S hybrid clones vary in genetic instability as measured by loss of heterozygosity at cyh2. There were two main classes of hybrids. The lawn hybrids have high genetic instability and generally become cyh2R/cyh2R homozygotes and lose the killer phenotype under nonselective conditions. The papilla hybrids have a much lower rate of loss of heterozygosity and maintain the killer phenotype. The genetic instability in lawn hybrids is 3 to 5 orders of magnitude greater than the highest loss-of-heterozygosity rates previously reported. Molecular mechanisms such as DNA repair by break-induced replication might account for the asymmetrical loss of heterozygosity. This loss-of-heterozygosity phenomenon could be economically important if it causes sudden phenotype changes in industrial or pathogenic yeasts and of more basic importance to the degree that it influences the evolution of naturally occurring yeast populations. PMID:15294803

  20. MAP kinase pathways in the yeast Saccharomyces cerevisiae

    NASA Technical Reports Server (NTRS)

    Gustin, M. C.; Albertyn, J.; Alexander, M.; Davenport, K.; McIntire, L. V. (Principal Investigator)

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area.

  1. MAP Kinase Pathways in the Yeast Saccharomyces cerevisiae

    PubMed Central

    Gustin, Michael C.; Albertyn, Jacobus; Alexander, Matthew; Davenport, Kenneth

    1998-01-01

    A cascade of three protein kinases known as a mitogen-activated protein kinase (MAPK) cascade is commonly found as part of the signaling pathways in eukaryotic cells. Almost two decades of genetic and biochemical experimentation plus the recently completed DNA sequence of the Saccharomyces cerevisiae genome have revealed just five functionally distinct MAPK cascades in this yeast. Sexual conjugation, cell growth, and adaptation to stress, for example, all require MAPK-mediated cellular responses. A primary function of these cascades appears to be the regulation of gene expression in response to extracellular signals or as part of specific developmental processes. In addition, the MAPK cascades often appear to regulate the cell cycle and vice versa. Despite the success of the gene hunter era in revealing these pathways, there are still many significant gaps in our knowledge of the molecular mechanisms for activation of these cascades and how the cascades regulate cell function. For example, comparison of different yeast signaling pathways reveals a surprising variety of different types of upstream signaling proteins that function to activate a MAPK cascade, yet how the upstream proteins actually activate the cascade remains unclear. We also know that the yeast MAPK pathways regulate each other and interact with other signaling pathways to produce a coordinated pattern of gene expression, but the molecular mechanisms of this cross talk are poorly understood. This review is therefore an attempt to present the current knowledge of MAPK pathways in yeast and some directions for future research in this area. PMID:9841672

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

    PubMed

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

    2014-09-01

    The universal genetic code is used by all life forms to encode biological information. It can also be used to encrypt semantic messages and convey them within organisms without anyone but the sender and recipient knowing, i.e., as a means of steganography. Several theoretical, but comparatively few experimental, approaches have been dedicated to this subject, so far. Here, we describe an experimental system to stably integrate encrypted messages within the yeast genome using a polymerase chain reaction (PCR)-based, one-step homologous recombination system. Thus, DNA sequences encoding alphabetical and/or numerical information will be inherited by yeast propagation and can be sent in the form of dried yeast. Moreover, due to the availability of triple shuttle vectors, Saccharomyces cerevisiae can also be used as an intermediate construction device for transfer of information to either Drosophila or mammalian cells as steganographic containers. Besides its classical use in alcoholic fermentation and its modern use for heterologous gene expression, we here show that baker's yeast can thus be employed in a novel Saccharomyces application (NSA) as a simple steganographic container to hide and convey messages. PMID:25238077

  3. Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence.

    PubMed Central

    Seifert, H S; Ajioka, R S; Paruchuri, D; Heffron, F; So, M

    1990-01-01

    The method of shuttle mutagenesis has been extended to Neisseria gonorrhoeae. We have constructed a defective mini-Tn3 derivative that encodes chloramphenicol resistance in both N. gonorrhoeae and Escherichia coli and selected for mutations in the chloramphenicol resistance gene that express higher levels of antibiotic resistance in N. gonorrhoeae. Isogenic N. gonorrhoeae strains that differ only in pilin expression were constructed and used to test the effect of pilin null mutations on DNA transformation competence. PMID:2152910

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

    PubMed Central

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

    1990-01-01

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

  5. Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

    PubMed Central

    Su, J Y; Sclafani, R A

    1991-01-01

    (Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity. Images PMID:2017365

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

    PubMed Central

    Contamine, Véronique; Picard, Marguerite

    2000-01-01

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

  7. Isolation and Characterization of Isogenic Pairs of Domed Hemolytic and Flat Nonhemolytic Colony Types of Bordetella pertussis

    PubMed Central

    Peppler, Mark S.

    1982-01-01

    Four different serotype strains of Bordetella pertussis, 3779BL2S4, Tohama I, 353/Z, and 2753, were plated on Bordet-Gengou agar, where they grew as domed, hemolytic (D+H+) wild-type colonies. Cloned D+H+ colony types of all four strains were passed onto modified Stainer-Scholte medium solidified with 1% Noble Agar. Colonies were selected from Stainer-Scholte agar, and these subsequently grew as flat, nonhemolytic (D−H−) colonies when transferred back onto Bordet-Gengou agar. The frequency of D−H− organisms within a population of cloned D+H+ was determined to be between 5 × 10−5 and 5 × 10−6. The D−H− colony types maintained their flat, nonhemolytic characteristics for over 80 single-colony passages on Bordet-Gengou agar. The isogenic pairs of D+H+ and D−H− colony types from the four strains were compared for hemagglutination titer, lymphocytosis-promoting activity, adenylate cyclase activity, and presence of agglutinogens by agglutination. In all cases the D−H− colony types showed reduced activities or amounts of antigen compared with their D+H+ parents. Freely diffusible antigens were markedly different between the two phenotypes as noted by double diffusion of antisera added to plates on which colonies of the variants were growing. Antigens solubilized from the two colony types by Triton X-100 were also markedly different as judged by radial immunodiffusion with antifimbrial hemagglutinin, antilymphocytosis-promoting factor, and anti-353/Z adsorbed with autoclaved 353/Z. In addition, autoradiographs of 125I-surface-labeled whole cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed unique banding patterns for each colony type. Since all organisms, regardless of colony type, were grown on Bordet-Gengou agar, the differences reported could not be due to medium composition. Differences between phenotypes were also independent of passage number on Bordet-Gengou agar. By analogy to previous studies, the D

  8. Yeast Genetics and Biotechnological Applications

    NASA Astrophysics Data System (ADS)

    Mishra, Saroj; Baranwal, Richa

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

  9. Construction of cell surface-engineered yeasts displaying antigen to detect antibodies by immunofluorescence and yeast-ELISA.

    PubMed

    Tang, Yu Qian; Han, Shuang Yan; Zheng, Hong; Wu, Lin; Ueda, Mitsuyoshi; Wang, Xiao Ning; Lin, Ying

    2008-07-01

    In order to detect monoclonal antibodies (MAbs) from insufficient and unavailable human proteins, yeast cells were engineered to display human antigens on their surface and consequently endowed with the ability to specifically bind antibodies. Thus, a fusion gene for the expression of the human proteasome subunit alpha 6 (hPSA6) and human profilin I (hProI) were assembled, respectively, with a His.tag marker at the C-terminal and displayed on yeast surface. With anti-His.tag MAb as the primary antibody and the fluorescein isothiocyanate-conjugated goat anti-mouse Immunoglobulin G as the second antibody, the surface display of hPSA6 and hProI were verified by immunofluorescence labeling. The antigen-displayed yeast particles were used for MAbs detection from ascites through both immunofluorescence and yeast-enzyme-linked immunosorbent assay (ELISA) methods. The results were verified by Western blotting and indirect ELISA. By improving the sensitivity, the novel MAbs detection can be applied in the generation and screening of positive hybridoma. It is suggested that by combining the DNA immunization, the present study can evolve into a quick and protein-free way of MAbs production for insufficient and unavailable antigen. PMID:18542951

  10. Molecular Genetic Tools and Techniques in Fission Yeast.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    The molecular genetic tools used in fission yeast have generally been adapted from methods and approaches developed for use in the budding yeast, Saccharomyces cerevisiae Initially, the molecular genetics of Schizosaccharomyces pombe was developed to aid gene identification, but it is now applied extensively to the analysis of gene function and the manipulation of noncoding sequences that affect chromosome dynamics. Much current research using fission yeast thus relies on the basic processes of introducing DNA into the organism and the extraction of DNA for subsequent analysis. Targeted integration into specific genomic loci is often used to create site-specific mutants or changes to noncoding regulatory elements for subsequent phenotypic analysis. It is also regularly used to introduce additional sequences that generate tagged proteins or to create strains in which the levels of wild-type protein can be manipulated through transcriptional regulation and/or protein degradation. Here, we draw together a collection of core molecular genetic techniques that underpin much of modern research using S. pombe We summarize the most useful methods that are routinely used and provide guidance, learned from experience, for the successful application of these methods. PMID:27140925

  11. Candida bituminiphila, a novel anamorphic species of yeast.

    PubMed

    Robert, V; Bonjean, B; Karutz, M; Paschold, H; Peeters, W; Wubbolts, M G

    2001-11-01

    A novel anamorphic species of yeast belonging to the genus Candida was isolated from tar in Canada. Morphological and physiological observations, as well as phylogenetic analyses, were performed. Conidiophores were produced, were usually short and had sympodial growth, numerous bud scars and a rachis-like structure. They bore one or more conidia. Pseudomycelium was scarcely produced and true mycelium was sparse. No sexual reproduction was observed on corn meal, malt, Gorodkowa, Dextrose Yeast Peptone or V8 agars. Zygoascus hellenicus was physiologically the most closely related species, but it differed from the novel species by its ability to assimilate D-galacturonate and L-rhamnose, ferment sucrose and grow at 37 degrees C. From sequence analysis of the 26S rDNA D1/D2 region, Z. hellenicus and Candida bertae var. bertae were the closest species with 54 and 56 bp substitutions, respectively. Similar results have been obtained from analysis of the 18S rDNA. All these data support the hypothesis that the yeast, named Candida bituminiphila, is a novel species closely related to Z. hellenicus. The holotype and only isolate of C. bituminiphila is strain CBS 8813T (= MUCL 41424T). PMID:11760960

  12. High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification.

    PubMed

    Nie, Minghua; Vashisht, Ajay A; Wohlschlegel, James A; Boddy, Michael N

    2015-01-01

    Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO "cloud" phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function. PMID:26404184

  13. High Confidence Fission Yeast SUMO Conjugates Identified by Tandem Denaturing Affinity Purification

    PubMed Central

    Nie, Minghua; Vashisht, Ajay A.; Wohlschlegel, James A.; Boddy, Michael N.

    2015-01-01

    Covalent attachment of the small ubiquitin-like modifier (SUMO) to key targets in the proteome critically regulates the evolutionarily conserved processes of cell cycle control, transcription, DNA replication and maintenance of genome stability. The proteome-wide identification of SUMO conjugates in budding yeast has been invaluable in helping to define roles of SUMO in these processes. Like budding yeast, fission yeast is an important and popular model organism; however, the fission yeast Schizosaccharomyces pombe community currently lacks proteome-wide knowledge of SUMO pathway targets. To begin to address this deficiency, we adapted and used a highly stringent Tandem Denaturing Affinity Purification (TDAP) method, coupled with mass spectrometry, to identify fission yeast SUMO conjugates. Comparison of our data with that compiled in budding yeast reveals conservation of SUMO target enrichment in nuclear and chromatin-associated processes. Moreover, the SUMO “cloud” phenomenon, whereby multiple components of a single protein complex are SUMOylated, is also conserved. Overall, SUMO TDAP provides both a key resource of high confidence SUMO-modified target proteins in fission yeast, and a robust method for future analyses of SUMO function. PMID:26404184

  14. Genetic Analysis Using an Isogenic Mating Pair of Aspergillus fumigatus Identifies Azole Resistance Genes and Lack of MAT Locus's Role in Virulence.

    PubMed

    Losada, Liliana; Sugui, Janyce A; Eckhaus, Michael A; Chang, Yun C; Mounaud, Stephanie; Figat, Abigail; Joardar, Vinita; Pakala, Suman B; Pakala, Suchitra; Venepally, Pratap; Fedorova, Natalie; Nierman, William C; Kwon-Chung, Kyung J

    2015-04-01

    Invasive aspergillosis (IA) due to Aspergillus fumigatus is a major cause of mortality in immunocompromised patients. The discovery of highly fertile strains of A. fumigatus opened the possibility to merge classical and contemporary genetics to address key questions about this pathogen. The merger involves sexual recombination, selection of desired traits, and genomics to identify any associated loci. We constructed a highly fertile isogenic pair of A. fumigatus strains with opposite mating types and used them to investigate whether mating type is associated with virulence and to find the genetic loci involved in azole resistance. The pair was made isogenic by 9 successive backcross cycles of the foundational strain AFB62 (MAT1-1) with a highly fertile (MAT1-2) progeny. Genome sequencing showed that the F9 MAT1-2 progeny was essentially identical to the AFB62. The survival curves of animals infected with either strain in three different animal models showed no significant difference, suggesting that virulence in A. fumigatus was not associated with mating type. We then employed a relatively inexpensive, yet highly powerful strategy to identify genomic loci associated with azole resistance. We used traditional in vitro drug selection accompanied by classical sexual crosses of azole-sensitive with resistant isogenic strains. The offspring were plated under varying drug concentrations and pools of resulting colonies were analyzed by whole genome sequencing. We found that variants in 5 genes contributed to azole resistance, including mutations in erg11A (cyp51A), as well as multi-drug transporters, erg25, and in HMG-CoA reductase. The results demonstrated that with minimal investment into the sequencing of three pools from a cross of interest, the variation(s) that contribute any phenotype can be identified with nucleotide resolution. This approach can be applied to multiple areas of interest in A. fumigatus or other heterothallic pathogens, especially for virulence

  15. Detection and quantitative determination by PIXE of the mutagen Sn 2+ in yeast cells

    NASA Astrophysics Data System (ADS)

    Viau, C. M.; Yoneama, M.-L.; Dias, J. F.; Pungartnik, C.; Brendel, M.; Henriques, J. A. P.

    2006-08-01

    The main goal of this work was to determine the concentration of Sn2+ ions in cells of the yeast Saccharomyces cerevisiae and to correlate their quantity with the genotoxicity of intracellularly accumulated metal ions. The intracellular metal content of yeast cells was determined by PIXE (particle-induced X-ray emission) after cell exposure to SnCl2. To that end, a thick target protocol was developed for PIXE analysis. The samples were irradiated with a 2 MeV proton beam, while the induced X-rays were detected with a high-purity germanium detector. The results of the toxicity of SnCl2 and the PIXE analysis performed with two different yeast strains (haploid and diploid) suggest that the exposure of haploid and diploid yeast to Sn2+ induces DNA lesions and that the absorption depends on the genetic background of each strain.

  16. Chemical synthesis of a gene for human epidermal growth factor urogastrone and its expression in yeast.

    PubMed Central

    Urdea, M S; Merryweather, J P; Mullenbach, G T; Coit, D; Heberlein, U; Valenzuela, P; Barr, P J

    1983-01-01

    We have chemically synthesized and expressed in yeast a gene coding for human epidermal growth factor (urogastrone), a 53-amino-acid polypeptide that has been shown to promote epithelial cell proliferation and to inhibit gastric acid secretion. The synthetic gene, consisting of 170 base pairs, was designed with yeast-preferred codons and assembled by enzymatic ligation of synthetic fragments produced by phosphoramidite chemistry. The DNA synthesis protocol used allows for facile synthesis of oligonucleotides larger than 50 bases. Yeast cells were transformed with plasmids containing the synthetic gene under control of a yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter and were shown to synthesize a biologically active human epidermal growth factor. Images PMID:6369317

  17. Expression of the Major Surface Antigen of Plasmodium knowlesi Sporozoites in Yeast

    NASA Astrophysics Data System (ADS)

    Sharma, Shobhona; Godson, G. Nigel

    1985-05-01

    The circumsporozoite protein, a surface antigen of the sporozoite stage of the monkey malarial parasite Plasmodium knowlesi, was expressed in the yeast Saccharomyces cerevisiae by using an expression vector containing the 5' regulatory region of the yeast alcohol dehydrogenase I gene. It was necessary to eliminate the entire 5' upstream region of the parasite DNA to obtain the expression of this protein. Only the circumsporozoite precursor protein was produced by the yeast transformants, as detected by immunoblotting. About 55 and 20 percent of the circumsporozoite protein produced in yeast was associated with the 25,000g and 150,000g particulate fractions, respectively. The protein could be solubilized in Triton X-100 and was stable in solubilized extracts.

  18. Can the grey mould disease of the grape-vine be controlled by yeast?

    PubMed

    Masih, E I; Alie, I; Paul, B

    2000-08-15

    Botrytis cinerea has been found to be highly pathogenic to 'Chardonnay' and 'Pinot noir' cultivars of the grape-vine producing the characteristic grey mould symptoms within 7 days of inoculation to the vitro-plants. The yeast Pichia anomala (strain FY-102), isolated from apple skin, was found to be antagonistic to B. cinerea as it completely inhibited the appearance of the grey mould symptoms when grown together. The yeast was responsible for morphological changes such as coagulation and leakage of the cytoplasm of B. cinerea. The pathogen, when applied together with P. anomala, failed to bring about the grey mould symptoms on the grape-vine, suggesting that the yeast could control the expression of this disease. An account of the interaction between B. cinerea and P. anomala, as well as the sequences of the complete ITS region of the ribosomal DNA of the yeast are described here. PMID:10930744

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

    SciTech Connect

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

    1987-01-01

    Yeast strains capable of direct fermentation of manioc starch were developed by hybridizing strains of Saccharomyces diastaticus and Saccharomyces cerevisiae. Hybrids were evaluated for speed of alcohol production, and yields and speed of formation of glycoamylase. Up to 6% solutions of Lintner starch could be fermented directly with about 80% conversion to alcohol. Pretreatment of crude 40% manioc starch suspensions with alpha-amylase, followed by fermentations with a starch-fermenting yeast strain, permitted accumulation of 12% ethanol within three days.