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Richard Y Zhao - One of the best experts on this subject based on the ideXlab platform.
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Molecular Cloning and Characterization of Small Viral Genome in Fission Yeast.
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Richard Y ZhaoAbstract:Fission Yeast is a single-cell eukaryote that has been used extensively as a model organism to study cell biology and virology of higher eukaryotes including plants and humans. In particular, it is a very well-tested model to study evolutionary highly conserved cellular activities such as cell proliferation, cell cycle regulation, and cell death. Some of the advantages of using Fission Yeast as a surrogate system: easy to carry out functional and genome-wide analysis of small viral genome, easy to maintain in the laboratory with a relatively short doubling time. It is genetically amendable and can be used to test the effect of gain-of-function or loss-of-function of a gene product. Here, we describe a streamlined and large-scale molecular cloning strategy for genome-wide characterization of small viruses in Fission Yeast.
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HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe
PloS one, 2016Co-Authors: Zsigmond Benko, Robert T. Elder, Dong Liang, Richard Y ZhaoAbstract:Background HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a Fission Yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. Results A Fission Yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in Fission Yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in Fission Yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. Conclusions This is the first report to show that HIV-1 protease is functional as an enzyme in Fission Yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in Fission Yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.
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A Fission Yeast protein kinase Hhp2 suppresses PR activities.
2016Co-Authors: Zsigmond Benko, Robert T. Elder, Dong Liang, Richard Y ZhaoAbstract:(A) Fission Yeast genome-wide searches for multicopy suppressors of PR-induced cell death of Fission Yeast cells revealed six unique cDNA clones out of ten isolates with overlapping open reading frame (ORF) of the hhp2 gene. The numbering and relative positions of each nucleotide were derived from the SPAC23C4 cosmid of Fission Yeast. (B) Overexpression of hhp2 restored colony formation in PR-expressing Fission Yeast cells. Expression of the hhp2 gene was induced at lower levels with 1 nM thiamine added to the media. Under this condition, HIV-1 PR still prevented colony formation as shown. Ctr, an empty pYZ1N plasmid control. Gene-off, no HIV-1 PR or Hhp2 protein production; Gene-on, i.e., PR protein production in the presence of Hhp2 or an empty plasmid vector. Agar plates were incubated at 30°C under the indicated conditions for 6 days before pictures were taken. (C) Overexpression of hhp2 reduced the percentage of cells showing the GFP pattern over time. The GFP-p6-Vpr fusion product was used here. The Hhp2 effect was measured over time after gene induction as indicated. The percentage of GFP pattern, i.e., the putative protein cleavage of GFP-p6-Vpr fusion construct by HIV-1 PR was quantified and is shown in (b). Error bars shown in (C) represent results of at least three different experiments with an average of 100–200 cells counted at each time point.
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Molecular characterization of HIV-1 genome in Fission Yeast Schizosaccharomyces pombe
Cell & bioscience, 2015Co-Authors: Joseph Nkeze, Zsigmond Benko, Richard Y ZhaoAbstract:The human immunodeficiency virus type 1 (HIV-1) genome (~9 kb RNA) is flanked by two long terminal repeats (LTR) promoter regions with nine open reading frames, which encode Gag, Pol and Env polyproteins, four accessory proteins (Vpu, Vif, Vpr, Nef) and two regulatory proteins (Rev, Tat). In this study, we carried out a genome-wide and functional analysis of the HIV-1 genome in Fission Yeast (Schizosaccharomyces pombe). Each one of the HIV-1 genes was cloned and expressed individually in Fission Yeast. Subcellular localization of each viral protein was first examined. The effect of protein expression on cellular proliferation and colony formations, an indication of cytotoxicity, were observed. Overall, there is a general correlation of subcellular localization of each viral protein between Fission Yeast and mammalian cells. Three viral proteins, viral protein R (Vpr), protease (PR) and regulator of expression of viral protein (Rev), were found to inhibit cellular proliferation. Rev was chosen for further analysis in Fission Yeast and mammalian cells. Consistent with the observation in Fission Yeast, expression of HIV-1 rev gene also caused growth retardation in mammalian cells. However, the observed growth delay was neither due to the cytotoxic effect nor due to alterations in cell cycling. Mechanistic testing of the Rev effect suggests it triggers transient induction of cellular oxidative stress. Some of the behavioral and functional similarities of Rev between Fission Yeast and mammalian cells suggest Fission Yeast might be a useful model system for further studies of molecular functions of Rev and other HIV-1 viral proteins.
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zeocin for selection of blemx6 resistance in Fission Yeast
BioTechniques, 2011Co-Authors: Zsigmond Benko, Richard Y ZhaoAbstract:Complementation of auxotrophic nutrient deficiencies in minimal media is widely used for selection of exogenous gene introduction to Fission Yeast. However, only a limited number of such selection ...
Zsigmond Benko - One of the best experts on this subject based on the ideXlab platform.
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HIV-1 Protease in the Fission Yeast Schizosaccharomyces pombe
PloS one, 2016Co-Authors: Zsigmond Benko, Robert T. Elder, Dong Liang, Richard Y ZhaoAbstract:Background HIV-1 protease (PR) is an essential viral enzyme. Its primary function is to proteolyze the viral Gag-Pol polyprotein for production of viral enzymes and structural proteins and for maturation of infectious viral particles. Increasing evidence suggests that PR cleaves host cellular proteins. However, the nature of PR-host cellular protein interactions is elusive. This study aimed to develop a Fission Yeast (Schizosaccharomyces pombe) model system and to examine the possible interaction of HIV-1 PR with cellular proteins and its potential impact on cell proliferation and viability. Results A Fission Yeast strain RE294 was created that carried a single integrated copy of the PR gene in its chromosome. The PR gene was expressed using an inducible nmt1 promoter so that PR-specific effects could be measured. HIV-1 PR from this system cleaved the same indigenous viral p6/MA protein substrate as it does in natural HIV-1 infections. HIV-1 PR expression in Fission Yeast cells prevented cell proliferation and induced cellular oxidative stress and changes in mitochondrial morphology that led to cell death. Both these PR activities can be prevented by a PR-specific enzymatic inhibitor, indinavir, suggesting that PR-mediated proteolytic activities and cytotoxic effects resulted from enzymatic activities of HIV-1 PR. Through genome-wide screening, a serine/threonine kinase, Hhp2, was identified that suppresses HIV-1 PR-induced protease cleavage and cell death in Fission Yeast and in mammalian cells, where it prevented PR-induced apoptosis and cleavage of caspase-3 and caspase-8. Conclusions This is the first report to show that HIV-1 protease is functional as an enzyme in Fission Yeast, and that it behaves in a similar manner as it does in HIV-1 infection. HIV-1 PR-induced cell death in Fission Yeast could potentially be used as an endpoint for mechanistic studies, and this system could be used for developing a high-throughput system for drug screenings.
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A Fission Yeast protein kinase Hhp2 suppresses PR activities.
2016Co-Authors: Zsigmond Benko, Robert T. Elder, Dong Liang, Richard Y ZhaoAbstract:(A) Fission Yeast genome-wide searches for multicopy suppressors of PR-induced cell death of Fission Yeast cells revealed six unique cDNA clones out of ten isolates with overlapping open reading frame (ORF) of the hhp2 gene. The numbering and relative positions of each nucleotide were derived from the SPAC23C4 cosmid of Fission Yeast. (B) Overexpression of hhp2 restored colony formation in PR-expressing Fission Yeast cells. Expression of the hhp2 gene was induced at lower levels with 1 nM thiamine added to the media. Under this condition, HIV-1 PR still prevented colony formation as shown. Ctr, an empty pYZ1N plasmid control. Gene-off, no HIV-1 PR or Hhp2 protein production; Gene-on, i.e., PR protein production in the presence of Hhp2 or an empty plasmid vector. Agar plates were incubated at 30°C under the indicated conditions for 6 days before pictures were taken. (C) Overexpression of hhp2 reduced the percentage of cells showing the GFP pattern over time. The GFP-p6-Vpr fusion product was used here. The Hhp2 effect was measured over time after gene induction as indicated. The percentage of GFP pattern, i.e., the putative protein cleavage of GFP-p6-Vpr fusion construct by HIV-1 PR was quantified and is shown in (b). Error bars shown in (C) represent results of at least three different experiments with an average of 100–200 cells counted at each time point.
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Molecular characterization of HIV-1 genome in Fission Yeast Schizosaccharomyces pombe
Cell & bioscience, 2015Co-Authors: Joseph Nkeze, Zsigmond Benko, Richard Y ZhaoAbstract:The human immunodeficiency virus type 1 (HIV-1) genome (~9 kb RNA) is flanked by two long terminal repeats (LTR) promoter regions with nine open reading frames, which encode Gag, Pol and Env polyproteins, four accessory proteins (Vpu, Vif, Vpr, Nef) and two regulatory proteins (Rev, Tat). In this study, we carried out a genome-wide and functional analysis of the HIV-1 genome in Fission Yeast (Schizosaccharomyces pombe). Each one of the HIV-1 genes was cloned and expressed individually in Fission Yeast. Subcellular localization of each viral protein was first examined. The effect of protein expression on cellular proliferation and colony formations, an indication of cytotoxicity, were observed. Overall, there is a general correlation of subcellular localization of each viral protein between Fission Yeast and mammalian cells. Three viral proteins, viral protein R (Vpr), protease (PR) and regulator of expression of viral protein (Rev), were found to inhibit cellular proliferation. Rev was chosen for further analysis in Fission Yeast and mammalian cells. Consistent with the observation in Fission Yeast, expression of HIV-1 rev gene also caused growth retardation in mammalian cells. However, the observed growth delay was neither due to the cytotoxic effect nor due to alterations in cell cycling. Mechanistic testing of the Rev effect suggests it triggers transient induction of cellular oxidative stress. Some of the behavioral and functional similarities of Rev between Fission Yeast and mammalian cells suggest Fission Yeast might be a useful model system for further studies of molecular functions of Rev and other HIV-1 viral proteins.
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zeocin for selection of blemx6 resistance in Fission Yeast
BioTechniques, 2011Co-Authors: Zsigmond Benko, Richard Y ZhaoAbstract:Complementation of auxotrophic nutrient deficiencies in minimal media is widely used for selection of exogenous gene introduction to Fission Yeast. However, only a limited number of such selection ...
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Zeocin for selection of bleMX6 resistance in Fission Yeast
BioTechniques, 2011Co-Authors: Zsigmond Benko, Richard Y ZhaoAbstract:Complementation of auxotrophic nutrient deficiencies in minimal media is widely used for selection of exogenous gene introduction to Fission Yeast. However, only a limited number of such selection markers are available. Antibiotic resistance markers are good alternatives, but they typically work well in complete rich medium but not in minimal defined Edinburgh minimal medium (EMM). It would be ideal if both the auxotrophic and antibiotic resistance markers can be used together for molecular genetic analysis. Here we describe the use of Zeocin in Pombe minimal glutamate (PMG) media for selection and maintenance of bleMX6 resistance with a LEU2 auxotrophic marker in Fission Yeast.
Thomas D. Pollard - One of the best experts on this subject based on the ideXlab platform.
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understanding cytokinesis lessons from Fission Yeast
Nature Reviews Molecular Cell Biology, 2010Co-Authors: Thomas D. PollardAbstract:For decades after the discovery that a contractile ring made of actin filaments and myosin II produces the force to constrict the cleavage furrow of animal cells, the complexity of cytokinesis has slowed progress in understanding the mechanism. Mechanistic insights, however, have been obtained by genetic, biochemical, microscopic and mathematical modelling approaches in the Fission Yeast Schizosaccharomyces pombe. Many features that have been identified in Fission Yeast are probably shared with animal cells, as both inherited many cytokinesis genes from their common ancestor about one billion years ago.
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Incompatibility with Formin Cdc12p Prevents Human Profilin from Substituting for Fission Yeast Profilin : INSIGHTS FROM CRYSTAL STRUCTURES OF Fission Yeast PROFILIN
The Journal of biological chemistry, 2008Co-Authors: Obidimma C. Ezezika, David R. Kovar, Noah S. Younger, Donald A. Kaiser, Zachary Corbin, Bradley J. Nolen, Thomas D. PollardAbstract:Expression of human profilin-I does not complement the temperature-sensitive cdc3-124 mutation of the single profilin gene in Fission Yeast Schizosaccharomyces pombe, resulting in death from cytokinesis defects. Human profilin-I and S. pombe profilin have similar affinities for actin monomers, the FH1 domain of Fission Yeast formin Cdc12p and poly-L-proline (Lu, J., and Pollard, T. D. (2001) Mol. Biol. Cell 12, 1161-1175), but human profilin-I does not stimulate actin filament elongation by formin Cdc12p like S. pombe profilin. Two crystal structures of S. pombe profilin and homology models of S. pombe profilin bound to actin show how the two profilins bind to identical surfaces on animal and Yeast actins even though 75% of the residues on the profilin side of the interaction differ in the two profilins. Overexpression of human profilin-I in Fission Yeast expressing native profilin also causes cytokinesis defects incompatible with viability. Human profilin-I with the R88E mutation has no detectable affinity for actin and does not have this dominant overexpression phenotype. The Y6D mutation reduces the affinity of human profilin-I for poly-l-proline by 1000-fold, but overexpression of Y6D profilin in Fission Yeast is lethal. The most likely hypotheses to explain the incompatibility of human profilin-I with Cdc12p are differences in interactions with the proline-rich sequences in the FH1 domain of Cdc12p and wider "wings" that interact with actin.
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Molecular basis of cytokinesis in Fission Yeast
The FASEB Journal, 2008Co-Authors: Thomas D. Pollard, Dimitrios Vavylonis, Ben O'shaughnessyAbstract:Cell division by cytokinesis in animals and fungi depends on a contractile ring of actin filaments and the motor protein myosin-II. Fluorescence microscopy of live Fission Yeast cells reveals that ...
Toru M. Nakamura - One of the best experts on this subject based on the ideXlab platform.
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Telomere Regulation During the Cell Cycle in Fission Yeast
Methods in molecular biology (Clifton N.J.), 2014Co-Authors: Bettina A. Moser, Ya Ting Chang, Toru M. NakamuraAbstract:The Fission Yeast Schizosaccharomyces pombe has emerged as a useful model organism to study telomere maintenance mechanisms. In this chapter, we provide detailed protocols for quantitative ChIP and BrdU incorporation analyses to investigate how Fission Yeast telomeres are regulated during the cell cycle by utilizing cdc25-22 synchronized cell cultures.
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Protection and replication of telomeres in Fission Yeast
Biochemistry and cell biology = Biochimie et biologie cellulaire, 2009Co-Authors: Bettina A. Moser, Toru M. NakamuraAbstract:Telomeres, the natural ends of linear chromosomes, must be protected and completely replicated to guarantee genomic stability in eukaryotic cells. However, the protected state of telomeres is not compatible with recruitment of telomerase, an enzyme responsible for extending telomeric G-rich repeats during S-phase; thus, telomeres must undergo switches from a protected state to an accessible state during the cell cycle. In this minireview, we will summarize recent advances in our understanding of proteins involved in the protection and replication of telomeres, and the way these factors are dynamically recruited to telomeres during the cell cycle. We will focus mainly on recent results from Fission Yeast Schizosaccharomyces pombe, and compare them with results from budding Yeast Saccharomyces cerevisiae and mammalian cell studies. In addition, a model for the way in which Fission Yeast cells replicate telomeres will be presented.
Masayuki Yamamato - One of the best experts on this subject based on the ideXlab platform.
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TOR signaling in Fission Yeast
Critical Reviews in Biochemistry and Molecular Biology, 2008Co-Authors: Yoko Otsubo, Masayuki YamamatoAbstract:Fission Yeast has two TOR kinases, Tor1 and Tor2. Recent studies have indicated that this microbe has a TSC/Rheb/TOR pathway like higher eukaryotes. Two TOR complexes, namely TORC1 and TORC2, have been identified in this Yeast, as in budding Yeast and mammals. Fission Yeast TORC1, which contains Tor2, and TORC2, which contains Tor1, apparently have opposite functions with regard to the promotion of G1 arrest and sexual development. Rapamycin does not inhibit growth of wild-type Fission Yeast cells, unlike other eukaryotic cells, but precise analyses have revealed that rapamycin affects certain cellular functions involving TOR in this Yeast. It appears that Fission Yeast has a potential to be an ideal model system to investigate the TOR signaling pathways.