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Sample records for cerevisiae checkpoint mutants

  1. The anaphase inhibitor of Saccharomyces cerevisiae Pds1p is a target of the DNA damage checkpoint pathway

    International Nuclear Information System (INIS)

    Inhibition of DNA replication and physical DNA damage induce checkpoint responses that arrest cell cycle progression at two different stages. In Saccharomyces cerevisiae, the execution of both checkpoint responses requires the Mec1 and Rad53 proteins. This observation led to the suggestion that these checkpoint responses are mediated through a common signal transduction pathway. However, because the checkpoint-induced arrests occur at different cell cycle stages, the downstream effectors mediating these arrests are likely to be distinct. We have previously shown that the S. cerevisiae protein Pds1p is an anaphase inhibitor and is essential for cell cycle arrest in mitosis in the presence DNA damage. Herein we show that DNA damage, but not inhibition of DNA replication, induces the phosphorylation of Pds1p. Analyses of Pds1p phosphorylation in different checkpoint mutants reveal that in the presence of DNA damage, Pds1p is phosphorylated in a Mec1p- and Rad9p-dependent hut Rad53p-independent manner. Our data place Pds1p and Rad53p on parallel branches of the DNA damage checkpoint pathway. We suggest that Pds1p is a downstream target of the DNA damage checkpoint pathway and that it is involved in implementing the DNA damage checkpoint arrest specifically in mitosis

  2. Interactions of checkpoint-genes RAD9, RAD17, RAD24 and RAD53 determining radioresistance of Yeast Saccharomyces Cerevisiae

    International Nuclear Information System (INIS)

    The mechanisms of genetic control of progress through the division cell cycle (checkpoint-control) in yeast Saccharomyces cerevisiae have been studied intensively. To investigate the role of checkpoint-genes RAD9, RAD17, RAD24, RAD53 in cell radioresistance we have investigated cell sensitivity of double mutants to γ-ray. Double mutants involving various combinations with rad9Δ show epistatic interactions, i.e. the sensitivity of the double mutants to γ-ray was no greater than that of more sensitive of the two single mutants. This suggests that all these genes govern the same pathway. This group of genes was named RAD9-epistasis group. It is interesting to note that the genes RAD9 and RAD53 have positive effect but RAD17 and RAD24 have negative effect on radiosensitivity of yeast cells. Interactions between mutations may differ depending on the agent γ-ray or UV-light, for example mutations rad9Δ and rad24Δ show additive effect for γ-ray and epistatic effect for UV-light

  3. CDC28, NETI, and HFII are required for checkpoints in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The involvement of SRM genes selected as genes affecting genetic stability and radiosensitivity in a cell cycle arrest under the action of damaging agents was studied. It was shown that the srm5/cdc28-srm, srm8/netI-srm, and srmI2/hfiI-srm mutations prevent checkpoint activation by DNA damage, particularly the G0/S (srm5, srm8), G1/S (srm5, srm8, srm12), S (srm8, srm12) and S/G2 (srm5) checkpoints. It seems that in budding yeast the CDC28, HFII/ADAI, and NETI genes mediate cellular response induced by DNA damage with checkpoint control. The well-known checkpoint-genes RAD9, RAD17, RAD24, and RAD53, and the genes CDC28, and NETI have been found to belong to one epistasis group named RAD9-group as regards cell sensitivity to γ radiation. An analysis of the radiosensitivity of double mutants has revealed that the mutation cdc-28-srm is hypostatic to each of mutations rad9Δ, and rad24Δ, and additive to rad17Δ. The mutation netI-srm is hypostatic to the mutations rad9Δ but additive to rad17Δ, rad24Δ, and rad53. The mutation hfiI-srm has an additive effect in compound with the mutations rad24Δ and rad9Δ. So, investigations of epistatic interactions have demonstrated a branched RAD9-dependent pathway. The analyzed genes can also participate in a minor mechanism involved in determining cell radiation sensitivity independently of the mentioned RAD9-dependent pathway

  4. Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae.

    OpenAIRE

    Letts, V A; Henry, S. A.

    1985-01-01

    chol mutants of Saccharomyces cerevisiae are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. chol mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). We exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. ...

  5. Expression of native and mutant extracellular lipases fromYarrowia lipolytica in Saccharomyces cerevisiae

    OpenAIRE

    Darvishi, Farshad

    2012-01-01

    Summary Saccharomyces cerevisiae cannot produce extracellular lipase and utilize low‐cost lipid substrates. This study aimed to express extracellular lipase from Yarrowia lipolytica in S. cerevisiae, construct recombinant oily substrate consumer strains, and compare the roles of native and mutant Y. lipolytica extracellular lipases in S. cerevisiae. The LIP2 gene of Y. lipolytica DSM3286 and its mutant Y. lipolytica U6 were isolated and cloned by expression vector in S. cerevisiae. New recomb...

  6. The checkpoint Saccharomyces cerevisiae Rad9 protein contains a tandem tudor domain that recognizes DNA.

    OpenAIRE

    Lancelot, Nathalie; Charier, Gaëlle; Couprie, Joël; Duband-Goulet, Isabelle; Alpha-Bazin, Béatrice; Quémeneur, Eric; Ma, Emilie; Marsolier-Kergoat, Marie-Claude; Ropars, Virginie; Charbonnier, Jean-Baptiste; Miron, Simona; Craescu, Constantin,; Callebaut, Isabelle; Gilquin, Bernard; Zinn-Justin, Sophie

    2007-01-01

    DNA damage checkpoints are signal transduction pathways that are activated after genotoxic insults to protect genomic integrity. At the site of DNA damage, 'mediator' proteins are in charge of recruiting 'signal transducers' to molecules 'sensing' the damage. Budding yeast Rad9, fission yeast Crb2 and metazoan 53BP1 are presented as mediators involved in the activation of checkpoint kinases. Here we show that, despite low sequence conservation, Rad9 exhibits a tandem tudor domain structurally...

  7. Sudden Telomere Lengthening Triggers a Rad53-dependent Checkpoint in Saccharomyces cerevisiae

    OpenAIRE

    Viscardi, Valeria; Baroni, Enrico; Romano, Michele; Lucchini, Giovanna; Longhese, Maria Pia

    2003-01-01

    Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent...

  8. An apoptotic cell cycle mutant in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Villadsen, Ingrid

    1996-01-01

    which apoptosis can be studied using the novel, temperature sensitive mutant, cdc77. The cdc77 cells are defective in a G1 process, and die show the characteristc signs of apoptosis: condensation of the chromatin, degradation of the inner nuclear membrane, dilation of the space between the nuclear...... membranes, condensation of the cytoplasm and degradation of DNA to 50kb fragmensts. It should be noted that in yeast, in contrast to higher eukaryotes, the nuclear membrane remain intact and the chromosomes remain uncondensed and invisible during mitosis. The cdc77 mutant exhibit a defect in initiation of...

  9. Ethanol from Whey: Continuous Fermentation with a Catabolite Repression-Resistant Saccharomyces cerevisiae Mutant

    OpenAIRE

    Terrell, Scott L.; Bernard, Alain; Bailey, Richard B.

    1984-01-01

    An alternative method for the conversion of cheese whey lactose into ethanol has been demonstrated. With the help of continuous-culture technology, a catabolite repression-resistant mutant of Saccharomyces cerevisiae completely fermented equimolar mixtures of glucose and galactose into ethanol. The first step in this process was a computer-controlled fed-batch operation based on the carbon dioxide evolution rate of the culture. In the absence of inhibitory ethanol concentrations, this step al...

  10. Repair of UV-irradiated plasmid DNA in excision repair deficient mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The repair of UV-irradiated DNA of plasmid YEp13 was studied in the incision defective strains by measurement of cell transformation frequency. In Saccharomyces cerevisiae, rad1,2,3 and 4 mutants could repair UV-damaged plasmid DNA. In Escherichia coli, uvrA mutant was unable to repair UV-damaged plasmid DNA; however, pretreatment of the plasmid with Micrococcus luteus endonuclease increased repair. It was concluded that all the mutations of yeast were probably limited only to the nuclear DNA. (author)

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

    Science.gov (United States)

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

    2016-01-01

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

  12. Regulation of phospholipid synthesis in phosphatidylserine synthase-deficient (chol) mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Saccharomyces cerevisiae mutants, chol, are deficient in the synthesis of the phospholipid phosphatidylserine owing to lowered activity of the membrane-associated enzyme phosphatidylserine synthase. These mutants are auxotrophic for ethanolamine or choline and, in the absence of these supplements, cannot synthesize phosphatidylethanolamine or phosphatidylcholine (PC). The authors exploited these characteristics of the chol mutants to examine the regulation of phospholipid metabolism in S. cerevisiae. Macromolecular synthesis and phospholipid metabolism were examined in chol cells starved for ethanolamine. Coupled to the decline in PC biosynthesis was a simultaneous decrease in the overall rate of phospholipid synthesis. In particular, the rate of synthesis of phosphatidylinositol decreased in parallel with the decline in PC biosynthesis. However, under conditions of ethanolamine deprivation in chol cells, the cytoplasmic enzyme inositol-1-phosphate synthase could not be repressed by exogenous inositol, and the endogenous synthesis of the phospholipid precursor inositol appeared to be elevated. The implications of these findings with respect to the coordinated regulation of phospholipid synthesis are discussed

  13. Multiple Defects of Cell Cycle Checkpoints in U937-ASPI3K, an U937 Cell Mutant Stably Expressing Anti-Sense ATM Gene cDNA

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    (Ataxia-telangiectasia mutated gene (ATM) functions in control of cell cycle checkpoints in responding to DNA damage and protects cells from undergoing apoptosis. Knock-out within tumor cells of endogenous ATM will achieve therapeutic benefits and nable a better understanding of the decisive mechanisms of cell death or survival in response to DNA damaging agents. ) In present paper, we sought to characterize the cell cycle checkpoint profiles in U937-ASPI3K, a U937 cell mutant that was previously established with endogenous ATM knock-out phenotype. Synchronized U937-ASPI3K was exposed to 137Cs irradiation, G1, S, G2/M cell cycle checkpoint profiles were evaluated by determining cell cycle kinetics, p53/p21 protein, cyclin dependent kinase 2 (CDK2) and p34CDC2 kinase activity in response to irradiation. U937-ASPI3K exhibited multiple defects in cell cycle checkpoints as defined by failing to arrest cells upon irradiation. The accumulation of cellular p53/p21 protein and inhibition of CDK kinase was also abolished in U937-ASPI3K. It was concluded that the stable expression of anti-sense PI3K cDNA fragment completely abolished multiple cell cycle checkpoints in U937-ASPI3K, and hence U937-ASPI3K with an AT-like phenotype could serves as a valuable model system for investigating the signal transduction pathway in responding to DNA damaging-based cancer therapy.

  14. Sodium Orthovanadate-Resistant Mutants of Saccharomyces Cerevisiae Show Defects in Golgi-Mediated Protein Glycosylation, Sporulation and Detergent Resistance

    OpenAIRE

    Kanik-Ennulat, C.; Montalvo, E.; Neff, N

    1995-01-01

    Orthovanadate is a small toxic molecule that competes with the biologically important oxyanion orthophosphate. Orthovanadate resistance arises spontaneously in Saccharomyces cerevisiae haploid cells by mutation in a number of genes. Mutations selected at 3 mM sodium orthovanadate have different degrees of vanadate resistance, hygromycin sensitivity, detergent sensitivity and sporulation defects. Recessive vanadate-resistant mutants belong to at least six genetic loci. Most mutants are defecti...

  15. Ultraviolet-endonuclease activity in cell extracts of Saccharomyces cerevisiae mutants defective in excision of pyrimidine dimers

    International Nuclear Information System (INIS)

    Cell-free extracts of ultraviolet-sensitive mutants of Saccharomyces cerevisiae defective in excision of pyrimidine dimers, rad1, rad2, rad3, rad4, rad10, and rad16, as well as the extracts of the wild-type strain RAD+, display ultraviolet-endonuclease activity

  16. Pleurotus sajor-caju HSP100 complements a thermotolerance defect in hsp104 mutant Saccharomyces cerevisiae

    Indian Academy of Sciences (India)

    Jin-Ohk Lee; Mi-Jeong Jeong; Tack-Ryun Kwon; Seung-Kon Lee; Myung-Ok Byun; Ill-Min Chung; Soo-Chul Park

    2006-06-01

    A putative Hsp100 gene was cloned from the fungus Pleurotus sajor-caju. mRNA expression studies demonstrated that this gene (designated PsHsp100) is highly induced by high temperature, induced less strongly by exposure to ethanol, and not induced by drought or salinity. Heat shock induction is detectable at 37°C and reaches a maximum level at 42°C. PsHsp100 mRNAlevels sharply increased within 15 min of exposure to high temperature, and reached a maximum expression level at 2 h that was maintained for several hours. These results indicate that PsHsp100 could work at an early step in thermotolerance. To examine its function, PsHsp100 was transformed into a temperature-sensitive hsp104 deletion mutant Saccharomyces cerevisiae strain to test the hypothesis that PsHSP100 is an protein that functions in thermotolerance. Overexpression of PsHSP100 complemented the thermotolerance defect of the hsp104 mutant yeast, allowing them survive even at 50°C for 4 h. These results indicate that PsHSP100 protein is functional as an HSP100 in yeast and could play an important role in thermotolerance in P. sajor-caju.

  17. Response to different oxidants of Saccharomyces cerevisiae ure2Delta mutant.

    Science.gov (United States)

    Todorova, Tatina T; Petrova, Ventsislava Y; Vuilleumier, Stéphane; Kujumdzieva, Anna V

    2009-11-01

    Growth of Saccharomyces cerevisiae ure2Delta mutant strain was investigated in the presence of diverse oxidant compounds. The inability of the strain to grow on a medium supplemented with H(2)O(2) was confirmed and a relationship between diminishing levels of glutathione (GSH) and peroxide sensitivity was established. Data for the lack of significant effect of URE2 disruption on the cellular growth in the presence of paraquat and menadione were obtained. The possible role of Ure2p in acquiring sensitivity to oxidative stress by means of its regulatory role in the GATA signal transduction pathway was discussed. It was suggested that the susceptibility of ure2Delta mutant to the exogenous hydrogen peroxide can result from increased GSH degradation due to the deregulated localization of the gamma-glutamyl transpeptidase activating factors Gln3/Gat1. The important role of Ure2p in in vivo glutathione-mediated reactive oxygen species (ROS) scavenging was shown by measuring the activity of antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD) and catalase in an URE2 disrupted strain. A time-dependent increase in SOD and catalase activity was observed. More importantly, it was shown that the ure2 mutation could cause significant disturbance in cellular oxidant balance and increased ROS level. PMID:19777209

  18. Pleurotus sajor-caju HSP100 complements a thermotolerance defect in hsp104 mutant Saccharomyces cerevisiae

    Indian Academy of Sciences (India)

    Jin-Ohk Lee; Mi-Jeong Jeong; Tack-Ryun Kwon; Seung-Kon Lee; Myung-Ok Byun; Ill-Min Chung; Soo-Chul Park

    2006-09-01

    A putative Hsp100 gene was cloned from the fungus Pleurotus sajor-caju. mRNA expression studies demonstrated that this gene (designated PsHsp100) is highly induced by high temperature, induced less strongly by exposure to ethanol, and not induced by drought or salinity. Heat shock induction is detectable at 37°C and reaches a maximum level at 42°C. PsHsp100 mRNAlevels sharply increased within 15 min of exposure to high temperature, and reached a maximum expression level at 2 h that was maintained for several hours. These results indicate that PsHsp100 could work at an early step in thermotolerance. To examine its function, PsHsp100 was transformed into a temperature-sensitive hsp104 deletion mutant Saccharomyces cerevisiae strain to test the hypothesis that PsHSP100 is an protein that functions in thermotolerance. Overexpression of PsHSP100 complemented the thermotolerance defect of the hsp104 mutant yeast, allowing them survive even at 50°C for 4 h. These results indicate that PsHSP100 protein is functional as an HSP100 in yeast and could play an important role in thermotolerance in P. sajor-caju.

  19. A novel role for the GTPase-activating protein Bud2 in the spindle position checkpoint.

    Directory of Open Access Journals (Sweden)

    Scott A Nelson

    Full Text Available The spindle position checkpoint (SPC ensures correct mitotic spindle position before allowing mitotic exit in the budding yeast Saccharomyces cerevisiae. In a candidate screen for checkpoint genes, we identified bud2Δ as deficient for the SPC. Bud2 is a GTPase activating protein (GAP, and the only known substrate of Bud2 was Rsr1/Bud1, a Ras-like GTPase and a central component of the bud-site-selection pathway. Mutants lacking Rsr1/Bud1 had no checkpoint defect, as did strains lacking and overexpressing Bud5, a guanine-nucleotide exchange factor (GEF for Rsr1/Bud1. Thus, the checkpoint function of Bud2 is distinct from its role in bud site selection. The catalytic activity of the Bud2 GAP domain was required for the checkpoint, based on the failure of the known catalytic point mutant Bud2(R682A to function in the checkpoint. Based on assays of heterozygous diploids, bud2(R682A, was dominant for loss of checkpoint but recessive for bud-site-selection failure, further indicating a separation of function. Tem1 is a Ras-like protein and is the critical regulator of mitotic exit, sitting atop the mitotic exit network (MEN. Tem1 is a likely target for Bud2, supported by genetic analyses that exclude other Ras-like proteins.

  20. Partial Rescue of pos5 Mutants by YEF1 and UTR1 Genes in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    Yong-Fu LI; Feng SHI

    2006-01-01

    Three NAD kinase homologs, encoded by UTR1, POS5 and YEF1 genes, are found in the yeast Saccharomyces cerevisiae and proven to be important sources of NADPH for the cell. Pos5p, existing in the mitochondrial matrix, is critical for higher temperature endurance and mitochondrial functions, such as glycerol usability and arginine biosynthesis. Through constructing the high-copy expression plasmids of YEF1 and UTR1, which contained the green fluorescent protein reporter tag at their 3' terminus, and introducing them into POS5 gene deletion mutants (i.e. pos5, utr1pos5, yef1pos5 and utr1yef1pos5), the high-copy YEF1 and UTR1 plasmids carrying transformants for pos5 mutants were obtained. Their temperature sensitivity and growth phenotype on media with glycerol as the sole carbon source, or on media without arginine, were checked. Results showed the partial rescue of mitochondrial dysfunctions and temperature sensitivity of pos5 mutants by the high-copy YEF1 gene, and of glycerol growth defect and temperature sensitivity by the high-copy UTR1 gene, which confirmed the potential supplying ability of Yef1p and Utr1p for mitochondrial NADP(H) and implied the weak transport of NADP from cytosol to mitochondria. However, even through the green fluorescent protein reporter label, the subcellular localization of Yef1p and Utr1p in yeast cells could not be observed, which indicated the low expression level of these two NAD kinase homologs.

  1. Rsp5 ubiquitin ligase is required for protein trafficking in Saccharomyces cerevisiae COPI mutants.

    Directory of Open Access Journals (Sweden)

    Katarzyna Jarmoszewicz

    Full Text Available Retrograde trafficking from the Golgi to the endoplasmic reticulum (ER depends on the formation of vesicles coated with the multiprotein complex COPI. In Saccharomyces cerevisiae ubiquitinated derivatives of several COPI subunits have been identified. The importance of this modification of COPI proteins is unknown. With the exception of the Sec27 protein (β'COP neither the ubiquitin ligase responsible for ubiquitination of COPI subunits nor the importance of this modification are known. Here we find that the ubiquitin ligase mutation, rsp5-1, has a negative effect that is additive with ret1-1 and sec28Δ mutations, in genes encoding α- and ε-COP, respectively. The double ret1-1 rsp5-1 mutant is also more severely defective in the Golgi-to-ER trafficking compared to the single ret1-1, secreting more of the ER chaperone Kar2p, localizing Rer1p mostly to the vacuole, and increasing sensitivity to neomycin. Overexpression of ubiquitin in ret1-1 rsp5-1 mutant suppresses vacuolar accumulation of Rer1p. We found that the effect of rsp5 mutation on the Golgi-to-ER trafficking is similar to that of sla1Δ mutation in a gene encoding actin cytoskeleton proteins, an Rsp5p substrate. Additionally, Rsp5 and Sla1 proteins were found by co-immunoprecipitation in a complex containing COPI subunits. Together, our results show that Rsp5 ligase plays a role in regulating retrograde Golgi-to-ER trafficking.

  2. Metabolic suppressors of trimethoprim and ultraviolet light sensitivities of Saccharomyces cerevisiae rad6 mutants

    International Nuclear Information System (INIS)

    Dominant mutations at two newly identified loci, designated SRS1 and SRS2, that metabolically suppress the trimethoprim sensitivity of rad6 and rad18 strains, have been isolated from trimethorprim-resistant mutants arising spontaneously in rad6-1 rad18-2 strains of the yeast Saccharomyces cerevisiae. The SRS2 mutations also efficiently suppress the ultraviolet light sensitivity of the parent strains. They do not, however, suppress their sensitivity to ionizing radiation or their deficiency with respect to induced mutagenesis and sporulation. Such observations support the hypothesis that RAD6-dependent activities can be separated into two functionally distinct groups: a group of error-free repair activities that are responsible for a large amount of the radiation resistance of wild-type strains and also for their resistance to trimethoprim, and a group of error-prone activities that are responsible for induced mutagenesis and are also important in sporulation, but which account at best for only a very small amount of wild-type recovery

  3. Assessment of the toxicity of CuO nanoparticles by using Saccharomyces cerevisiae mutants with multiple genes deleted.

    Science.gov (United States)

    Bao, Shaopan; Lu, Qicong; Fang, Tao; Dai, Heping; Zhang, Chao

    2015-12-01

    To develop applicable and susceptible models to evaluate the toxicity of nanoparticles, the antimicrobial effects of CuO nanoparticles (CuO-NPs) on various Saccharomyces cerevisiae (S. cerevisiae) strains (wild type, single-gene-deleted mutants, and multiple-gene-deleted mutants) were determined and compared. Further experiments were also conducted to analyze the mechanisms associated with toxicity using copper salt, bulk CuO (bCuO), carbon-shelled copper nanoparticles (C/Cu-NPs), and carbon nanoparticles (C-NPs) for comparisons. The results indicated that the growth inhibition rates of CuO-NPs for the wild-type and the single-gene-deleted strains were comparable, while for the multiple-gene deletion mutant, significantly higher toxicity was observed (P CuO-NPs to yeast cells was compared with the toxicities of copper salt and bCuO, we concluded that the toxicity of CuO-NPs should be attributed to soluble copper rather than to the nanoparticles. The striking difference in adverse effects of C-NPs and C/Cu-NPs with equivalent surface areas also proved this. A toxicity assay revealed that the multiple-gene-deleted mutant was significantly more sensitive to CuO-NPs than the wild type. Specifically, compared with the wild-type strain, copper was readily taken up by mutant strains when cell permeability genes were knocked out, and the mutants with deletions of genes regulated under oxidative stress (OS) were likely producing more reactive oxygen species (ROS). Hence, as mechanism-based gene inactivation could increase the susceptibility of yeast, the multiple-gene-deleted mutants should be improved model organisms to investigate the toxicity of nanoparticles. PMID:26386067

  4. Transcriptional expression of selected genes associated with excretion of carboxylic acids from aci mutants of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Ewa Boniewska-Bernacka

    2013-04-01

    Full Text Available Introduction: Saccharomyces cerevisiae is an excellent model organism for studies of transcriptional regulation of metabolic processes in other eukaryotic cells including human cells. Cellular acid-base balance can be disturbed in pathologic situations such as renal acidosis or cancer. The extracellular pH of malignant solid tumors is acidic in the range of 6.5-6.9. EG07 and EG37 aci mutants of Saccharomyces cerevisiae excessively excrete carboxylic acids to glucose-containing media or distilled water. The excreted acids are Krebs and/or glyoxylate cycle intermediates. The genes restoring the wild-type phenotype have function that does not easily explain theAci phenotype.Material/Methods: In this study, using real-time PCR we measured relative mRNA expression, in the mutants compared to the wild-type strain, of selected genes associated with both carboxylic acid cycles and two cell transporters, Pma1 and Pdr12, of organic acids. Results: Unexpectedly, we found that the relative expression of the selected Krebs cycle and glyoxylate cycle genes did not change significantly. However, the expression of the two transporter genes was strongly elevated in EG37 and moderately increased in EG07.Conclusion: These results indicate that the induction of the two cell transporterg enes plays an important role in acid excretion by the aci mutants.

  5. The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint

    OpenAIRE

    Marquitz, Aron R.; Harrison, Jacob C.; Bose, Indrani; Zyla, Trevin R.; McMillan, John N.; Lew, Daniel J.

    2002-01-01

    The Saccharomyces cerevisiae morphogenesis checkpoint delays mitosis in response to insults that impair actin organization and/or bud formation. The delay is due to accumulation of the inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. Having screened through a panel of yeast mutants with defects in cell morphogenesis, we report here that the polarity establishment protein Bem2p is required for the checkpoint response. Bem2p is a Rho-GTPase activating protein (G...

  6. Expression of the denV gene of coliphage T4 in UV-sensitive rad mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    A plasmid containing the denV gene from bacteriophage T4, under the control of the yeast alcohol dehydrogenase I (ADC1) promoter, conferred a substantial increase in UV resistance in the UV-sensitive Saccharomyces cerevisiae mutants rad1-2 and rad3-2. The UV resistance of the denV+ yeast cells was cell cycle dependent and correlated well with the level of the denV gene product as measured by immunoblotting and by a photoreversal assay for pyrimidine dimer-DNA glycosylase activity

  7. Hyper- and hyporesponsive mutant forms of the Saccharomyces cerevisiae Ssy1 amino acid sensor

    DEFF Research Database (Denmark)

    Poulsen, Peter; Gaber, Richard F.; Kielland-Brandt, Morten

    2008-01-01

    The Saccharomyces cerevisiae integral membrane protein Ssy1p functions with Ssy5p and Ptr3p to sense extracellular amino acids. Signal transduction leads to processing and nuclear localization of Stp1p and Stp2p, transcriptional activators of many amino acid transporter genes. Ssy1p is structurally...

  8. Growth study of radio-mutant saccharomyce cerevisiae K 1,5 on irradiated molases media

    International Nuclear Information System (INIS)

    The application of the radiopasteurization method for alcoholic fermentation of molases media have been studied which compared to heat pasteurization. The molases samples were obtained from sugar industry in Cirebon, Yogyakarta, and Lawang, used as a samples for gamma irradiation, doses of 3 kGy, 6 kGy and heat pasteurization 80 Celcius centigrade for 30 minutes, which compared to untreated molases. Innculum yeast was S. Cerevisiae K 1.5 which was resulted by irradiation mutation. The results showed that gamma irradiation dose of 3 kGy have pasteurization effect better than 6 kGy and heat pasteurization 80 Celcius centigrade, 30 minutes. Total cells count of microflora per gram samples (% survivors) on molasses media which has been heat pasteurized, decreased to be 70%, 10% for irradiated molasses 3 kGy; and 1% for molasses irradiated 6 kGy, but it did not have significant effect on the growth capacity of S. cerevisiae K 1.5 on that molasses media. Microflora isolated from molasses samples obtained from Cirebon, Yogyakarta, and Lawang, generally from Bacillus subtilis, Lactobacillus sp., Corynebacterium sp., and Rhizopus oligosporus, although was detected but not grows well on molasses media. The growth of S. cerevisiae K 1.5 on fermentation media suplemented with trace elements nitrogen and phosphor resulted difference on fermentation rate i.e.: in irradiated molasses 3 kGy and 6 kGy showed a higher rate, which compared to heat pasteurization and controle. In the environment condition study on molasses media shows the yeast S. cerevisiae K 1.5 have optimal growth at the pH 5.5, specific growth rate 0.3-0.5 per hour, the saturation constant 0.5 - 0.60 g/l, temperature 30 +/- 2 Celcius centigrade with sugar : nitrogen : phosphor ratio = 100 : 5 : 1. The nitrogen and phosphor sources are ammonium sulphate and sodium hidrogen phosphate respectively. (author). 6 refs, 2 figs, 2 tabs

  9. A Genetics Laboratory Module Involving Selection and Identification of Lysine Synthesis Mutants in the Yeast Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Jill B. Keeney

    2009-12-01

    Full Text Available We have developed a laboratory exercise, currently being used with college sophomores, which uses the yeast Saccharomyces cerevisiae to convey the concepts of amino acid biosynthesis, mutation, and gene complementation. In brief, selective medium is used to isolate yeast cells carrying a mutation in the lysine biosynthesis pathway. A spontaneous mutation in any one of three separate genetic loci will allow for growth on selective media; however, the frequency of mutations isolated from each locus differs. Following isolation of a mutated strain, students use complementation analysis to identify which gene contains the mutation. Since the yeast genome has been mapped and sequenced, students with access to the Internet can then research and develop hypotheses to explain the differences in frequencies of mutant genes obtained.

  10. Ethanol Production Kinetics by a Thermo-Tolerant Mutant of Saccharomyces Cerevisiae from Starch Industry Waste (Hydrol

    Directory of Open Access Journals (Sweden)

    Farman Ali Shah

    2010-06-01

    Full Text Available A thermo-tolerant and deoxyglucose-resistant mutant of Saccharomyces cerevisiae was developed and employed to convert them to fuel ethanol in a 150 litre fermenter. Maximum ethanol production was achieved when fermentation of dextrozyme- treated hydrol was carried out for about 36 hours under optimized fermenting conditions. The maximum specific ethanol production rate (qP, and overall ethanol yield (YP/S were found to be 2.82 g L-1 h-1 and 0.49 g/g respectively. Determination of activation energy for cell growth (Ea= 20.8 kJ/mol and death (Ed = 19.1 kJ/mol and product formation and inactivation (EP=35.8 kJ/mol and Edp = 33.5 kJ/mol revealed the thermo-stability of the organism for up to 47°C.

  11. Ethanol production kinetics by a thermo-tolerant mutant of saccharomyces cerevisiae from starch industry waste (hydrol)

    International Nuclear Information System (INIS)

    A thermo-tolerant and deoxyglucose-resistant mutant of Saccharomyces cerevisiae was developed and employed to convert them to fuel ethanol in a 150 litre fermenter. Maximum ethanol production was achieved when fermentation of dextrozyme- treated hydrol was carried out for about 36 hours under optimized fermenting conditions. The maximum specific ethanol production rate (qP), and overall ethanol yield (YP/S) were found to be 2.82 g L/sup -1/ h/sup -1/ and 0.49 g/g respectively. Determination of activation energy for cell growth (Ea= 20.8 kJ/mol) and death (Ed = 19.1 kJ/mol) and product formation and inactivation (EP=35.8 kJ/mol and Edp = 33.5 kJ/mol) revealed the thermo-stability of the organism for up to 47 deg. C. (author)

  12. Anaerobic and aerobic batch cultivations of Saccharomyces cerevisiae mutants impaired in glycerol synthesis

    DEFF Research Database (Denmark)

    Nissen, Torben Lauesgaard; Hamann, Claus Wendelboe; Kielland-Brandt, M. C.; Nielsen, Jens; Villadsen, John

    2000-01-01

    Glycerol is formed as a by-product in production of ethanol and baker's yeast during fermentation of Saccharomyces cerevisiae under anaerobic and aerobic growth conditions, respectively. One physiological role of glycerol formation by yeast is to reoxidize NADH, formed in synthesis of biomass and...... secondary fermentation products, to NAD(+). The objective of this study was to evaluate whether introduction of a new pathway for reoxidation of NADH, in a yeast strain where glycerol synthesis had been impaired, would result in elimination of glycerol production and lead to increased yields of ethanol and...

  13. Rapid Purification and Characterization of Mutant Origin Recognition Complexes in Saccharomyces cerevisiae

    Science.gov (United States)

    Kawakami, Hironori; Ohashi, Eiji; Tsurimoto, Toshiki; Katayama, Tsutomu

    2016-01-01

    Purification of the origin recognition complex (ORC) from wild-type budding yeast cells more than two decades ago opened up doors to analyze the initiation of eukaryotic chromosomal DNA replication biochemically. Although revised methods to purify ORC from overproducing cells were reported later, purification of mutant proteins using these systems still depends on time-consuming processes including genetic manipulation to construct and amplify mutant baculoviruses or yeast strains as well as several canonical protein fractionations. Here, we present a streamlined method to construct mutant overproducers, followed by purification of mutant ORCs. Use of mammalian cells co-transfected with conveniently mutagenized plasmids bearing a His tag excludes many of the construction and fractionation steps. Transfection is highly efficient. All the six subunits of ORC are overexpressed at a considerable level and isolated as a functional heterohexameric complex. Furthermore, use of mammalian cells prevents contamination of wild-type ORC from yeast cells. The method is applicable to wild-type and at least three mutant ORCs, and the resultant purified complexes show expected biochemical activities. The rapid acquisition of mutant ORCs using this system will boost systematic biochemical dissection of ORC and can be even applied to the purification of protein complexes other than ORC. PMID:27148210

  14. Checkpoint Kinases Regulate a Global Network of Transcription Factors in Response to DNA Damage

    Directory of Open Access Journals (Sweden)

    Eric J. Jaehnig

    2013-07-01

    Full Text Available DNA damage activates checkpoint kinases that induce several downstream events, including widespread changes in transcription. However, the specific connections between the checkpoint kinases and downstream transcription factors (TFs are not well understood. Here, we integrate kinase mutant expression profiles, transcriptional regulatory interactions, and phosphoproteomics to map kinases and downstream TFs to transcriptional regulatory networks. Specifically, we investigate the role of the Saccharomyces cerevisiae checkpoint kinases (Mec1, Tel1, Chk1, Rad53, and Dun1 in the transcriptional response to DNA damage caused by methyl methanesulfonate. The result is a global kinase-TF regulatory network in which Mec1 and Tel1 signal through Rad53 to synergistically regulate the expression of more than 600 genes. This network involves at least nine TFs, many of which have Rad53-dependent phosphorylation sites, as regulators of checkpoint-kinase-dependent genes. We also identify a major DNA damage-induced transcriptional network that regulates stress response genes independently of the checkpoint kinases.

  15. A genome-wide deletion mutant screen identifies pathways affected by nickel sulfate in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Dai Wei

    2009-11-01

    Full Text Available Abstract Background The understanding of the biological function, regulation, and cellular interactions of the yeast genome and proteome, along with the high conservation in gene function found between yeast genes and their human homologues, has allowed for Saccharomyces cerevisiae to be used as a model organism to deduce biological processes in human cells. Here, we have completed a systematic screen of the entire set of 4,733 haploid S. cerevisiae gene deletion strains (the entire set of nonessential genes for this organism to identify gene products that modulate cellular toxicity to nickel sulfate (NiSO4. Results We have identified 149 genes whose gene deletion causes sensitivity to NiSO4 and 119 genes whose gene deletion confers resistance. Pathways analysis with proteins whose absence renders cells sensitive and resistant to nickel identified a wide range of cellular processes engaged in the toxicity of S. cerevisiae to NiSO4. Functional categories overrepresented with proteins whose absence renders cells sensitive to NiSO4 include homeostasis of protons, cation transport, transport ATPases, endocytosis, siderophore-iron transport, homeostasis of metal ions, and the diphthamide biosynthesis pathway. Functional categories overrepresented with proteins whose absence renders cells resistant to nickel include functioning and transport of the vacuole and lysosome, protein targeting, sorting, and translocation, intra-Golgi transport, regulation of C-compound and carbohydrate metabolism, transcriptional repression, and chromosome segregation/division. Interactome analysis mapped seven nickel toxicity modulating and ten nickel-resistance networks. Additionally, we studied the degree of sensitivity or resistance of the 111 nickel-sensitive and 72 -resistant strains whose gene deletion product has a similar protein in human cells. Conclusion We have undertaken a whole genome approach in order to further understand the mechanism(s regulating the cell

  16. Dpb11/TopBP1 plays distinct roles in DNA replication, checkpoint response and homologous recombination

    DEFF Research Database (Denmark)

    Germann, Susanne Manuela; Østergaard, Vibe Hallundbæk; Haas, Caroline;

    2011-01-01

    DPB11/TopBP1 is an essential evolutionarily conserved gene involved in initiation of DNA replication and checkpoint signaling. Here, we show that Saccharomyces cerevisiae Dpb11 forms nuclear foci that localize to sites of DNA damage in G1, S and G2 phase, a recruitment that is conserved for its...... and Tel1, and of the checkpoint mediator Rad9. In a site-directed mutagenesis screen, we identify a separation-of-function mutant, dpb11-PF, that is sensitive to DSB-inducing agents yet remains proficient for DNA replication and the S-phase checkpoint at the permissive temperature. The dpb11-PF mutant...... homologue TopBP1 in Gallus gallus. Damage-induced Dpb11 foci are distinct from Sld3 replication initiation foci. Further, Dpb11 foci are dependent on the checkpoint proteins Mec3 (9-1-1 complex) and Rad24, and require the C-terminal domain of Dpb11. Dpb11 foci are independent of the checkpoint kinases Mec1...

  17. CLD1 Reverses the Ubiquinone Insufficiency of Mutant cat5/coq7 in a Saccharomyces cerevisiae Model System.

    Science.gov (United States)

    Kar, Adwitiya; Beam, Haley; Borror, Megan B; Luckow, Michael; Gao, Xiaoli; Rea, Shane L

    2016-01-01

    Ubiquinone (Qn) functions as a mobile electron carrier in mitochondria. In humans, Q biosynthetic pathway mutations lead to Q10 deficiency, a life threatening disorder. We have used a Saccharomyces cerevisiae model of Q6 deficiency to screen for new modulators of ubiquinone biosynthesis. We generated several hypomorphic alleles of coq7/cat5 (clk-1 in Caenorhabditis elegans) encoding the penultimate enzyme in Q biosynthesis which converts 5-demethoxy Q6 (DMQ6) to 5-demethyl Q6, and screened for genes that, when overexpressed, suppressed their inability to grow on non-fermentable ethanol-implying recovery of lost mitochondrial function. Through this approach we identified Cardiolipin-specific Deacylase 1 (CLD1), a gene encoding a phospholipase A2 required for cardiolipin acyl remodeling. Interestingly, not all coq7 mutants were suppressed by Cld1p overexpression, and molecular modeling of the mutant Coq7p proteins that were suppressed showed they all contained disruptions in a hydrophobic α-helix that is predicted to mediate membrane-binding. CLD1 overexpression in the suppressible coq7 mutants restored the ratio of DMQ6 to Q6 toward wild type levels, suggesting recovery of lost Coq7p function. Identification of a spontaneous Cld1p loss-of-function mutation illustrated that Cld1p activity was required for coq7 suppression. This observation was further supported by HPLC-ESI-MS/MS profiling of monolysocardiolipin, the product of Cld1p. In summary, our results present a novel example of a lipid remodeling enzyme reversing a mitochondrial ubiquinone insufficiency by facilitating recovery of hypomorphic enzymatic function. PMID:27603010

  18. Checkpoints Studies Using the Budding Yeast Saccharomyces cerevisiae: Analysis of changes in protein level and subcellular localization during cell cycle progression

    OpenAIRE

    Wu, Xiaorong; Liu, Lili; HUANG, Mingxia

    2011-01-01

    Methods are described here to monitor changes in protein level and subcellular localization during the cell cycle progression in the budding yeast S. cerevisiae. Cell synchronization is achieved by an α-factor mediated block-and-release protocol. Cells are collected at different time points for the first two cell cycles upon release. Cellular DNA contents are analyzed by flow cytometry. Trichloroacetic acid protein precipitates are prepared for monitoring levels of cell cycle regulated protei...

  19. Crystallization and preliminary X-ray analysis of a decameric form of cytosolic thioredoxin peroxidase 1 (Tsa1), C47S mutant, from Saccharomyces cerevisiae

    OpenAIRE

    de Oliveira, Marcos Antonio; Genu, Victor; Discola, Karen Fulan; Alves, Simone Vidigal; Netto, Luis Eduardo Soares; Guimarães, Beatriz Gomes

    2007-01-01

    A recombinant mutant (C47S) of cytosolic thioredoxin peroxidase 1 from S. cerevisiae was expressed, purified and crystallized by the hanging-drop vapour-diffusion method from protein previously treated with 1,4-dithiothreitol. The crystals belong to the monoclinic space group C2 and diffraction data were collected to 2.8 Å resolution using a synchrotron-radiation source.

  20. Immune checkpoints

    OpenAIRE

    Chawla, Akhil; Philips, Anne V; Alatrash, Gheath; Mittendorf, Elizabeth,

    2014-01-01

    Early clinical trials investigating monoclonal antibodies targeting the T-cell inhibitory receptor programmed cell death 1 (PD-1) and its ligand PD-L1 have shown efficacy in melanoma, non-small cell lung cancer and renal cell carcinoma. We recently demonstrated PD-L1 expression in 20% of triple negative breast cancers suggesting that targeting the PD-1/PD-L1 immune checkpoint may be an effective treatment modality in patients with this disease.

  1. Post-transcriptional regulation in the myo1Δ mutant of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Akamine Pearl

    2010-12-01

    Full Text Available Abstract Background Saccharomyces cerevisiae myosin type II-deficient (myo1Δ strains remain viable and divide, despite the absence of a cytokinetic ring, by activation of the PKC1-dependent cell wall integrity pathway (CWIP. Since the myo1Δ transcriptional fingerprint is a subset of the CWIP fingerprint, the myo1Δ strain may provide a simplified paradigm for cell wall stress survival. Results To explore the post-transcriptional regulation of the myo1Δ stress response, 1,301 differentially regulated ribosome-bound mRNAs were identified by microarray analysis of which 204 were co-regulated by transcription and translation. Four categories of mRNA were significantly affected - protein biosynthesis, metabolism, carbohydrate metabolism, and unknown functions. Nine genes of the 20 CWIP fingerprint genes were post-transcriptionally regulated. Down and up regulation of selected ribosomal protein and cell wall biosynthesis mRNAs was validated by their distribution in polysomes from wild type and myo1Δ strains. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2α-P and a reduction in the steady state levels of the translation initiation factor eIF4Gp in myo1Δ strains. Deletion of GCN2 in myo1Δ abolished eIF2αp phosphorylation, and showed a severe growth defect. The presence of P-bodies in myo1Δ strains suggests that the process of mRNA sequestration is active, however, the three representative down regulated RP mRNAs, RPS8A, RPL3 and RPL7B were present at equivalent levels in Dcp2p-mCh-positive immunoprecipitated fractions from myo1Δ and wild type cells. These same RP mRNAs were also selectively co-precipitated with eIF2α-P in myo1Δ strains. Conclusions Quantitative analysis of ribosome-associated mRNAs and their polyribosome distributions suggests selective regulation of mRNA translation efficiency in myo1Δ strains. Inhibition of translation initiation factor eIF2α (e

  2. Thermo tolerant and ethanol producing saccharomyces cerevisiae mutants using gamma radiation

    International Nuclear Information System (INIS)

    Gene manipulation now plays the main role in fermentation industries. However, throughout ethanol production processes, it appeared the requirements for the selection of higher-producing isolate(s) associated, at the same time, with heat-resistant to overcome higher degrees above 30-35 degree, a step which, actually, will reduce final - producing costs. A total of 43 yeast isolates were selected, after exposure of the strain saccharomyces cervisiae to different doses of gamma radiation. Isolated varied in colony size from the original strain as well as among themselves. These isolates were screened for their ability to grow on glucose and supplemented cane molasses media at 30 degree and 40 degree. Out fo them, only 13 isolates proved to grow well on 40 degree. Furthermore, determination of ethanol production by each of these mutants revealed that yielded in general, 16 to 52.0% increase in alcohol production at 40 degree on cane molasses medium (17.5% w/v initial sugars), compared to the original strain. At 40 degree, maximum ethanol yield was 0.63 coupled with 9.5% ethanol concentration and 85.1% sugar conversion which represents 40, 46.2 and 3.4% increase, respectively from the parental strain

  3. The Rho-GAP Bem2p plays a GAP-independent role in the morphogenesis checkpoint

    Science.gov (United States)

    Marquitz, Aron R.; Harrison, Jacob C.; Bose, Indrani; Zyla, Trevin R.; McMillan, John N.; Lew, Daniel J.

    2002-01-01

    The Saccharomyces cerevisiae morphogenesis checkpoint delays mitosis in response to insults that impair actin organization and/or bud formation. The delay is due to accumulation of the inhibitory kinase Swe1p, which phosphorylates the cyclin-dependent kinase Cdc28p. Having screened through a panel of yeast mutants with defects in cell morphogenesis, we report here that the polarity establishment protein Bem2p is required for the checkpoint response. Bem2p is a Rho-GTPase activating protein (GAP) previously shown to act on Rho1p, and we now show that it also acts on Cdc42p, the GTPase primarily responsible for establishment of cell polarity in yeast. Whereas the morphogenesis role of Bem2p required GAP activity, the checkpoint role of Bem2p did not. Instead, this function required an N-terminal Bem2p domain. Thus, this single protein has a GAP-dependent role in promoting cell polarity and a GAP-independent role in responding to defects in cell polarity by enacting the checkpoint. Surprisingly, Swe1p accumulation occurred normally in bem2 cells, but they were nevertheless unable to promote Cdc28p phosphorylation. Therefore, Bem2p defines a novel pathway in the morphogenesis checkpoint. PMID:12145202

  4. Crystallization and preliminary X-ray analysis of a decameric form of cytosolic thioredoxin peroxidase 1 (Tsa1), C47S mutant, from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    A recombinant mutant (C47S) of cytosolic thioredoxin peroxidase 1 from S. cerevisiae was expressed, purified and crystallized by the hanging-drop vapour-diffusion method from protein previously treated with 1,4-dithiothreitol. The crystals belong to the monoclinic space group C2 and diffraction data were collected to 2.8 Å resolution using a synchrotron-radiation source. Saccharomyces cerevisiae cytosolic thioredoxin peroxidase 1 (cTPxI or Tsa1) is a bifunctional enzyme with protective roles in cellular defence against oxidative and thermal stress that exhibits both peroxidase and chaperone activities. Protein overoxidation and/or high temperatures induce great changes in its quaternary structure and lead to its assembly into large complexes that possess chaperone activity. A recombinant mutant of Tsa1 from S. cerevisiae, with Cys47 substituted by serine, was overexpressed in Escherichia coli as a His6-tagged fusion protein and purified by nickel-affinity chromatography. Crystals were obtained from protein previously treated with 1,4-dithiothreitol by the hanging-drop vapour-diffusion method using PEG 3000 as precipitant and sodium fluoride as an additive. Diffraction data were collected to 2.8 Å resolution using a synchrotron-radiation source. The crystal structure was solved by molecular-replacement methods and structure refinement is currently in progress

  5. Effects of rpt1, rpt4 and rpt6 td mutants on GAL1/10 gene expression in Saccharomyces cerevisiae

    OpenAIRE

    Román, Lorena Casado 1991

    2012-01-01

    The aim of this project was to study the roles of Rpt1, Rpt4 and Rpt6 in transcriptional regulation of the GAL network. Therefore, three temperature sentitive degron (td) mutants were created by integrating recombinant plasmids into Saccharomyces cerevisiae chromosomes. Under galactose induction, degradation of Rpt4 caused a decrease in GAL1 and GAL10 mRNA levels, Rpt1 degradation did not cause any detectable effect and Rpt6 degradation caused an increase in GAL1 and GAL10 mRNA transcribed...

  6. Effect of bacterial RecA expression on DNA repair in the rad51 and rad52 on DNA mutants of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Molecular and functional homology between yeast proteins pRad51 and pRad52 and Escherichia coli p RecA involved in re combinational DNA repair led us to investigate possible effects of reA gene expression on DNA repair in rad51 and rad52 mutants of Saccharomyces cerevisiae. The mutant cells were subjected to one of the following treatments: preincubation with 8-methoxypsoralen and subsequent irradiation with 360-nm ultraviolet (UVA) (8-MOP + UVA), irradiation with 254-nm UV light or treatment with methyl methane sulfonate (MMS). While RecA expression did not repair lethal DNA lesions in mutant rad51, it was able to partially restore resistance to 8-MOP + UVA and MMS in rad52. Expression of recA could not complement the sensitivity of rad51rad52 double mutants, indicating that pRad51 may be essential for the repair-stimulating activity of pRecA in the rad52 mutant. Spontaneous mutagenesis was increased, and 8-MOP-photoinduced mutagenesis in rad51. Thus, pRecA may function in yeast DNA repair either as a member of a protein complex or as an individual protein that binds to mutagen-damaged DNA. (author)

  7. Chemogenetic profiling identifies RAD17 as synthetically lethal with checkpoint kinase inhibition.

    Science.gov (United States)

    Shen, John Paul; Srivas, Rohith; Gross, Andrew; Li, Jianfeng; Jaehnig, Eric J; Sun, Su Ming; Bojorquez-Gomez, Ana; Licon, Katherine; Sivaganesh, Vignesh; Xu, Jia L; Klepper, Kristin; Yeerna, Huwate; Pekin, Daniel; Qiu, Chu Ping; van Attikum, Haico; Sobol, Robert W; Ideker, Trey

    2015-11-01

    Chemical inhibitors of the checkpoint kinases have shown promise in the treatment of cancer, yet their clinical utility may be limited by a lack of molecular biomarkers to identify specific patients most likely to respond to therapy. To this end, we screened 112 known tumor suppressor genes for synthetic lethal interactions with inhibitors of the CHEK1 and CHEK2 checkpoint kinases. We identified eight interactions, including the Replication Factor C (RFC)-related protein RAD17. Clonogenic assays in RAD17 knockdown cell lines identified a substantial shift in sensitivity to checkpoint kinase inhibition (3.5-fold) as compared to RAD17 wild-type. Additional evidence for this interaction was found in a large-scale functional shRNA screen of over 100 genotyped cancer cell lines, in which CHEK1/2 mutant cell lines were unexpectedly sensitive to RAD17 knockdown. This interaction was widely conserved, as we found that RAD17 interacts strongly with checkpoint kinases in the budding yeast Saccharomyces cerevisiae. In the setting of RAD17 knockdown, CHEK1/2 inhibition was found to be synergistic with inhibition of WEE1, another pharmacologically relevant checkpoint kinase. Accumulation of the DNA damage marker γH2AX following chemical inhibition or transient knockdown of CHEK1, CHEK2 or WEE1 was magnified by knockdown of RAD17. Taken together, our data suggest that CHEK1 or WEE1 inhibitors are likely to have greater clinical efficacy in tumors with RAD17 loss-of-function. PMID:26437225

  8. Effect of bacterial recA expression on DNA repair in the rad51 and rad52 mutants of Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    M.A. Morais Jr.

    1998-03-01

    Full Text Available Molecular and functional homology between yeast proteins pRad51 and pRad52 and Escherichia coli pRecA involved in recombinational DNA repair led us to investigate possible effects of recA gene expression on DNA repair in rad51 and rad52 mutants of Saccharomyces cerevisiae. The mutant cells were subjected to one of the following treatments: preincubation with 8-methoxypsoralen and subsequent irradiation with 360-nm ultraviolet (UVA (8-MOP + UVA, irradiation with 254-nm UV light or treatment with methyl methane sulfonate (MMS. While recA expression did not repair lethal DNA lesions in mutant rad51, it was able to partially restore resistance to 8-MOP + UVA and MMS in rad52. Expression of recA could not complement the sensitivity of rad51rad52 double mutants, indicating that pRad51 may be essential for the repair-stimulating activity of pRecA in the rad52 mutant. Spontaneous mutagenesis was increased, and 8-MOP-photoinduced mutagenesis was decreased by the presence of pRecA in rad52, whereas pRecA decreased UV-induced mutagenesis in rad51. Thus, pRecA may function in yeast DNA repair either as a member of a protein complex or as an individual protein that binds to mutagen-damaged DNA.A homologia tanto a nível molecular como funcional entre as proteínas de leveduras pRad51 e pRad52 envolvidas na reparação de DNA tipo recombinacional e pRecA de E. coli nos levou a analisar os possíveis efeitos da expressão do gene recA sobre a reparação de DNA nos mutantes rad51 e rad52 de S. cerevisiae após tratamento com 8-MOP + UVA, com UV e com MMS. A expressão de recA não foi capaz de restaurar a reparação das lesões induzidas no DNA do mutante rad51 após tratamento com esses agentes, entretanto ela restaurou parcialmente a resistência ao 8-MOP + UVA e ao MMS no mutante rad52. A expressão de recA não complementou a sensibilidade do duplo mutante rad51rad52, indicando que pRad51 pode ser essencial para estimular a atividade de reparação da p

  9. Cyclin-dependent kinase CDK1/CDC28 and checkpoints

    International Nuclear Information System (INIS)

    The genetic instability induced by defects in the cell cycle progression contributes to different human diseases, particularly neoplastic transformation. The control mechanisms of correct cell cycle progression are the most studied in the yeast Saccharomyces cerevisiae, in which checkpoint was first discovered. Many components of these processes have been identified by now. Here, the role of the central kinase of cell cycle CDK1/CDC28 is considered in checkpoint in different phases.

  10. Genome-Wide Analysis of the Effects of Heat Shock on a Saccharomyces cerevisiae Mutant With a Constitutively Activated cAMP-Dependent Pathway

    Directory of Open Access Journals (Sweden)

    Lubomira Stateva

    2006-04-01

    Full Text Available We have used DNA microarray technology and 2-D gel electrophoresis combined with mass spectrometry to investigate the effects of a drastic heat shock from 30℃ to 50℃ on a genome-wide scale. This experimental condition is used to differentiate between wild-type cells and those with a constitutively active cAMP-dependent pathway in Saccharomyces cerevisiae. Whilst more than 50% of the former survive this shock, almost all of the latter lose viability. We compared the transcriptomes of the wildtype and a mutant strain deleted for the gene PDE2, encoding the high-affinity cAMP phosphodiesterase before and after heat shock treatment. We also compared the two heat-shocked samples with one another, allowing us to determine the changes that occur in the pde2Δ mutant which cause such a dramatic loss of viability after heat shock. Several genes involved in ergosterol biosynthesis and carbon source utilization had altered expression levels, suggesting that these processes might be potential factors in heat shock survival. These predictions and also the effect of the different phases of the cell cycle were confirmed by biochemical and phenotypic analyses. 146 genes of previously unknown function were identified amongst the genes with altered expression levels and deletion mutants in 13 of these genes were found to be highly sensitive to heat shock. Differences in response to heat shock were also observed at the level of the proteome, with a higher level of protein degradation in the mutant, as revealed by comparing 2-D gels of wild-type and mutant heat-shocked samples and mass spectrometry analysis of the differentially produced proteins.

  11. Anaphase onset before complete DNA replication with intact checkpoint responses

    DEFF Research Database (Denmark)

    Torres-Rosell, Jordi; De Piccoli, Giacomo; Cordon-Preciado, Violeta;

    2007-01-01

    Cellular checkpoints prevent mitosis in the presence of stalled replication forks. Whether checkpoints also ensure the completion of DNA replication before mitosis is unknown. Here, we show that in yeast smc5-smc6 mutants, which are related to cohesin and condensin, replication is delayed, most...

  12. Physiology of inactivation of microbial cells by near-ultraviolet light: mode of action and application for the enrichment of mutants of Escherichia coli and saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The mode of action of near-ultraviolet (NUV) light was studied in Escherichia coli. NUV light (maximum emission at 365 nm) caused the photodestruction of ribonucleoside diphosphate (RDP) reductase activity in vivo. Evidence was presented for a model suggesting that the loss of RDP-reductase resulted in a metabolic state analogous to that produced during starvation for thymine. Some important properties of cells irradiated by NUV light, cell death, loss of the ability to support the replication of DNA phages and a delay in the onset of cell division in sublethally irradiated cells, were accounted for in terms of photoinactivation of RDP-reductase. Conditions were described under which NUV light was an effective counterselective agent for the enrichment of mutants of Escherichia coli and Saccharomyces cerevisiae

  13. Systematic strain construction and process development: Xylitol production by Saccharomyces cerevisiae expressing Candida tenuis xylose reductase in wild-type or mutant form.

    Science.gov (United States)

    Pratter, S M; Eixelsberger, T; Nidetzky, B

    2015-12-01

    A novel Saccharomyces cerevisiae whole-cell biocatalyst for xylitol production based on Candida tenuis xylose reductase (CtXR) is presented. Six recombinant strains expressing wild-type CtXR or an NADH-specific mutant were constructed and evaluated regarding effects of expression mode, promoter strength, biocatalyst concentration and medium composition. Intracellular XR activities ranged from 0.09 U mgProt(-1) to 1.05 U mgProt(-1) but did not correlate with the strains' xylitol productivities, indicating that other factors limited xylose conversion in the high-activity strains. The CtXR mutant decreased the biocatalyst's performance, suggesting use of the NADPH-preferring wild-type enzyme when (semi-)aerobic conditions are applied. In a bioreactor process, the best-performing strain converted 40 g L(-1) xylose with an initial productivity of 1.16 g L(-1)h(-1) and a xylitol yield of 100%. The obtained results underline the potential of CtXR wild-type for xylose reduction and point out parameters to improve "green" xylitol production. PMID:26452180

  14. Comparative studies of genome-wide maps of nucleosomes between deletion mutants of elp3 and hos2 genes of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Takashi Matsumoto

    Full Text Available In order to elucidate the influence of histone acetylation upon nucleosomal DNA length and nucleosome position, we compared nucleosome maps of the following three yeast strains; strain BY4741 (control, the elp3 (one of histone acetyltransferase genes deletion mutant, and the hos2 (one of histone deactylase genes deletion mutant of Saccharomyces cerevisiae. We sequenced mononucleosomal DNA fragments after treatment with micrococcal nuclease. After mapping the DNA fragments to the genome, we identified the nucleosome positions. We showed that the distributions of the nucleosomal DNA lengths of the control and the hos2 disruptant were similar. On the other hand, the distribution of the nucleosomal DNA lengths of the elp3 disruptant shifted toward shorter than that of the control. It strongly suggests that inhibition of Elp3-induced histone acetylation causes the nucleosomal DNA length reduction. Next, we compared the profiles of nucleosome mapping numbers in gene promoter regions between the control and the disruptant. We detected 24 genes with low conservation level of nucleosome positions in promoters between the control and the elp3 disruptant as well as between the control and the hos2 disruptant. It indicates that both Elp3-induced acetylation and Hos2-induced deacetylation influence the nucleosome positions in the promoters of those 24 genes. Interestingly, in 19 of the 24 genes, the profiles of nucleosome mapping numbers were similar between the two disruptants.

  15. Conditional Mutants of Rpc160, the Gene Encoding the Largest Subunit of RNA Polymerase C in Saccharomyces Cerevisiae

    OpenAIRE

    Gudenus, R; Mariotte, S; Moenne, A; Ruet, A; Memet, S; Buhler, J M; Sentenac, A; Thuriaux, P

    1988-01-01

    A 18.4-kb fragment of the yeast genome containing the gene of the largest subunit of RNA polymerase C (RPC160) was cloned by hybridization to a previously isolated fragment of that gene. RPC160 maps on chromosome XV, tightly linked but not allelic to the essential gene TSM8740. Temperature sensitive (ts) mutant alleles were constructed by in vitro mutagenesis with NaHSO(3) and substituted for the wild-type allele on the chromosome. Four of them were unambiguously identified as rpc160 mutants ...

  16. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1.

    Science.gov (United States)

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D

    2016-01-29

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  17. Regulation of Sphingolipid Biosynthesis by the Morphogenesis Checkpoint Kinase Swe1*

    Science.gov (United States)

    Chauhan, Neha; Han, Gongshe; Somashekarappa, Niranjanakumari; Gable, Kenneth; Dunn, Teresa; Kohlwein, Sepp D.

    2016-01-01

    Sphingolipid (SL) biosynthesis is negatively regulated by the highly conserved endoplasmic reticulum-localized Orm family proteins. Defective SL synthesis in Saccharomyces cerevisiae leads to increased phosphorylation and inhibition of Orm proteins by the kinase Ypk1. Here we present evidence that the yeast morphogenesis checkpoint kinase, Swe1, regulates SL biosynthesis independent of the Ypk1 pathway. Deletion of the Swe1 kinase renders mutant cells sensitive to serine palmitoyltransferase inhibition due to impaired sphingoid long-chain base synthesis. Based on these data and previous results, we suggest that Swe1 kinase perceives alterations in SL homeostasis, activates SL synthesis, and may thus represent the missing regulatory link that controls the SL rheostat during the cell cycle. PMID:26634277

  18. Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Kishino, Hideyuki; Eguchi, Hiroki; Takagi, Keiko; Horiuchi, Hiroyuki; Fukuda, Ryouichi; Ohta, Akinori, E-mail: aaohta@isc.chubu.ac.jp

    2014-03-07

    Highlights: • Dioctanoyl-PC (diC8PC) supported growth of a yeast mutant defective in PC synthesis. • diC8PC was converted to PC species containing longer acyl residues in the mutant. • Both acyl residues of diC8PC were replaced by longer fatty acids in vitro. • This system will contribute to the elucidation of the acyl chain remodeling of PC. - Abstract: A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of {sup 13}C-labeled diC8PC ((methyl-{sup 13}C){sub 3}-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-{sup 13}C){sub 3}-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast.

  19. Acyl-chain remodeling of dioctanoyl-phosphatidylcholine in Saccharomyces cerevisiae mutant defective in de novo and salvage phosphatidylcholine synthesis

    International Nuclear Information System (INIS)

    Highlights: • Dioctanoyl-PC (diC8PC) supported growth of a yeast mutant defective in PC synthesis. • diC8PC was converted to PC species containing longer acyl residues in the mutant. • Both acyl residues of diC8PC were replaced by longer fatty acids in vitro. • This system will contribute to the elucidation of the acyl chain remodeling of PC. - Abstract: A yeast strain, in which endogenous phosphatidylcholine (PC) synthesis is controllable, was constructed by the replacement of the promoter of PCT1, encoding CTP:phosphocholine cytidylyltransferase, with GAL1 promoter in a double deletion mutant of PEM1 and PEM2, encoding phosphatidylethanolamine methyltransferase and phospholipid methyltransferase, respectively. This mutant did not grow in the glucose-containing medium, but the addition of dioctanoyl-phosphatidylcholine (diC8PC) supported its growth. Analyses of the metabolism of 13C-labeled diC8PC ((methyl-13C)3-diC8PC) in this strain using electrospray ionization tandem mass spectrometry revealed that it was converted to PC species containing acyl residues of 16 or 18 carbons at both sn-1 and sn-2 positions. In addition, both acyl residues of (methyl-13C)3-diC8PC were replaced with 16:1 acyl chains in the in vitro reaction using the yeast cell extract in the presence of palmitoleoyl-CoA. These results indicate that PC containing short acyl residues was remodeled to those with acyl chains of physiological length in yeast

  20. A novel mutant of the Sup35 protein of Saccharomyces cerevisiae defective in translation termination and in GTPase activity still supports cell viability

    Directory of Open Access Journals (Sweden)

    Gillet Sylvie

    2008-02-01

    Full Text Available Abstract Background When a stop codon is located in the ribosomal A-site, the termination complex promotes release of the polypeptide and dissociation of the 80S ribosome. In eukaryotes two proteins eRF1 and eRF3 play a crucial function in the termination process. The essential GTPase Sup35p, the eRF3 release factor of Saccharomyces cerevisiae is highly conserved. In particular, we observed that all eRF3 homologs share a potential phosphorylation site at threonine 341, suggesting a functional role for this residue. The goal of this study was to determine whether this residue is actually phosphorylated in yeast and if it is involved in the termination activity of the protein. Results We detected no phosphorylation of the Sup35 protein in vivo. However, we show that it is phosphorylated by the cAMP-dependent protein kinase A on T341 in vitro. T341 was mutated to either alanine or to aspartic acid to assess the role of this residue in the activity of the protein. Both mutant proteins showed a large decrease of GTPase activity and a reduced interaction with eRF1/Sup45p. This was correlated with an increase of translational readthrough in cells carrying the mutant alleles. We also show that this residue is involved in functional interaction between the N- and C-domains of the protein. Conclusion Our results point to a new critical residue involved in the translation termination activity of Sup35 and in functional interaction between the N- and C-domains of the protein. They also raise interesting questions about the relation between GTPase activity of Sup35 and its essential function in yeast.

  1. Engineering of chromosomal wax ester synthase integrated Saccharomyces cerevisiae mutants for improved biosynthesis of fatty acid ethyl esters.

    Science.gov (United States)

    Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2014-09-01

    In recent years, significant advances have been made to engineer robust microbes for overproducing biochemical products from renewable resources. These accomplishments have to a large extend been based on plasmid based methods. However, plasmid maintenance may cause a metabolic burden on the host cell and plasmid-based overexpression of genes can result in genetically unstable strains, which contributes to loss in productivity. Here, a chromosome engineering method based on delta integration was applied in Saccharomyces cerevisiae for the production of fatty acid ethyl esters (FAEEs), which can be directly used as biodiesel and would be a possible substitute for conventional petroleum-based diesel. An integration construct was designed and integrated into chromosomal delta sequences by repetitive transformation, which resulted in 1-6 copies of the integration construct per genome. The corresponding FAEE production increased up to 34 mg/L, which is an about sixfold increase compared to the equivalent plasmid-based producer. The integrated cassette in the yeast genome was stably maintained in nonselective medium after deletion of RAD52 which is essential for efficient homologous recombination. To obtain a further increase of FAEE production, genes encoding endogenous acyl-CoA binding protein (ACB1) and a bacterial NADP(+)-dependent glyceraldehyde-3-phosphate dehydrogenase (gapN) were overexpressed in the final integration strain, which resulted in another 40% percent increase in FAEE production. Our integration strategy enables easy engineering of strains with adjustable gene copy numbers integrated into the genome and this allows for an easy evaluation of the effect of the gene copy number on pathway flux. It therefore represents a valuable tool for introducing and expressing a heterologous pathway in yeast. PMID:24752598

  2. Cardiolipin Molecular Species with Shorter Acyl Chains Accumulate in Saccharomyces cerevisiae Mutants Lacking the Acyl Coenzyme A-binding Protein Acb1p

    Science.gov (United States)

    Rijken, Pieter J.; Houtkooper, Riekelt H.; Akbari, Hana; Brouwers, Jos F.; Koorengevel, Martijn C.; de Kruijff, Ben; Frentzen, Margrit; Vaz, Frédéric M.; de Kroon, Anton I. P. M.

    2009-01-01

    The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates. PMID:19656950

  3. The SFP1 gene product of Saccharomyces cerevisiae regulates G2/M transitions during the mitotic cell cycle and DNA-damage response

    International Nuclear Information System (INIS)

    In eukaryotic cells, checkpoint pathways arrest cell-cycle progression if a particular event has failed to complete appropriately or if an important intracellular structure is defective or damaged. Saccharomyces cerevisiae strains that lack the SFP1 gene fail to arrest at the G2 DNA-damage checkpoint in response to genomic injury, but maintain their ability to arrest at the replication and spindle-assembly checkpoints. sfp1D mutants are characterized by a premature entrance into mitosis during a normal (undamaged) cell cycle, while strains that overexpress Sfp1p exhibit delays in G2. Sfp1p therefore acts as a repressor of the G2/M transition, both in the normal cell cycle and in the G2 checkpoint pathway. Sfp1 is a nuclear protein with two Cys2His2 zinc-finger domains commonly found in transcription factors. We propose that Sfp1p regulates the expression of gene products involved in the G2/M transition during the mitotic cell cycle and the DNA-damage response. In support of this model, overexpression of Sfp1p induces the expression of the PDS1 gene, which is known to encode a protein that regulates the G2 checkpoint. (author)

  4. A role for Ddc1 in signaling meiotic double-strand breaks at the pachytene checkpoint

    OpenAIRE

    Hong, Eun-Jin Erica; Roeder, G. Shirleen

    2002-01-01

    The pachytene checkpoint prevents meiotic cell cycle progression in response to unrepaired recombination intermediates. We show that Ddc1 is required for the pachytene checkpoint in Saccharomyces cerevisiae. During meiotic prophase, Ddc1 localizes to chromosomes and becomes phosphorylated; these events depend on the formation and processing of double-strand breaks (DSBs). Ddc1 colocalizes with Rad51, a DSB-repair protein, indicating that Ddc1 associates with sites of DSB repair. The Rad24 che...

  5. An organic acid-tolerant HAA1-overexpression mutant of an industrial bioethanol strain of Saccharomyces cerevisiae and its application to the production of bioethanol from sugarcane molasses

    OpenAIRE

    Inaba, Takuya; Watanabe, Daisuke; Yoshiyama, Yoko; Tanaka,Koichi; Ogawa, Jun; Takagi, Hiroshi; Shimoi, Hitoshi; Shima, Jun

    2013-01-01

    Bacterial contamination is known as a major cause of the reduction in ethanol yield during bioethanol production by Saccharomyces cerevisiae. Acetate is an effective agent for the prevention of bacterial contamination, but it negatively affects the fermentation ability of S. cerevisiae. We have proposed that the combined use of organic acids including acetate and lactate and yeast strains tolerant to organic acids may be effective for the elimination of principally lactic acid bacterial (LAB)...

  6. Complementation of a Saccharomyces cerevisiae ERG11/CYP51 (Sterol 14α-Demethylase) Doxycycline-Regulated Mutant and Screening of the Azole Sensitivity of Aspergillus fumigatus Isoenzymes CYP51A and CYP51B▿

    OpenAIRE

    Martel, Claire M.; Parker, Josie E.; Warrilow, Andrew G. S.; Rolley, Nicola J.; Kelly, Steven L.; Kelly, Diane E.

    2010-01-01

    Aspergillus fumigatus sterol 14α-demethylase isoenzymes CYP51A and CYP51B were heterologously expressed in a Saccharomyces cerevisiae mutant (YUG37-erg11), wherein native ERG11/CYP51 expression is controlled using a doxycycline-regulatable promoter. When cultured in the presence of doxycycline, recombinant YUG37-pcyp51A and YUG37-pcyp51B yeasts were able to synthesize ergosterol and grow; a control strain harboring reverse-oriented cyp51A could not. YUG37-pcyp51A and YUG37-pcyp51B constructs ...

  7. Checkpoint control and cancer

    Czech Academy of Sciences Publication Activity Database

    Medema, R.H.; Macůrek, Libor

    2012-01-01

    Roč. 31, č. 21 (2012), s. 2601-2613. ISSN 0950-9232 R&D Projects: GA ČR GAP301/10/1525; GA ČR GPP305/10/P420 Institutional support: RVO:68378050 Keywords : DNA damage * checkpoint control * anticancer strategies Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.357, year: 2012

  8. Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Bojsen, Rasmus K; Andersen, Kaj Scherz; Regenberg, Birgitte

    2012-01-01

    Microbial biofilms can be defined as multi-cellular aggregates adhering to a surface and embedded in an extracellular matrix (ECM). The nonpathogenic yeast, Saccharomyces cerevisiae, follows the common traits of microbial biofilms with cell-cell and cell-surface adhesion. S. cerevisiae is shown t...... cues, cell-to-cell variation and niches in S. cerevisiae biofilm. Being closely related to Candida species, S. cerevisiae is a model to investigate biofilms of pathogenic yeast....

  9. 2-DE based proteomic analysis of Saccharomyces cerevisiae wild and K+ transport-affected mutant (trk1,2) strains at the growth exponential and stationary phases

    Czech Academy of Sciences Publication Activity Database

    Curto, M.; Valledor, L.; Navarrete, C.; Gutiérrez, D.; Sychrová, Hana; Ramos, J.; Jorrin, J.

    2010-01-01

    Roč. 73, č. 12 (2010), s. 2316-2335. ISSN 1874-3919 Institutional research plan: CEZ:AV0Z50110509 Keywords : potassium starvation * S. cerevisiae * proteom Subject RIV: CE - Biochemistry Impact factor: 5.074, year: 2010

  10. Profiling of the toxicity mechanisms of coated and uncoated silver nanoparticles to yeast Saccharomyces cerevisiae BY4741 using a set of its 9 single-gene deletion mutants defective in oxidative stress response, cell wall or membrane integrity and endocytosis.

    Science.gov (United States)

    Käosaar, Sandra; Kahru, Anne; Mantecca, Paride; Kasemets, Kaja

    2016-09-01

    The widespread use of nanosilver in various antibacterial, antifungal, and antiviral products warrants the studies of the toxicity pathways of nanosilver-enabled materials toward microbes and viruses. We profiled the toxicity mechanisms of uncoated, casein-coated, and polyvinylpyrrolidone-coated silver nanoparticles (AgNPs) using Saccharomyces cerevisiae wild-type (wt) and its 9 single-gene deletion mutants defective in oxidative stress (OS) defense, cell wall/membrane integrity, and endocytosis. The 48-h growth inhibition assay in organic-rich growth medium and 24-h cell viability assay in deionized (DI) water were applied whereas AgNO3, H2O2, and SDS served as positive controls. Both coated AgNPs (primary size 8-12nm) were significantly more toxic than the uncoated (~85nm) AgNPs. All studied AgNPs were ~30 times more toxic if exposed to yeast cells in DI water than in the rich growth medium: the IC50 based on nominal concentration of AgNPs in the growth inhibition test ranged from 77 to 576mg Ag/L and in the cell viability test from 2.7 to 18.7mg Ag/L, respectively. Confocal microscopy showed that wt but not endocytosis mutant (end3Δ) internalized AgNPs. Comparison of toxicity patterns of wt and mutant strains defective in OS defense and membrane integrity revealed that the toxicity of the studied AgNPs to S. cerevisiae was not caused by the OS or cell wall/membrane permeabilization. PMID:27260961

  11. An organic acid-tolerant HAA1-overexpression mutant of an industrial bioethanol strain of Saccharomyces cerevisiae and its application to the production of bioethanol from sugarcane molasses.

    Science.gov (United States)

    Inaba, Takuya; Watanabe, Daisuke; Yoshiyama, Yoko; Tanaka, Koichi; Ogawa, Jun; Takagi, Hiroshi; Shimoi, Hitoshi; Shima, Jun

    2013-01-01

    Bacterial contamination is known as a major cause of the reduction in ethanol yield during bioethanol production by Saccharomyces cerevisiae. Acetate is an effective agent for the prevention of bacterial contamination, but it negatively affects the fermentation ability of S. cerevisiae. We have proposed that the combined use of organic acids including acetate and lactate and yeast strains tolerant to organic acids may be effective for the elimination of principally lactic acid bacterial (LAB) contamination. In a previous study employing laboratory S. cerevisiae strains, we showed that overexpression of the HAA1 gene, which encodes a transcriptional activator, could be a useful molecular breeding method for acetate-tolerant yeast strains. In the present study, we constructed a HAA1-overexpressing diploid strain (MATa/α, named ER HAA1-OP) derived from the industrial bioethanol strain Ethanol Red (ER). ER HAA1-OP showed tolerance not only to acetate but also to lactate, and this tolerance was dependent on the increased expression of HAA1 gene. The ethanol production ability of ER HAA1-OP was almost equivalent to that of the parent strain during the bioethanol production process from sugarcane molasses in the absence of acetate. The addition of acetate at 0.5% (w/v, pH 4.5) inhibited the fermentation ability of the parent strain, but such an inhibition was not observed in the ethanol production process using ER HAA1-OP. PMID:24373204

  12. Checkpoint inhibition in meningiomas.

    Science.gov (United States)

    Bi, Wenya Linda; Wu, Winona W; Santagata, Sandro; Reardon, David A; Dunn, Ian F

    2016-06-01

    Meningiomas are increasingly appreciated to share similar features with other intra-axial central nervous system neoplasms as well as systemic cancers. Immune checkpoint inhibition has emerged as a promising therapy in a number of cancers, with durable responses of years in a subset of patients. Several lines of evidence support a role for immune-based therapeutic strategies in the management of meningiomas, especially high-grade subtypes. Meningiomas frequently originate juxtaposed to venous sinuses, where an anatomic conduit for lymphatic drainage resides. Multiple populations of immune cells have been observed in meningiomas. PD-1/PD-L1 mediated immunosuppression has been implicated in high-grade meningiomas, with association between PD-L1 expression with negative prognostic outcome. These data point to the promise of future combinatorial therapeutic strategies in meningioma. PMID:27197540

  13. In situ assay for 5-aminolevulinate dehydratase and application to the study of a catabolite repression-resistant Saccharomyces cerevisiae mutant.

    Science.gov (United States)

    Borralho, L M; Panek, A D; Malamud, D R; Sanders, H K; Mattoon, J R

    1983-10-01

    To facilitate the study of the effects of carbon catabolite repression and mutations on 5-aminolevulinate dehydratase (EC 4.2.1.24) from Saccharomyces cerevisiae, a sensitive in situ assay was developed, using cells permeabilized by five cycles of freezing and thawing. Enzymatic activity was measured by colorimetric determination of porphobilinogen with a modified Ehrlich reagent. For normal strains, porphobilinogen production was linear for 15 min, and the reaction rate was directly proportional to the permeabilized cell concentration up to 20 mg (dry weight) per ml. The reaction exhibited Michaelis-Menten-type kinetics, and an apparent Km of 2.6 mM was obtained for 5-aminolevulinic acid. This value is only slightly higher than the value of 1.8 mM obtained for the enzyme assayed in cell extracts. The in situ assay was used to assess catabolite repression-dependent changes in 5-aminolevulinate dehydratase during batch culture on glucose medium. In normal S. cerevisiae cells, the enzyme is strongly repressed as long as glucose is present in the medium. In contrast, a strain bearing the hex2-3 mutation exhibits derepressed levels of enzyme activity during growth on glucose. Synthesis of cytochromes by this strain is also resistant to catabolite repression. Similar studies employing a strain containing the glc1 mutation, which enhances porphyrin accumulation, did not reveal any significant phenotypic change in catabolite regulation of 5-aminolevulinate dehydratase. PMID:6352674

  14. Growth-inhibitory activity of the D-mannan of Saccharomyces cerevisiae X2180-1A-5 mutant strain against mouse-implanted sarcoma 180 and Ehrlich-carcinoma solid tumor.

    Science.gov (United States)

    Matsumoto, T; Takanohashi, M; Okubo, Y; Suzuki, M; Suzuki, S

    1980-08-15

    The D-mannan of Saccharomyces cerevisiae X2180-1A-5 mutant strain, which possesses a main chain composed of alpha-(1 yields 6) linked D-mannopyranosyl residues and a small proportion of branches composed of alpha-(1 yields 2)- and alpha-(1 yields 3)-linked D-mannopyranosyl residues, showed strong growth-inhibitory activity against mouse-implanted Sarcoma 180 and Ehrlich-carcinoma solid tumor. The observation that the level of this activity was nearly identical with that of the D-mannan of a wild-type strain of bakers' yeast, which possesses a high proportion of branches composed of alpha-(1 yields 2)-and alpha-(1 yields 3)-linked D-mannopyranosyl residues, suggests that the branches are not essential for antitumor activity. The partial acid-degradation products of both D-mannans, the molecular weight of which was one-third of that of each parent D-mannan, had only one half of the antitumor activity of the parent D-mannans. This suggests that molecular size is the most important factor for the differences in acitvity of the polysaccharides of wild and mutant strains. PMID:6996813

  15. Preserving Yeast Genetic Heritage through DNA Damage Checkpoint Regulation and Telomere Maintenance

    Directory of Open Access Journals (Sweden)

    Huilin Zhou

    2012-10-01

    Full Text Available In order to preserve genome integrity, extrinsic or intrinsic DNA damages must be repaired before they accumulate in cells and trigger other mutations and genome rearrangements. Eukaryotic cells are able to respond to different genotoxic stresses as well as to single DNA double strand breaks (DSBs, suggesting highly sensitive and robust mechanisms to detect lesions that trigger a signal transduction cascade which, in turn, controls the DNA damage response (DDR. Furthermore, cells must be able to distinguish natural chromosomal ends from DNA DSBs in order to prevent inappropriate checkpoint activation, DDR and chromosomal rearrangements. Since the original discovery of RAD9, the first DNA damage checkpoint gene identified in Saccharomyces cerevisiae, many genes that have a role in this pathway have been identified, including MRC1, MEC3, RAD24, RAD53, DUN1, MEC1 and TEL1. Extensive studies have established most of the genetic basis of the DNA damage checkpoint and uncovered its different functions in cell cycle regulation, DNA replication and repair, and telomere maintenance. However, major questions concerning the regulation and functions of the DNA damage checkpoint remain to be answered. First, how is the checkpoint activity coupled to DNA replication and repair? Second, how do cells distinguish natural chromosome ends from deleterious DNA DSBs? In this review we will examine primarily studies performed using Saccharomyces cerevisiae as a model system.

  16. The pachytene checkpoint prevents accumulation and phosphorylation of the meiosis-specific transcription factor Ndt80

    OpenAIRE

    Tung, Kuei-Shu; Hong, Eun-Jin Erica; Roeder, G. Shirleen

    2000-01-01

    In budding yeast, many mutants defective in meiotic recombination and chromosome synapsis undergo checkpoint-mediated arrest at the pachytene stage of meiotic prophase. We recovered the NDT80 gene in a screen for genes whose overexpression bypasses the pachytene checkpoint. Ndt80 is a meiosis-specific transcription factor that promotes expression of genes required for exit from pachytene and entry into meiosis I. Herein, we show that the Ndt80 protein accumulates a...

  17. Toward an Optimal Online Checkpoint Solution under a Two-Level HPC Checkpoint Model

    OpenAIRE

    Di, Sheng; Robert, Yves; Vivien, Frédéric; Cappello, Franck

    2016-01-01

    The traditional single-level checkpointing method suffers from significantoverhead on large-scale platforms. Hence, multilevel checkpointing protocols have been studied extensively in recent years. The multilevel checkpoint approach allows different levels of checkpoints to be set (each with different checkpoint overheads and recovery abilities), in order to further improve the fault tolerance performance of extreme-scale HPC applications. How to optimize the checkpoint intervals for each lev...

  18. A septin from the filamentous fungus A. nidulans induces atypical pseudohyphae in the budding yeast S. cerevisiae

    Science.gov (United States)

    Septins were first discovered in Saccharomyces cerevisiae where they form a scaffold that organizes the bud site and are a component of the morphogenesis checkpoint that coordinates budding with mitosis. Five of the seven S. cerevisiae septins (Cdc3, Cdc10, Cdc11, Cdc12 and Shs1) colocalize as a rin...

  19. Meiotic gene conversion mutants in Saccharomyces cerevisiae. I. Isolation and characterization of PMS1-1 and PMS1-2

    International Nuclear Information System (INIS)

    The PMS1 mutants, isolated on the basis of sharply elevated meiotic prototroph frequencies for two closely linked HIS4 alleles, display pleiotropic phenotypes in meiotic and mitotic cells. Two isolates carrying recessive mutations in PMS1 were characterized. They identify a function required to maintain low postmeiotic segregation (PMS) frequencies at many heterozygous sites. In addition, they are mitotic mutators. In mutant diploids, spore viability is reduced, and among survivors, gene conversion and postmeiotic segregation frequencies are increased, but reciprocal exchange frequencies are not affected. The conversion event pattern is also dramatically changed in multiply marked regions in PMS1 homozygotes. The PMS1 locus maps near MET4 on chromosome XIV. The PMS1 gene may identify an excision-resynthesis long patch mismatch correction function or a function that facilitates correction tract elongation. The PMS1 gene product may also play an important role in spontaneous mitotic mutation avoidance and correction of mismatches in heteroduplex DNA formed during spontaneous and UV-induced mitotic recombination. Based on meiotic recombination models emphasizing mismatch correction in heteroduplex DNA intermediates, this interpretation is favored, but alternative interpretations involving longer recombination intermediates in the mutants are also considered

  20. Three Candida albicans potassium uptake systems differ in their ability to provide Saccharomyces cerevisiae trk1trk2 mutants with necessary potassium.

    Science.gov (United States)

    Elicharová, Hana; Hušeková, Barbora; Sychrová, Hana

    2016-06-01

    Yeasts usually have one or two high-affinity potassium transporters. Two complete and one interrupted gene encoding three types of putative potassium uptake system exist in Candida albicans SC5314. As high intracellular potassium is essential for many yeast cell functions, the existence of three transporters with differing transport mechanisms (Trk uniporter, Hak cation-proton symporter, Acu ATPase) may help pathogenic C. albicans cells to acquire the necessary potassium in various organs and tissues of the host. When expressed in Saccharomyces cerevisiae lacking their own potassium uptake systems, all three putative transporters were able to provide cells with the ability to grow with low amounts of potassium over a broad range of external pH. Only CaTrk1 was properly recognized and secreted to the plasma membrane. Nevertheless, even the small number of CaHak1 and mainly CaAcu1 molecules which reached the plasma membrane resulted in an improved growth of cells in low potassium concentrations, suggesting a high affinity and capacity of the transporters. A single-point mutation restored the complete CaACU1 gene, and the resulting protein not only provided cells with the necessary potassium but also improved their tolerance to toxic lithium. In contrast to its known homologues, CaAcu1 did not seem to transport sodium. PMID:27189364

  1. Characterization of the S. cerevisiae inp51 mutant links phosphatidylinositol 4,5-bisphosphate levels with lipid content, membrane fluidity and cold growth.

    Science.gov (United States)

    Córcoles-Sáez, Isaac; Hernández, Maria Luisa; Martínez-Rivas, Jose Manuel; Prieto, Jose A; Randez-Gil, Francisca

    2016-03-01

    Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its derivatives diphosphoinositol phosphates (DPIPs) play key signaling and regulatory roles. However, a direct function of these molecules in lipid and membrane homeostasis remains obscure. Here, we have studied the cold tolerance phenotype of yeast cells lacking the Inp51-mediated phosphoinositide-5-phosphatase. Genetic and biochemical approaches showed that increased metabolism of PI(4,5)P2 reduces the activity of the Pho85 kinase by increasing the levels of the DPIP isomer 1-IP7. This effect was key in the cold tolerance phenotype. Indeed, pho85 mutant cells grew better than the wild-type at 15 °C, and lack of this kinase abolished the inp51-mediated cold phenotype. Remarkably, reduced Pho85 function by loss of Inp51 affected the activity of the Pho85-regulated target Pah1, the yeast phosphatidate phosphatase. Cells lacking Inp51 showed reduced Pah1 abundance, derepression of an INO1-lacZ reporter, decreased content of triacylglycerides and elevated levels of phosphatidate, hallmarks of the pah1 mutant. However, the inp51 phenotype was not associated to low Pah1 activity since deletion of PAH1 caused cold sensitivity. In addition, the inp51 mutant exhibited features not shared by pah1, including a 40%-reduction in total lipid content and decreased membrane fluidity. These changes may influence the activity of membrane-anchored and/or associated proteins since deletion of INP51 slows down the transit to the vacuole of the fluorescent dye FM4-64. In conclusion, our work supports a model in which changes in the PI(4,5)P2 pool affect the 1-IP7 levels modulating the activity of Pho85, Pah1 and likely additional Pho85-controlled targets, and regulate lipid composition and membrane properties. PMID:26724696

  2. Cardiolipin molecular species with shorter acyl chains accumulate in Saccharomyces cerevisiae mutants lacking the acyl coenzyme A-binding protein Acb1p: new insights into acyl chain remodeling of cardiolipin.

    Science.gov (United States)

    Rijken, Pieter J; Houtkooper, Riekelt H; Akbari, Hana; Brouwers, Jos F; Koorengevel, Martijn C; de Kruijff, Ben; Frentzen, Margrit; Vaz, Frédéric M; de Kroon, Anton I P M

    2009-10-01

    The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates. PMID:19656950

  3. Efficient Incremental Checkpointing of Java Programs

    DEFF Research Database (Denmark)

    Lawall, Julia Laetitia; Muller, Gilles

    2000-01-01

    This paper investigates the optimization of language-level checkpointing of Java programs. First, we describe how to systematically associate incremental checkpoints with Java classes. While being safe, the genericness of this solution induces substantial execution overhead. Second, to solve...

  4. Cambridge checkpoint English workbook 1

    CERN Document Server

    Reynolds, John

    2013-01-01

    This Workbook supports our bestselling Checkpoint English series, with exercises specifically matched to the Cambridge Progression tests and the Checkpoint English tests. - Offers plenty of additional questions for use in class or as homework. - Includes clearly identified questions on grammar and punctuation, comprehension, use of language and essay planning. - Follows the structure of the relevant textbook to ensure a thorough understanding of all aspects of the course. - Provides a space for Students to write their answers. This Workbook is matched to the Cambridge Secondary 1 Curriculum Fr

  5. Network support for system initiated checkpoints

    Science.gov (United States)

    Chen, Dong; Heidelberger, Philip

    2013-01-29

    A system, method and computer program product for supporting system initiated checkpoints in parallel computing systems. The system and method generates selective control signals to perform checkpointing of system related data in presence of messaging activity associated with a user application running at the node. The checkpointing is initiated by the system such that checkpoint data of a plurality of network nodes may be obtained even in the presence of user applications running on highly parallel computers that include ongoing user messaging activity.

  6. Cell size checkpoint control by the retinoblastoma tumor suppressor pathway.

    Directory of Open Access Journals (Sweden)

    Su-Chiung Fang

    2006-10-01

    Full Text Available Size control is essential for all proliferating cells, and is thought to be regulated by checkpoints that couple cell size to cell cycle progression. The aberrant cell-size phenotypes caused by mutations in the retinoblastoma (RB tumor suppressor pathway are consistent with a role in size checkpoint control, but indirect effects on size caused by altered cell cycle kinetics are difficult to rule out. The multiple fission cell cycle of the unicellular alga Chlamydomonas reinhardtii uncouples growth from division, allowing direct assessment of the relationship between size phenotypes and checkpoint function. Mutations in the C. reinhardtii RB homolog encoded by MAT3 cause supernumerous cell divisions and small cells, suggesting a role for MAT3 in size control. We identified suppressors of an mat3 null allele that had recessive mutations in DP1 or dominant mutations in E2F1, loci encoding homologs of a heterodimeric transcription factor that is targeted by RB-related proteins. Significantly, we determined that the dp1 and e2f1 phenotypes were caused by defects in size checkpoint control and were not due to a lengthened cell cycle. Despite their cell division defects, mat3, dp1, and e2f1 mutants showed almost no changes in periodic transcription of genes induced during S phase and mitosis, many of which are conserved targets of the RB pathway. Conversely, we found that regulation of cell size was unaffected when S phase and mitotic transcription were inhibited. Our data provide direct evidence that the RB pathway mediates cell size checkpoint control and suggest that such control is not directly coupled to the magnitude of periodic cell cycle transcription.

  7. Checkpointing for a hybrid computing node

    Science.gov (United States)

    Cher, Chen-Yong

    2016-03-08

    According to an aspect, a method for checkpointing in a hybrid computing node includes executing a task in a processing accelerator of the hybrid computing node. A checkpoint is created in a local memory of the processing accelerator. The checkpoint includes state data to restart execution of the task in the processing accelerator upon a restart operation. Execution of the task is resumed in the processing accelerator after creating the checkpoint. The state data of the checkpoint are transferred from the processing accelerator to a main processor of the hybrid computing node while the processing accelerator is executing the task.

  8. Defective DNA repair increases susceptibility to senescence through extension of Chk1-mediated G2 checkpoint activation.

    Science.gov (United States)

    Johmura, Yoshikazu; Yamashita, Emiri; Shimada, Midori; Nakanishi, Keiko; Nakanishi, Makoto

    2016-01-01

    Susceptibility to senescence caused by defective DNA repair is a major hallmark of progeroid syndrome patients, but molecular mechanisms of how defective DNA repair predisposes to senescence are largely unknown. We demonstrate here that suppression of DNA repair pathways extends the duration of Chk1-dependent G2 checkpoint activation and sensitizes cells to senescence through enhancement of mitosis skipping. Extension of G2 checkpoint activation by introduction of the TopBP1 activation domain and the nondegradable mutant of Claspin sensitizes cells to senescence. In contrast, a shortening of G2 checkpoint activation by expression of SIRT6 or depletion of OTUB2 reduces susceptibility to senescence. Fibroblasts from progeroid syndromes tested shows a correlation between an extension of G2 checkpoint activation and an increase in the susceptibility to senescence. These results suggest that extension of G2 checkpoint activation caused by defective DNA repair is critical for senescence predisposition in progeroid syndrome patients. PMID:27507734

  9. DNA damage checkpoint recovery and cancer development

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Haiyong [First affiliated hospital, Zhejiang University, School of medicine, Cancer Center, 79 Qingchun Road, Hangzhou 310003 (China); Zhang, Xiaoshan [Department of Genetics, University of Texas M.D. Anderson Cancer Center, Department of Genetics Unit 1010, 1515 Holcombe Blvd. Houston, TX 77030 (United States); Teng, Lisong, E-mail: lsteng@zju.edu.cn [First affiliated hospital, Zhejiang University, School of medicine, Cancer Center, 79 Qingchun Road, Hangzhou 310003 (China); Legerski, Randy J., E-mail: rlegersk@mdanderson.org [Department of Genetics, University of Texas M.D. Anderson Cancer Center, Department of Genetics Unit 1010, 1515 Holcombe Blvd. Houston, TX 77030 (United States)

    2015-06-10

    Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observed to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint

  10. DNA damage checkpoint recovery and cancer development

    International Nuclear Information System (INIS)

    Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observed to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint

  11. RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

    International Nuclear Information System (INIS)

    Highlights: ► RNAi is linked to the cell cycle checkpoint in fission yeast. ► Ptr1 co-purifies with Ago1. ► The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. ► ago1+ and ptr1+ regulate the cell cycle checkpoint via the same pathway. ► Mutations in ago1+ and ptr1+ lead to the nuclear accumulation of poly(A)+ RNAs. -- Abstract: Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1+, the overexpression of ago1+ alleviated the cell cycle defect in dcr1Δ. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1+ is dependent on ptr1+. Nuclear accumulation of poly(A)+ RNAs was detected in mutants of ago1+ and ptr1+, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

  12. Cenp-meta is required for sustained spindle checkpoint

    Directory of Open Access Journals (Sweden)

    Thomas Rubin

    2014-05-01

    Full Text Available Cenp-E is a kinesin-like motor protein required for efficient end-on attachment of kinetochores to the spindle microtubules. Cenp-E immunodepletion in Xenopus mitotic extracts results in the loss of mitotic arrest and massive chromosome missegregation, whereas its depletion in mammalian cells leads to chromosome segregation defects despite the presence of a functional spindle assembly checkpoint (SAC. Cenp-meta has previously been reported to be the Drosophila homolog of vertebrate Cenp-E. In this study, we show that cenp-metaΔ mutant neuroblasts arrest in mitosis when treated with colchicine. cenp-metaΔ mutant cells display a mitotic delay. Yet, despite the persistence of the two checkpoint proteins Mad2 and BubR1 on unattached kinetochores, these cells eventually enter anaphase and give rise to highly aneuploid daughter cells. Indeed, we find that cenp-metaΔ mutant cells display a slow but continuous degradation of cyclin B, which eventually triggers the mitotic exit observed. Thus, our data provide evidence for a role of Cenp-meta in sustaining the SAC response.

  13. Visualizing the spindle checkpoint in Drosophila spermatocytes

    OpenAIRE

    Rebollo, Elena; González, Cayetano

    2000-01-01

    The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint ma...

  14. Isolation of the catalase T structural gene of Saccharomyces cerevisiae by functional complementation.

    OpenAIRE

    Spevak, W; Fessl, F; Rytka, J; Traczyk, A; Skoneczny, M; Ruis, H

    1983-01-01

    The catalase T structural gene of Saccharomyces cerevisiae was cloned by functional complementation of a mutation causing specific lack of the enzyme (cttl). Catalase T-deficient mutants were obtained by UV mutagenesis of an S. cerevisiae strain bearing the cas1 mutation, which causes insensitivity of catalase T to glucose repression. Since the second catalase protein of S. cerevisiae, catalase A, is completely repressed on 10% glucose, catalase T-deficient mutant colonies could be detected u...

  15. Yeast mutants auxotrophic for choline or ethanolamine.

    OpenAIRE

    Atkinson, K D; Jensen, B.; Kolat, A I; Storm, E M; Henry, S. A.; Fogel, S

    1980-01-01

    Three mutants of the yeast Saccharomyces cerevisiae which require exogenous ethanolamine or choline were isolated. The mutants map to a single locus (cho1) on chromosome V. The lipid composition suggests that cho1 mutants do not synthesize phosphatidylserine under any growth conditions. If phosphatidylethanolamine or phosphatidylcholine, which are usually derived from phosphatidylserine, were synthesized from exogenous ethanolamine or choline, the mutants grew and divided relatively normally....

  16. GRID COMPUTING AND CHECKPOINT APPROACH

    Directory of Open Access Journals (Sweden)

    Pankaj gupta

    2011-05-01

    Full Text Available Grid computing is a means of allocating the computational power of alarge number of computers to complex difficult computation or problem. Grid computing is a distributed computing paradigm thatdiffers from traditional distributed computing in that it is aimed toward large scale systems that even span organizational boundaries. In this paper we investigate the different techniques of fault tolerance which are used in many real time distributed systems. The main focus is on types of fault occurring in the system, fault detection techniques and the recovery techniques used. A fault can occur due to link failure, resource failure or by any other reason is to be tolerated for working the system smoothly and accurately. These faults can be detected and recovered by many techniques used accordingly. An appropriate fault detector can avoid loss due to system crash and reliable fault tolerance technique can save from system failure. This paper provides how these methods are applied to detect and tolerate faults from various Real Time Distributed Systems. The advantages of utilizing the check pointing functionality are obvious; however so far the Grid community has notdeveloped a widely accepted standard that would allow the Gridenvironment to consciously utilize low level check pointing packages.Therefore, such a standard named Grid Check pointing Architecture isbeing designed. The fault tolerance mechanism used here sets the jobcheckpoints based on the resource failure rate. If resource failureoccurs, the job is restarted from its last successful state using acheckpoint file from another grid resource. A critical aspect for anautomatic recovery is the availability of checkpoint files. A strategy to increase the availability of checkpoints is replication. Grid is a form distributed computing mainly to virtualizes and utilize geographically distributed idle resources. A grid is a distributed computational and storage environment often composed of

  17. Isolation of autophagocytosis mutants of Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Thumm, M.; Egner, R.; Schlumpberger, M.; Straub, M.; Veenhuis, M.; Wolf, D.H.

    1994-01-01

    Protein degradation in the vacuole (lysosome) is an important event in cellular regulation. In yeast, as in mammalian cells, a major route of protein uptake for degradation into the vacuole (lysosome) has been found to be autophagocytosis. The discovery of this process in yeast enables the elucidati

  18. Determinants of Swe1p Degradation in Saccharomyces cerevisiae

    Science.gov (United States)

    McMillan, John N.; Theesfeld, Chandra L.; Harrison, Jacob C.; Bardes, Elaine S. G.; Lew, Daniel J.

    2002-01-01

    Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the relocalization of Swe1p from the nucleus to the mother-bud neck, and neck targeting requires the Swe1p-interacting protein Hsl7p. In addition, Swe1p degradation is stimulated by its substrate, cyclin/Cdc28p, and Swe1p is thought to be a target of the ubiquitin ligase SCFMet30 acting with the ubiquitin-conjugating enzyme Cdc34p. The basis for regulation of Swe1p degradation by the morphogenesis checkpoint remains unclear, and in order to elucidate that regulation we have dissected the Swe1p degradation pathway in more detail, yielding several novel findings. First, we show here that Met30p (and by implication SCFMet30) is not, in fact, required for Swe1p degradation. Second, cyclin/Cdc28p does not influence Swe1p neck targeting, but can directly phosphorylate Swe1p, suggesting that it acts downstream of neck targeting in the Swe1p degradation pathway. Third, a screen for functional but nondegradable mutants of SWE1 identified two small regions of Swe1p that are key to its degradation. One of these regions mediates interaction of Swe1p with Hsl7p, showing that the Swe1p-Hsl7p interaction is critical for Swe1p neck targeting and degradation. The other region did not appear to affect interactions with known Swe1p regulators, suggesting that other as-yet-unknown regulators exist. PMID:12388757

  19. Overlapped checkpointing with hardware assist

    Energy Technology Data Exchange (ETDEWEB)

    Mitchell, Christopher J [Los Alamos National Laboratory; Nunez, James A [Los Alamos National Laboratory; Wang, Jun [U. OF CENTRAL FLORIDA (UCF)

    2009-01-01

    We present a new approach to handling the demanding I/O workload incurred during checkpoint writes encountered in High Performance Computing. Prior efforts to improve performance have been primarily bound by mechanical limitations of the hard drive. Our research surpasses this limitation by providing a method to: (1) write checkpoint data to a high-speed, non-volatile buffer, and (2) asynchronously write this data to permanent storage while resuming computation. This removes the hard drive from the critical data path because our I/O node based buffers isolate the compute nodes from the storage servers. This solution is feasible because of industry declines in cost for high-capacity, non-volatile storage technologies. Testing was conducted on a small-scale cluster to prove the design, and then scaled at Los Alamos National Laboratory. Results show a definitive speedup factor for select workloads over writing directly to a typical global parallel file system; the Panasas ActiveScale File System.

  20. Kinase signaling in the spindle checkpoint.

    Science.gov (United States)

    Kang, Jungseog; Yu, Hongtao

    2009-06-01

    The spindle checkpoint is a cell cycle surveillance system that ensures the fidelity of chromosome segregation. In mitosis, it elicits the "wait anaphase" signal to inhibit the anaphase-promoting complex or cyclosome until all chromosomes achieve bipolar microtubule attachment and align at the metaphase plate. Because a single kinetochore unattached to microtubules activates the checkpoint, the wait anaphase signal is thought to be generated by this kinetochore and is then amplified and distributed throughout the cell to inhibit the anaphase-promoting complex/cyclosome. Several spindle checkpoint kinases participate in the generation and amplification of this signal. Recent studies have begun to reveal the activation mechanisms of these checkpoint kinases. Increasing evidence also indicates that the checkpoint kinases not only help to generate the wait anaphase signal but also actively correct kinetochore-microtubule attachment defects. PMID:19228686

  1. Radiation-sensitive mutants of yeast

    International Nuclear Information System (INIS)

    Nomenclature for various radiosensitive mutants of Saccharomyces cerevisiae is briefly discussed. Tables are presented to show results of allelism tests of most of the radiosensitive mutants isolated by various investigators together with a standardized rad locus designation and map positions of a number of rad loci in yeast

  2. Visualizing the spindle checkpoint in Drosophila spermatocytes

    Science.gov (United States)

    Rebollo, Elena; González, Cayetano

    2000-01-01

    The spindle assembly checkpoint detects defects in spindle structure or in the alignment of the chromosomes on the metaphase plate and delays the onset of anaphase until defects are corrected. Thus far, the evidence regarding the presence of a spindle checkpoint during meiosis in male Drosophila has been indirect and contradictory. On the one hand, chromosomes without pairing partners do not prevent meiosis progression. On the other hand, some conserved components of the spindle checkpoint machinery are expressed in these cells and behave as their homologue proteins do in systems with an active spindle checkpoint. To establish whether the spindle checkpoint is active in Drosophila spermatocytes we have followed meiosis progression by time-lapse microscopy under conditions where the checkpoint is likely to be activated. We have found that the presence of a relatively high number of misaligned chromosomes or a severe disruption of the meiotic spindle results in a significant delay in the time of entry into anaphase. These observations provide the first direct evidence substantiating the activity of a meiotic spindle checkpoint in male Drosophila. PMID:11256627

  3. BIOSORPTION OF CHROMIUM (VI FROM INDUSTRIAL EFFLUENT BY WILD ANDMUTANT TYPE STRAIN OF SACCHAROMYCES CEREVISIAE AND ITS IMMOBILIZED FORM

    Directory of Open Access Journals (Sweden)

    K Selvam, K Arungandhi, B Vishnupriya, T Shanmuga priya and M Yamuna

    2013-01-01

    Full Text Available Biosorption of chromium was studied by wild type Saccharomyces cerevisiae strain, mutant strain, immobilized-wild type and mutant strain. Chromium absorption pattern was observed in all experimental conditions. Hexavalent chromium (VI was analyzed by diphenyl carbazide method, by oxidizing the trivalent chromium (III. The percentage efficiency of wild type S. cerevisiae and its mutant strain, immobilized-wild type and mutant strain were 94.8%, 98.7%, 97.4% and 100% respectively. S. cerevisiae mutant strain and their immobilized form was found to be effective in biosorption of chromium (VI than the wild type forms.

  4. BIOSORPTION OF CHROMIUM (VI) FROM INDUSTRIAL EFFLUENT BY WILD ANDMUTANT TYPE STRAIN OF SACCHAROMYCES CEREVISIAE AND ITS IMMOBILIZED FORM

    OpenAIRE

    K Selvam, K Arungandhi, B Vishnupriya, T Shanmuga priya and M Yamuna

    2013-01-01

    Biosorption of chromium was studied by wild type Saccharomyces cerevisiae strain, mutant strain, immobilized-wild type and mutant strain. Chromium absorption pattern was observed in all experimental conditions. Hexavalent chromium (VI) was analyzed by diphenyl carbazide method, by oxidizing the trivalent chromium (III). The percentage efficiency of wild type S. cerevisiae and its mutant strain, immobilized-wild type and mutant strain were 94.8%, 98.7%, 97.4% and 100% respectively. S. cerevisi...

  5. REVIEW OF CHECKPOINTING ALGORITHMS IN DISTRIBUTED SYSTEMS

    Directory of Open Access Journals (Sweden)

    Poonam Gahlan

    2010-06-01

    Full Text Available Checkpointing is the process of saving the status information. Checkpoint is defined as a designated place in a program at which normal processing is interrupted specifically to preserve the status information necessary to allow resumption of processing at a later time. Mobile computing raises many new issues such as lack of stablestorage, low bandwidth of wireless channel, high mobility, and limited battery life. Coordinated checkpointing is an attractive approach for transparently adding fault tolerance to distributed applications since it avoids domino effects and minimizes the stable storage requirement. This paper presents the review of the algorithms,which have been reported in the literature for checkpointing. This paper also covers backward error recovery techniques for distributed systems specially the distributed mobile systems.

  6. A Nonblocking Coordinated Checkpointing Algorithm for Mobile Computing Systems

    Directory of Open Access Journals (Sweden)

    Rachit Garg

    2010-05-01

    Full Text Available A checkpoint algorithm for mobile computing systems needs to handle many new issues like: mobility, low bandwidth of wireless channels, lack of stable storage on mobile nodes, disconnections, limited battery power and high failure rate of mobile nodes. These issues make traditional checkpointing techniques unsuitable for such environments. Minimum-process coordinated checkpointing is an attractive approach to introduce fault tolerance in mobile distributed systems transparently. This approach is domino-free, requires at most two checkpoints of a process on stable storage, and forces only a minimum number of processes to checkpoint. But, it requires extra synchronization messages, blocking of the underlying computation or taking some useless checkpoints. In this paper, we propose a nonblocking coordinated checkpointing algorithm for mobile computing systems, which requires only a minimum number of processes to take permanent checkpoints. We reduce the message complexity as compared to the Cao-Singhal algorithm [4], while keeping the number of useless checkpoints unchanged. We also address the related issues like: failures during checkpointing, disconnections, concurrent initiations of the algorithm and maintaining exact dependencies among processes. Finally, the paper presents an optimization technique, which significantly reduces the number of useless checkpoints at the cost of minor increase in the message complexity. In coordinated checkpointing, if a single process fails to take its tentative checkpoint; all the checkpoint effort is aborted. We try to reduce this effort by taking soft checkpoints in the first phase at Mobile Hosts.

  7. Structural and functional analysis of the Crb2–BRCT2 domain reveals distinct roles in checkpoint signaling and DNA damage repair

    OpenAIRE

    Kilkenny, Mairi L.; Doré, Andrew S.; Roe, S. Mark; Nestoras, Konstantinos; Ho, Jenny C. Y.; Watts, Felicity Z.; Pearl, Laurence H.

    2008-01-01

    Schizosaccharomyces pombe Crb2 is a checkpoint mediator required for the cellular response to DNA damage. Like human 53BP1 and Saccharomyces cerevisiae Rad9 it contains Tudor2 and BRCT2 domains. Crb2-Tudor2 domain interacts with methylated H4K20 and is required for recruitment to DNA dsDNA breaks. The BRCT2 domain is required for dimerization, but its precise role in DNA damage repair and checkpoint signaling is unclear. The crystal structure of the Crb2–BRCT2 domain, alone and in complex wit...

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

    Science.gov (United States)

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

    2008-12-01

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

  9. Asynchronous Checkpoint Migration with MRNet in the Scalable Checkpoint / Restart Library

    Energy Technology Data Exchange (ETDEWEB)

    Mohror, K; Moody, A; de Supinski, B R

    2012-03-20

    Applications running on today's supercomputers tolerate failures by periodically saving their state in checkpoint files on stable storage, such as a parallel file system. Although this approach is simple, the overhead of writing the checkpoints can be prohibitive, especially for large-scale jobs. In this paper, we present initial results of an enhancement to our Scalable Checkpoint/Restart Library (SCR). We employ MRNet, a tree-based overlay network library, to transfer checkpoints from the compute nodes to the parallel file system asynchronously. This enhancement increases application efficiency by removing the need for an application to block while checkpoints are transferred to the parallel file system. We show that the integration of SCR with MRNet can reduce the time spent in I/O operations by as much as 15x. However, our experiments exposed new scalability issues with our initial implementation. We discuss the sources of the scalability problems and our plans to address them.

  10. RNA interference regulates the cell cycle checkpoint through the RNA export factor, Ptr1, in fission yeast

    Energy Technology Data Exchange (ETDEWEB)

    Iida, Tetsushi, E-mail: tiida@nig.ac.jp [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012 (Japan); Iida, Naoko [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Tsutsui, Yasuhiro [Department of Life Science, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuda-cho, Midori-ku, Yokohama 226-8501 (Japan); Yamao, Fumiaki [Division of Mutagenesis, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan); Kobayashi, Takehiko [Division of Cytogenetics, National Institute of Genetics, Mishima, 1111 Yata, Mishima 411-8540 (Japan); The Graduate University for Advanced Studies, Sokendai, Mishima, 1111 Yata, Mishima 411-8540 (Japan)

    2012-10-12

    Highlights: Black-Right-Pointing-Pointer RNAi is linked to the cell cycle checkpoint in fission yeast. Black-Right-Pointing-Pointer Ptr1 co-purifies with Ago1. Black-Right-Pointing-Pointer The ptr1-1 mutation impairs the checkpoint but does not affect gene silencing. Black-Right-Pointing-Pointer ago1{sup +} and ptr1{sup +} regulate the cell cycle checkpoint via the same pathway. Black-Right-Pointing-Pointer Mutations in ago1{sup +} and ptr1{sup +} lead to the nuclear accumulation of poly(A){sup +} RNAs. -- Abstract: Ago1, an effector protein of RNA interference (RNAi), regulates heterochromatin silencing and cell cycle arrest in fission yeast. However, the mechanism by which Ago1 controls cell cycle checkpoint following hydroxyurea (HU) treatment has not been elucidated. In this study, we show that Ago1 and other RNAi factors control cell cycle checkpoint following HU treatment via a mechanism independent of silencing. While silencing requires dcr1{sup +}, the overexpression of ago1{sup +} alleviated the cell cycle defect in dcr1{Delta}. Ago1 interacted with the mRNA export factor, Ptr1. The ptr1-1 mutation impaired cell cycle checkpoint but gene silencing was unaffected. Genetic analysis revealed that the regulation of cell cycle checkpoint by ago1{sup +} is dependent on ptr1{sup +}. Nuclear accumulation of poly(A){sup +} RNAs was detected in mutants of ago1{sup +} and ptr1{sup +}, suggesting there is a functional link between the cell cycle checkpoint and RNAi-mediated RNA quality control.

  11. PAS3, a Saccharomyces cerevisiae Gene Encoding a Peroxisomal Integral Membrane Protein Essential for Peroxisome Biogenesis

    NARCIS (Netherlands)

    Höhfeld, Jörg; Veenhuis, Marten; Kunau, Wolf-H.

    1991-01-01

    Saccharomyces cerevisiae pas3-mutants are described which conform the pas-phenotype recently reported for the peroxisomal assembly mutants pas1-1 and pas2 (Erdmann, R., M. Veenhuis, D. Mertens, and W.-H Kunau. 1989. Proc. Natl. Acad. Sci. USA. 86:5419-5423). The isolation of pas3-mutants enabled us

  12. Stationary phase in the yeast Saccharomyces cerevisiae.

    OpenAIRE

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

    1993-01-01

    Growth and proliferation of microorganisms such as the yeast Saccharomyces cerevisiae are controlled in part by the availability of nutrients. When proliferating yeast cells exhaust available nutrients, they enter a stationary phase characterized by cell cycle arrest and specific physiological, biochemical, and morphological changes. These changes include thickening of the cell wall, accumulation of reserve carbohydrates, and acquisition of thermotolerance. Recent characterization of mutant c...

  13. Detoxification of Aflatoxin B1 by Saccharomyces cerevisiae Mutants of Anti-Oxidative Relating Genes%酿酒酵母抗氧化相关基因突变体对黄曲霉毒素B1的清除作用

    Institute of Scientific and Technical Information of China (English)

    史锋; 黄宇啸; 李永富

    2012-01-01

    黄曲霉毒素(AF)是粮食作物和饲料原料中容易污染的一种强毒性和强致癌性物质,酿酒酵母具有毒素清除功能.利用HPLC分析了酿酒酵母野生菌BY4742及三株关键的抗氧化相关基因缺失茵zwf1△、sod2△、glr1△对黄曲霉毒素B1的清除能力.结果表明,在PBS缓冲液中存活和死亡的细胞对AFB1的清除率分别为74%~76%和71%~73%,说明酵母细胞对AFB1的清除以生物吸附作用为主.在培养基中,3种突变菌活细胞对AFB1的清除率发生不同程度的降低,其中glr1△的AFB1清除能力下降最明显,其次是sod2△,而zwf1△下降最少,说明这些关键的抗氧化基因的缺失会影响细胞在生长状态下对AFB1的清除作用.%Aflatoxins are a group of mycotoxins with strong mutagenic and carcinogenic properties. Various commodities including crop and feed materials are easy to be contaminated with aflatoxin. Saccharomyces cerevisiae have been reported to bind or degrade aflatoxin. Here, detoxification of aflatoxin B1 (AFB1 ) by wild-type strain of S. cerevisiae (BY4742) and three mutants of anti-oxidative relating genes (zw/l△, sod2△and girl △) were determined by HPLC. In PBS buffer, AFBi binding abilities of viable and dead cells were 74% -76% and 71% — 73%, respectively, indicating AFB1 was removed by yeast cells mainly through cell adsorption. In YPD medium, clearance of AFB1 by three mutant viable cells reduced, while that by wild-type BY4742 remaining high. AFB1 binding ability of g/rlA decreased most seriously, then was that of sod2△ and zwfl△. Thus, the deletion of critical anti-oxidative relating genes would decrease the AFB1 binding ability of S. cerevisiae growing cells.

  14. Regulators of ribonucleotide reductase inhibit Ty1 mobility in saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    O'Donnell John P

    2010-11-01

    Full Text Available Abstract Background Ty1 is a long terminal repeat retrotransposon of Saccharomyces cerevisiae, with a replication cycle similar to retrovirus replication. Structurally, Ty1 contains long terminal repeat (LTR regions flanking the gag and pol genes that encode for the proteins that enable Ty1 mobility. Reverse transcriptase produces Ty1 complementary (cDNA that can either be integrated back into the genome by integrase or recombined into the yeast genome through homologous recombination. The frequency of Ty1 mobility is temperature sensitive, with optimum activity occurring at 24-26°C. Results In this study, we identified two host genes that when deleted allow for high temperature Ty1 mobility: RFX1 and SML1. The protein products of these genes are both negative regulators of the enzyme ribonucleotide reductase, a key enzyme in regulating deoxyribonucleotide triphosphate (dNTP levels in the cell. Processing of Ty1 proteins is defective at high temperature, and processing is not improved in either rfx1 or sml1 deletion strains. Ty1 mobility at high temperature is mediated by homologous recombination of Ty1 cDNA to Ty1 elements within the yeast genome. We quantified cDNA levels in wild type, rfx1 and sml1 deletion background strains at different temperatures. Southern blot analysis demonstrated that cDNA levels were not markedly different between the wild type and mutant strains as temperatures increased, indicating that the increased Ty1 mobility is not a result of increased cDNA synthesis in the mutant strains. Homologous recombination efficiency was increased in both rfx1 and sml1 deletion strains at high temperatures; the rfx1 deletion strain also had heightened homologous recombination efficiency at permissive temperatures. In the presence of the dNTP reducing agent hydroxyurea at permissive temperatures, Ty1 mobility was stimulated in the wild type and sml1 deletion strains but not in the rfx1 deletion strain. Mobility frequency was greatly

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

    International Nuclear Information System (INIS)

    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

  16. Checkpoint triggering in a computer system

    Energy Technology Data Exchange (ETDEWEB)

    Cher, Chen-Yong

    2016-09-06

    According to an aspect, a method for triggering creation of a checkpoint in a computer system includes executing a task in a processing node of the computer system and determining whether it is time to read a monitor associated with a metric of the task. The monitor is read to determine a value of the metric based on determining that it is time to read the monitor. A threshold for triggering creation of the checkpoint is determined based on the value of the metric. Based on determining that the value of the metric has crossed the threshold, the checkpoint including state data of the task is created to enable restarting execution of the task upon a restart operation.

  17. Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Kron, S J; Styles, C. A.; Fink, G R

    1994-01-01

    Laboratory strains of Saccharomyces cerevisiae are dimorphic; in response to nitrogen starvation they switch from a yeast form (YF) to a filamentous pseudohyphal (PH) form. Time-lapse video microscopy of dividing cells reveals that YF and PH cells differ in their cell cycles and budding polarity. The YF cell cycle is controlled at the G1/S transition by the cell-size checkpoint Start. YF cells divide asymmetrically, producing small daughters from full-sized mothers. As a result, mothers and d...

  18. Determinants of Swe1p Degradation in Saccharomyces cerevisiae

    OpenAIRE

    McMillan, John N.; Theesfeld, Chandra L.; Harrison, Jacob C.; Bardes, Elaine S.G.; Lew, Daniel J.

    2002-01-01

    Swe1p, the sole Wee1-family kinase in Saccharomyces cerevisiae, is synthesized during late G1 and is then degraded as cells proceed through the cell cycle. However, Swe1p degradation is halted by the morphogenesis checkpoint, which responds to insults that perturb bud formation. The Swe1p stabilization promotes cell cycle arrest through Swe1p-mediated inhibitory phosphorylation of Cdc28p until the cells can recover from the perturbation and resume bud formation. Swe1p degradation involves the...

  19. Vanadate-resistant yeast mutants are defective in protein glycosylation.

    OpenAIRE

    Ballou, L; Hitzeman, R A; Lewis, M. S.; Ballou, C E

    1991-01-01

    Spontaneous recessive orthovanadate-resistant mutants of Saccharomyces cerevisiae were obtained in five complementation groups, and all show defects in protein glycosylation that mimic the previously isolated mnn mutants. Three of the groups are allelic to the known mnn8, mnn9, and mnn10 mutants, whereas the other two groups show other glycosylation defects. The vanadate-resistant phenotype was associated with enhanced hygromycin B sensitivity. The glycosylation phenotypes of the mutants are ...

  20. Garbage Collection in Uncoordinated Checkpointing Algorithms

    Institute of Scientific and Technical Information of China (English)

    LIU Yunlong; CHEN Junliang

    1999-01-01

    In this paper, the hard problem of thethorough garbage collection in uncoordinated checkpointing algorithms isstudied. After introduction of the traditional garbage collectingscheme, with which only obsolete checkpoints can be discarded, it isshown that this kind of traditional method may fail to discard anycheckpoint in some special cases, and it is necessary and urgent to finda thorough garbage collecting method, with which all the checkpointsuseless for any future rollback-recovery including the obsolete ones canbe discarded. Then, the Thorough Garbage Collection Theorem is proposedand proved, which ensures the feasibility of the thorough garbagecollection, and gives the method to calculate the set of the usefulcheckpoints as well.

  1. Checkpointing and Recovery in Distributed and Database Systems

    Science.gov (United States)

    Wu, Jiang

    2011-01-01

    A transaction-consistent global checkpoint of a database records a state of the database which reflects the effect of only completed transactions and not the results of any partially executed transactions. This thesis establishes the necessary and sufficient conditions for a checkpoint of a data item (or the checkpoints of a set of data items) to…

  2. Methionine catabolism in Saccharomyces cerevisiae.

    Science.gov (United States)

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

    2006-01-01

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

  3. Targeting the Checkpoint to Kill Cancer Cells

    Czech Academy of Sciences Publication Activity Database

    Benada, Jan; Macůrek, Libor

    2015-01-01

    Roč. 6, č. 3 (2015), s. 1912-1937. ISSN 2218-273X R&D Projects: GA ČR(CZ) GA14-34264S Institutional support: RVO:68378050 Keywords : checkpoint * DNA damage response * cancer Subject RIV: EB - Genetics ; Molecular Biology

  4. The tumor suppressor homolog in fission yeast, myh1+, displays a strong interaction with the checkpoint gene rad1+

    International Nuclear Information System (INIS)

    The DNA glycosylase MutY is strongly conserved in evolution, and homologs are found in most eukaryotes and prokaryotes examined. This protein is implicated in repair of oxidative DNA damage, in particular adenine mispaired opposite 7,8-dihydro-8-oxoguanine. Previous investigations in Escherichia coli, fission yeast, and mammalian cells show an association of mutations in MutY homologs with a mutator phenotype and carcinogenesis. Eukaryotic MutY homologs physically associate with several proteins with a role in replication, DNA repair, and checkpoint signaling, specifically the trimeric 9-1-1 complex. In a genetic investigation of the fission yeast MutY homolog, myh1+, we show that the myh1 mutation confers a moderately increased UV sensitivity alone and in combination with mutations in several DNA repair genes. The myh1 rad1, and to a lesser degree myh1 rad9, double mutants display a synthetic interaction resulting in enhanced sensitivity to DNA damaging agents and hydroxyurea. UV irradiation of myh1 rad1 double mutants results in severe chromosome segregation defects and visible DNA fragmentation, and a failure to activate the checkpoint. Additionally, myh1 rad1 double mutants exhibit morphological defects in the absence of DNA damaging agents. We also found a moderate suppression of the slow growth and UV sensitivity of rhp51 mutants by the myh1 mutation. Our results implicate fission yeast Myh1 in repair of a wider range of DNA damage than previously thought, and functionally link it to the checkpoint pathway

  5. Genetic Basis for Saccharomyces cerevisiae Biofilm in Liquid Medium

    DEFF Research Database (Denmark)

    Andersen, Kaj Scherz; Bojsen, Rasmus Kenneth; Gro Rejkjær Sørensen, Laura; Weiss Nielsen, Martin; Lisby, Michael; Folkesson, Sven Anders; Regenberg, Birgitte

    2014-01-01

    than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the S1278b background and found 71 genes that were essential for biofilm development. Quantitative...

  6. Replication licensing and the DNA damage checkpoint

    OpenAIRE

    Cook, Jeanette Gowen

    2009-01-01

    Accurate and timely duplication of chromosomal DNA requires that replication be coordinated with processes that ensure genome integrity. Significant advances in determining how the earliest steps in DNA replication are affected by DNA damage have highlighted some of the mechanisms to establish that coordination. Recent insights have expanded the relationship between the ATM and ATR-dependent checkpoint pathways and the proteins that bind and function at replication origins. These findings sug...

  7. Immune Checkpoint Inhibitors in Older Adults.

    Science.gov (United States)

    Elias, Rawad; Morales, Joshua; Rehman, Yasser; Khurshid, Humera

    2016-08-01

    Cancer is primarily a disease of older adults. The treatment of advanced stage tumors usually involves the use of systemic agents that may be associated with significant risk of toxicity, especially in older patients. Immune checkpoint inhibitors are newcomers to the oncology world with improved efficacy and better safety profiles when compared to traditional cytotoxic drugs. This makes them an attractive treatment option. While there are no elderly specific trials, this review attempts to look at the current available data from a geriatric oncology perspective. We reviewed data from phase III studies that led to newly approved indications of checkpoint inhibitors in non-small cell lung cancer, melanoma, and renal cell cancer. Data were reviewed with respect to response, survival, and toxicity according to three groups: 75 years. Current literature does not allow one to draw definitive conclusions regarding the role of immune checkpoint inhibitors in older adults. However, they may offer a potentially less toxic but equally efficacious treatment option for the senior adult oncology patient. PMID:27287329

  8. Drosophila MOF controls Checkpoint protein2 and regulates genomic stability during early embryogenesis

    Directory of Open Access Journals (Sweden)

    Pushpavalli Sreerangam NCVL

    2013-01-01

    Full Text Available Abstract Background In Drosophila embryos, checkpoints maintain genome stability by delaying cell cycle progression that allows time for damage repair or to complete DNA synthesis. Drosophila MOF, a member of MYST histone acetyl transferase is an essential component of male X hyperactivation process. Until recently its involvement in G2/M cell cycle arrest and defects in ionizing radiation induced DNA damage pathways was not well established. Results Drosophila MOF is highly expressed during early embryogenesis. In the present study we show that haplo-insufficiency of maternal MOF leads to spontaneous mitotic defects like mitotic asynchrony, mitotic catastrophe and chromatid bridges in the syncytial embryos. Such abnormal nuclei are eliminated and digested in the yolk tissues by nuclear fall out mechanism. MOF negatively regulates Drosophila checkpoint kinase 2 tumor suppressor homologue. In response to DNA damage the checkpoint gene Chk2 (Drosophila mnk is activated in the mof mutants, there by causing centrosomal inactivation suggesting its role in response to genotoxic stress. A drastic decrease in the fall out nuclei in the syncytial embryos derived from mof1/+; mnkp6/+ females further confirms the role of DNA damage response gene Chk2 to ensure the removal of abnormal nuclei from the embryonic precursor pool and maintain genome stability. The fact that mof mutants undergo DNA damage has been further elucidated by the increased number of single and double stranded DNA breaks. Conclusion mof mutants exhibited genomic instability as evidenced by the occurance of frequent mitotic bridges in anaphase, asynchronous nuclear divisions, disruption of cytoskeleton, inactivation of centrosomes finally leading to DNA damage. Our findings are consistent to what has been reported earlier in mammals that; reduced levels of MOF resulted in increased genomic instability while total loss resulted in lethality. The study can be further extended using

  9. An Anti-Checkpoint Activity for Rif1

    OpenAIRE

    Xue, Yuan; Rushton, Michael D.; Maringele, Laura

    2011-01-01

    Cells accumulate single-stranded DNA (ssDNA) when telomere capping, DNA replication, or DNA repair is impeded. This accumulation leads to cell cycle arrest through activating the DNA–damage checkpoints involved in cancer protection. Hence, ssDNA accumulation could be an anti-cancer mechanism. However, ssDNA has to accumulate above a certain threshold to activate checkpoints. What determines this checkpoint-activation threshold is an important, yet unanswered question. Here we identify Rif1 (R...

  10. A Monitor for Bud Emergence in the Yeast Morphogenesis Checkpoint

    OpenAIRE

    Theesfeld, Chandra L.; Zyla, Trevin R.; Bardes, Elaine G.S.; Lew, Daniel J.

    2003-01-01

    Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon...

  11. A Cdc2 dependent checkpoint maintains diploidy in Drosophila.

    Science.gov (United States)

    Hayashi, S

    1996-04-01

    DNA replication in G2 does not normally occur due to the checkpoint control. To elucidate its mechanism, the functions of the escargot and Dmcdc2 genes of Drosophila were studied. When escargot function was eliminated, diploid imaginal cells that were arrested in G2 lost Cyclin A, a regulatory subunit of G2/M cdk, and entered an endocycle. escargot genetically interacted with Dmcdc2 which encodes a catalytic subunit of G2/M cdk. The mutant phenotypes of Dmcdc2 itself was similar to those of escargot: many diploid cells in imaginal discs, salivary glands and the central nervous system entered an endocycle and sometimes formed polytene chromosomes. Since mitotically quiescent abdominal histoblasts still required Dmcdc2 to remain diploid, the inhibitory activity of G2/M cdk on DNA replication appeared to be separable from its activity as the mitosis promoting factor. These results suggest that in G2, escargot is required to maintain a high level of G2/M cdk that actively inhibits the entry into S phase. PMID:8620832

  12. Direct and indirect control of the initiation of meiotic recombination by DNA damage checkpoint mechanisms in budding yeast.

    Directory of Open Access Journals (Sweden)

    Bilge Argunhan

    Full Text Available Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs. The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI whereas no significant reduction was found in smaller chromosomes (III and VI. On the other hand, the absence of Rad17 (a critical component of the ATR pathway lead to an increase in DSB formation (chromosomes VII and II were tested. We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.

  13. Photorepair mutants of Arabidopsis

    International Nuclear Information System (INIS)

    UV radiation induces two major DNA damage products, the cyclobutane pyrimidine dimer (CPD) and, at a lower frequency, the pyrimidine (6-4) pyrimidinone dimer (6-4 product). Although Escherichia coli and Saccharomyces cerevisiae produce a CPD-specific photolyase that eliminates only this class of dimer, Arabidopsis thaliana, Drosophila melanogaster, Crotalus atrox, and Xenopus laevis have recently been shown to photoreactivate both CPDs and 6-4 products. We describe the isolation and characterization of two new classes of mutants of Arabidopsis, termed uvr2 and uvr3, that are defective in the photoreactivation of CPDs and 6-4 products, respectively. We demonstrate that the CPD photolyase mutation is genetically linked to a DNA sequence encoding a type II (metazoan) CPD photolyase. In addition, we are able to generate plants in which only CPDs or 6-4 products are photoreactivated in the nuclear genome by exposing these mutants to UV light and then allowing them to repair one or the other class of dimers. This provides us with a unique opportunity to study the biological consequences of each of these two major UV-induced photoproducts in an intact living system

  14. Rad4 mainly functions in Chk1-mediated DNA damage checkpoint pathway as a scaffold protein in the fission yeast Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ming Yue

    Full Text Available Rad4/Cut5 is a scaffold protein in the Chk1-mediated DNA damage checkpoint in S. pombe. However, whether it contains a robust ATR-activation domain (AAD required for checkpoint signaling like its orthologs TopBP1 in humans and Dpb11 in budding yeast has been incompletely clear. To identify the putative AAD in Rad4, we carried out an extensive genetic screen looking for novel mutants with an enhanced sensitivity to replication stress or DNA damage in which the function of the AAD can be eliminated by the mutations. Two new mutations near the N-terminus were identified that caused significantly higher sensitivities to DNA damage or chronic replication stress than all previously reported mutants, suggesting that most of the checkpoint function of the protein is eliminated. However, these mutations did not affect the activation of Rad3 (ATR in humans yet eliminated the scaffolding function of the protein required for the activation of Chk1. Several mutations were also identified in or near the recently reported AAD in the C-terminus of Rad4. However, all mutations in the C-terminus only slightly sensitized the cells to DNA damage. Interestingly, a mutant lacking the whole C-terminus was found resistant to DNA damage and replication stress almost like the wild type cells. Consistent with the resistance, all known Rad3 dependent phosphorylations of checkpoint proteins remained intact in the C-terminal deletion mutant, indicating that unlike that in Dpb11, the C-terminus of Rad4 does not contain a robust AAD. These results, together with those from the biochemical studies, show that Rad4 mainly functions as a scaffold protein in the Chk1, not the Cds1(CHK2 in humans, checkpoint pathway. It plays a minor role or is functionally redundant with an unknown factor in Rad3 activation.

  15. Immune checkpoint inhibition in lymphoid disease.

    Science.gov (United States)

    Eyre, Toby A; Collins, Graham P

    2015-08-01

    It has long been understood that the immune system has intrinsic anti-tumour activity in humans, and that a key mechanism of tumour progression is the ability of a tumour to escape this immune surveillance. A number of attempts have been made to harness this anti-tumour immunity in both solid tumour oncology and haematological malignancies with variable success. Examples include the use of allogeneic stem cell transplantation and donor lymphocyte infusion in haematological cancer and vaccine studies in solid tumours. Enhanced signalling of the Programmed cell death-1 (PDCD1, PD-1)/cytotoxic T-lymphocyte-associated protein 4 (CTLA4) 'immune checkpoint' pathway has emerged recently as a critical mechanism by which tumours can escape the natural anti-tumour immune response. As such, novel therapies have been developed to help enhance this natural immunity by switching off the PDCD1/CTLA4 immune checkpoint pathway. The following review will discuss the pathobiology of these pathways and the exciting new data now available in lymphoid malignancies. PMID:25824455

  16. Developmental checkpoints guarded by regulated necrosis.

    Science.gov (United States)

    Dillon, Christopher P; Tummers, Bart; Baran, Katherine; Green, Douglas R

    2016-06-01

    The process of embryonic development is highly regulated through the symbiotic control of differentiation and programmed cell death pathways, which together sculpt tissues and organs. The importance of programmed necrotic (RIPK-dependent necroptosis) cell death during development has recently been recognized as important and has largely been characterized using genetically engineered animals. Suppression of necroptosis appears to be essential for murine development and occurs at three distinct checkpoints, E10.5, E16.5, and P1. These distinct time points have helped delineate the molecular pathways and regulation of necroptosis. The embryonic lethality at E10.5 seen in knockouts of caspase-8, FADD, or FLIP (cflar), components of the extrinsic apoptosis pathway, resulted in pallid embryos that did not exhibit the expected cellular expansions. This was the first suggestion that these factors play an important role in the inhibition of necroptotic cell death. The embryonic lethality at E16.5 highlighted the importance of TNF engaging necroptosis in vivo, since elimination of TNFR1 from casp8 (-/-), fadd (-/-), or cflar (-/-), ripk3 (-/-) embryos delayed embryonic lethality from E10.5 until E16.5. The P1 checkpoint demonstrates the dual role of RIPK1 in both the induction and inhibition of necroptosis, depending on the upstream signal. This review summarizes the role of necroptosis in development and the genetic evidence that helped detail the molecular mechanisms of this novel pathway of programmed cell death. PMID:27056574

  17. Immune checkpoints in cancer clinical trials

    Institute of Scientific and Technical Information of China (English)

    Elad Sharon; Howard Streicher; Priscila Goncalves; Helen XChen

    2014-01-01

    Immunology-based therapy is rapidly developing into an effective treatment option for a surprising range of cancers. We have learned over the last decade that powerful immunologic effector cells may be blocked by inhibitory regulatory pathways controlled by specific molecules often called“immune checkpoints.” These checkpoints serve to control or turn off the immune response when it is no longer needed to prevent tissue injury and autoimmunity. Cancer cells have learned or evolved to use these mechanisms to evade immune control and elimination. The development of a new therapeutic class of drugs that inhibit these inhibitory pathways has recently emerged as a potent strategy in oncology. Three sets of agents have emerged in clinical trials exploiting this strategy. These agents are antibody-based therapies targeting cytotoxic T-lymphocyte antigen4 (CTLA4), programmed cell death1 (PD-1), and programmed cell death ligand 1 (PD-L1). These inhibitors of immune inhibition have demonstrated extensive activity as single agents and in combinations. Clinical responses have been seen in melanoma, renal cellcarcinoma, non-smal celllung cancer, and several other tumor types. Despite the autoimmune or inflammatory immune-mediated adverse effects which have been seen, the responses and overall survival benefits exhibited thus far warrant further clinical development.

  18. Checkpoint kinase 1 negatively regulates somatic hypermutation.

    Science.gov (United States)

    Frankenberger, Samantha; Davari, Kathrin; Fischer-Burkart, Sabine; Böttcher, Katrin; Tomi, Nils-Sebastian; Zimber-Strobl, Ursula; Jungnickel, Berit

    2014-04-01

    Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis. PMID:24423870

  19. DMTCP: Scalable User-Level Transparent Checkpointing for Cluster Computations

    CERN Document Server

    Ansel, Jason; Arya, Kapil

    2008-01-01

    As the size of clusters increases, failures are becoming increasingly frequent. Applications must become fault tolerant if they are to run for extended periods of time. We present DMTCP (Distributed MultiThreaded CheckPointing), the first user-level distributed checkpointing package not dependent on a specific message passing library. This contrasts with existing approaches either specific to libraries such as MPI or requiring kernel modification. DMTCP provides fault tolerance through checkpointing. DMTCP transparently checkpoints general cluster computations consisting of many nodes, processes, and threads. DMTCP automatically accounts for TCP/IP sockets, UNIX domain sockets, pipes, ptys (pseudo-terminals), signal handlers, ordinary file descriptors, shared file descriptors, and other operating system artifacts. We demonstrate checkpointing and restart of applications communicating through MPICH2, OpenMPI, and sockets directly. These applications were written with a variety of languages including Fortran, C...

  20. Optimization of feeding strategy for the ergosterol production by yeasts saccharomyces cerevisiae

    OpenAIRE

    Mojmir Rychtera; Josef Cermak; Jaroslav Votruba; Jan Nahlik; Karel Melzoch; Christopher A. Kent; Estela Escalante, Waldir D.

    2010-01-01

    Objective of this study was to optimize ergosterol production by yeast strain Saccharomyces cerevisiae with the use of computer controlled feeding of cultivation medium. Baker´s yeasts strain of Saccharomyces cerevisiae originally modified and selected as mutant D7 was further applied in an industrial scale and also in this investigation. Composition of cultivation medium was optimized with the use of a modified Rosenbrock´s method with regard to following components: glucose, yeast extract, ...

  1. Paclitaxel-induced microtubule stabilization causes mitotic block and apoptotic-like cell death in a paclitaxel-sensitive strain of Saccharomyces cerevisiae

    OpenAIRE

    Foland, Travis B.; Dentler, William L.; SUPRENANT, KATHY A.; Gupta, Mohan L.; Himes, Richard H.

    2005-01-01

    Wild-type Saccharomyces cerevisiae tubulin does not bind the anti-mitotic microtubule stabilizing agent paclitaxel. Previously, we introduced mutations into the S. cerevisiae gene for β-tubulin that imparted paclitaxel binding to the protein, but the mutant strain was not sensitive to paclitaxel and other microtubule-stabilizing agents, due to the multiple ABC transporters in the membranes of budding yeast. Here, we introduced the mutated β-tubulin gene into a S. cerevisiae strain with dimini...

  2. FORMAL VERIFICATION OF DISTRIBUTED CHECKPOINTING USING EVENT-B

    Directory of Open Access Journals (Sweden)

    Girish Chandra

    2015-10-01

    Full Text Available The development of complex system makes challenging task for correct software development. Due to faulty specification, software may involve errors. The traditional testing methods are not sufficient to verify the correctness of such complex system. In order to capture correct system requirements and rigorous reasoning about the problems, formal methods are required. Formal methods are mathematical techniques that provide precise specification of problems with their solutions and proof of correctness. In this paper, we have done formal verification of check pointing process in a distributed database system using Event B. Event-B is an event driven formal method which is used to develop formal models of distributed database systems. In a distributed database system, the database is stored at different sites that are connected together through the network. Checkpoint is a recovery point which contains the state information about the site. In order to do recovery of a distributed transaction a global checkpoint number (GCPN is required. A global checkpoint number decides which transaction will be included for recovery purpose. All transactions whose timestamp are less than global checkpoint number will be marked as before checkpoint transaction (BCPT and will be considered for recovery purpose. The transactions whose timestamp are greater than GCPN will be marked as after checkpoint transaction (ACPT and will be part of next global checkpoint number.

  3. Keeping checkpoint/restart viable for exascale systems.

    Energy Technology Data Exchange (ETDEWEB)

    Riesen, Rolf E.; Bridges, Patrick G. (IBM Research, Ireland, Mulhuddart, Dublin); Stearley, Jon R.; Laros, James H., III; Oldfield, Ron A.; Arnold, Dorian (University of New Mexico, Albuquerque, NM); Pedretti, Kevin Thomas Tauke; Ferreira, Kurt Brian; Brightwell, Ronald Brian

    2011-09-01

    Next-generation exascale systems, those capable of performing a quintillion (10{sup 18}) operations per second, are expected to be delivered in the next 8-10 years. These systems, which will be 1,000 times faster than current systems, will be of unprecedented scale. As these systems continue to grow in size, faults will become increasingly common, even over the course of small calculations. Therefore, issues such as fault tolerance and reliability will limit application scalability. Current techniques to ensure progress across faults like checkpoint/restart, the dominant fault tolerance mechanism for the last 25 years, are increasingly problematic at the scales of future systems due to their excessive overheads. In this work, we evaluate a number of techniques to decrease the overhead of checkpoint/restart and keep this method viable for future exascale systems. More specifically, this work evaluates state-machine replication to dramatically increase the checkpoint interval (the time between successive checkpoint) and hash-based, probabilistic incremental checkpointing using graphics processing units to decrease the checkpoint commit time (the time to save one checkpoint). Using a combination of empirical analysis, modeling, and simulation, we study the costs and benefits of these approaches on a wide range of parameters. These results, which cover of number of high-performance computing capability workloads, different failure distributions, hardware mean time to failures, and I/O bandwidths, show the potential benefits of these techniques for meeting the reliability demands of future exascale platforms.

  4. Protein phosphatases acting on the replication checkpoint

    OpenAIRE

    Conde, Rui Miguel Esteves Antunes Seabra

    2010-01-01

    A génese de um cancro está dependente da acumulação de mutações genéticas que dão origem a instabilidade genómica, que por sua vez resulta na proliferação descontrolada. Para prevenir a acumulação destas mutações, as células têm mecanismos de controlo (checkpoints) que suspendem o ciclo celular e accionam as vias de reparação do ADN. Estes eventos são muitas vezes regulados por dinâmicas de (des)fosforilação de proteínas. As proteínas fosfatases (PPs), enzimas responsáveis p...

  5. Managing Adverse Events With Immune Checkpoint Agents.

    Science.gov (United States)

    Dadu, Ramona; Zobniw, Chrystia; Diab, Adi

    2016-01-01

    Immune checkpoint inhibitors (anti-cytotoxic T-lymphocyte antigen 4 and anti programmed cell death 1/programmed cell death 1 ligand antibodies) have shown impressive clinical activity in multiple cancer types. Despite achieving great clinical success, challenges and limitations of these drugs as monotherapy or various combinational strategies include the development of a unique set of immune-related adverse events (irAEs) that can be severe and even fatal. Therefore, identification of patients at risk, prevention, consistent communication between patients and medical team, rapid recognition, and treatment of irAEs are critical in optimizing treatment outcomes. This review focuses on the description of more common irAEs and provides a suggested approach for management of specific irAEs. PMID:27111908

  6. Repression of nitrogen catabolic genes by ammonia and glutamine in nitrogen-limited continuous cultures of Saccharomyces cerevisiae

    NARCIS (Netherlands)

    ter Schure, E G; Silljé, H H; Vermeulen, E E; Kalhorn, J W; Verkleij, A J; Boonstra, J; Verrips, C T

    1998-01-01

    Growth of Saccharomyces cerevisiae on ammonia and glutamine decreases the expression of many nitrogen catabolic genes to low levels. To discriminate between ammonia- and glutamine-driven repression of GAP1, PUT4, GDH1 and GLN1, a gln1-37 mutant was used. This mutant is not able to convert ammonia in

  7. SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae

    OpenAIRE

    Gale, Cheryl A.; Leonard, Michelle D.; Finley, Kenneth R.; Christensen, Leah; McClellan, Mark; Abbey, Darren; Kurischko, Cornelia; Bensen, Eric; Tzafrir, Iris; Kauffman, Sarah; Becker, Jeff; Berman, Judith

    2009-01-01

    The early endocytic patch protein Sla2 is important for morphogenesis and growth rates in Saccharomyces cerevisiae and Candida albicans, but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2 or ScSLA2 are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other c...

  8. Targeting lung cancer through inhibition of checkpoint kinases

    OpenAIRE

    Syljuåsen, Randi G; Hasvold, Grete; Hauge, Sissel; Helland, Åslaug

    2015-01-01

    Inhibitors of checkpoint kinases ATR, Chk1, and Wee1 are currently being tested in preclinical and clinical trials. Here, we review the basic principles behind the use of such inhibitors as anticancer agents, and particularly discuss their potential for treatment of lung cancer. As lung cancer is one of the most deadly cancers, new treatment strategies are highly needed. We discuss how checkpoint kinase inhibition in principle can lead to selective killing of lung cancer cells while sparing t...

  9. A New Adaptive Checkpointing Strategy for Mobile Computing

    Institute of Scientific and Technical Information of China (English)

    MENChaoguang; ZUODecheng; YANGXiaozong

    2005-01-01

    Adaptive checkpointing strategy is an efficient recovery scheme, which is suitable for mobile computing system. However, all existing adaptive checkpointing schemes are not correct to recover system when failure occurs in some special period. In this paper, the issues that will lead to system inconsistency are first discussed and then a new adaptive strategy that can recover system to correct consistent state is proposed. Our algorithm improves system recovery performance because only failure process needs rollback through logging.

  10. A Hierarchical Checkpointing Protocol for Parallel Applications in Cluster Federations

    OpenAIRE

    Monnet, Sébastien; Morin, Christine; Badrinath, Ramamurthy

    2004-01-01

    Code coupling applications can be divided into communicating modules, that may be executed on different clusters in a cluster federation. As a cluster federation comprises of a large number of nodes, there is a high probability of a node failure. We propose a hierarchical checkpointing protocol that combines a synchronized checkpointing technique inside clusters and a communication-induced technique between clusters. This protocol fits to the characteristics of a cluster federation (large num...

  11. DMTCP: bringing interactive checkpoint-restart to Python

    Science.gov (United States)

    Arya, Kapil; Cooperman, Gene

    2015-01-01

    DMTCP (Distributed MultiThreaded CheckPointing) is a mature checkpoint-restart package. It operates in user space without kernel privilege, and adapts to application-specific requirements through plugins. While DMTCP has been able to checkpoint Python and IPython ‘from the outside’ for many years, a Python module has recently been created to support DMTCP. IPython support is included through a new DMTCP plugin. A checkpoint can be requested interactively within a Python session or under the control of a specific Python program. Further, the Python program can execute specific Python code prior to checkpoint, upon resuming (within the original process) and upon restarting (from a checkpoint image). Applications of DMTCP are demonstrated for: (i) Python-based graphics using virtual network client, (ii) a fast/slow technique to use multiple hosts or cores to check one (Cython Behnel S et al 2011 Comput. Sci. Eng. 13 31-39) computation in parallel, and (iii) a reversible debugger, FReD, with a novel reverse-expression watchpoint feature for locating the cause of a bug.

  12. A survey of checkpointing algorithms for parallel and distributed computers

    Indian Academy of Sciences (India)

    S Kalaiselvi; V Rajaraman

    2000-10-01

    Checkpoint is defined as a designated place in a program at which normal processing is interrupted specifically to preserve the status information necessary to allow resumption of processing at a later time. Checkpointing is the process of saving the status information. This paper surveysthe algorithms which have been reported in the literature for checkpointing parallel/distributed systems. It has been observed that most of the algorithms published for checkpointing in message passing systems are based on the seminal article by Chandy and Lamport. A large number of articles have been published in this area by relaxing the assumptions made in this paper and by extending it to minimise the overheads of coordination and context saving. Checkpointing for sharedmemory systems primarily extend cache coherence protocolstomaintain a consistent memory. All of them assume that the main memory is safe for storing the context. Recently algorithms have been published for distributed shared memory systems, which extend the cache coherence protocols used in shared memory systems. They however also include methods for storing the status of distributed memory in stable storage. Most of the algorithms assume that there is no knowledge about the programs being executed.It is howeverfelt that in development of parallel programs the user has to do a fair amount of work in distributing tasks and this information can be effectively used to simplify checkpointing and rollback recovery.

  13. An Analysis of Checkpointing Algorithms for Distributed Mobile Systems

    Directory of Open Access Journals (Sweden)

    Ajay Khunteta

    2010-07-01

    Full Text Available Distributed snapshots are an important building block for distributed systems, and are useful for constructing efficient checkpointing protocols, among other uses. Direct application of these algorithms to mobile systems is not easible, however, due to differences in the environment in which mobile systems operate, relative to general distributed systems. The mobile computing environment introduces newchallenges in the area of fault-tolerant computing. Compared to traditional distributed environments, wireless networks are typically slower, providing lower throughput and latency, comparing to wireline networks. In addition, the mobile hosts have limited computation esources, are often exposed to harsh operating environment that makes them more likely to fail, and can roam while operating. Over the past two decades, intensive research work has been carried out on providing efficient checkpointing protocols in traditional distributed computing. Recently, more attention has been paid to providing checkpointing protocols for mobile systems. Some of these protocols have been adapted from the traditional distributed environment; others have been created from scratch for mobile systems. Checkpoint is defined as a designated place in a program at which normal processing is interrupted specifically to preserve the status information necessary to allow resumption of processing at alater time. Checkpointing is the process of saving the status information. This paper surveys the algorithms which have been reported in the literature for checkpointing in Mobile Distributed systems.

  14. A Tunable Checkpointing Algorithm for Distributed Mobile Applications

    Directory of Open Access Journals (Sweden)

    Sungchae Lim

    2011-11-01

    Full Text Available The aim of a distributed checkpointing algorithm is to efficiently restore the execution state of distributed applications in face of hardware or software failures. Originally, such algorithms were devised for fixed networking systems, of which computing components communicate with each other via wired networks. Therefore, those algorithms usually suffer from heavy networking costs coming from frequent data transits over wireless networks, if they are used in the wireless computing environment. In this paper, to reduce usage of wireless communications, our checkpointing algorithm allows the distributed mobile application to tune the level of its checkpointing strictness. The strictness is defined by the maximum rollback distance (MRD that says how many recent local checkpoints can be rolled back in the worst case. Since our algorithm have more flexibility in checkpointing schedule due to the use of MRD, it is possible to reduce the number of enforced local checkpointing. In particular, the amount of data transited on wirelesses networks becomes smaller than in earlier methods; thus, our algorithm provides less communication cost and shortened blocking time.

  15. Review of Some Checkpointing Schemes for Distributed and Mobile Computing Environments

    Directory of Open Access Journals (Sweden)

    Mr Raman Kumar

    2015-05-01

    Full Text Available Fault Tolerance Techniques facilitate systems to carry out tasks in the incidence of faults. A checkpoint is a local state of a process saved on stable storage. In a distributed system, since the processes in the system do not share memory; a global state of the system is defined as a combination of local states, one from each process. In case of a fault in distributed systems, checkpointing enables the execution of a program to be resumed from a previous consistent global state rather than resuming the execution from the commencement. In this way, the sum of constructive processing vanished because of the fault is appreciably reduced. In this paper, we talk about various issues related to the checkpointing for distributed systems and mobile computing environments. We also confer various types of checkpointing: coordinated checkpointing, asynchronous checkpointing, communication induced checkpointing and message logging based checkpointing. We also present a survey of some checkpointing algorithms for distributed systems.

  16. Old Yellow Enzymes Protect against Acrolein Toxicity in the Yeast Saccharomyces cerevisiae

    OpenAIRE

    Trotter, Eleanor W; Collinson, Emma J.; Dawes, Ian W.; Grant, Chris M.

    2006-01-01

    Acrolein is a ubiquitous reactive aldehyde which is formed as a product of lipid peroxidation in biological systems. In this present study, we screened the complete set of viable deletion strains in Saccharomyces cerevisiae for sensitivity to acrolein to identify cell functions involved in resistance to reactive aldehydes. We identified 128 mutants whose gene products are localized throughout the cell. Acrolein-sensitive mutants were distributed among most major biological processes but parti...

  17. Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

    OpenAIRE

    Boeckmann, Lars; Takahashi, Yoshimitsu; Au, Wei-Chun; Prashant K. Mishra; Choy, John S.; Dawson, Anthony R.; Szeto, May Y.; Waybright, Timothy J.; Heger, Christopher; McAndrew, Christopher; Goldsmith, Paul K.; VEENSTRA, TIMOTHY D.; Baker, Richard E.; Basrai, Munira A.

    2013-01-01

    The centromeric histone H3 variant (CenH3) is essential for chromosome segregation in eukaryotes. We identify posttranslational modifications of Saccharomyces cerevisiae CenH3, Cse4. Functional characterization of cse4 phosphorylation mutants shows growth and chromosome segregation defects when combined with kinetochore mutants okp1 and ame1. Using a phosphoserine-specific antibody, we show that the association of phosphorylated Cse4 with centromeres increases in response to defective microtu...

  18. Berkeley lab checkpoint/restart (BLCR) for Linux clusters

    Science.gov (United States)

    Hargrove, Paul H.; Duell, Jason C.

    2006-09-01

    This article describes the motivation, design and implementation of Berkeley Lab Checkpoint/Restart (BLCR), a system-level checkpoint/restart implementation for Linux clusters that targets the space of typical High Performance Computing applications, including MPI. Application-level solutions, including both checkpointing and fault-tolerant algorithms, are recognized as more time and space efficient than system-level checkpoints, which cannot make use of any application-specific knowledge. However, system-level checkpointing allows for preemption, making it suitable for responding to ''fault precursors'' (for instance, elevated error rates from ECC memory or network CRCs, or elevated temperature from sensors). Preemption can also increase the efficiency of batch scheduling; for instance reducing idle cycles (by allowing for shutdown without any queue draining period or reallocation of resources to eliminate idle nodes when better fitting jobs are queued), and reducing the average queued time (by limiting large jobs to running during off-peak hours, without the need to limit the length of such jobs). Each of these potential uses makes BLCR a valuable tool for efficient resource management in Linux clusters.

  19. Message Efficient Checkpointing and Rollback Recovery in Heterogeneous Mobile Networks

    Science.gov (United States)

    Jaggi, Parmeet Kaur; Singh, Awadhesh Kumar

    2016-06-01

    Heterogeneous networks provide an appealing way of expanding the computing capability of mobile networks by combining infrastructure-less mobile ad-hoc networks with the infrastructure-based cellular mobile networks. The nodes in such a network range from low-power nodes to macro base stations and thus, vary greatly in their capabilities such as computation power and battery power. The nodes are susceptible to different types of transient and permanent failures and therefore, the algorithms designed for such networks need to be fault-tolerant. The article presents a checkpointing algorithm for the rollback recovery of mobile hosts in a heterogeneous mobile network. Checkpointing is a well established approach to provide fault tolerance in static and cellular mobile distributed systems. However, the use of checkpointing for fault tolerance in a heterogeneous environment remains to be explored. The proposed protocol is based on the results of zigzag paths and zigzag cycles by Netzer-Xu. Considering the heterogeneity prevalent in the network, an uncoordinated checkpointing technique is employed. Yet, useless checkpoints are avoided without causing a high message overhead.

  20. Berkeley lab checkpoint/restart (BLCR) for Linux clusters

    International Nuclear Information System (INIS)

    This article describes the motivation, design and implementation of Berkeley Lab Checkpoint/Restart (BLCR), a system-level checkpoint/restart implementation for Linux clusters that targets the space of typical High Performance Computing applications, including MPI. Application-level solutions, including both checkpointing and fault-tolerant algorithms, are recognized as more time and space efficient than system-level checkpoints, which cannot make use of any application-specific knowledge. However, system-level checkpointing allows for preemption, making it suitable for responding to ''fault precursors'' (for instance, elevated error rates from ECC memory or network CRCs, or elevated temperature from sensors). Preemption can also increase the efficiency of batch scheduling; for instance reducing idle cycles (by allowing for shutdown without any queue draining period or reallocation of resources to eliminate idle nodes when better fitting jobs are queued), and reducing the average queued time (by limiting large jobs to running during off-peak hours, without the need to limit the length of such jobs). Each of these potential uses makes BLCR a valuable tool for efficient resource management in Linux clusters

  1. The blockade of immune checkpoints in cancer immunotherapy

    Science.gov (United States)

    Pardoll, Drew M.

    2016-01-01

    Among the most promising approaches to activating therapeutic antitumour immunity is the blockade of immune checkpoints. Immune checkpoints refer to a plethora of inhibitory pathways hardwired into the immune system that are crucial for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues in order to minimize collateral tissue damage. It is now clear that tumours co-opt certain immune-checkpoint pathways as a major mechanism of immune resistance, particularly against T cells that are specific for tumour antigens. Because many of the immune checkpoints are initiated by ligand–receptor interactions, they can be readily blocked by antibodies or modulated by recombinant forms of ligands or receptors. Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibodies were the first of this class of immunotherapeutics to achieve US Food and Drug Administration (FDA) approval. Preliminary clinical findings with blockers of additional immune-checkpoint proteins, such as programmed cell death protein 1 (PD1), indicate broad and diverse opportunities to enhance antitumour immunity with the potential to produce durable clinical responses. PMID:22437870

  2. TagSmart: analysis and visualization for yeast mutant fitness data measured by tag microarrays

    OpenAIRE

    Xie Dan; Dorer Russell; Kim Sangkyum; Kim Chulyun; Han Jiawei; Zhong Sheng

    2007-01-01

    Abstract Background A nearly complete collection of gene-deletion mutants (96% of annotated open reading frames) of the yeast Saccharomyces cerevisiae has been systematically constructed. Tag microarrays are widely used to measure the fitness of each mutant in a mutant mixture. The tag array experiments can have a complex experimental design, such as time course measurements and drug treatment with multiple dosages. Results TagSmart is a web application for analysis and visualization of Sacch...

  3. Checkpoint Inhibitors for the Treatment of Hodgkin Lymphoma.

    Science.gov (United States)

    Bennani-Baiti, Nabila; Thanarajasingam, Gita; Ansell, Stephen

    2016-06-01

    Hodgkin lymphoma's (HL) tumor composition is characterized by a paucity of malignant cells and a preponderance of immune and stromal cells. Despite the rich immune milieu within the tumor microenvironment, malignant cells are able to effectively evade the immune system and use immune support to promote lymphoma cell growth and proliferation. Recognizing this has led to the identification of checkpoint inhibitory signals that enable immune evasion and to opening the door to therapeutic strategies on how to exploit the immune system in targeting tumor cells. We discuss herein some of the tumor evasion mechanisms in HL with a particular focus on the immune checkpoint pathways and focus on recent clinical data of checkpoint blockade in HL treatment. PMID:26818843

  4. Immune checkpoint blockade therapy for bladder cancer treatment.

    Science.gov (United States)

    Kim, Jayoung

    2016-06-01

    Bladder cancer remains the most immunogenic and expensive malignant tumor in the United States today. As the 4th leading cause of death from cancer in United States, Immunotherapy blocking immune checkpoints have been recently been applied to many aggressive cancers and changed interventions of urological cancers including advanced bladder cancer. The applied inhibition of PD-1-PD-L1 interactions can restore antitumor T-cell activity and enhance the cellular immune attack on antigens. The overall goals of this short review article are to introduce current cancer immunotherapy and immune checkpoint inhibitors, and to provide new insight into the underlying mechanisms that block immune checkpoints in tumor microenvironment. Furthermore, this review will address the preclinical and clinical trials to determine whether bladder cancer patients could benefit from this new cancer therapy in near future. PMID:27326412

  5. Low-Overhead Non-Blocking Checkpointing Scheme for Mobile Computing Systems

    Institute of Scientific and Technical Information of China (English)

    MEN Chaoguang; CAO Liujuan; WANG Liwen; XU Zhenpeng

    2007-01-01

    When applied to mobile computing systems, checkpoint protocols for distributed computing systems would face many new challenges, such as low wireless bandwidth, frequent disconnections, and lack of stable storage at mobile hosts. This paper proposes a novel checkpoint protocol to effectively reduce the coordinating overhead. By using a communication vector, only a few processes participate in the checkpointing event. During checkpointing, the scheme can save the time used to trace the dependency tree by sending checkpoint requests to dependent processes at once. In addition, processes are non-blocking in this scheme, since the inconsistency is resolved by the piggyback technique. Hence the unnecessary and orphan messages can be avoided. Compared with the traditional coordinated checkpoint approach, the proposed non-blocking algorithm obtains a minimal number of processes to take checkpoints. It also reduces the checkpoint latency, which brings less overhead to mobile host with limited resources.

  6. Keeping it together in times of stress: checkpoint function at stalled replication forks

    OpenAIRE

    Berens, Theresa J.; David P Toczyski

    2012-01-01

    In this issue, De Piccoli et al. (2012) show that, contrary to current models of DNA replication checkpoint function, replication proteins remain associated with each other and with replicating DNA when replication is stressed in checkpoint-deficient cells.

  7. [Involvement of PHO80 and PHO85 genes in Saccharomyces cerevisiae ion tolerance].

    Science.gov (United States)

    Mao, Xi-Cheng; Xia, Yu-Lei; Hu, Ya-Fang; Lu, Chang-De

    2003-01-01

    PHO85 is a versatile gene in Saccharomyces cerevisiae, which is involved in metabolism of inorganic phosphate and usage of carbon source, accumulation of glycogen, regulation of protein stability and cell cycle control. The viability of wild type budding yeast strain YPH499 and its derivative pho85Delta mutant, pho80 mutant, and pap1(pcl-7)Delta mutant in different cations were investigated and their tolerance to the cations(LC(50)) was measured. The results showed that the deletion of PHO85 or PHO80 gene both increased sensibility of Sacchromyces cerevisiae to ions K(+), Mg(2+), Zn(2+), Ca(2+) and Mn(2+), while the deletion of pap1(pcl-7) gene did not lead to such phenotype. The difference between the patterns of relative growth curve of the mutants and wild type strain in the above ions also implied that PHO80 was the unique PCLs in complex with PHO85 CDK, that were contributed to K(+) and Mg(2+) ion homeostasis control and there were some other PCLs besides PHO80 that were involved in Zn(2+), Ca(2+) and Mn(2+) tolerance regulation as cyclin of PHO85 CDK. Furthermore, the amount of the total cellular calcium of pho85Delta mutant, pho80Delta mutant and YPH499 indicated that the ability of calcium accumulation of pho85 mutant and pho80Delta mutant was impaired. PMID:12518234

  8. Metabolomic approach for improving ethanol stress tolerance in Saccharomyces cerevisiae.

    Science.gov (United States)

    Ohta, Erika; Nakayama, Yasumune; Mukai, Yukio; Bamba, Takeshi; Fukusaki, Eiichiro

    2016-04-01

    The budding yeast Saccharomyces cerevisiae is widely used for brewing and ethanol production. The ethanol sensitivity of yeast cells is still a serious problem during ethanol fermentation, and a variety of genetic approaches (e.g., random mutant screening under selective pressure of ethanol) have been developed to improve ethanol tolerance. In this study, we developed a strategy for improving ethanol tolerance of yeast cells based on metabolomics as a high-resolution quantitative phenotypic analysis. We performed gas chromatography-mass spectrometry analysis to identify and quantify 36 compounds on 14 mutant strains including knockout strains for transcription factor and metabolic enzyme genes. A strong relation between metabolome of these mutants and their ethanol tolerance was observed. Data mining of the metabolomic analysis showed that several compounds (such as trehalose, valine, inositol and proline) contributed highly to ethanol tolerance. Our approach successfully detected well-known ethanol stress related metabolites such as trehalose and proline thus, to further prove our strategy, we focused on valine and inositol as the most promising target metabolites in our study. Our results show that simultaneous deletion of LEU4 and LEU9 (leading to accumulation of valine) or INM1 and INM2 (leading to reduction of inositol) significantly enhanced ethanol tolerance. This study shows the potential of the metabolomic approach to identify target genes for strain improvement of S. cerevisiae with higher ethanol tolerance. PMID:26344121

  9. Characterization of transcription site-associated mRNP retention in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    In a variety of S. cerevisiae mutants with defective mRNP maturation and/or export, heat shock (hs) mRNPs are retained at or near their sites of transcription. For example, mutants of the THO complex display an intense hs-mRNA FISH signal, which co-localizes with the hs-gene after transcriptional...... underrepresented in recovered fractions from mutant cells. This bias is abolished when a THO mutation is combined with a second site mutation alleviating the mRNA export block. Thus, the bias parallels transcription-site retention of the mRNP and suggests the existence of a complex specifically formed at the 3...

  10. 21 CFR 866.5785 - Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems.

    Science.gov (United States)

    2010-04-01

    ...) antibody (ASCA) test systems. 866.5785 Section 866.5785 Food and Drugs FOOD AND DRUG ADMINISTRATION... Immunological Test Systems § 866.5785 Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test systems. (a) Identification. The Anti-Saccharomyces cerevisiae (S. cerevisiae) antibody (ASCA) test system...

  11. Heat shock response improves heterologous protein secretion in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hou, Jin; Österlund, Tobias; Liu, Zihe;

    2013-01-01

    The yeast Saccharomyces cerevisiae is a widely used platform for the production of heterologous proteins of medical or industrial interest. However, heterologous protein productivity is often low due to limitations of the host strain. Heat shock response (HSR) is an inducible, global, cellular...... the accumulation of mis-folded or aggregated proteins. In this work, we over-expressed a mutant HSF1 gene HSF1-R206S which can constitutively activate HSR, so the heat shock response was induced at different levels, and we studied the impact of HSR on heterologous protein secretion. We found that moderate and high...

  12. Immune-Checkpoint Blockade and Active Immunotherapy for Glioma

    Energy Technology Data Exchange (ETDEWEB)

    Ahn, Brian J. [Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States); Brain Tumor Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Pollack, Ian F. [Brain Tumor Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States); Okada, Hideho, E-mail: okadah@upmc.edu [Department of Immunology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States); Brain Tumor Program, University of Pittsburgh Cancer Institute, Pittsburgh, PA 15213 (United States); Department of Neurological Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States); Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213 (United States)

    2013-11-01

    Cancer immunotherapy has made tremendous progress, including promising results in patients with malignant gliomas. Nonetheless, the immunological microenvironment of the brain and tumors arising therein is still believed to be suboptimal for sufficient antitumor immune responses for a variety of reasons, including the operation of “immune-checkpoint” mechanisms. While these mechanisms prevent autoimmunity in physiological conditions, malignant tumors, including brain tumors, actively employ these mechanisms to evade from immunological attacks. Development of agents designed to unblock these checkpoint steps is currently one of the most active areas of cancer research. In this review, we summarize recent progresses in the field of brain tumor immunology with particular foci in the area of immune-checkpoint mechanisms and development of active immunotherapy strategies. In the last decade, a number of specific monoclonal antibodies designed to block immune-checkpoint mechanisms have been developed and show efficacy in other cancers, such as melanoma. On the other hand, active immunotherapy approaches, such as vaccines, have shown encouraging outcomes. We believe that development of effective immunotherapy approaches should ultimately integrate those checkpoint-blockade agents to enhance the efficacy of therapeutic approaches. With these agents available, it is going to be quite an exciting time in the field. The eventual success of immunotherapies for brain tumors will be dependent upon not only an in-depth understanding of immunology behind the brain and brain tumors, but also collaboration and teamwork for the development of novel trials that address multiple layers of immunological challenges in gliomas.

  13. The absence of a DNA replication checkpoint in porcine zygotes

    Czech Academy of Sciences Publication Activity Database

    Vacková, I.; Křen, Radomír; Loi, P.; Krylov, V.; Fulka Jr., J.

    2006-01-01

    Roč. 14, 1 (2006), s. 33-37. ISSN 0967-1994 Institutional research plan: CEZ:AV0Z50450515 Keywords : checkpoint * DNA replication * fertilization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 0.782, year: 2006

  14. Immune-Checkpoint Blockade and Active Immunotherapy for Glioma

    Directory of Open Access Journals (Sweden)

    Brian J. Ahn

    2013-11-01

    Full Text Available Cancer immunotherapy has made tremendous progress, including promising results in patients with malignant gliomas. Nonetheless, the immunological microenvironment of the brain and tumors arising therein is still believed to be suboptimal for sufficient antitumor immune responses for a variety of reasons, including the operation of “immune-checkpoint” mechanisms. While these mechanisms prevent autoimmunity in physiological conditions, malignant tumors, including brain tumors, actively employ these mechanisms to evade from immunological attacks. Development of agents designed to unblock these checkpoint steps is currently one of the most active areas of cancer research. In this review, we summarize recent progresses in the field of brain tumor immunology with particular foci in the area of immune-checkpoint mechanisms and development of active immunotherapy strategies. In the last decade, a number of specific monoclonal antibodies designed to block immune-checkpoint mechanisms have been developed and show efficacy in other cancers, such as melanoma. On the other hand, active immunotherapy approaches, such as vaccines, have shown encouraging outcomes. We believe that development of effective immunotherapy approaches should ultimately integrate those checkpoint-blockade agents to enhance the efficacy of therapeutic approaches. With these agents available, it is going to be quite an exciting time in the field. The eventual success of immunotherapies for brain tumors will be dependent upon not only an in-depth understanding of immunology behind the brain and brain tumors, but also collaboration and teamwork for the development of novel trials that address multiple layers of immunological challenges in gliomas.

  15. Attachment issues : kinetochore transformations and spindle checkpoint silencing

    NARCIS (Netherlands)

    Etemad, Banafsheh; Kops, Geert Jpl

    2016-01-01

    Cell division culminates in the segregation of duplicated chromosomes in opposite directions prior to cellular fission. This process is guarded by the spindle assembly checkpoint (SAC), which prevents the anaphase of cell division until stable connections between spindle microtubules and the kinetoc

  16. Immune-Checkpoint Blockade and Active Immunotherapy for Glioma

    International Nuclear Information System (INIS)

    Cancer immunotherapy has made tremendous progress, including promising results in patients with malignant gliomas. Nonetheless, the immunological microenvironment of the brain and tumors arising therein is still believed to be suboptimal for sufficient antitumor immune responses for a variety of reasons, including the operation of “immune-checkpoint” mechanisms. While these mechanisms prevent autoimmunity in physiological conditions, malignant tumors, including brain tumors, actively employ these mechanisms to evade from immunological attacks. Development of agents designed to unblock these checkpoint steps is currently one of the most active areas of cancer research. In this review, we summarize recent progresses in the field of brain tumor immunology with particular foci in the area of immune-checkpoint mechanisms and development of active immunotherapy strategies. In the last decade, a number of specific monoclonal antibodies designed to block immune-checkpoint mechanisms have been developed and show efficacy in other cancers, such as melanoma. On the other hand, active immunotherapy approaches, such as vaccines, have shown encouraging outcomes. We believe that development of effective immunotherapy approaches should ultimately integrate those checkpoint-blockade agents to enhance the efficacy of therapeutic approaches. With these agents available, it is going to be quite an exciting time in the field. The eventual success of immunotherapies for brain tumors will be dependent upon not only an in-depth understanding of immunology behind the brain and brain tumors, but also collaboration and teamwork for the development of novel trials that address multiple layers of immunological challenges in gliomas

  17. Checkpointing for graceful degradation in distributed embedded systems

    Science.gov (United States)

    Sababha, Belal Hussein

    Graceful degradation is an approach to developing dependable safety-critical embedded applications, where redundant active or standby resources are used to cope with faults through a system reconfiguration at run-time. Compared to traditional hardware and software redundancy, it is a promising technique that may achieve dependability with a significant reduction in cost, size, weight, and power requirements. Reconfiguration at run-time necessitates using proper checkpointing protocols to support state reservation to ensure correct task restarts after a system reconfiguration. One of the most common checkpointing protocols are communication induced checkpointing (CIC) protocols, which are well developed and understood for large parallel and information systems, but not much has been done for resource limited embedded systems. This work implements and evaluates some of the most common CIC protocols in a periodic resource constrained distributed embedded system for graceful degradation purposes. A test-bed has been developed and used for the evaluation of the various protocols. The implemented protocols are thoroughly studied and performances are contrasted. Specifically the periodicity property and how it benefits checkpointing in embedded systems is investigated. This work introduces a unique effort of CIC protocol implementation and evaluation in the field of distributed embedded systems. Other than providing a test-bed for graceful degradation support, this work shows that some checkpointing protocols that are not efficient in large information systems and supercomputers perform well in embedded systems. We show that a simple index-based CIC protocol, such as the BCS protocol, is more appropriate in embedded system applications compared to other protocols that piggyback a significant amount of information to reduce the number of forced checkpoints. Finally, this work proposes a whole graceful degradation approach to achieve fault tolerance in resource constrained

  18. Membrane Trafficking in the Yeast Saccharomyces cerevisiae Model

    Directory of Open Access Journals (Sweden)

    Serge Feyder

    2015-01-01

    Full Text Available The yeast Saccharomyces cerevisiae is one of the best characterized eukaryotic models. The secretory pathway was the first trafficking pathway clearly understood mainly thanks to the work done in the laboratory of Randy Schekman in the 1980s. They have isolated yeast sec mutants unable to secrete an extracellular enzyme and these SEC genes were identified as encoding key effectors of the secretory machinery. For this work, the 2013 Nobel Prize in Physiology and Medicine has been awarded to Randy Schekman; the prize is shared with James Rothman and Thomas Südhof. Here, we present the different trafficking pathways of yeast S. cerevisiae. At the Golgi apparatus newly synthesized proteins are sorted between those transported to the plasma membrane (PM, or the external medium, via the exocytosis or secretory pathway (SEC, and those targeted to the vacuole either through endosomes (vacuolar protein sorting or VPS pathway or directly (alkaline phosphatase or ALP pathway. Plasma membrane proteins can be internalized by endocytosis (END and transported to endosomes where they are sorted between those targeted for vacuolar degradation and those redirected to the Golgi (recycling or RCY pathway. Studies in yeast S. cerevisiae allowed the identification of most of the known effectors, protein complexes, and trafficking pathways in eukaryotic cells, and most of them are conserved among eukaryotes.

  19. Genetic Basis for Saccharomyces cerevisiae Biofilm in Liquid Medium

    DEFF Research Database (Denmark)

    Andersen, Kaj Scherz; Bojsen, Rasmus Kenneth; Gro Rejkjær Sørensen, Laura; Weiss Nielsen, Martin; Lisby, Michael; Folkesson, Sven Anders; Regenberg, Birgitte

    2014-01-01

    Biofilm-forming microorganisms switch between two forms: free-living planktonic and sessile multicellular. Sessile communities of yeast biofilms in liquid medium provide a primitive example of multicellularity and are clinically important because biofilms tend to have other growth characteristics...... than free-living cells. We investigated the genetic basis for yeast, Saccharomyces cerevisiae, biofilm on solid surfaces in liquid medium by screening a comprehensive deletion mutant collection in the S1278b background and found 71 genes that were essential for biofilm development. Quantitative...... functioned specifically in biofilm and mat formation. In a tpk3 mutant, transcription of FLO11 was induced three-fold compared with wild-type, but biofilm development and cell–cell adhesion was absent, suggesting that Tpk3p regulates FLO11 positive posttranscriptionally and negative transcriptionally. The...

  20. Stress Granule-Defective Mutants Deregulate Stress Responsive Transcripts

    OpenAIRE

    Yang, Xiaoxue; Shen, Yi; Garre, Elena; Hao, Xinxin; Krumlinde, Daniel; Cvijović, Marija; Arens, Christina; Nyström, Thomas; Liu, Beidong; Sunnerhagen, Per

    2014-01-01

    To reduce expression of gene products not required under stress conditions, eukaryotic cells form large and complex cytoplasmic aggregates of RNA and proteins (stress granules; SGs), where transcripts are kept translationally inert. The overall composition of SGs, as well as their assembly requirements and regulation through stress-activated signaling pathways remain largely unknown. We have performed a genome-wide screen of S. cerevisiae gene deletion mutants for defects in SG formation upon...

  1. A novel allele of Saccharomyces cerevisiae RFA1 that is deficient in recombination and repair and suppressible by RAD52.

    OpenAIRE

    Firmenich, A A; Elias-Arnanz, M; Berg, P

    1995-01-01

    To understand the mechanisms involved in homologous recombination, we have performed a search for Saccharomyces cerevisiae mutants unable to carry out plasmid-to-chromosome gene conversion. For this purpose, we have developed a colony color assay in which recombination is induced by the controlled delivery of double-strand breaks (DSBs). Recombination occurs between a chromosomal mutant ade2 allele and a second plasmid-borne ade2 allele where DSBs are introduced via the site-specific HO endon...

  2. Review of some Minimum-Process Synchronous Checkpointing Schemes for Mobile Distributed Systems

    Directory of Open Access Journals (Sweden)

    Preeti Gupta,

    2010-07-01

    Full Text Available The term Distributed Systems is used to describe a system with the following characteristics: i it consists of several computers that do not share memory or a clock, ii the computers communicate with each other by exchanging messages over a communication network, iii each computer has its own memory and runs its own operating system. A mobile computing system is a distributed system where some of processes are running on mobile hosts (MHs, whose location in the network changes with time. The number of processes that take checkpoints is minimized to 1 avoid awakening of MHs in doze mode of operation, 2 minimize thrashing of MHs with checkpointing activity, 3 save limited battery life of MHs and low bandwidth of wireless channels. In minimum-process checkpointing protocols, some useless checkpoints are taken or blocking of processes takes place. To take a checkpoint, an MH has to transfer a large amount of checkpoint data to its local MSS over the wireless network. Since the wireless network has low bandwidth and MHs have low computation power, all-process checkpointing will waste the scarce resources of the mobile system on every checkpoint.Minimum-process coordinated checkpointing is a preferred approach for mobile distributed systems. In this paper, we discuss various existing minimum-process checkpointing protocols for mobile distributed systems.

  3. Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions.

    Science.gov (United States)

    Ólafsson, Guðjón; Thorpe, Peter H

    2016-01-01

    The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2. PMID:27280788

  4. Epigenetic modifiers in immunotherapy: a focus on checkpoint inhibitors.

    Science.gov (United States)

    Terranova-Barberio, Manuela; Thomas, Scott; Munster, Pamela N

    2016-06-01

    Immune surveillance should be directed to suppress tumor development and progression, involving a balance of coinhibitory and costimulatory signals that amplify immune response without overwhelming the host. Immunotherapy confers durable clinical benefit in 'immunogenic tumors', whereas in other tumors the responses are modest. Thus, immune checkpoint inhibitors may need to be combined with strategies to boost immune response or increase the tumor immune profile. Epigenetic aberrations contribute significantly to carcinogenesis. Recent findings suggest that epigenetic drugs prime the immune response by increasing expression of tumor-associated antigens and immune-related genes, as well as modulating chemokines and cytokines involved in immune system activation. This review describes our current understanding regarding epigenetic and immunotherapy combination, focusing on immune response priming to checkpoint blockade. PMID:27197539

  5. Rapid and efficient galactose fermentation by engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Quarterman, Josh; Skerker, Jeffrey M; Feng, Xueyang; Liu, Ian Y; Zhao, Huimin; Arkin, Adam P; Jin, Yong-Su

    2016-07-10

    In the important industrial yeast Saccharomyces cerevisiae, galactose metabolism requires energy production by respiration; therefore, this yeast cannot metabolize galactose under strict anaerobic conditions. While the respiratory dependence of galactose metabolism provides benefits in terms of cell growth and population stability, it is not advantageous for producing fuels and chemicals since a substantial fraction of consumed galactose is converted to carbon dioxide. In order to force S. cerevisiae to use galactose without respiration, a subunit (COX9) of a respiratory enzyme was deleted, but the resulting deletion mutant (Δcox9) was impaired in terms of galactose assimilation. Interestingly, after serial sub-cultures on galactose, the mutant evolved rapidly and was able to use galactose via fermentation only. The evolved strain (JQ-G1) produced ethanol from galactose with a 94% increase in yield and 6.9-fold improvement in specific productivity as compared to the wild-type strain. (13)C-metabolic flux analysis demonstrated a three-fold reduction in carbon flux through the TCA cycle of the evolved mutant with redirection of flux toward the fermentation pathway. Genome sequencing of the JQ-G1 strain revealed a loss of function mutation in a master negative regulator of the Leloir pathway (Gal80p). The mutation (Glu348*) in Gal80p was found to act synergistically with deletion of COX9 for efficient galactose fermentation, and thus the double deletion mutant Δcox9Δgal80 produced ethanol 2.4 times faster and with 35% higher yield than a single knockout mutant with deletion of GAL80 alone. When we introduced a functional COX9 cassette back into the JQ-G1 strain, the JQ-G1-COX9 strain showed a 33% reduction in specific galactose uptake rate and a 49% reduction in specific ethanol production rate as compared to JQ-G1. The wild-type strain was also subjected to serial sub-cultures on galactose but we failed to isolate a mutant capable of utilizing galactose without

  6. Vacuolar compartmentation of the cadmium-glutathione complex protects Saccharomyces cerevisiae from mutagenesis.

    Science.gov (United States)

    Adamis, Paula D B; Panek, Anita D; Eleutherio, Elis C A

    2007-08-30

    In the yeast Saccharomyces cerevisiae, gamma-glutamyl transferase (gamma-GT; EC 2.3.2.2) is a vacuolar-membrane bound enzyme. In this work we verified that S. cerevisiae cells deficient in gamma-GT absorbed almost 2.5-fold as much cadmium as the wild-type (wt) cells, suggesting that this enzyme might be responsible for the recycle of cadmium-glutathione complex stored in the vacuole. The mutant strain showed difficulty in keeping constant levels of glutathione (GSH) during the stress, although the GSH-reductase activity was practically the same in both wt and mutant strains, before and after metal stress. This difficulty to maintain the GSH levels in the gamma-GT mutant strain led to high levels of lipid peroxidation and carbonyl proteins in response to cadmium, higher than in the wt, but lower than in a mutant deficient in GSH synthesis. Although the increased levels of oxidative stress, gamma-GT mutant strain showed to be tolerant to cadmium and showed similar mutation rates to the wt, indicating that the compartmentation of the GSH-cadmium complex in vacuole protects cells against the mutagenic action of the metal. Confirming this hypothesis, a mutant strain deficient in Ycf1, which present high concentrations of GSH-cadmium in cytoplasm due to its deficiency in transport the complex to vacuole, showed increased mutation rates. PMID:17644279

  7. Dynamin-related proteins Vps1p and Dnm1p control peroxisome abundance in Saccharomyces cerevisiae

    NARCIS (Netherlands)

    Kuravi, Kasinath; Nagotu, Shirisha; Krikken, Arjen M; Sjollema, Klaas; Deckers, Markus; Erdmann, Ralf; Veenhuis, Marten; van der Klei, Ida J

    2006-01-01

    Saccharomyces cerevisiae contains three dynamin-related-proteins, Vps1p, Dnm1p and Mgm1p. Previous data from glucose-grown VPS1 and DNM1 null mutants suggested that Vps1p, but not Dnm1p, plays a role in regulating peroxisome abundance. Here we show that deletion of DNM1 also results in reduction of

  8. Checkpoint signaling from a single DNA interstrand crosslink

    OpenAIRE

    Ben-Yehoyada, Merav; Wang, Lily C; Kozekov, Ivan D.; Rizzo, Carmelo J.; Gottesman, Max E.; Gautier, Jean

    2009-01-01

    DNA interstrand crosslinks (ICLs) are the most toxic lesions induced by chemotherapeutic agents such as Mitomycin C and Cisplatin. By covalently linking both DNA strands, ICLs prevent DNA melting, transcription, and replication. Studies on ICL signaling and repair have been limited because these drugs generate additional DNA lesions that trigger checkpoint signaling. Here, we monitor sensing, signaling from and repairing of a single, site-specific ICL in cell-free extract derived from Xenopus...

  9. Checkpointing for the RESTART problem in Markov networks

    OpenAIRE

    Lipsky, Lester; Doran, Derek; Gokhale, Swapna

    2011-01-01

    We apply the known formulae of the RESTART problem to Markov models of software (and many other) systems, and derive new equations. We show how checkpoints might be included, with their resultant performance under RESTART. The result is a complete procedure for finding the mean, variance, and tail behavior of the job completion time as a function of the failure rate. We also provide a detailed example.

  10. A Monitor for Bud Emergence in the Yeast Morphogenesis Checkpoint

    Science.gov (United States)

    Theesfeld, Chandra L.; Zyla, Trevin R.; Bardes, Elaine G.S.; Lew, Daniel J.

    2003-01-01

    Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation. PMID:12925763

  11. Potential biomarker for checkpoint blockade immunotherapy and treatment strategy.

    Science.gov (United States)

    Dong, Zhong-Yi; Wu, Si-Pei; Liao, Ri-Qiang; Huang, Shu-Mei; Wu, Yi-Long

    2016-04-01

    Programmed cell death protein-1 (PD-1) and ligand (PD-L1) provide an important escape mechanism from immune attack, and blockade therapy of these proteins show promising clinical benefits in many types of cancer. PD-L1 can be induced by interferon-gamma (IFN-γ), hypoxia, or toll-like receptor (TLR)-mediated pathways that confer adaptive immune resistance, or upregulated by oncogenic signals leading to constitutive expression and resulting in intrinsic immune resistance. The PD-1/PD-L1 checkpoint blockade, which targets regulatory pathways in T cells to overcome immune resistance, is correlated to PD-L1 expression pattern and the presence of tumor-infiltrating lymphocytes (TILs). Meanwhile, immunogenic mutation loads show significant response to checkpoint blockade, which is probably due to PD-1/L1 status and TIL content. Finally, the clinical strategies to design effective checkpoint-targeting immunotherapies are based on the classification of inducible/constitutive expression of PD-L1 and the presence of TILs. PMID:26779629

  12. Complex Commingling: Nucleoporins and the Spindle Assembly Checkpoint

    Directory of Open Access Journals (Sweden)

    Ikram Mossaid

    2015-11-01

    Full Text Available The segregation of the chromosomes during mitosis is an important process, in which the replicated DNA content is properly allocated into two daughter cells. To ensure their genomic integrity, cells present an essential surveillance mechanism known as the spindle assembly checkpoint (SAC, which monitors the bipolar attachment of the mitotic spindle to chromosomes to prevent errors that would result in chromosome mis-segregation and aneuploidy. Multiple components of the nuclear pore complex (NPC, a gigantic protein complex that forms a channel through the nuclear envelope to allow nucleocytoplasmic exchange of macromolecules, were shown to be critical for faithful cell division and implicated in the regulation of different steps of the mitotic process, including kinetochore and spindle assembly as well as the SAC. In this review, we will describe current knowledge about the interconnection between the NPC and the SAC in an evolutional perspective, which primarily relies on the two mitotic checkpoint regulators, Mad1 and Mad2. We will further discuss the role of NPC constituents, the nucleoporins, in kinetochore and spindle assembly and the formation of the mitotic checkpoint complex during mitosis and interphase.

  13. Learning common lessons and checkpoints form human error incidents. Maintenance

    International Nuclear Information System (INIS)

    CRIEPI has been conducting detailed and structured analyses of all human error incidents collected from Japanese Licensee Event Reports using J-HPES (Japanese version of HPES) as an analysis method. Results obtained by the analyses have been stored in J-HPES database. This paper described the process to analyze J-HPES data concerning maintenance systematically to extract problems identified in the process of error action and checkpoints for preventing errors. Human error actions of these J-HPES data are classified by viewpoints of error mode and work type. As to each of these error categories, problems are extracted based on J-HPES causal relation charts in following three viewpoints: acts at workplace, activities, and preconditions of job. Moreover, checkpoints for preventing errors are developed referring proposed countermeasures in J-HPES database. In order to share these results, we started to issue 4-pages booklets 'Catch the Point' periodically. In future, based on these results, we will publish a teaching material. We also have a plan to store the contents of Catch the Point in a database, which facilitate users to find necessary checkpoints and hazards before they start their activities. (author)

  14. Transcription-Dependent DNA Transactions in the Mitochondrial Genome of a Yeast Hypersuppressive Petite Mutant

    OpenAIRE

    Van Dyck, Eric; Clayton, David A.

    1998-01-01

    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...

  15. Three peroxisome protein packaging pathways suggested by selective permeabilization of yeast mutants defective in peroxisome biogenesis.

    OpenAIRE

    Zhang, J W; Luckey, C; Lazarow, P B

    1993-01-01

    We have identified five complementation groups of peroxisome biogenesis (peb) mutants in Saccharomyces cerevisiae by a positive selection procedure. Three of these contained morphologically recognizable peroxisomes, and two appeared to lack the organelle altogether. The packaging of peroxisomal proteins in these mutants has been analyzed with a new gentle cell fractionation procedure. It employs digitonin titration for the selective permeabilization of yeast plasma and intracellular membranes...

  16. Emodnet Med Sea Check-Point - Indicators for decision- maker

    Science.gov (United States)

    Besnard, Sophie; Claverie, Vincent; Blanc, Frédérique

    2015-04-01

    The Emodnet Checkpoint projects aim is to assess the cost-effectiveness, reliability and utility of the existing monitoring at the sea basin level. This involves the development of monitoring system indicators and a GIS Platform to perform the assessment and make it available. Assessment or production of Check-Point information is made by developing targeted products based on the monitoring data and determining whether the products are meeting the needs of industry and public authorities. Check-point users are the research community, the 'institutional' policy makers for IMP and MSFD implementation, the 'intermediate users', i.e., users capable to understand basic raw data but that benefit from seeing the Checkpoint targeted products and the assessment of the fitness for purpose. We define assessment criteria aimed to characterize/depict the input datasets in terms of 3 territories capable to show performance and gaps of the present monitoring system, appropriateness, availability and fitness for purpose. • Appropriateness: What is made available to users? What motivate/decide them to select this observation rather than this one. • Availability: How this is made available to the user? Place to understand the readiness and service performance of the EU infrastructure • Fitness for use / fitness for purpose: Ability for non-expert user to appreciate the data exploitability (feedback on efficiency & reliability of marine data) For each territory (appropriateness, Availability and Fitness for purpose / for use), we define several indicators. For example, for Availability we define Visibility, Accessibility and Performance. And Visibility is itself defined by "Easily found" and "EU service". So these indicators can be classified according to their territory and sub-territory as seen above, but also according to the complexity to build them. Indicators are built from raw descriptors in 3 stages:  Stage 1: to give a neutral and basic status directly computed from

  17. Loss of yeast peroxiredoxin Tsa1p induces genome instability through activation of the DNA damage checkpoint and elevation of dNTP levels.

    Directory of Open Access Journals (Sweden)

    Hei-Man Vincent Tang

    2009-10-01

    Full Text Available Peroxiredoxins are a family of antioxidant enzymes critically involved in cellular defense and signaling. Particularly, yeast peroxiredoxin Tsa1p is thought to play a role in the maintenance of genome integrity, but the underlying mechanism is not understood. In this study, we took a genetic approach to investigate the cause of genome instability in tsa1Delta cells. Strong genetic interactions of TSA1 with DNA damage checkpoint components DUN1, SML1, and CRT1 were found when mutant cells were analyzed for either sensitivity to DNA damage or rate of spontaneous base substitutions. An elevation in intracellular dNTP production was observed in tsa1Delta cells. This was associated with constitutive activation of the DNA damage checkpoint as indicated by phosphorylation of Rad9/Rad53p, reduced steady-state amount of Sml1p, and induction of RNR and HUG1 genes. In addition, defects in the DNA damage checkpoint did not modulate intracellular level of reactive oxygen species, but suppressed the mutator phenotype of tsa1Delta cells. On the contrary, overexpression of RNR1 exacerbated this phenotype by increasing dNTP levels. Taken together, our findings uncover a new role of TSA1 in preventing the overproduction of dNTPs, which is a root cause of genome instability.

  18. Checkpoint Defects Leading to Premature Mitosis Also Cause Endoreplication of DNA in Aspergillus nidulans

    OpenAIRE

    De Souza, Colin P. C.; Ye, Xiang S.; Osmani, Stephen A.

    1999-01-01

    The G2 DNA damage and slowing of S-phase checkpoints over mitosis function through tyrosine phosphorylation of NIMXcdc2 in Aspergillus nidulans. We demonstrate that breaking these checkpoints leads to a defective premature mitosis followed by dramatic rereplication of genomic DNA. Two additional checkpoint functions, uvsB and uvsD, also cause the rereplication phenotype after their mutation allows premature mitosis in the presence of low concentrations of hydroxyurea. uvsB is shown to encode ...

  19. The Dynamics of the Unreplicated DNA Checkpoint in Xenopus laevis Embryos and Extracts.

    OpenAIRE

    Adjerid, Nassiba

    2008-01-01

    When unreplicated or damaged DNA is present, cell cycle checkpoint pathways cause cell cycle arrest by inhibiting cyclin-dependent kinases (Cdks). In Xenopus laevis, early embryonic development consists of twelve rapid cleavage cycles between DNA replication (S) and mitosis (M) without checkpoints or gap phases. However, checkpoints are engaged in Xenopus once the embryo reaches the midblastula transition (MBT). At this point, the embryo initiates transcription, acquires gap phases between S...

  20. Assays Used to Study the DNA Replication Checkpoint in Fission Yeast

    OpenAIRE

    Noguchi, Eishi; Ansbach, Alison B.; Noguchi, Chiaki; Russell, Paul

    2009-01-01

    The DNA replication checkpoint, also known as the intra-S or S-phase checkpoint, plays a central role in ensuring the accuracy of DNA replication. When replication is impeded by DNA damage or other conditions, this checkpoint delays cell cycle progression and coordinates resumption of replication with DNA repair pathways. One of its critical functions is to stabilize stalled replication forks in a replication-competent state, presumably by maintaining proper assembly of replisome components a...

  1. GIT1, a gene encoding a novel transporter for glycerophosphoinositol in Saccharomyces cerevisiae.

    OpenAIRE

    Patton-Vogt, J L; Henry, S A

    1998-01-01

    Phosphatidylinositol catabolism in Saccharomyces cerevisiae cells cultured in media containing inositol results in the release of glycerophosphoinositol (GroPIns) into the medium. As the extracellular concentration of inositol decreases with growth, the released GroPIns is transported back into the cell. Exploiting the ability of the inositol auxotroph, ino1, to use exogenous GroPIns as an inositol source, we have isolated mutants (Git-) defective in the uptake and metabolism of GroPIns. One ...

  2. Improvement of ethanol production by ethanol-tolerant Saccharomyces cerevisiae UVNR56

    OpenAIRE

    Thammasittirong, Sutticha Na-Ranong; Thirasaktana, Thanawan; Thammasittirong, Anon; Srisodsuk, Malee

    2013-01-01

    Ethanol tolerance is one of the important characteristics of ethanol-producing yeast. This study focused on the improvement of ethanol tolerance of Saccharomyces cerevisiae NR1 for enhancing ethanol production by random UV-C mutagenesis. One ethanol-tolerant mutant, UVNR56, displayed a significantly improved ethanol tolerance in the presence of 15% (v/v) ethanol and showed a considerably higher viability during ethanol fermentation from sugarcane molasses and sugarcane molasses with initial e...

  3. Nucleosome alterations caused by mutations at modifiable histone residues in Saccharomyces cerevisiae

    OpenAIRE

    Hongde Liu; Pingyan Wang; Lingjie Liu; Zhu Min; Kun Luo; Yakun Wan

    2015-01-01

    Nucleosome organization exhibits dynamic properties depending on the cell state and environment. Histone proteins, fundamental components of nucleosomes, are subject to chemical modifications on particular residues. We examined the effect of substituting modifiable residues of four core histones with the non-modifiable residue alanine on nucleosome dynamics. We mapped the genome-wide nucleosomes in 22 histone mutants of Saccharomyces cerevisiae and compared the nucleosome alterations relative...

  4. Transposon Mutagenesis To Improve the Growth of Recombinant Saccharomyces cerevisiae on d-Xylose▿

    OpenAIRE

    Ni, Haiying; Laplaza, José M.; Jeffries, Thomas W.

    2007-01-01

    Saccharomyces cerevisiae L2612 transformed with genes for xylose reductase and xylitol dehydrogenase (XYL1 and XYL2) grows well on glucose but very poorly on d-xylose. When a gene for d-xylulokinase (XYL3 or XKS1) is overexpressed, growth on glucose is unaffected, but growth on xylose is blocked. Spontaneous or chemically induced mutants of this engineered yeast that would grow on xylose could, however, be obtained. We therefore used insertional transposon mutagenesis to identify two loci tha...

  5. Interactions between mutations for sensitivity to psoralen photoaddition (pso) and to radiation (rad) in Saccharomyces cerevisiae.

    OpenAIRE

    Henriques, J A; Moustacchi, E

    1981-01-01

    The mode of interaction in haploid Saccharomyces cerevisiae of two pso mutations with each other and with rad mutations affected in their excision-resynthesis (rad3), error-prone (rad6), and deoxyribonucleic acid double-strand break (rad52) repair pathways was determined for various double mutant combinations. Survival data for 8-methoxypsoralen photoaddition, 254-nm ultraviolet light and gamma rays are presented. For 8-methoxypsoralen photoaddition, which induces both deoxyribonucleic acid i...

  6. Oxidative stress is involved in heat-induced cell death in Saccharomyces cerevisiae.

    OpenAIRE

    Davidson, J. F.; Whyte, B.; Bissinger, P H; Schiestl, R H

    1996-01-01

    The cause for death after lethal heat shock is not well understood. A shift from low to intermediate temperature causes the induction of heat-shock proteins in most organisms. However, except for HSP104, a convincing involvement of heat-shock proteins in the development of stress resistance has not been established in Saccharomyces cerevisiae. This paper shows that oxidative stress and antioxidant enzymes play a major role in heat-induced cell death in yeast. Mutants deleted for the antioxida...

  7. The role of cytoplasmic catalase in dehydration tolerance of Saccharomyces cerevisiae

    OpenAIRE

    França, Mauro Braga; Panek, Anita Dolly; Eleutherio, Elis Cristina Araujo

    2005-01-01

    In this study, we investigated the role played by cytoplasmic catalase (Ctt1) in resistance against water loss using the yeast Saccharomyces cerevisiae as eukaryotic cell model. Comparing a mutant possessing a specific lesion in CTT1 with its parental strain, it was observed that both control and ctt1 strains exhibited increased levels of lipid peroxidation after dehydration, suggesting that catalase does not protect membranes during drying. Although the ctt1 strain has only 1 catalase isofor...

  8. Identification and analysis of a Saccharomyces cerevisiae copper homeostasis gene encoding a homeodomain protein.

    OpenAIRE

    Knight, S A; Tamai, K T; Kosman, D J; Thiele, D J

    1994-01-01

    Yeast metallothionein, encoded by the CUP1 gene, and its copper-dependent transcriptional activator ACE1 play a key role in mediating copper resistance in Saccharomyces cerevisiae. Using an ethyl methanesulfonate mutant of a yeast strain in which CUP1 and ACE1 were deleted, we isolated a gene, designated CUP9, which permits yeast cells to grow at high concentrations of environmental copper, most notably when lactate is the sole carbon source. Disruption of CUP9, which is located on chromosome...

  9. Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

    OpenAIRE

    Bulter, Thomas; Alcalde, Miguel; Sieber, Volker; Meinhold, Peter; Schlachtbauer, Christian; Arnold, Frances H

    2003-01-01

    Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in kcat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutat...

  10. Comprehensive reanalysis of transcription factor knockout expression data in Saccharomyces cerevisiae reveals many new targets

    OpenAIRE

    Reimand, Jüri; Vaquerizas, Juan M.; Todd, Annabel E.; Vilo, Jaak; Luscombe, Nicholas M.

    2010-01-01

    Transcription factor (TF) perturbation experiments give valuable insights into gene regulation. Genome-scale evidence from microarray measurements may be used to identify regulatory interactions between TFs and targets. Recently, Hu and colleagues published a comprehensive study covering 269 TF knockout mutants for the yeast Saccharomyces cerevisiae. However, the information that can be extracted from this valuable dataset is limited by the method employed to process the microarray data. Here...

  11. Members of the Hsp70 family of proteins in the cell wall of Saccharomyces cerevisiae.

    OpenAIRE

    López-Ribot, J L; Chaffin, W L

    1996-01-01

    Western blot (immunoblot) analysis of cell wall and cytosolic extracts obtained from parental and ssa1 and ssa2 single- and double-mutant strains of Saccharomyces cerevisiae showed that the heat shock protein 70 (Hsp70) products of these genes, previously thought to be restricted to the cell interior, are also present in the cell wall. A cell wall location was further confirmed by indirect immunofluorescence with intact cells and biotinylation of extracellular Hsp70. Hsp70s have been implicat...

  12. Action-oriented use of ergonomic checkpoints for healthy work design in different settings.

    Science.gov (United States)

    Kogi, Kazutaka

    2007-12-01

    Recent experiences in the action-oriented use of ergonomic checkpoints in different work settings are reviewed. The purpose is to know what features are useful for healthy work design adjusted to each local situation. Based on the review results, common features of ergonomic checkpoints used in participatory training programs for improving workplace conditions in small enterprises, construction sites, home work and agriculture in industrially developing countries in Asia are discussed. These checkpoints generally compile practical improvement options in a broad range of technical areas, such as materials handling, workstation design, physical environment and work organization. Usually, "action checklists" comprising the tiles of the checkpoints are used together. A clear focus is placed on readily applicable low-cost options. Three common features of these various checkpoints appear to be important. First, the checkpoints represent typical good practices in multiple areas. Second, each how-to section of these checkpoints presents simple improvements reflecting basic ergonomic principles. Examples of these principles include easy reach, fewer and faster transport, elbow-level work, coded displays, isolated or screened hazards and shared teamwork. Third, the illustrated checkpoints accompanied by corresponding checklists are used as group work tools in short-term training courses. Many practical improvements achieved are displayed in websites for inter-country work improvement networks. It is suggested to promote the use of locally adjusted checkpoints in various forms of participatory action-oriented training in small-scale workplaces and in agriculture particularly in industrially developing countries. PMID:18572793

  13. Cystathionine accumulation in Saccharomyces cerevisiae.

    OpenAIRE

    Ono, B; Suruga, T; Yamamoto, M.; Yamamoto, S.; Murata, K; Kimura, A; Shinoda, S; Ohmori, S.

    1984-01-01

    A cysteine-dependent strain of Saccharomyces cerevisiae and its prototrophic revertants accumulated cystathionine in cells. The cystathionine accumulation was caused by a single mutation having a high incidence of gene conversion. The mutation was designated cys3 and was shown to cause loss of gamma-cystathionase activity. Cysteine dependence of the initial strain was determined by two linked and interacting mutations, cys3 and cys1 . Since cys1 mutations cause a loss of serine acetyltransfer...

  14. RAD24 (=R1/sup S/) gene product of Saccharomyces cerevisiae participates in two different pathways of DNA repair

    International Nuclear Information System (INIS)

    The moderately UV- and X-ray-sensitive mutant of Saccharomyces cerevisiae originally designated r1/sup s/ complements all rad and mms mutants available. Therefore, the new nomination rad24-1 according to the RAD nomenclature is suggested. RAD24 maps on chromosome V, close to RAD3 (1.3 cM). In order to associate the RAD24 gene with one of the three repair pathways, double mutants of rad24 and various representative genes of each pathway were constructed. The UV and X-ray sensitivities of the double mutants compared to the single mutants indicate that RAD24 is involved in excision repair of UV damage (RAD3 epistasis group), as well as in recombination repair of UV and X-ray damage (RAD52 epistasis group). Properties of the mutant are discussed which hint at the control of late steps in the pathways

  15. Ectopic Expression of Arabidopsis Phospholipase A Genes Elucidates Role of Phospholipase Bs in S. cerevisiae Cells

    OpenAIRE

    Zhang, Meng; Zhang, Yan; Giblin, E Michael; Taylor, David C.

    2009-01-01

    In S. cerevisiae neither disruption of the phospholipase B triple knockout mutant (plb1plb2plb3; plb123) nor over-expression of phospholipase Bs (PLBs) result in a phenotype different from wild type. In performing experiments to characterize candidate plant phospholipase (PLA) genes, we found, surprisingly, that ectopic expression of either of two different A. thaliana PLA2 or PLA1 genes in the yeast plb123 mutant completely inhibited cell growth. We proposed that while PLBs might not be esse...

  16. Fin1-PP1 Helps Clear Spindle Assembly Checkpoint Protein Bub1 from Kinetochores in Anaphase.

    Science.gov (United States)

    Bokros, Michael; Gravenmier, Curtis; Jin, Fengzhi; Richmond, Daniel; Wang, Yanchang

    2016-02-01

    The spindle assembly checkpoint (SAC) monitors chromosome attachment defects, and the assembly of SAC proteins at kinetochores is essential for its activation, but the SAC disassembly process remains unknown. We found that deletion of a 14-3-3 protein, Bmh1, or hyperactivation of Cdc14 early anaphase release (FEAR) allows premature SAC silencing in budding yeast, which depends on a kinetochore protein Fin1 that forms a complex with protein phosphatase PP1. Previous works suggest that FEAR-dependent Fin1 dephosphorylation promotes Bmh1-Fin1 dissociation, which enables kinetochore recruitment of Fin1-PP1. We found persistent kinetochore association of SAC protein Bub1 in fin1Δ mutants after anaphase entry. Therefore, we revealed a mechanism that clears SAC proteins from kinetochores. After anaphase entry, FEAR activation promotes kinetochore enrichment of Fin1-PP1, resulting in SAC disassembly at kinetochores. This mechanism is required for efficient SAC silencing after SAC is challenged, and untimely Fin1-kinetochore association causes premature SAC silencing and chromosome missegregation. PMID:26832405

  17. Recognition of a Single Transmembrane Degron by Sequential Quality Control Checkpoints

    Science.gov (United States)

    Fayadat, Laurence; Kopito, Ron R.

    2003-01-01

    To understand the relationship between conformational maturation and quality control–mediated proteolysis in the secretory pathway, we engineered the well-characterized degron from the α-subunit of the T-cell antigen receptor (TCRα) into the α-helical transmembrane domain of homotrimeric type I integral membrane protein, influenza hemagglutinin (HA). Although the membrane degron does not appear to interfere with acquisition of native secondary structure, as assessed by the formation of native intrachain disulfide bonds, only ∼50% of nascent mutant HA chains (HA++) become membrane-integrated and acquire complex N-linked glycans indicative of transit to a post-ER compartment. The remaining ∼50% of nascent HA++ chains fail to integrate into the lipid bilayer and are subject to proteasome-dependent degradation. Site-specific cleavage by extracellular trypsin and reactivity with conformation-specific monoclonal antibodies indicate that membrane-integrated HA++ molecules are able to mature to the plasma membrane with a conformation indistinguishable from that of HAwt. These apparently native HA++ molecules are, nevertheless, rapidly degraded by a process that is insensitive to proteasome inhibitors but blocked by lysosomotropic amines. These data suggest the existence in the secretory pathway of at least two sequential quality control checkpoints that recognize the same transmembrane degron, thereby ensuring the fidelity of protein deployment to the plasma membrane. PMID:12631739

  18. Tetrandrine: A Potent Abrogator of G2 Checkpoint Function in Tumor Cells and Its Mechanism

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To assess the ability of tetrandrine (Tet) to enhance the sensitivity to irradiation and its mechanism in cell lines of human breast cancer p53-mutant MCF-7/ADR, p53-wild-type MCF-7 and human colon carcinoma p53-mutant HT-29 as well as in C26 colorectal carcinoma-bearing BALB/c mice. Methods MCF-7/ADR, HT-29 and MCF-7 cells were exposed to irradiation in the absence or presence of tetrandrine. The effect of Tet on the cytotoxicity of X-irradiation in these three cells was determined and the effect of tetrandrine on cell cycle arrest induced by irradiation in its absence or presence was studied by flow cytometry, Moreover, mitotic index measurement determined mitosis of cells to enter mitosis. Western blotting was employed to detect cyclin B1 and Cdc2 proteins in extracts from irradiated or non-irradiated cells of MCF-7/ADR, HT-29 and MCF-7 treated with tetrandrine at various concentrations. Tumor growth delay assay was conducted to determine the radio-sensitization of tetrandrine in vivo. Results Clonogenic assay showed that tetrandrine markedly enhanced the lethal effect of X-rays on p53-mutant MCF-7/ADR and HT-29 cells and the sensitization enhancement ratio (SER) of tetrandrine was 1.51 and 1.63, but its SER was only 1.1 in p53-wt MCF-7 cells. Irradiated p53-mutant MCF-7/ADR and HT-29 cells were only arrested in G2/M phase while MCF-7 cells were arrested in G1 and G2/M phases. Radiation-induced G2 phase arrests were abrogated by tetrandrine in a concentration-dependent manner in MCF-7/ADR and HT-29 cells,whereas redistribution within MCF-7 cell cycle changed slightly. The proportion of cells in M phase increased from 1.3% to 14.7% in MCF-7/ADR cells, and from 1.5% to 13.2% in HT-29 cells, but 2.4% to 7.1% in MCF-7 cells. Furthermore, the levels of cyclin B 1 and Cdc2 expression decreased after X-irradiation in MCF-7/ADR and HT-29 cells, and the mitotic index was also lower. Tet could reverse the decrease and induce the irradiated cells to enter mitosis

  19. Molecular cloning of eucaryotic genes required for excision repair of UV-irradiated DNA: isolation and partial characterization of the RAD3 gene of Saccharomyces cerevisiae.

    OpenAIRE

    Naumovski, L; Friedberg, E C

    1982-01-01

    We describe the molecular cloning of a 6-kilobase (kb) fragment of yeast chromosomal DNA containing the RAD3 gene of Saccharomyces cerevisiae. When present in the autonomously replicating yeast cloning vector YEp24, this fragment transformed two different UV-sensitive, excision repair-defective rad3 mutants of S. cerevisiae to UV resistance. The same result was obtained with a variety of other plasmids containing a 4.5-kb subclone of the 6-kb fragment. The UV sensitivity of mutants defective ...

  20. DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

    Science.gov (United States)

    Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

    2016-08-23

    Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production. PMID:27503884

  1. PHO13 deletion-induced transcriptional activation prevents sedoheptulose accumulation during xylose metabolism in engineered Saccharomyces cerevisiae.

    Science.gov (United States)

    Xu, Haiqing; Kim, Sooah; Sorek, Hagit; Lee, Youngsuk; Jeong, Deokyeol; Kim, Jungyeon; Oh, Eun Joong; Yun, Eun Ju; Wemmer, David E; Kim, Kyoung Heon; Kim, Soo Rin; Jin, Yong-Su

    2016-03-01

    The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae. PMID:26724864

  2. Rif1 regulates initiation timing of late replication origins throughout the S. cerevisiae genome.

    Directory of Open Access Journals (Sweden)

    Jared M Peace

    Full Text Available Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1's role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4Δ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1Δ cells, and checkpoint-defective mec1Δ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Raymond T Suhandynata

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

  5. The CRO-1 gene of Saccharomyces cerevisiae controls mitotic crossing over, chromosomal stability and sporulation

    International Nuclear Information System (INIS)

    The properties of a novel temperature-sensitive recombination-defective mutant of Saccharomyces cerevisiae, cro1-1 is described. The cro1-1 mutant is the first instance of a rec mutation that reduces drastically the rates of spontaneous mitotic crossing-over events but not those of gene conversional events. The cro1-1 mutation thus provides evidence that mitotic crossing-over is dependent upon gene products that are not essential for gene conversional events. The cro1-1 mutation also results in enhanced mitotic-chromosomal instability and MATa/MATα cro1-1/cro1-1 mutants are sporulation deficient. These phenotypes indicate that the CRO1 gene modulates mitotic chromosomal integrity and is essential for normal meiosis. The cro1-1 mutant possesses Holliday junction resolvase activity, hence its recombinational defect does not involve failure to execute this putative final recombinational step. 7 refs., 1 fig., 5 tabs

  6. Caracterización de nuevos reguladores de los mecanismos de tolerancia al daño en el DNA en Saccharomyces cerevisiae

    OpenAIRE

    Gallego Sánchez, Alfonso

    2014-01-01

    [ES]Tanto los mecanismos de tolerancia al daño en el DNA como los checkpoints de integridad del material genético son sistemas esenciales en el mantenimiento de la estabilidad genómica en los organismos eucariotas, concepto este a su vez ligado con campos de estudio tan diversos como la oncogénesis o la evolución de las especies. Empleando la levadura de gemación, Saccharomyces cerevisiae, este trabajo de tesis doctoral ha contribuido, en primer lugar y mediante una aproximación genética, al ...

  7. Activation of repair and checkpoints by double-strand breaks of DNA. Activational cascade of protein phosphorylation

    International Nuclear Information System (INIS)

    Molecular mechanisms of double-strand breaks repair and checkpoints include phosphorylations of repair and checkpoint-proteins by protein kinases. Chemical modification of proteins has different consequences including activation, changing of affinity to proteins and localization

  8. Centrosome-associated regulators of the G2/M checkpoint as targets for cancer therapy

    Directory of Open Access Journals (Sweden)

    Broaddus Russell R

    2009-02-01

    Full Text Available Abstract In eukaryotic cells, control mechanisms have developed that restrain cell-cycle transitions in response to stress. These regulatory pathways are termed cell-cycle checkpoints. The G2/M checkpoint prevents cells from entering mitosis when DNA is damaged in order to afford these cells an opportunity to repair the damaged DNA before propagating genetic defects to the daughter cells. If the damage is irreparable, checkpoint signaling might activate pathways that lead to apoptosis. Since alteration of cell-cycle control is a hallmark of tumorigenesis, cell-cycle regulators represent potential targets for therapy. The centrosome has recently come into focus as a critical cellular organelle that integrates G2/M checkpoint control and repairs signals in response to DNA damage. A growing number of G2/M checkpoint regulators have been found in the centrosome, suggesting that centrosome has an important role in G2/M checkpoint function. In this review, we discuss centrosome-associated regulators of the G2/M checkpoint, the dysregulation of this checkpoint in cancer, and potential candidate targets for cancer therapy.

  9. Twitter as a Tool to Warn Others about Sobriety Checkpoints: A Pilot Observational Study

    Science.gov (United States)

    Seitz, Christopher M.; Orsini, Muhsin Michael; Fearnow-Kenney, Melodie; Hatzudis, Kiki; Wyrick, David L.

    2012-01-01

    Anecdotal evidence suggests that young people use the website Twitter as a tool to warn drivers about the locations of sobriety checkpoints. Researchers investigated this claim by independently analyzing the website's content regarding a sample of 10 sobriety checkpoints that were conducted in cities throughout the United States during the weekend…

  10. Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A

    NARCIS (Netherlands)

    Wolthuis, Rob; Clay-Farrace, Lori; van Zon, Wouter; Yekezare, Mona; Koop, Lars; Ogink, Janneke; Medema, Rene; Pines, Jonathon

    2008-01-01

    Successful mitosis requires the right protein be degraded at the right time. Central to this is the spindle checkpoint that prevents the destruction of securin and cyclin 131 when there are improperly attached chromosomes. The principal target of the checkpoint is Cdc20, which activates the anaphase

  11. Space Reclamation for Uncoordinated Checkpointing in Message-Passing Systems. Ph.D. Thesis

    Science.gov (United States)

    Wang, Yi-Min

    1993-01-01

    Checkpointing and rollback recovery are techniques that can provide efficient recovery from transient process failures. In a message-passing system, the rollback of a message sender may cause the rollback of the corresponding receiver, and the system needs to roll back to a consistent set of checkpoints called recovery line. If the processes are allowed to take uncoordinated checkpoints, the above rollback propagation may result in the domino effect which prevents recovery line progression. Traditionally, only obsolete checkpoints before the global recovery line can be discarded, and the necessary and sufficient condition for identifying all garbage checkpoints has remained an open problem. A necessary and sufficient condition for achieving optimal garbage collection is derived and it is proved that the number of useful checkpoints is bounded by N(N+1)/2, where N is the number of processes. The approach is based on the maximum-sized antichain model of consistent global checkpoints and the technique of recovery line transformation and decomposition. It is also shown that, for systems requiring message logging to record in-transit messages, the same approach can be used to achieve optimal message log reclamation. As a final topic, a unifying framework is described by considering checkpoint coordination and exploiting piecewise determinism as mechanisms for bounding rollback propagation, and the applicability of the optimal garbage collection algorithm to domino-free recovery protocols is demonstrated.

  12. Cyclin F suppresses B-Myb activity to promote cell cycle checkpoint control

    DEFF Research Database (Denmark)

    Klein, Ditte Kjærsgaard; Hoffmann, Saskia; Ahlskog, Johanna K;

    2015-01-01

    an important role in checkpoint control following ionizing radiation. Cyclin F-depleted cells initiate checkpoint signalling after ionizing radiation, but fail to maintain G2 phase arrest and progress into mitosis prematurely. Importantly, cyclin F suppresses the B-Myb-driven transcriptional programme...

  13. Priming the pancreatic cancer tumor microenvironment for checkpoint-inhibitor immunotherapy

    OpenAIRE

    Lutz, Eric R.; Kinkead, Heather; Jaffee, Elizabeth M.; Zheng, Lei

    2014-01-01

    Single agent immunotherapy is effective against several cancers, but has failed against poorly immunogenic cancers, including pancreatic cancer. Evaluation of pancreatic tumors following treatment with an experimental vaccine (Lutz et al. Cancer Immunology Research 2014) suggests that vaccination primes the tumor microenvironment (TME) for checkpoint-inhibitor immunotherapy, and supports a new platform for evaluating checkpoint-inhibitors in poorly immunogenic cancers.

  14. Progress in Metabolic Engineering of Saccharomyces cerevisiae

    OpenAIRE

    Nevoigt, Elke

    2008-01-01

    Summary: The traditional use of the yeast Saccharomyces cerevisiae in alcoholic fermentation has, over time, resulted in substantial accumulated knowledge concerning genetics, physiology, and biochemistry as well as genetic engineering and fermentation technologies. S. cerevisiae has become a platform organism for developing metabolic engineering strategies, methods, and tools. The current review discusses the relevance of several engineering strategies, such as rational and inverse metabolic...

  15. Identification of inhibitors of checkpoint kinase 1 through template screening.

    Science.gov (United States)

    Matthews, Thomas P; Klair, Suki; Burns, Samantha; Boxall, Kathy; Cherry, Michael; Fisher, Martin; Westwood, Isaac M; Walton, Michael I; McHardy, Tatiana; Cheung, Kwai-Ming J; Van Montfort, Rob; Williams, David; Aherne, G Wynne; Garrett, Michelle D; Reader, John; Collins, Ian

    2009-08-13

    Checkpoint kinase 1 (CHK1) is an oncology target of significant current interest. Inhibition of CHK1 abrogates DNA damage-induced cell cycle checkpoints and sensitizes p53 deficient cancer cells to genotoxic therapies. Using template screening, a fragment-based approach to small molecule hit generation, we have identified multiple CHK1 inhibitor scaffolds suitable for further optimization. The sequential combination of in silico low molecular weight template selection, a high concentration biochemical assay and hit validation through protein-ligand X-ray crystallography provided 13 template hits from an initial in silico screening library of ca. 15000 compounds. The use of appropriate counter-screening to rule out nonspecific aggregation by test compounds was essential for optimum performance of the high concentration bioassay. One low molecular weight, weakly active purine template hit was progressed by iterative structure-based design to give submicromolar pyrazolopyridines with good ligand efficiency and appropriate CHK1-mediated cellular activity in HT29 colon cancer cells. PMID:19572549

  16. The budding yeast protein kinase Ipl1/Aurora allows the absence of tension to activate the spindle checkpoint

    OpenAIRE

    Biggins, Sue; Murray, Andrew W.

    2001-01-01

    The spindle checkpoint prevents cell cycle progression in cells that have mitotic spindle defects. Although several spindle defects activate the spindle checkpoint, the exact nature of the primary signal is unknown. We have found that the budding yeast member of the Aurora protein kinase family, Ipl1p, is required to maintain a subset of spindle checkpoint arrests. Ipl1p is required to maintain the spindle checkpoint that is induced by overexpression of the protein kinase Mps1. Inactivating I...

  17. Fungal genomics beyond Saccharomyces cerevisiae?

    DEFF Research Database (Denmark)

    Hofmann, Gerald; Mcintyre, Mhairi; Nielsen, Jens

    2003-01-01

    Fungi are used extensively in both fundamental research and industrial applications. Saccharomyces cerevisiae has been the model organism for fungal research for many years, particularly in functional genomics. However, considering the diversity within the fungal kingdom, it is obvious that the...... application of the existing methods of genome, transcriptome, proteome and metabolome analysis to other fungi has enormous potential, especially for the production of food and food ingredients. The developments in the past year demonstrate that we have only just started to exploit this potential....

  18. Detailed Modeling, Design, and Evaluation of a Scalable Multi-level Checkpointing System

    Energy Technology Data Exchange (ETDEWEB)

    Moody, A T; Bronevetsky, G; Mohror, K M; de Supinski, B R

    2010-04-09

    High-performance computing (HPC) systems are growing more powerful by utilizing more hardware components. As the system mean-time-before-failure correspondingly drops, applications must checkpoint more frequently to make progress. However, as the system memory sizes grow faster than the bandwidth to the parallel file system, the cost of checkpointing begins to dominate application run times. A potential solution to this problem is to use multi-level checkpointing, which employs multiple types of checkpoints with different costs and different levels of resiliency in a single run. The goal is to design light-weight checkpoints to handle the most common failure modes and rely on more expensive checkpoints for less common, but more severe failures. While this approach is theoretically promising, it has not been fully evaluated in a large-scale, production system context. To this end we have designed a system, called the Scalable Checkpoint/Restart (SCR) library, that writes checkpoints to storage on the compute nodes utilizing RAM, Flash, or disk, in addition to the parallel file system. We present the performance and reliability properties of SCR as well as a probabilistic Markov model that predicts its performance on current and future systems. We show that multi-level checkpointing improves efficiency on existing large-scale systems and that this benefit increases as the system size grows. In particular, we developed low-cost checkpoint schemes that are 100x-1000x faster than the parallel file system and effective against 85% of our system failures. This leads to a gain in machine efficiency of up to 35%, and it reduces the the load on the parallel file system by a factor of two on current and future systems.

  19. Nucleotide-excision repair of DNA in cell-free extracts of the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    A wide spectrum of DNA lesions are repaired by the nucleotide-excision repair (NER) pathway in both eukaryotic and prokaryotic cells. We have developed a cell-free system in Saccharomyces cerevisiae that supports NER. NER was monitored by measuring repair synthesis in DNA treated with cisplatin or with UV radiation. Repair synthesis in vitro was defective in extracts of rad1, rad2, and rad10 mutant cells, all of which have mutations in genes whose products are known to be required for NER in vivo. Additionally, repair synthesis was complemented by mixing different mutant extracts, or by adding purified Rad1 or Rad10 protein to rad1 or rad10 mutant extracts, respectively. The latter observation demonstrates that the Rad1 and Rad10 proteins directly participate in the biochemical pathway of NER. NER supported by nuclear extracts requires ATP and Mg2+ and is stimulated by polyethylene glycol and by small amounts of whole cell extract containing overexpressed Rad2 protein. The nuclear extracts also contain base-excision repair activity that is present at wild-type levels in rad mutant extracts. This cell-free system is expected to facilitate studies on the biochemical pathway of NER in S. cerevisiae

  20. Photodynamic DNA damage induced by phycocyanin and its repair in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    M. Pádula

    1999-09-01

    Full Text Available In the present study, we analyzed DNA damage induced by phycocyanin (PHY in the presence of visible light (VL using a set of repair endonucleases purified from Escherichia coli. We demonstrated that the profile of DNA damage induced by PHY is clearly different from that induced by molecules that exert deleterious effects on DNA involving solely singlet oxygen as reactive species. Most of PHY-induced lesions are single strand breaks and, to a lesser extent, base oxidized sites, which are recognized by Nth, Nfo and Fpg enzymes. High pressure liquid chromatography coupled to electrochemical detection revealed that PHY photosensitization did not induce 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo at detectable levels. DNA repair after PHY photosensitization was also investigated. Plasmid DNA damaged by PHY photosensitization was used to transform a series of Saccharomyces cerevisiae DNA repair mutants. The results revealed that plasmid survival was greatly reduced in rad14 mutants, while the ogg1 mutation did not modify the plasmid survival when compared to that in the wild type. Furthermore, plasmid survival in the ogg1 rad14 double mutant was not different from that in the rad14 single mutant. The results reported here indicate that lethal lesions induced by PHY plus VL are repaired differently by prokaryotic and eukaryotic cells. Morever, nucleotide excision repair seems to play a major role in the recognition and repair of these lesions in Saccharomyces cerevisiae.

  1. Vanillin Inhibits Translation and Induces Messenger Ribonucleoprotein (mRNP) Granule Formation in Saccharomyces cerevisiae: Application and Validation of High-Content, Image-Based Profiling

    OpenAIRE

    Iwaki, Aya; Ohnuki, Shinsuke; Suga, Yohei; Izawa, Shingo; Ohya, Yoshikazu

    2013-01-01

    Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ri...

  2. The F-Box Protein Rcy1p Is Involved in Endocytic Membrane Traffic and Recycling Out of an Early Endosome in Saccharomyces cerevisiae

    OpenAIRE

    Wiederkehr, Andreas; Avaro, Sandrine; Prescianotto-Baschong, Cristina; Haguenauer-Tsapis, Rosine; Riezman, Howard

    2000-01-01

    In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the ac...

  3. Parallelization and checkpointing of GPU applications through program transformation

    Energy Technology Data Exchange (ETDEWEB)

    Solano-Quinde, Lizandro Damian [Iowa State Univ., Ames, IA (United States)

    2012-01-01

    GPUs have emerged as a powerful tool for accelerating general-purpose applications. The availability of programming languages that makes writing general-purpose applications for running on GPUs tractable have consolidated GPUs as an alternative for accelerating general purpose applications. Among the areas that have benefited from GPU acceleration are: signal and image processing, computational fluid dynamics, quantum chemistry, and, in general, the High Performance Computing (HPC) Industry. In order to continue to exploit higher levels of parallelism with GPUs, multi-GPU systems are gaining popularity. In this context, single-GPU applications are parallelized for running in multi-GPU systems. Furthermore, multi-GPU systems help to solve the GPU memory limitation for applications with large application memory footprint. Parallelizing single-GPU applications has been approached by libraries that distribute the workload at runtime, however, they impose execution overhead and are not portable. On the other hand, on traditional CPU systems, parallelization has been approached through application transformation at pre-compile time, which enhances the application to distribute the workload at application level and does not have the issues of library-based approaches. Hence, a parallelization scheme for GPU systems based on application transformation is needed. Like any computing engine of today, reliability is also a concern in GPUs. GPUs are vulnerable to transient and permanent failures. Current checkpoint/restart techniques are not suitable for systems with GPUs. Checkpointing for GPU systems present new and interesting challenges, primarily due to the natural differences imposed by the hardware design, the memory subsystem architecture, the massive number of threads, and the limited amount of synchronization among threads. Therefore, a checkpoint/restart technique suitable for GPU systems is needed. The goal of this work is to exploit higher levels of parallelism and

  4. Genome-Wide Transposon Mutagenesis in Saccharomyces cerevisiae and Candida albicans

    Science.gov (United States)

    Xu, Tao; Bharucha, Nikë; Kumar, Anuj

    2016-01-01

    Transposon mutagenesis is an effective method for generating large sets of random mutations in target DNA, with applicability toward numerous types of genetic screens in prokaryotes, single-celled eukaryotes, and metazoans alike. Relative to methods of random mutagenesis by chemical/UV treatment, transposon insertions can be easily identified in mutants with phenotypes of interest. The construction of transposon insertion mutants is also less labor-intensive on a genome-wide scale than methods for targeted gene replacement, although transposon insertions are not precisely targeted to a specific residue, and thus coverage of the target DNA can be problematic. The collective advantages of transposon mutagenesis have been well demonstrated in studies of the budding yeast Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans, as transposon mutagenesis has been used extensively for phenotypic screens in both yeasts. Consequently, we present here protocols for the generation and utilization of transposon-insertion DNA libraries in S. cerevisiae and C. albicans. Specifically, we present methods for the large-scale introduction of transposon insertion alleles in a desired strain of S. cerevisiae. Methods are also presented for transposon mutagenesis of C. albicans, encompassing both the construction of the plasmid-based transposon-mutagenized DNA library and its introduction into a desired strain of Candida. In total, these methods provide the necessary information to implement transposon mutagenesis in yeast, enabling the construction of large sets of identifiable gene disruption mutations, with particular utility for phenotypic screening in nonstandard genetic backgrounds. PMID:21815095

  5. Xylose utilizing recombinant Saccharomyces cerevisiae strains

    Energy Technology Data Exchange (ETDEWEB)

    Walfridsson, M.

    1996-04-01

    Through metabolic engineering, S. cerevisiae was provided with the necessary enzymes required for xylose utilisation during ethanolic fermentation of xylose-rich lignocellulose raw materials. For xylitol production, S. cerevisiae was provided with the Pichia stipitis XYL1 gene encoding xylose reductase (XR). The in-vivo reduction and the following excretion of xylitol, requires a co-substrate for maintenance and cofactor regeneration. Xylitol yields close to 100% were obtained with the XYL1 containing S. cerevisiae. Introducing P. stipitis XYL1 and XYL2 genes, encoding XR and xylitol dehydrogenase (XDH), respectively, enabled S. cerevisiae to convert xylose to xylulose, via xylitol. During the screening work of P. stipitis XDH gene, another gene encoding a polyol dehydrogenase was isolated and cloned in S. cerevisiae. The gene was identified as a D-arabinitol dehydrogenase gene. In P. stipitis it may function as a redox sink by reducing D-ribulose to D-arabinitol. The metabolism through the pentose phosphate pathway (PPP) was enhanced by over-expressing the native genes TKL1 and TAL1 encoding transketolase and transaldolase, respectively, resulting in improved xylose utilisation. The XR and XDH activities in recombinant S. cerevisiae were produced at different levels by constructing yeast vectors in which the PGK1 and ADHI promoters controlled XYL1 and XYL2. With higher XDH than XR activities, less by-products, in the form of xylitol and glycerol, were formed by the recombinant S. cerevisiae strains. The Thermus thermophilus xylA gene encoding a thermostable xylose isomerase was cloned and expressed in S. cerevisiae. The recombinant xylose isomerase was actively produced and a new functional metabolic pathway was established in S. cerevisiae resulting in ethanol production from xylose. 150 refs, 3 figs, 4 tabs

  6. Fueling the engine and releasing the break:combinational therapy of cancer vaccines and immune checkpoint inhibitors

    Institute of Scientific and Technical Information of China (English)

    Jennifer Kleponis; Richard Skelton; Lei Zheng

    2015-01-01

    Immune checkpoint inhibitors are increasingly drawing much attention in the therapeutic development for cancer treatment. However, many cancer patients do not respond to treatments with immune checkpoint inhibitors, partly because of the lack of tumor-inifltrating effector T cells. Cancer vaccines may prime patients for treatments with immune checkpoint inhibitors by inducing effector T-cell infiltration into the tumors and immune checkpoint signals. The combination of cancer vaccine and an immune checkpoint inhibitor may function synergistically to induce more effective antitumor immune responses, and clinical trials to test the combination are currently ongoing.

  7. Probabilistic Checkpointing Protocol to Sensor Network Fault-Tolerant

    Directory of Open Access Journals (Sweden)

    Titouna Faiza

    2012-09-01

    Full Text Available A wireless sensor network WSN is a collection of autonomous sensors nodes organized into a cooperative network. A sensor node transmits the data quantity to the sink. Indeed, a failed sink may abort the overall mission of the network. Due to their crucial functions, sinks must be designed and maintained to be robust enough in order to face trouble coming from the harsh environment. Thus, as a keystone of a WSN, a sink has to be provided with ability to recover from failures. In this paper, we propose a new protocol avoiding to the sink to be a central point of failure. First, we model a sensor node failure estimation problem through a causal network. Then, we show how the checkpointing process ensures the recovery of the network. This approach reduces both energy consumption and communication bandwidth requirements, and prolongs the lifetime of WSN. Interesting results are given by simulation

  8. EMODnet MedSea Checkpoint for sustainable Blue Growth

    Science.gov (United States)

    Moussat, Eric; Pinardi, Nadia; Manzella, Giuseppe; Blanc, Frederique

    2016-04-01

    The EMODNET checkpoint is a wide monitoring system assessment activity aiming to support the sustainable Blue Growth at the scale of the European Sea Basins by: 1) Clarifying the observation landscape of all compartments of the marine environment including Air, Water, Seabed, Biota and Human activities, pointing out to the existing programs, national, European and international 2) Evaluating fitness for use indicators that will show the accessibility and usability of observation and modeling data sets and their roles and synergies based upon selected applications by the European Marine Environment Strategy 3) Prioritizing the needs to optimize the overall monitoring Infrastructure (in situ and satellite data collection and assembling, data management and networking, modeling and forecasting, geo-infrastructure) and release recommendations for evolutions to better meet the application requirements in view of sustainable Blue Growth The assessment is designed for : - Institutional stakeholders for decision making on observation and monitoring systems - Data providers and producers to know how their data collected once for a given purpose could fit other user needs - End-users interested in a regional status and possible uses of existing monitoring data Selected end-user applications are of paramount importance for: (i) the blue economy sector (offshore industries, fisheries); (ii) marine environment variability and change (eutrophication, river inputs and ocean climate change impacts); (iii) emergency management (oil spills); and (iv) preservation of natural resources and biodiversity (Marine Protected Areas). End-user applications generate innovative products based on the existing observation landscape. The fitness for use assessment is made thanks to the comparison of the expected product specifications with the quality of the product derived from the selected data. This involves the development of checkpoint information and indicators based on Data quality and

  9. Attachment issues: kinetochore transformations and spindle checkpoint silencing.

    Science.gov (United States)

    Etemad, Banafsheh; Kops, Geert J P L

    2016-04-01

    Cell division culminates in the segregation of duplicated chromosomes in opposite directions prior to cellular fission. This process is guarded by the spindle assembly checkpoint (SAC), which prevents the anaphase of cell division until stable connections between spindle microtubules and the kinetochores of all chromosomes are established. The anaphase inhibitor is generated at unattached kinetochores and inhibitor production is prevented when microtubules are captured. Understanding the molecular changes in the kinetochore that are evoked by microtubule attachments is crucial for understanding the mechanisms of SAC signaling and silencing. Here, we highlight the most recent findings on these events, pinpoint some remaining mysteries, and argue for incorporating holistic views of kinetochore dynamics in order to understand SAC silencing. PMID:26947988

  10. Checkpoint inhibition for colorectal cancer: progress and possibilities.

    Science.gov (United States)

    Paul, Barry; O'Neil, Bert H; McRee, Autumn J

    2016-06-01

    Colorectal cancer (CRC) remains the third most common cause of cancer death in the USA. Despite an increase in the repertoire of treatment options available for CRC, median overall survival has plateaued at approximately 2.5 years. Strategies that engage the patient's native immune system to overcome checkpoint inhibition have proven to be promising in subsets of CRCs, specifically those with mismatch repair deficiency. Further studies are required to determine combinations of standard therapies with immunotherapy drugs and to discover the best biomarkers to predict response. This review provides insight into the progress made in treating patients with advanced CRC with immunotherapeutics and the areas that demand further research to make these drugs more effective in this patient population. PMID:27197538

  11. Checkpoint Blockade in Cancer Immunotherapy: Squaring the Circle

    Directory of Open Access Journals (Sweden)

    Maria A.V. Marzolini

    2015-03-01

    Full Text Available Manipulating the complex interaction between the immune system and tumour cells has been the focus of cancer research for many years, but it is only in the past decade that significant progress has been made in the field of cancer immunotherapy resulting in clinically effective treatments. The blockade of co-inhibitory immune checkpoints, essential for maintaining lymphocyte homeostasis and self-tolerance, by immunomodulatory monoclonal antibodies has resulted in the augmentation of anti-tumour responses. The greatest successes so far have been seen with the blockade of cytotoxic T lymphocyte associated antigen-4, which has resulted in the first Phase III clinical trial showing an overall survival benefit in metastatic melanoma, and in the blockade of the programmed cell death protein-1 axis. This concise review will focus on the clinical advances made by the blockade of these two pathways and their role in current cancer treatment strategies.

  12. Deleting the para-nitrophenyl phosphatase (pNPPase), PHO13, in recombinant Saccharomyces cerevisiae improves growth and ethanol production on D-xylose

    DEFF Research Database (Denmark)

    Van Vleet, Jennifer; Jeffries, T.W.; Olsson, Lisbeth

    2008-01-01

    Overexpression of D-xylulokinase in Saccharomyces cerevisiae engineered for assimilation of xylose results in growth inhibition that is more pronounced at higher xylose concentrations. Mutants deficient in the para-nitrophenyl phosphatase, PHO13, resist growth inhibition on xylose. We studied thi...

  13. Probing the structure and function of the estrogen receptor ligand binding domain by analysis of mutants with altered transactivation characteristics.

    OpenAIRE

    Eng, F C; Lee, H.S.; Ferrara, J; Willson, T M; White, J H

    1997-01-01

    We have developed a genetic screen for the yeast Saccharomyces cerevisiae to isolate estrogen receptor (ER) mutants with altered transactivation characteristics. Use of a "reverse" ER, in which the mutagenized ligand binding domain was placed at the N terminus of the receptor, eliminated the isolation of truncated constitutively active mutants. A library was screened with a low-affinity estrogen, 2-methoxyestrone (2ME), at concentrations 50-fold lower than those required for activation of the...

  14. Influence of α sex factor on the biosynthesis of the cell wall from Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Cells of Saccharomyces cerevisiae produce peptide hormones (a and α) which dramatically affect the physiology, structure, and behavior of cells from the opposite mating type, presumably in preparation for conjugation. Some cell division cycle mutants mimick several of the changes induced by α factor. Accordingly, conditional mutants cdc 28, cdc 36, cdc 37, and cdc 39 undergo arrest in G1, exhibit shmoo morphology and are able to mate when they are transferred to the restrictive temperature. Formation of shmoo cells would require increased synthesis of glycosyl transferases involved in the biosynthesis of cell wall polysaccharides. Accordingly, the authors investigated the effect of G1 arrest on the chemical composition of the cell wall and on the levels of glycosyl transferases. Arrest in G1 was obtained by two methods: addition of α factor, and transfer of a cdc 28 mutant to the restrictive temperature

  15. A mitotic phosphorylation feedback network connects Cdk1, Plk1, 53BP1, and Chk2 to inactivate the G(2)/M DNA damage checkpoint

    DEFF Research Database (Denmark)

    van Vugt, Marcel A T M; Gardino, Alexandra K; Linding, Rune; Ostheimer, Gerard J; Reinhardt, H Christian; Ong, Shao-En; Tan, Chris S; Miao, Hua; Keezer, Susan M; Li, Jeijin; Pawson, Tony; Lewis, Timothy A; Carr, Steven A; Smerdon, Stephen J; Brummelkamp, Thijn R; Yaffe, Michael B

    2010-01-01

    DNA damage checkpoints arrest cell cycle progression to facilitate DNA repair. The ability to survive genotoxic insults depends not only on the initiation of cell cycle checkpoints but also on checkpoint maintenance. While activation of DNA damage checkpoints has been studied extensively, molecular...

  16. DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

    Directory of Open Access Journals (Sweden)

    Katherine S Lawrence

    2015-04-01

    Full Text Available Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR, which monitors DNA integrity, and the spindle assembly checkpoint (SAC, which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

  17. NEK11: linking CHK1 and CDC25A in DNA damage checkpoint signaling

    DEFF Research Database (Denmark)

    Sørensen, Claus Storgaard; Melixetian, Marina; Klein, Ditte Kjaersgaard;

    2010-01-01

    The DNA damage induced G(2)/M checkpoint is an important guardian of the genome that prevents cell division when DNA lesions are present. The checkpoint prevents cells from entering mitosis by degrading CDC25A, a key CDK activator. CDC25A proteolysis is controlled by direct phosphorylation events...... required for beta-TrCP mediated CDC25A polyubiquitylation and degradation. The activity of NEK11 is in turn controlled by CHK1 that activates NEK11 via phosphorylation on serine 273. Since inhibition of NEK11 activity forces checkpoint-arrested cells into mitosis and cell death, NEK11 is, like CHK1, a...

  18. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

    OpenAIRE

    Ivanova, Tsvetomira Georgieva, 1978-; Alves-Rodrigues, Isabel; G??mez Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A.; Rhind, Nick; Hidalgo Hernando, Elena; Ayt?? del Olmo, Jos??

    2013-01-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint....

  19. Glycolipids of Saccharomyces cerevisiae Cell

    Directory of Open Access Journals (Sweden)

    Renuka Malhotra

    2005-01-01

    Full Text Available Total lipids of Saccharomyces cerevisiae were isolated by chloroform and methanol (2:1. Glycolipids were separated from total lipids by silicic acid chromatography. Glycolipid’s constituent sugars and fatty acids were analyzed by using Gas Liquid Chromatography. Galactose was the prominent sugar followed by mannose. Relative concentrations of fucose, mannose, galactose and glucose in the glycolipid were 5.3, 35.2, 55.1 and 4.2%. 16:0, 18:0, 18:1, 18:2 and 18:3 were the major fatty acids of the total glycolipids. Oleic acid was the dominating fatty acid followed by linoliec acid. They were separated into different fractions by using DEAE-Sephadex ion exchange chromatography. Glycolipids were fractionated and identified as cerebrosides, ceramide polyhexosides, sulfatides, monoglucosyldiglycerides and diglucosyldiglycerides. Ceramide polyhexosides were present in higher concentration as compared to other fractions.

  20. Glucose repression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kayikci, Omur; Nielsen, Jens

    2015-01-01

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

  1. Localization of nuclear retained mRNAs in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Thomsen, Rune; Libri, Domenico; Boulay, Jocelyne;

    2003-01-01

    In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated in retain......In the yeast Saccharomyces cerevisiae, a common conditional phenotype associated with deletion or mutation of genes encoding mRNA export factors is the rapid accumulation of mRNAs in intranuclear foci, suggested to be near transcription sites. The nuclear RNA exosome has been implicated...... in retaining RNAs in these foci; on deletion of the exosome component Rrp6p, the RNA is released. To determine the exact nuclear location of retained as well as released mRNAs, we have used mRNA export mutant strains to analyze the spatial relationship between newly synthesized heat shock mRNA, the chromosomal...

  2. Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Weinert, Brian Tate; Iesmantavicius, Vytautas; Moustafa, Tarek; Schölz, Christian; Wagner, Sebastian A; Magnes, Christoph; Zechner, Rudolf; Choudhary, Chuna Ram

    2014-01-01

    Lysine acetylation is a frequently occurring posttranslational modification; however, little is known about the origin and regulation of most sites. Here we used quantitative mass spectrometry to analyze acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. We found that acetylation...

  3. TOTAL ANTIOXIDANT ACTIVITY OF YEAST SACCHAROMYCES CEREVISIAE

    OpenAIRE

    Blažena Lavová; Dana Urminská

    2013-01-01

    Antioxidants are health beneficial compounds that can protect cells and macromolecules (e.g. fats, lipids, proteins and DNA) from the damage of reactive oxygen species (ROS). Sacchamomyces cerevisiae are know as organisms with very important antioxidative enzyme systems such as superoxide dismutase or catalase. The total antioxidant activity (mmol Trolox equivalent – TE.g-1 d.w.) of Saccharomyces cerevisiae was measured by 2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) during the yeas...

  4. mRNA quality control pathways in Saccharomyces cerevisiae

    Indian Academy of Sciences (India)

    Satarupa Das; Biswadip Das

    2013-09-01

    Efficient production of translation-competent mRNAs involves processing and modification events both in the nucleus and cytoplasm which require a number of complex machineries at both co-transcriptional and post-transcriptional levels. Mutations in the genomic sequence sometimes result in the formation of mutant non-functional defective messages. In addition, the enormous amounts of complexities involved in the biogenesis of mRNPs in the nucleus very often leads to the formation of aberrant and faulty messages along with their functional counterpart. Subsequent translation of these mutant and defective populations of messenger RNAs could possibly result in the unfaithful transmission of genetic information and thus is considered a threat to the survival of the cell. To prevent this possibility, mRNA quality control systems have evolved both in the nucleus and cytoplasm in eukaryotes to scrutinize various stages of mRNP biogenesis and translation. In this review, we will focus on the physiological role of some of these mRNA quality control systems in the simplest model eukaryote Saccharomyces cerevisiae.

  5. Distinct Subsets of Sit4 Holophosphatases Are Required for Inhibition of Saccharomyces cerevisiae Growth by Rapamycin and Zymocin ▿ †

    OpenAIRE

    Jablonowski, Daniel; Täubert, Jens-Eike; Bär, Christian; Stark, Michael J. R.; Schaffrath, Raffael

    2009-01-01

    Protein phosphatase Sit4 is required for growth inhibition of Saccharomyces cerevisiae by the antifungals rapamycin and zymocin. Here, we show that the rapamycin effector Tap42, which interacts with Sit4, is dispensable for zymocin action. Although Tap42 binding-deficient sit4 mutants are resistant to zymocin, these mutations also block interaction between Sit4 and the Sit4-associating proteins Sap185 and Sap190, previously shown to mediate zymocin toxicity. Among the four different SAP genes...

  6. Defining the optimal murine models to investigate immune checkpoint blockers and their combination with other immunotherapies.

    Science.gov (United States)

    Sanmamed, M F; Chester, C; Melero, I; Kohrt, H

    2016-07-01

    The recent success of checkpoint blockers to treat cancer has demonstrated that the immune system is a critical player in the war against cancer. Historically, anticancer therapeutics have been tested in syngeneic mouse models (with a fully murine immune system) or in immunodeficient mice that allow the engraftment of human xenografts. Animal models with functioning human immune systems are critically needed to more accurately recapitulate the complexity of the human tumor microenvironment. Such models are integral to better predict tumor responses to both immunomodulatory agents and directly antineoplastic therapies. In this regard, the development of humanized models is a promising, novel strategy that offers the possibility of testing checkpoint blockers' capacity and their combination with other antitumor drugs. In this review, we discuss the strengths and weaknesses of the available animal models regarding their capacity to evaluate checkpoint blockers and checkpoint blocker-based combination immunotherapy. PMID:26912558

  7. Coupling end resection with the checkpoint response at DNA double-strand breaks.

    Science.gov (United States)

    Villa, Matteo; Cassani, Corinne; Gobbini, Elisa; Bonetti, Diego; Longhese, Maria Pia

    2016-10-01

    DNA double-strand breaks (DSBs) are a nasty form of damage that needs to be repaired to ensure genome stability. The DSB ends can undergo a strand-biased nucleolytic processing (resection) to generate 3'-ended single-stranded DNA (ssDNA) that channels DSB repair into homologous recombination. Generation of ssDNA also triggers the activation of the DNA damage checkpoint, which couples cell cycle progression with DSB repair. The checkpoint response is intimately linked to DSB resection, as some checkpoint proteins regulate the resection process. The present review will highlight recent works on the mechanism and regulation of DSB resection and its interplays with checkpoint activation/inactivation in budding yeast. PMID:27141941

  8. Characterization of the Viable but Nonculturable (VBNC State in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Mohammad Salma

    Full Text Available The Viable But Non Culturable (VBNC state has been thoroughly studied in bacteria. In contrast, it has received much less attention in other microorganisms. However, it has been suggested that various yeast species occurring in wine may enter in VBNC following sulfite stress.In order to provide conclusive evidences for the existence of a VBNC state in yeast, the ability of Saccharomyces cerevisiae to enter into a VBNC state by applying sulfite stress was investigated. Viable populations were monitored by flow cytometry while culturable populations were followed by plating on culture medium. Twenty-four hours after the application of the stress, the comparison between the culturable population and the viable population demonstrated the presence of viable cells that were non culturable. In addition, removal of the stress by increasing the pH of the medium at different time intervals into the VBNC state allowed the VBNC S. cerevisiae cells to "resuscitate". The similarity between the cell cycle profiles of VBNC cells and cells exiting the VBNC state together with the generation rate of cells exiting VBNC state demonstrated the absence of cellular multiplication during the exit from the VBNC state. This provides evidence of a true VBNC state. To get further insight into the molecular mechanism pertaining to the VBNC state, we studied the involvement of the SSU1 gene, encoding a sulfite pump in S. cerevisiae. The physiological behavior of wild-type S. cerevisiae was compared to those of a recombinant strain overexpressing SSU1 and null Δssu1 mutant. Our results demonstrated that the SSU1 gene is only implicated in the first stages of sulfite resistance but not per se in the VBNC phenotype. Our study clearly demonstrated the existence of an SO2-induced VBNC state in S. cerevisiae and that the stress removal allows the "resuscitation" of VBNC cells during the VBNC state.

  9. Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability

    Directory of Open Access Journals (Sweden)

    Doerfler Lillian

    2011-10-01

    Full Text Available Abstract Background Genome instability is associated with human cancers and chromosome breakage syndromes, including Bloom's syndrome, caused by inactivation of BLM helicase. Numerous mutations that lead to genome instability are known, yet how they interact genetically is poorly understood. Results We show that spontaneous translocations that arise by nonallelic homologous recombination in DNA-damage-checkpoint-defective yeast lacking the BLM-related Sgs1 helicase (sgs1Δ mec3Δ are inhibited if cells lack Mec1/ATR kinase. Tel1/ATM, in contrast, acts as a suppressor independently of Mec3 and Sgs1. Translocations are also inhibited in cells lacking Dun1 kinase, but not in cells defective in a parallel checkpoint branch defined by Chk1 kinase. While we had previously shown that RAD51 deletion did not inhibit translocation formation, RAD59 deletion led to inhibition comparable to the rad52Δ mutation. A candidate screen of other DNA metabolic factors identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1Δ mutant, becoming even more important for chromosomal stability upon MEC3 deletion. We determined that the C-terminal third of Exo1, harboring mismatch repair protein binding sites and phosphorylation sites, is dispensable for Exo1's roles in chromosomal rearrangement suppression, mutation avoidance and resistance to DNA-damaging agents. Conclusions Our findings suggest that translocations between related genes can form by Rad59-dependent, Rad51-independent homologous recombination, which is independently suppressed by Sgs1, Tel1, Mec3 and Exo1 but promoted by Dun1 and the telomerase-inhibitor Mec1. We propose a model for the functional interaction between mitotic recombination and the DNA-damage checkpoint in the suppression of chromosomal rearrangements in sgs1Δ cells.

  10. Cambridge checkpoint English revision guide for the Cambridge secondary 1 test

    CERN Document Server

    Reynolds, John

    2013-01-01

    With Checkpoint English Revision Guide for the Cambridge Secondary 1 test you can aim for the best grade with the help of relevant and accessible notes, examiner advice plus questions and answers on each key topic. - Clear explanations of every topic covered in the Cambridge Secondary 1 Checkpoint English syllabus. - Builds revision skills you need for success in the test. - Exam tips wirtten by test setters and examiners giving you their expert advice. This text has not been through the Cambridge endorsement process.

  11. Cambridge checkpoint maths revision guide for the Cambridge secondary 1 test

    CERN Document Server

    Smith, Alan

    2013-01-01

    With Checkpoint Maths Revision Guide for the Cambridge Secondary 1 test you can aim for the best grade with the help of relevant and accessible notes, examiner advice plus questions and answers on each key topic. - Clear explanations of every topic covered in the Cambridge Secondary 1 Checkpoint Maths syllabus. - Builds revision skills you need for success in the test. - Exam tips wirtten by test setters and examiners giving you their expert advice. This text has not been through the Cambridge endorsement process.

  12. A Minimum-Process Coordinated Checkpointing Protocol For Mobile Distributed System

    Directory of Open Access Journals (Sweden)

    Praveen Kumar

    2010-05-01

    Full Text Available While dealing with Mobile Distributed systems, we come across some issues like: mobility, low bandwidth of wireless channels and lack of stable storage on mobile nodes, disconnections, limited battery power and high failure rate of mobile nodes. These issues make traditional checkpointing techniques designed for Distributed systems unsuitable for Mobile environments. In this paper, we design a minimum process algorithm for Mobile Distributed systems, where no useless checkpoints are taken and an effort has been made to optimize the blocking of processes. We propose to delay the processing of selective messages at the receiver end only during the checkpointing period. A Process is allowed to perform its normal computations and send messages during its blocking period. In this way, we try to keep blocking of processes to bare minimum. We captured the transitive dependencies during the normal execution by piggybacking dependency vectors onto computational messages. In this way, we try to reduce the Checkpointing time by avoiding formation of Checkpointing tree. The Z-dependencies are well taken care of. The proposed scheme forces zero useless checkpoints at the cost of very small blocking.

  13. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

    Science.gov (United States)

    Gaspar, Miguel; Shenk, Thomas

    2006-02-01

    The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

  14. Ploidy influences cellular responses to gross chromosomal rearrangements in saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Lemoine Sophie

    2011-06-01

    Full Text Available Abstract Background Gross chromosomal rearrangements (GCRs such as aneuploidy are key factors in genome evolution as well as being common features of human cancer. Their role in tumour initiation and progression has not yet been completely elucidated and the effects of additional chromosomes in cancer cells are still unknown. Most previous studies in which Saccharomyces cerevisiae has been used as a model for cancer cells have been carried out in the haploid context. To obtain new insights on the role of ploidy, the cellular effects of GCRs were compared between the haploid and diploid contexts. Results A total number of 21 haploid and diploid S. cerevisiae strains carrying various types of GCRs (aneuploidies, nonreciprocal translocations, segmental duplications and deletions were studied with a view to determining the effects of ploidy on the cellular responses. Differences in colony and cell morphology as well as in the growth rates were observed between mutant and parental strains. These results suggest that cells are impaired physiologically in both contexts. We also investigated the variation in genomic expression in all the mutants. We observed that gene expression was significantly altered. The data obtained here clearly show that genes involved in energy metabolism, especially in the tricarboxylic acid cycle, are up-regulated in all these mutants. However, the genes involved in the composition of the ribosome or in RNA processing are down-regulated in diploids but up-regulated in haploids. Over-expression of genes involved in the regulation of the proteasome was found to occur only in haploid mutants. Conclusion The present comparisons between the cellular responses of strains carrying GCRs in different ploidy contexts bring to light two main findings. First, GCRs induce a general stress response in all studied mutants, regardless of their ploidy. Secondly, the ploidy context plays a crucial role in maintaining the stoichiometric balance

  15. Checkpoint Activation of an Unconventional DNA Replication Program in Tetrahymena.

    Science.gov (United States)

    Sandoval, Pamela Y; Lee, Po-Hsuen; Meng, Xiangzhou; Kapler, Geoffrey M

    2015-07-01

    The intra-S phase checkpoint kinase of metazoa and yeast, ATR/MEC1, protects chromosomes from DNA damage and replication stress by phosphorylating subunits of the replicative helicase, MCM2-7. Here we describe an unprecedented ATR-dependent pathway in Tetrahymena thermophila in which the essential pre-replicative complex proteins, Orc1p, Orc2p and Mcm6p are degraded in hydroxyurea-treated S phase cells. Chromosomes undergo global changes during HU-arrest, including phosphorylation of histone H2A.X, deacetylation of histone H3, and an apparent diminution in DNA content that can be blocked by the deacetylase inhibitor sodium butyrate. Most remarkably, the cell cycle rapidly resumes upon hydroxyurea removal, and the entire genome is replicated prior to replenishment of ORC and MCMs. While stalled replication forks are elongated under these conditions, DNA fiber imaging revealed that most replicating molecules are produced by new initiation events. Furthermore, the sole origin in the ribosomal DNA minichromosome is inactive and replication appears to initiate near the rRNA promoter. The collective data raise the possibility that replication initiation occurs by an ORC-independent mechanism during the recovery from HU-induced replication stress. PMID:26218270

  16. SACCHAROMYCES CEREVISIAE AND ITS VALIDATION

    Directory of Open Access Journals (Sweden)

    Miroslav Ondrejovič

    2015-02-01

    Full Text Available The aim of this study was to optimize of independent variables as temperature, time and reaction ratio to output parameter of simultaneous enzyme saccharification and fermentation by Saccharomyces cerevisiae of pretreated wheat straw as model substrate via RSM (response surface methodology approach. As dependent variable, it was chosen ethanol yields characterizing effectivity of process. The optimal conditions were approximately temperature 100 °C, time 1 hour and reaction ratio 26 mL to 1 g of treated wheat straw with ethanol yields 141.9 mg.g-1. After calculating the optimal values, the validation analyze was carried out and it was found out that the predicted and experimentally verified dependent variable was in agreement with the optimal parameters (~ 95 %. Proposed model was tested for three lignocellulosic materials (winter wheat straw, alfalfa hay and maize straw as wheat straw used as model substrate and it was confirmed the possibility of its use for other agricultural residues with similar content of lignocellulose.

  17. Functional and Physical Interactions among Saccharomyces cerevisiae α-Factor Receptors

    OpenAIRE

    Gehret, Austin U.; Connelly, Sara M.; Dumont, Mark E.

    2012-01-01

    The α-factor receptor Ste2p is a G protein-coupled receptor (GPCR) expressed on the surface of MATa haploid cells of the yeast Saccharomyces cerevisiae. Binding of α-factor to Ste2p results in activation of a heterotrimeric G protein and of the pheromone response pathway. Functional interactions between α-factor receptors, such as dominant-negative effects and recessive behavior of constitutive and hypersensitive mutant receptors, have been reported previously. We show here that dominant-nega...

  18. Specialization of B-Type Cyclins for Mitosis or Meiosis in S. Cerevisiae

    OpenAIRE

    Dahmann, C.; Futcher, B.

    1995-01-01

    The CLB1, CLB2, and CLB3 genes encode B-type cyclins important for mitosis in Saccharomyces cerevisiae, while a fourth B-type cyclin gene, CLB4, has no clear role. The effects of homozygous clb mutations on meiosis were examined. Mutants homozygous for clb1 clb3, or for clb1 clb4, gave high levels of sporulation, but produced mainly two-spored asci instead of four-spored asci. The cells had completed meiosis I but not meiosis II, producing viable diploid ascospores. CLB1 and CLB4 seem to be m...

  19. Improved Production of a Heterologous Amylase in Saccharomyces cerevisiae by Inverse Metabolic Engineering

    DEFF Research Database (Denmark)

    Liu, Zihe; Liu, Lifang; Osterlund, Tobias;

    2014-01-01

    The increasing demand for industrial enzymes and biopharmaceutical proteins relies on robust production hosts with high protein yield and productivity. Being one of the best-studied model organisms and capable of performing posttranslational modifications, the yeast Saccharomyces cerevisiae is...... engineering to identify novel targets for improving protein secretion. Screening that combined UV-random mutagenesis and selection for growth on starch was performed to find mutant strains producing heterologous amylase 5-fold above the level produced by the reference strain. Genomic mutations that could be...

  20. Optimization of ordered plasmid assembly by gap repair in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Eckert-Boulet, Nadine Valerie; Pedersen, Mette Louise; Krogh, Berit Olsen;

    2012-01-01

    Combinatorial genetic libraries are powerful tools for diversifying and optimizing biomolecules. The process of library assembly is a major limiting factor for library complexity and quality. Gap repair by homologous recombination in Saccharomyces cerevisiae can facilitate in vivo assembly of DNA...... in mutants carrying a deletion of the SGS1 helicase-encoding gene. Using our experimental conditions, a gap-repair efficiency of > 10(6) plasmid-harbouring colonies/µg gapped vector DNA is obtained in a single transformation, with a recombination fidelity > 90%....

  1. Translational control of catalase synthesis by hemin in the yeast Saccharomyces cerevisiae

    OpenAIRE

    Hamilton, Barbara; Hofbauer, Reinhold; Ruis, Helmut

    1982-01-01

    mRNA-dependent cell-free protein synthesis systems were prepared from a heme-deficient ole3 mutant of the yeast Saccharomyces cerevisiae grown either in the absence or in the presence of the heme precursor δ-aminolevulinate. When supplemented with total yeast mRNA, the two systems—from heme-deficient and from heme-containing cells—translate most mRNAs with comparable efficiencies. mRNAs coding for the hemoproteins catalase T and catalase A, however, are translated at a low rate by the system ...

  2. Role of Saccharomyces cerevisiae ISA1 and ISA2 in Iron Homeostasis

    OpenAIRE

    Jensen, Laran T.; Culotta, Valeria Cizewski

    2000-01-01

    The budding yeast Saccharomyces cerevisiae contains two homologues of bacterial IscA proteins, designated Isa1p and Isa2p. Bacterial IscA is a product of the isc (iron-sulfur cluster) operon and has been suggested to participate in Fe-S cluster formation or repair. To test the function of yeast Isa1p and Isa2p, single or combinatorial disruptions were introduced in ISA1 and ISA2. The resultant isaΔ mutants were viable but exhibited a dependency on lysine and glutamate for growth and a respira...

  3. Exogenous Valine Reduces Conversion of Leucine to 3-Methyl-1-Butanol in Saccharomyces cerevisiae

    OpenAIRE

    1983-01-01

    Mutant strains of the yeast Saccharomyces cerevisiae that require branched-chain amino acids must be supplemented with large concentrations (up to 10 mM) of these amino acids to satisfy their nutritional requirement. The utilization of one branched-chain amino acid, leucine, was examined in several leul strains of yeast grown aerobically in a glucose-ammonium salts minimal medium containing a limiting concentration (0.2 mM) of leucine. In this medium, the leucine requirement of the auxotrophi...

  4. Compositions and methods for modeling Saccharomyces cerevisiae metabolism

    DEFF Research Database (Denmark)

    2012-01-01

    The invention provides an in silica model for determining a S. cerevisiae physiological function. The model includes a data structure relating a plurality of S. cerevisiae reactants to a plurality of S. cerevisiae reactions, a constraint set for the plurality of S. cerevisiae reactions, and...... commands for determining a distribution of flux through the reactions that is predictive of a S. cerevisiae physiological function. A model of the invention can further include a gene database containing information characterizing the associated gene or genes. The invention further provides methods for...... making an in silica S. cerevisiae model and methods for determining a S. cerevisiae physiological function using a model of the invention. The invention provides an in silica model for determining a S. cerevisiae physiological function. The model includes a data structure relating a plurality of S...

  5. Genetic redundancy between SPT23 and MGA2: regulators of Ty-induced mutations and Ty1 transcription in Saccharomyces cerevisiae.

    OpenAIRE

    S. Zhang; Burkett, T J; Yamashita, I; Garfinkel, D J

    1997-01-01

    SPT23 was isolated as a dosage-dependent suppressor of Ty-induced mutations in Saccharomyces cerevisiae. SPT23 shows considerable sequence homology with MGA2, a gene identified as a dosage-dependent suppressor of a snf2-imposed block on STA1 transcription in S. cerevisiae var. diastaticus. Although single mutations in either of these genes have only modest effects on cell growth, spt23 mga2 double mutants are inviable. Unlike SPT23, multicopy expression of a truncated form of MGA2 suppresses ...

  6. Web application for genetic modification flux with database to estimate metabolic fluxes of genetic mutants.

    Science.gov (United States)

    Mohd Ali, Noorlin; Tsuboi, Ryo; Matsumoto, Yuta; Koishi, Daisuke; Inoue, Kentaro; Maeda, Kazuhiro; Kurata, Hiroyuki

    2016-07-01

    Computational analysis of metabolic fluxes is essential in understanding the structure and function of a metabolic network and in rationally designing genetically modified mutants for an engineering purpose. We had presented the genetic modification flux (GMF) that predicts the flux distribution of a broad range of genetically modified mutants. To enhance the feasibility and usability of GMF, we have developed a web application with a metabolic network database to predict a flux distribution of genetically modified mutants. One hundred and twelve data sets of Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Chinese hamster ovary were registered as standard models. PMID:26777238

  7. The Meiotic Recombination Checkpoint Suppresses NHK-1 Kinase to Prevent Reorganisation of the Oocyte Nucleus in Drosophila

    OpenAIRE

    Lancaster, Oscar M.; Breuer, Manuel; Cullen, C. Fiona; Ito, Takashi; Ohkura, Hiroyuki

    2010-01-01

    The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular ...

  8. Defects in Protein Folding Machinery Affect Cell Wall Integrity and Reduce Ethanol Tolerance in S. cerevisiae.

    Science.gov (United States)

    Narayanan, Aswathy; Pullepu, Dileep; Reddy, Praveen Kumar; Uddin, Wasim; Kabir, M Anaul

    2016-07-01

    The chaperonin complex CCT/TRiC (chaperonin containing TCP-1/TCP-1 ring complex) participates in the folding of many crucial proteins including actin and tubulin in eukaryotes. Mutations in genes encoding its subunits can affect protein folding and in turn, the physiology of the organism. Stress response in Saccharomyces cerevisiae is important in fermentation reactions and operates through overexpression and underexpression of genes, thus altering the protein profile. Defective protein folding machinery can disturb this process. In this study, the response of cct mutants to stress conditions in general and ethanol in specific was investigated. CCT1 mutants showed decreased resistance to different conditions tested including osmotic stress, metal ions, surfactants, reducing and oxidising agents. Cct1-3 mutant with the mutation in the conserved ATP-binding region showed irreversible defects than other mutants. These mutants were found to have inherent cell wall defects and showed decreased ethanol tolerance. This study reveals that cell wall defects and ethanol sensitivity are linked. Genetic and proteomic analyses showed that the yeast genes RPS6A (ribosomal protein), SCL1 (proteasomal subunit) and TDH3 (glyceraldehyde-3-phosphate dehydrogenase) on overexpression, improved the growth of cct1-3 mutant on ethanol. We propose the breakdown of common stress response pathways caused by mutations in CCT complex and the resulting scarcity of functional stress-responsive proteins, affecting the cell's defence against different stress agents in cct mutants. Defective cytoskeleton and perturbed cell wall integrity reduce the ethanol tolerance in the mutants which are rescued by the extragenic suppressors. PMID:26992923

  9. Smurf2 as a novel mitotic regulator: From the spindle assembly checkpoint to tumorigenesis

    Directory of Open Access Journals (Sweden)

    Moore Finola E

    2009-07-01

    Full Text Available Abstract The execution of the mitotic program with high fidelity is dependent upon precise spatiotemporal regulation of posttranslational protein modifications. For example, the timely polyubiquitination of critical mitotic regulators by Anaphase Promoting Complex/Cyclosome (APC/C is essential for the metaphase to anaphase transition and mitotic exit. The spindle assembly checkpoint prevents unscheduled activity of APC/C-Cdc20 in early mitosis, allowing bipolar attachment of kinetochores to mitotic spindle and facilitating equal segregation of sister chromatids. The critical effector of the spindle checkpoint, Mitotic arrest deficient 2 (Mad2, is recruited to unattached kinetochores forming a complex with other regulatory proteins to efficiently and cooperatively inhibit APC/C-Cdc20. A weakened and/or dysfunctional spindle checkpoint has been linked to the development of genomic instability in both cell culture and animal models, and evidence suggests that aberrant regulation of the spindle checkpoint plays a critical role in human carcinogenesis. Recent studies have illuminated a network of both degradative and non-degradative ubiquitination events that regulate the metaphase to anaphase transition and mitotic exit. Within this context, our recent work showed that the HECT (Homologous to E6-AP C-terminus-family E3 ligase Smurf2 (Smad specific ubiquitin regulatory factor 2, known as a negative regulator of transforming growth factor-beta (TGF-β signaling, is required for a functional spindle checkpoint by promoting the functional localization and stability of Mad2. Here we discuss putative models explaining the role of Smurf2 as a new regulator in the spindle checkpoint. The dynamic mitotic localization of Smurf2 to the centrosome and other critical mitotic structures provides implications about mitotic checkpoint control dependent on various ubiquitination events. Finally, deregulated Smurf2 activity may contribute to carcinogenesis by

  10. Productive mutants of niger

    International Nuclear Information System (INIS)

    Seeds of six niger (Guizotia abyssinica Cass.) varieties ('GA-10', 'ONS-8', 'IGP-72', 'N-71', 'NB-9' and 'UN-4') were treated with 0.5, 0.75 and 1% ethyl methanesulphonate. After four generations of selection, 29 mutant lines were developed and those were evaluated from 1990-92 during Kharif (July to October) and Rabi (December to March) seasons. Average plant characteristics and yield data of four high yielding mutants along with 'IGP-76' (National Check), GA-10 (Zonal Check) and 'Semiliguda Local' (Local Check) are presented

  11. Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Tani, Motohiro; Kuge, Osamu

    2012-12-01

    Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway. PMID:23062277

  12. In-silico modeling of the mitotic spindle assembly checkpoint.

    Directory of Open Access Journals (Sweden)

    Bashar Ibrahim

    Full Text Available BACKGROUND: The Mitotic Spindle Assembly Checkpoint ((MSAC is an evolutionary conserved mechanism that ensures the correct segregation of chromosomes by restraining cell cycle progression from entering anaphase until all chromosomes have made proper bipolar attachments to the mitotic spindle. Its malfunction can lead to cancer. PRINCIPLE FINDINGS: We have constructed and validated for the human (MSAC mechanism an in silico dynamical model, integrating 11 proteins and complexes. The model incorporates the perspectives of three central control pathways, namely Mad1/Mad2 induced Cdc20 sequestering based on the Template Model, MCC formation, and APC inhibition. Originating from the biochemical reactions for the underlying molecular processes, non-linear ordinary differential equations for the concentrations of 11 proteins and complexes of the (MSAC are derived. Most of the kinetic constants are taken from literature, the remaining four unknown parameters are derived by an evolutionary optimization procedure for an objective function describing the dynamics of the APC:Cdc20 complex. MCC:APC dissociation is described by two alternatives, namely the "Dissociation" and the "Convey" model variants. The attachment of the kinetochore to microtubuli is simulated by a switching parameter silencing those reactions which are stopped by the attachment. For both, the Dissociation and the Convey variants, we compare two different scenarios concerning the microtubule attachment dependent control of the dissociation reaction. Our model is validated by simulation of ten perturbation experiments. CONCLUSION: Only in the controlled case, our models show (MSAC behaviour at meta- to anaphase transition in agreement with experimental observations. Our simulations revealed that for (MSAC activation, Cdc20 is not fully sequestered; instead APC is inhibited by MCC binding.

  13. Evolved hexose transporter enhances xylose uptake and glucose/xylose co-utilization in Saccharomyces cerevisiae.

    Science.gov (United States)

    Reider Apel, Amanda; Ouellet, Mario; Szmidt-Middleton, Heather; Keasling, Jay D; Mukhopadhyay, Aindrila

    2016-01-01

    Enhancing xylose utilization has been a major focus in Saccharomyces cerevisiae strain-engineering efforts. The incentive for these studies arises from the need to use all sugars in the typical carbon mixtures that comprise standard renewable plant-biomass-based carbon sources. While major advances have been made in developing utilization pathways, the efficient import of five carbon sugars into the cell remains an important bottleneck in this endeavor. Here we use an engineered S. cerevisiae BY4742 strain, containing an established heterologous xylose utilization pathway, and imposed a laboratory evolution regime with xylose as the sole carbon source. We obtained several evolved strains with improved growth phenotypes and evaluated the best candidate using genome resequencing. We observed remarkably few single nucleotide polymorphisms in the evolved strain, among which we confirmed a single amino acid change in the hexose transporter HXT7 coding sequence to be responsible for the evolved phenotype. The mutant HXT7(F79S) shows improved xylose uptake rates (Vmax = 186.4 ± 20.1 nmol•min(-1)•mg(-1)) that allows the S. cerevisiae strain to show significant growth with xylose as the sole carbon source, as well as partial co-utilization of glucose and xylose in a mixed sugar cultivation. PMID:26781725

  14. UV- and gamma-radiation sensitive mutants of Arabidopsis thaliana

    International Nuclear Information System (INIS)

    Arabidopsis seedlings repair UV-induced DNA damage via light-dependent and -independent pathways. The mechanism of the ''dark repair'' pathway is still unknown. To determine the number of genes required for dark repair and to investigate the substrate-specificity of this process we isolated mutants with enhanced sensitivity to UV radiation in the absence of photoreactivating light. Seven independently derived UV sensitive mutants were isolated from an EMS-mutagenized population. These fell into six complementation groups, two of which (UVR1 and UVH1) have previously been defined. Four of these mutants are defective in the dark repair of UV-induced pyrimidine [6-4] pyrimidinone dimers. These four mutant lines are sensitive to the growth-inhibitory effects of gamma radiation, suggesting that this repair pathway is also involved in the repair of some type of gamma-induced DNA damage product. The requirement for the coordinate action of several different gene products for effective repair of pyrimidine dimers, as well as the nonspecific nature of the repair activity, is consistent with nucleotide excision repair mechanisms previously described in Saccharomyces cerevisiae and nonplant higher eukaryotes and inconsistent with substrate-specific base excision repair mechanisms found in some bacteria, bacteriophage, and fungi. (author)

  15. Barosensitivity in Saccharomyces cerevisiae is Closely Associated with a Deletion of the COX1 Gene.

    Science.gov (United States)

    Nomura, Kazuki; Iwahashi, Hitoshi; Iguchi, Akinori; Shigematsu, Toru

    2015-05-01

    High hydrostatic pressure causes physical stress to microorganisms; therefore, this technology may be applied to food pasteurization without introducing the unfavorable effects of thermal denaturation. However, its application is limited to high-value foods because the treatment requires a robust steel vessel and expensive pressurization equipment. To reduce these costs, we studied the pasteurization of Saccharomyces cerevisiae using relatively moderate high-pressure levels. A mutant strain isolated by ultraviolet mutagenesis showed significant loss of viability under high-pressure conditions. Gene expression analysis of the mutant strain revealed that it incurred a deletion of the COX1 gene. Our results suggest that the pressure-sensitivity can readily be introduced into industrial/food microorganisms by complementing a COX1 deleted mitochondria. PMID:25881710

  16. Anethole induces apoptotic cell death accompanied by reactive oxygen species production and DNA fragmentation in Aspergillus fumigatus and Saccharomyces cerevisiae.

    Science.gov (United States)

    Fujita, Ken-Ichi; Tatsumi, Miki; Ogita, Akira; Kubo, Isao; Tanaka, Toshio

    2014-02-01

    trans-Anethole (anethole), a major component of anise oil, has a broad antimicrobial spectrum, and antimicrobial activity that is weaker than that of other antibiotics on the market. When combined with polygodial, nagilactone E, and n-dodecanol, anethole has been shown to possess significant synergistic antifungal activity against a budding yeast, Saccharomyces cerevisiae, and a human opportunistic pathogenic yeast, Candida albicans. However, the antifungal mechanism of anethole has not been completely determined. We found that anethole stimulated cell death of a human opportunistic pathogenic fungus, Aspergillus fumigatus, in addition to S. cerevisiae. The anethole-induced cell death was accompanied by reactive oxygen species production, metacaspase activation, and DNA fragmentation. Several mutants of S. cerevisiae, in which genes related to the apoptosis-initiating execution signals from mitochondria were deleted, were resistant to anethole. These results suggest that anethole-induced cell death could be explained by oxidative stress-dependent apoptosis via typical mitochondrial death cascades in fungi, including A. fumigatus and S. cerevisiae. PMID:24393541

  17. Identification of amino acids involved in the Flo11p-mediated adhesion of Saccharomyces cerevisiae to a polystyrene surface using phage display with competitive elution

    DEFF Research Database (Denmark)

    Mortensen, Henrik Dam; Dupont, Kitt; Jespersen, Lene;

    2007-01-01

    . cerevisiae FLO11 wild-type (TBR1) cells had a higher consensus than those from competitive panning with S. cerevisiae flo11¿ mutant (TBR5) cells, suggesting that the wild-type cells interact with the plastic surface in a stronger and more similar way than the mutant cells. Tryptophan and proline were more...... a phage with a hydrophobic peptide containing no tryptophan and only two proline residues. Conclusions: Our results suggest a key role of tryptophan and proline in the hydrophobic interactions between Flo11p on the S. cerevisiae cell surface and the PolySorp surface. Significance and Impact of the Study......: Our study may contribute to the development of novel strategies to limit yeast infections in hospitals and other medical environments....

  18. Metabolic pathway engineering for fatty acid ethyl ester production in Saccharomyces cerevisiae using stable chromosomal integration.

    Science.gov (United States)

    de Jong, Bouke Wim; Shi, Shuobo; Valle-Rodríguez, Juan Octavio; Siewers, Verena; Nielsen, Jens

    2015-03-01

    Fatty acid ethyl esters are fatty acid derived molecules similar to first generation biodiesel (fatty acid methyl esters; FAMEs) which can be produced in a microbial cell factory. Saccharomyces cerevisiae is a suitable candidate for microbial large scale and long term cultivations, which is the typical industrial production setting for biofuels. It is crucial to conserve the metabolic design of the cell factory during industrial cultivation conditions that require extensive propagation. Genetic modifications therefore have to be introduced in a stable manner. Here, several metabolic engineering strategies for improved production of fatty acid ethyl esters in S. cerevisiae were combined and the genes were stably expressed from the organisms' chromosomes. A wax ester synthase (ws2) was expressed in different yeast strains with an engineered acetyl-CoA and fatty acid metabolism. Thus, we compared expression of ws2 with and without overexpression of alcohol dehydrogenase (ADH2), acetaldehyde dehydrogenase (ALD6) and acetyl-CoA synthetase (acs SE (L641P) ) and further evaluated additional overexpression of a mutant version of acetyl-CoA decarboxylase (ACC1 (S1157A,S659A) ) and the acyl-CoA binding protein (ACB1). The combined engineering efforts of the implementation of ws2, ADH2, ALD6 and acs SE (L641P) , ACC1 (S1157A,S659A) and ACB1 in a S. cerevisiae strain lacking storage lipid formation (are1Δ, are2Δ, dga1Δ and lro1Δ) and β-oxidation (pox1Δ) resulted in a 4.1-fold improvement compared with sole expression of ws2 in S. cerevisiae. PMID:25422103

  19. Immune checkpoints aberrations and gastric cancer; assessment of prognostic value and evaluation of therapeutic potentials.

    Science.gov (United States)

    Abdel-Rahman, Omar

    2016-01-01

    Till now, the prognosis of advanced gastric cancer looked dreadful; thus the search for newer better approaches for this lethal disease has been a strategic target for cancer researchers. In recent years, important immunobiological aspects of the tumor have been revealed with the subsequent proposal of immune check point inhibitors to target these pathways. Clinically, unselected use of immune checkpoint inhibitors in gastric cancer has been deemed with failure; in contrast to the clear success of more recent studies reporting on the use of pembrolizumab in molecularly selected patients. This may illustrate that any future use of immune checkpoint inhibitors in gastric cancer has to be molecularly supported. This review provides a delicate dissection of the clinical and immunobiological considerations underlying the use of these agents in addition to a thorough review of the published clinical data of immune checkpoint inhibitors in gastric cancer. PMID:26321371

  20. The Replication Checkpoint Prevents Two Types of Fork Collapse without Regulating Replisome Stability.

    Science.gov (United States)

    Dungrawala, Huzefa; Rose, Kristie L; Bhat, Kamakoti P; Mohni, Kareem N; Glick, Gloria G; Couch, Frank B; Cortez, David

    2015-09-17

    The ATR replication checkpoint ensures that stalled forks remain stable when replisome movement is impeded. Using an improved iPOND protocol combined with SILAC mass spectrometry, we characterized human replisome dynamics in response to fork stalling. Our data provide a quantitative picture of the replisome and replication stress response proteomes in 32 experimental conditions. Importantly, rather than stabilize the replisome, the checkpoint prevents two distinct types of fork collapse. Unsupervised hierarchical clustering of protein abundance on nascent DNA is sufficient to identify protein complexes and place newly identified replisome-associated proteins into functional pathways. As an example, we demonstrate that ZNF644 complexes with the G9a/GLP methyltransferase at replication forks and is needed to prevent replication-associated DNA damage. Our data reveal how the replication checkpoint preserves genome integrity, provide insights into the mechanism of action of ATR inhibitors, and will be a useful resource for replication, DNA repair, and chromatin investigators. PMID:26365379

  1. Blocking CHK1 Expression Induces Apoptosis and Abrogates the G2 Checkpoint Mechanism

    Directory of Open Access Journals (Sweden)

    Yan Luo

    2001-01-01

    Full Text Available Checkpoint kinase 1 (Chki is a checkpoint gene that is activated after DNA damage. It phosphorylates and inactivates the Cdc2 activating phosphatase Cdc25C. This in turn inactivates Cdc2, which leads to G2/M arrest. We report that blocking Chki expression by antisense or ribozymes in mammalian cells induces apoptosis and interferes with the G2/M arrest induced by adriamycin. The Chki inhibitor UCN-01 also blocks the G2 arrest after DNA damage and renders cells more susceptible to adriamycin. These results indicate that Chki is an essential gene for the checkpoint mechanism during normal cell proliferation as well as in the DNA damage response.

  2. Hierarchical Non-blocking Coordinated Checkpointing Algorithms for Mobile Distributed Computing

    Directory of Open Access Journals (Sweden)

    Parveen Kumar

    2010-01-01

    Full Text Available Mobile system typically uses wireless communication which is based on electromagnetic waves and utilizes a shared broadcast medium. This has made possible creating a mobile distributed computing environment and has brought us several new challenges in distributed protocol design. So many issues such as range of transmission, limited power supply due to battery capacity and mobility of processes. These new issue makes traditional recovery algorithm unsuitable. In this paper, we propose hierarchical non blocking coordinated checkpointing algorithms suitable for mobile distributed computing. The algorithm is non-blocking, requires minimum message logging, has minimum stable storage requirement and produce a consistent set of checkpoints. This algorithm requires minimum number of processes to take checkpoint.

  3. Combining targeted therapy and immune checkpoint inhibitors in the treatment of metastatic melanoma

    Institute of Scientific and Technical Information of China (English)

    Teresa Kim; Rodabe N Amaria; Christine Spencer; Alexandre Reuben; Zachary A Cooper; Jennifer A Wargo

    2014-01-01

    Melanoma is the deadliest form of skin cancer and has an incidence that is rising faster than any other solid tumor. Metastatic melanoma treatment has considerably progressed in the past ifve years with the introduction of targeted therapy (BARF and MEK inhibitors) and immune checkpoint blockade (anti-CTLA4, anti-PD-1, and anti-PD-L1). However, each treatment modality has limitations. Treatment with targeted therapy has been associated with a high response rate, but with short-term responses. Conversely, treatment with immune checkpoint blockade has a lower response rate, but with long-term responses. Targeted therapy affects antitumor immunity, and synergy may exist when targeted therapy is combined with immunotherapy. hTis article presents a brief review of the rationale and evidence for the potential synergy between targeted therapy and immune checkpoint blockade. Challenges and directions for future studies are also proposed.

  4. Use of checkpoint-restart for complex HEP software on traditional architectures and Intel MIC

    International Nuclear Information System (INIS)

    Process checkpoint-restart is a technology with great potential for use in HEP workflows. Use cases include debugging, reducing the startup time of applications both in offline batch jobs and the High Level Trigger, permitting job preemption in environments where spare CPU cycles are being used opportunistically and efficient scheduling of a mix of multicore and single-threaded jobs. We report on tests of checkpoint-restart technology using CMS software, Geant4-MT (multi-threaded Geant4), and the DMTCP (Distributed Multithreaded Checkpointing) package. We analyze both single- and multi-threaded applications and test on both standard Intel x86 architectures and on Intel MIC. The tests with multi-threaded applications on Intel MIC are used to consider scalability and performance. These are considered an indicator of what the future may hold for many-core computing

  5. Nitrogen Catabolite Repression in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hofman-Bang, H Jacob Peider

    1999-01-01

    In Saccharomyces cerevisiae the expression of all known nitrogen catabolite pathways are regulated by four regulators known as Gln3, Gat1, Da180, and Deh1. This is known as nitrogen catabolite repression (NCR). They bind to motifs in the promoter region to the consensus sequence S' GATAA 3'. Gln3...

  6. Oscillations in glycolysis in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kloster, Antonina; Olsen, Lars Folke

    Wehave investigated the glycolytic oscillations, measured as NADH autofluorescence, in the yeast Saccharomyces cerevisiae in a batch reactor. Specifically, we have tested the effect of cell density and a number of inhibitors or activators of ATPase activity on the amplitude of the oscillations. The...

  7. Tangential Ultrafiltration of Aqueous "Saccharomyces Cerevisiae" Suspensions

    Science.gov (United States)

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

    2008-01-01

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

  8. Construction of novel Saccharomyces cerevisiae strains for bioethanol active dry yeast (ADY production.

    Directory of Open Access Journals (Sweden)

    Daoqiong Zheng

    Full Text Available The application of active dry yeast (ADY in bioethanol production simplifies operation processes and reduces the risk of bacterial contamination. In the present study, we constructed a novel ADY strain with improved stress tolerance and ethanol fermentation performances under stressful conditions. The industrial Saccharomyces cerevisiae strain ZTW1 showed excellent properties and thus subjected to a modified whole-genome shuffling (WGS process to improve its ethanol titer, proliferation capability, and multiple stress tolerance for ADY production. The best-performing mutant, Z3-86, was obtained after three rounds of WGS, producing 4.4% more ethanol and retaining 2.15-fold higher viability than ZTW1 after drying. Proteomics and physiological analyses indicated that the altered expression patterns of genes involved in protein metabolism, plasma membrane composition, trehalose metabolism, and oxidative responses contribute to the trait improvement of Z3-86. This work not only successfully developed a novel S. cerevisiae mutant for application in commercial bioethanol production, but also enriched the current understanding of how WGS improves the complex traits of microbes.

  9. Ypq3p-dependent histidine uptake by the vacuolar membrane vesicles of Saccharomyces cerevisiae.

    Science.gov (United States)

    Manabe, Kunio; Kawano-Kawada, Miyuki; Ikeda, Koichi; Sekito, Takayuki; Kakinuma, Yoshimi

    2016-06-01

    The vacuolar membrane proteins Ypq1p, Ypq2p, and Ypq3p of Saccharomyces cerevisiae are known as the members of the PQ-loop protein family. We found that the ATP-dependent uptake activities of arginine and histidine by the vacuolar membrane vesicles were decreased by ypq2Δ and ypq3Δ mutations, respectively. YPQ1 and AVT1, which are involved in the vacuolar uptake of lysine/arginine and histidine, respectively, were deleted in addition to ypq2Δ and ypq3Δ. The vacuolar membrane vesicles isolated from the resulting quadruple deletion mutant ypq1Δypq2Δypq3Δavt1Δ completely lost the uptake activity of basic amino acids, and that of histidine, but not lysine and arginine, was evidently enhanced by overexpressing YPQ3 in the mutant. These results suggest that Ypq3p is specifically involved in the vacuolar uptake of histidine in S. cerevisiae. The cellular level of Ypq3p-HA(3) was enhanced by depletion of histidine from culture medium, suggesting that it is regulated by the substrate. PMID:26928127

  10. Non-enzymatic roles for the URE2 glutathione S-transferase in the response of Saccharomyces cerevisiae to arsenic.

    Science.gov (United States)

    Todorova, Tatina T; Kujumdzieva, Anna V; Vuilleumier, Stéphane

    2010-11-01

    The response of Saccharomyces cerevisiae to arsenic involves a large ensemble of genes, many of which are associated with glutathione-related metabolism. The role of the glutathione S-transferase (GST) product of the URE2 gene involved in resistance of S. cerevisiae to a broad range of heavy metals was investigated. Glutathione peroxidase activity, previously reported for the Ure2p protein, was unaffected in cell-free extracts of an ure2Δ mutant of S. cerevisiae. Glutathione levels in the ure2Δ mutant were lowered about threefold compared to the isogenic wild-type strain but, as in the wild-type strain, increased 2-2.5-fold upon addition of either arsenate (As(V)) or arsenite (As(III)). However, lack of URE2 specifically caused sensitivity to arsenite but not to arsenate. The protective role of URE2 against arsenite depended solely on the GST-encoding 3'-end portion of the gene. The nitrogen source used for growth was suggested to be an important determinant of arsenite toxicity, in keeping with non-enzymatic roles of the URE2 gene product in GATA-type regulation. PMID:20740275

  11. Morphological mutants of garlic

    Energy Technology Data Exchange (ETDEWEB)

    Choudhary, A.D.; Dnyansagar, V.R. (Nagpur Univ. (India). Dept. of Botany)

    1982-01-01

    Cloves of garlic (Allium sativuum Linn.) were exposed to gamma rays with various doses and different concentrations of ethylmethane sulphonate (EMS), diethyl sulphate (dES) and ethylene imine (EI). In the second and third generations, 16 types of morphological mutants were recorded with varied frequencies. Of all the mutagens used, gamma rays were found to be the most effective in inducing the maximum number of mutations followed EI, EMS and dES in that order.

  12. Morphological mutants of garlic

    International Nuclear Information System (INIS)

    Cloves of garlic (Allium sativuum Linn.) were exposed to gamma rays with various doses and different concentrations of ethylmethane sulphonate (EMS), diethyl sulphate (dES) and ethylene imine (EI). In the second and third generations, 16 types of morphological mutants were recorded with varied frequencies. Of all the mutagens used, gamma rays were found to be the most effective in inducing the maximum number of mutations followed EI, EMS and dES in that order. (author)

  13. Functions of spindle check-point and its relationship to chromosome instability

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    It is generally believed that the equal distribution of genetic materials to two daughter cells during mitosis is the key to cell health and development. During the dynamic process, spindle checkpoint plays a very important role in chromosome movements and final sister chromatid separation. The equal and precise segregation of chromosomes contributes to the genomic stability while aberrant separations result in chromosome instability that causes pathogenesis of certain diseases such as Down's syndrome and cancers. Kinetochore and its regulatory proteins consist of the spindle checkpoint and determine the spatial and temporal orders of chromosome segregation.

  14. Constitutive Cdk2 activity promotes aneuploidy while altering the spindle assembly and tetraploidy checkpoints

    DEFF Research Database (Denmark)

    Jahn, Stephan C; Corsino, Patrick E; Davis, Bradley J;

    2013-01-01

    The cell has many mechanisms for protecting the integrity of its genome. These mechanisms are often weakened or absent in many cancers, leading to high rates of chromosomal instability in tumors. Control of the cell cycle is crucial for the function of these checkpoints, and is frequently lost in...... instability. Expression of these complexes in the MCF10A cell line leads to retinoblastoma protein (Rb) hyperphosphorylation, a subsequent increase in proliferation rate, and increased expression of the spindle assembly checkpoint protein Mad2. This results in a strengthening of the spindle assembly...

  15. Role for Rif1 in the checkpoint response to damaged DNA in Xenopus egg extracts

    OpenAIRE

    Kumar, Sanjay; Yoo, Hae Yong; Kumagai, Akiko; Shevchenko, Anna; Shevchenko, Andrej; Dunphy, William G.

    2012-01-01

    TopBP1 is critical for both DNA replication and checkpoint regulation in vertebrate cells. In this study, we have identified Rif1 as a binding partner of TopBP1 in Xenopus egg extracts. In addition, Rif1 also interacts with both ATM and the Mre11-Rad50-Nbs1 (MRN) complex, which are key regulators of checkpoint responses to double-stranded DNA breaks (DSBs). Depletion of Rif1 from egg extracts compromises the activation of Chk1 in response to DSBs but not stalled replication forks. Removal of ...

  16. TagSmart: analysis and visualization for yeast mutant fitness data measured by tag microarrays

    Directory of Open Access Journals (Sweden)

    Xie Dan

    2007-04-01

    Full Text Available Abstract Background A nearly complete collection of gene-deletion mutants (96% of annotated open reading frames of the yeast Saccharomyces cerevisiae has been systematically constructed. Tag microarrays are widely used to measure the fitness of each mutant in a mutant mixture. The tag array experiments can have a complex experimental design, such as time course measurements and drug treatment with multiple dosages. Results TagSmart is a web application for analysis and visualization of Saccharomyces cerevisiae mutant fitness data measured by tag microarrays. It implements a robust statistical approach to assess the concentration differences among S. cerevisiae mutant strains. It also provides an interactive environment for data analysis and visualization. TagSmart has the following advantages over previously described analysis procedures: 1 it is user-friendly software rather than merely a description of analytical procedure; 2 It can handle complicated experimental designs, such as multiple time points and treatment with multiple dosages; 3 it has higher sensitivity and specificity; 4 It allows users to mask out "bad" tags in the analysis. Two biological tests were performed to illustrate the performance of TagSmart. First, we generated titration mixtures of mutant strains, in which the relative concentration of each strain was controlled. We used tag microarrays to measure the numbers of tag copies in each titration mixture. The data was analyzed with TagSmart and the result showed high precision and recall. Second, TagSmart was applied to a dataset in which heterozygous deletion strain mixture pools were treated with a new drug, Cincreasin. TagSmart identified 53 mutant strains as sensitive to Cincreasin treatment. We individually tested each identified mutant, and found 52 out of the 53 predicted mutants were indeed sensitive to Cincreasin. Conclusion TagSmart is provided "as is" to analyze tag array data produced by Affymetrix and Agilent

  17. Irradiation of mutants of rose

    International Nuclear Information System (INIS)

    Radiation-induced Reddish-orange (R) and Pink (P) flowered mutants of the rose cultivar Montezuma were subjected to a second treatment of gamma radiation. Effects of this treatment were recorded on bud-take, growth, survival, flowering and essential oil content. The P mutant was more radiosensitive than the R mutant. The occurrence of certain early flowering and flower yielding plants in the latter mutant proved the efficiency of this technique for inducing genetic variability in garden roses. (author)

  18. Connexin mutants and cataracts

    Directory of Open Access Journals (Sweden)

    EricCBeyer

    2013-04-01

    Full Text Available The lens is a multicellular, but avascular tissue that must stay transparent to allow normal transmission of light and focusing of it on the retina. Damage to lens cells and/or proteins can cause cataracts, opacities that disrupt these processes. The normal survival of the lens is facilitated by an extensive network of gap junctions formed predominantly of connexin46 and connexin50. Mutations of the genes that encode these connexins (GJA3 and GJA8 have been identified and linked to inheritance of cataracts in human families and mouse lines. In vitro expression studies of several of these mutants have shown that they exhibit abnormalities that may lead to disease. Many of the mutants reduce or modify intercellular communication due to channel alterations (including loss of function or altered gating or due to impaired cellular trafficking which reduces the number of gap junction channels within the plasma membrane. However, the abnormalities detected in studies of other mutants suggest that they cause cataracts through other mechanisms including gain of hemichannel function (leading to cell injury and death and formation of cytoplasmic accumulations (that may act as light scattering particles. These observations and the anticipated results of ongoing studies should elucidate the mechanisms of cataract development due to mutations of lens connexins and abnormalities of other lens proteins. They may also contribute to our understanding of the mechanisms of disease due to connexin mutations in other tissues.

  19. Evolutionary engineering of Saccharomyces cerevisiae for enhanced tolerance to hydrolysates of lignocellulosic biomass.

    Science.gov (United States)

    Almario, María P; Reyes, Luis H; Kao, Katy C

    2013-10-01

    Lignocellulosic biomass has become an important feedstock to mitigate current ethical and economical concerns related to the bio-based production of fuels and chemicals. During the pre-treatment and hydrolysis of the lignocellulosic biomass, a complex mixture of sugars and inhibitors are formed. The inhibitors interfere with microbial growth and product yields. This study uses an adaptive laboratory evolution method called visualizing evolution in real-time (VERT) to uncover the molecular mechanisms associated with tolerance to hydrolysates of lignocellulosic biomass in Saccharomyces cerevisiae. VERT enables a more rational scheme for isolating adaptive mutants for characterization and molecular analyses. Subsequent growth kinetic analyses of the mutants in individual and combinations of common inhibitors present in hydrolysates (acetic acid, furfural, and hydroxymethylfurfural) showed differential levels of resistance to different inhibitors, with enhanced growth rates up to 57%, 12%, 22%, and 24% in hydrolysates, acetic acid, HMF and furfural, respectively. Interestingly, some of the adaptive mutants exhibited reduced fitness in the presence of individual inhibitors, but showed enhanced fitness in the presence of combinations of inhibitors compared to the parental strains. Transcriptomic analysis revealed different mechanisms for resistance to hydrolysates and a potential cross adaptation between oxidative stress and hydrolysates tolerance in several of the mutants. PMID:23613173

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

    Science.gov (United States)

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

    2001-07-01

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

  1. Starvation induced cell death in autophagy-defective yeast mutants is caused by mitochondria dysfunction.

    Directory of Open Access Journals (Sweden)

    Sho W Suzuki

    Full Text Available Autophagy is a highly-conserved cellular degradation and recycling system that is essential for cell survival during nutrient starvation. The loss of viability had been used as an initial screen to identify autophagy-defective (atg mutants of the yeast Saccharomyces cerevisiae, but the mechanism of cell death in these mutants has remained unclear. When cells grown in a rich medium were transferred to a synthetic nitrogen starvation media, secreted metabolites lowered the extracellular pH below 3.0 and autophagy-defective mutants mostly died. We found that buffering of the starvation medium dramatically restored the viability of atg mutants. In response to starvation, wild-type (WT cells were able to upregulate components of the respiratory pathway and ROS (reactive oxygen species scavenging enzymes, but atg mutants lacked this synthetic capacity. Consequently, autophagy-defective mutants accumulated the high level of ROS, leading to deficient respiratory function, resulting in the loss of mitochondria DNA (mtDNA. We also showed that mtDNA deficient cells are subject to cell death under low pH starvation conditions. Taken together, under starvation conditions non-selective autophagy, rather than mitophagy, plays an essential role in preventing ROS accumulation, and thus in maintaining mitochondria function. The failure of response to starvation is the major cause of cell death in atg mutants.

  2. Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez, Pilar [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Barquinero, Joan Francesc [Unitat d' Antropologia Biologica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Duran, Assumpta [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Caballin, Maria Rosa [Unitat d' Antropologia Biologica, Departament de Biologia Animal, Biologia Vegetal i Ecologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain); Ribas, Montserrat [Servei de Radiofisica i Radioproteccio de l' Hospital de la Santa Creu i Sant Pau, 08025 Barcelona (Spain); Barrios, Leonardo, E-mail: Lleonard.Barrios@uab.cat [Unitat de Biologia Cel.lular, Departament de Biologia Cel.lular, Fisiologia i Immunologia, Universitat Autonoma de Barcelona, 08193 Bellaterra (Spain)

    2009-11-02

    Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of {gamma}-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

  3. Differential impact of diverse anticancer chemotherapeutics on the Cdc25A-degradation checkpoint pathway

    International Nuclear Information System (INIS)

    When exposed to DNA-damaging insults such as ionizing radiation (IR) or ultraviolet light (UV), mammalian cells activate checkpoint pathways to halt cell cycle progression or induce cell death. Here we examined the ability of five commonly used anticancer drugs with different mechanisms of action to activate the Chk1/Chk2-Cdc25A-CDK2/cyclin E cell cycle checkpoint pathway, previously shown to be induced by IR or UV. Whereas exposure of human cells to topoisomerase inhibitors camptothecin, etoposide, or adriamycin resulted in rapid (within 1 h) activation of the pathway including degradation of the Cdc25A phosphatase and inhibition of cyclin E/CDK2 kinase activity, taxol failed to activate this checkpoint even after a prolonged treatment. Unexpectedly, although the alkylating agent cisplatin also induced degradation of Cdc25A (albeit delayed, after 8-12 h), cyclin E/CDK2 activity was elevated and DNA synthesis continued, a phenomena that correlated with increased E2F1 protein levels and consequently enhanced expression of cyclin E. These results reveal a differential impact of various classes of anticancer chemotherapeutics on the Cdc25A-degradation pathway, and indicate that the kinetics of checkpoint induction, and the relative balance of key components within the DNA damage response network may dictate whether the treated cells arrest their cell cycle progression

  4. A phospho-proteomic screen identifies substrates of the checkpoint kinase Chk1

    DEFF Research Database (Denmark)

    Blasius, Melanie; Forment, Josep V; Thakkar, Neha;

    2011-01-01

    BACKGROUND: The cell-cycle checkpoint kinase Chk1 is essential in mammalian cells due to its roles in controlling processes such as DNA replication, mitosis and DNA-damage responses. Despite its paramount importance, how Chk1 controls these functions remains unclear, mainly because very few Chk1...

  5. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    Institute of Scientific and Technical Information of China (English)

    Hai Jiang; Jianchun Wu; Chen He; Wending Yang; Honglin Li

    2009-01-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdkl activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chkl and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  6. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

    DEFF Research Database (Denmark)

    McGranahan, Nicholas; Furness, Andrew J S; Rosenthal, Rachel;

    2016-01-01

    As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we...

  7. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor

    Science.gov (United States)

    Ivanova, Tsvetomira; Alves-Rodrigues, Isabel; Gómez-Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A.; Rhind, Nick; Hidalgo, Elena; Ayté, José

    2013-01-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)–dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex. PMID:24006488

  8. The DNA damage and the DNA replication checkpoints converge at the MBF transcription factor.

    Science.gov (United States)

    Ivanova, Tsvetomira; Alves-Rodrigues, Isabel; Gómez-Escoda, Blanca; Dutta, Chaitali; DeCaprio, James A; Rhind, Nick; Hidalgo, Elena; Ayté, José

    2013-11-01

    In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex. PMID:24006488

  9. Synthesis of Fault-Tolerant Embedded Systems with Checkpointing and Replication

    DEFF Research Database (Denmark)

    Izosimov, Viacheslav; Pop, Paul; Eles, Petru; Peng, Zebo

    We present an approach to the synthesis of fault-tolerant hard real-time systems for safety-critical applications. We use checkpointing with rollback recovery and active replication for tolerating transient faults. Processes are statically scheduled and communications are performed using the time...

  10. Cells bearing chromosome aberrations lacking one telomere are selectively blocked at the G2/M checkpoint

    International Nuclear Information System (INIS)

    Cell cycle checkpoints are part of the cellular mechanisms to maintain genomic integrity. After ionizing radiation exposure, the cells can show delay or arrest in their progression through the cell cycle, as well as an activation of the DNA repair machinery in order to reduce the damage. The G2/M checkpoint prevents G2 cells entering mitosis until the DNA damage has been reduced. The present study evaluates which G0 radiation-induced chromosome aberrations are negatively selected in the G2/M checkpoint. For this purpose, peripheral blood samples were irradiated at 1 and 3 Gy of γ-rays, and lymphocytes were cultured for 48 h. Calyculin-A and Colcemid were used to analyze, in the same slide, cells in G2 and M. Chromosome spreads were consecutively analyzed by solid stain, pancentromeric and pantelomeric FISH and mFISH. The results show that the frequency of incomplete chromosome elements, those lacking a telomeric signal at one end, decreases abruptly from G2 to M. This indicates that cells with incomplete chromosome elements can progress from G0 to G2, but at the G2/M checkpoint suffer a strong negative selection.

  11. Importance of immunopharmacogenomics in cancer treatment: Patient selection and monitoring for immune checkpoint antibodies.

    Science.gov (United States)

    Choudhury, Noura; Nakamura, Yusuke

    2016-02-01

    In the last 5 years, immune checkpoint antibodies have become established as anticancer agents for various types of cancer. These antibody drugs, namely cytotoxic T-lymphocyte-associated antigen, programmed death-1, and programmed death ligand-1 antibodies, have revealed relatively high response rates, the ability to induce durable responses, and clinical efficacy in malignancies not previously thought to be susceptible to immune-based strategies. However, because of its unique mechanisms of activating the host immune system against cancer as well as expensive cost, immune checkpoint blockade faces novel challenges in selecting appropriate patient populations, monitoring clinical responses, and predicting immune adverse events. The development of objective criteria for selecting patient populations that are likely to have benefit from these therapies has been vigorously investigated but still remains unclear. In this review, we describe immune checkpoint inhibition-specific challenges with patient selection and monitoring, and focus on approaches to remedy these challenges. We also discuss applications of the emerging field of immunopharmacogenomics for guiding selection and monitoring for anti-immune checkpoint treatment. PMID:26678880

  12. Preserved DNA Damage Checkpoint Pathway Protects against Complications in Long-Standing Type 1 Diabetes

    Energy Technology Data Exchange (ETDEWEB)

    Bhatt, Shweta; Gupta, Manoj; Khamaisi, Mogher; Martinez, Rachael; Gritsenko, Marina A.; Wagner, Bridget; Guye, Patrick; Busskamp, Volker; Shirakawa, Jun; Wu, Gongxiong; Liew, Chong Wee; Clauss, Therese RW; Valdez, Ivan; EL Ouaaman, Abdelfattah; Dirice, Ercument; Takatani, Tomozumi; Keenan, Hillary; Smith, Richard D.; Church, George; Weiss, Ron; Wagers, Amy J.; Qian, Weijun; King, George L.; Kulkami, Rohit N.

    2015-08-04

    Themechanisms underlying the development of complications in type 1 diabetes (T1D) are poorly understood. Disease modeling of induced pluripotent stem cells (iPSCs) from patients with longstanding T1D(disease durationR50 years) with severe (Medalist +C) or absent to mild complications (Medalist *C) revealed impaired growth, reprogramming, and differentiation in Medalist +C. Genomics and proteomics analyses suggested differential regulation of DNA damage checkpoint proteins favoring protection from cellular apoptosis in Medalist *C. In silico analyses showed altered expression patterns of DNA damage checkpoint factors among the Medalist groups to be targets of miR200, whose expression was significantly elevated in Medalist +C serum. Notably, neurons differentiated from Medalist +C iPSCs exhibited enhanced susceptibility to genotoxic stress that worsened upon miR200 overexpression. Furthermore, knockdown of miR200 in Medalist +C fibroblasts and iPSCs rescued checkpoint protein expression and reduced DNA damage.WeproposemiR200-regulated DNA damage checkpoint pathway as a potential therapeutic target for treating complications of diabetes.

  13. The effect of heavy ion on DNA damage checkpoint, and the discovery of its sensitizing compounds

    International Nuclear Information System (INIS)

    In the present study, we demonstrated that a persimmon leaf extract (PLE) promoted cytotoxic effect of cancer cells by chemotherapeutic agents inhibiting the DNA checkpoint activity. Therefore, PLE is a strong possibility of sensitizing agents for heavy ion cancer therapy. Herein, we investigate the mechanism of cellular responses to DNA damage induced by heavy ion radiation with PLE. Human adenocarcinoma A549 cells were pre-incubated with PLE (0, 1, 10, 30 μg/mL) at one hour. After pre-incubation, the cells were irradiated with carbon-ion beams [135C (linear energy transfer (LET) 70 Kev/um)]. The phosphorylation of p53, Chk1 and SMC1 was increased by 2.5 Gy heavy ion exposure. PLE decreased the phosphorylation of p53, Chk1 and SMC1 in cells damaged by heavy ion with PLE dose-dependent manner. G2/M checkpoint was investigated in percentage of mitotic cell. The percentage of mitotic cell decreased in heavy ion beam treatment. Interestingly, heavy ion with PLE treatment was significantly increased. The result indicated that PLE treatment disrupted the G2/M checkpoint activated by heavy ion. These results indicated that DNA damage checkpoint system in heavy ion exposed cells were abrogated by PLE treatment through the inhibition of ataxia telangiectasia mutated (ATM) and/or AT and Rad3 related (ATR)-dependent signaling pathway. (author)

  14. Important role of catalase in the cellular response of the budding yeast Saccharomyces cerevisiae exposed to ionizing radiation.

    Science.gov (United States)

    Nishimoto, Takuto; Furuta, Masakazu; Kataoka, Michihiko; Kishida, Masao

    2015-03-01

    Ionizing radiation indirectly causes oxidative stress in cells via reactive oxygen species (ROS), such as hydroxyl radicals (OH(-)) generated by the radiolysis of water. We investigated how the catalase function was affected by ionizing radiation and analyzed the phenotype of mutants with a disrupted catalase gene in Saccharomyces cerevisiae exposed to radiation. The wild-type yeast strain and isogenic mutants with disrupted catalase genes were exposed to various doses of (60)Co gamma-rays. There was no difference between the wild-type strain and the cta1 disruption mutant following exposure to gamma-ray irradiation. In contrast, there was a significant decrease in the ctt1 disruption mutant, suggesting that this strain exhibited decreased survival on gamma-ray exposure compared with other strains. In all three strains, stationary phase cells were more tolerant to the exposure of gamma-rays than exponential phase cells, whereas the catalase activity in the wild-type strain and cta1 disruption mutant was higher in the stationary phase than in the exponential phase. These data suggest a correlation between catalase activity and survival following gamma-ray exposure. However, this correlation was not clear in the ctt1 disruption mutant, suggesting that other factors are involved in the tolerance to ROS induced by irradiation. PMID:25416226

  15. TOTAL ANTIOXIDANT ACTIVITY OF YEAST SACCHAROMYCES CEREVISIAE

    Directory of Open Access Journals (Sweden)

    Blažena Lavová

    2013-02-01

    Full Text Available Antioxidants are health beneficial compounds that can protect cells and macromolecules (e.g. fats, lipids, proteins and DNA from the damage of reactive oxygen species (ROS. Sacchamomyces cerevisiae are know as organisms with very important antioxidative enzyme systems such as superoxide dismutase or catalase. The total antioxidant activity (mmol Trolox equivalent – TE.g-1 d.w. of Saccharomyces cerevisiae was measured by 2,2´-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid during the yeast cultivation. It was found that the total antioxidant activity was the highest (1.08 mmol TE.g-1 d.w. in the strain Kolín after 32 hours of cultivation and the lowest (0.26 mmol TE.g-1 d.w. in the strain Gyöng after 12 hours of cultivation.

  16. Genome-scale reconstruction of the Saccharomyces cerevisiae metabolic network

    DEFF Research Database (Denmark)

    Förster, Jochen; Famili, I.; Fu, P.; Palsson, B.O.; Nielsen, Jens

    2003-01-01

    The metabolic network in the yeast Saccharomyces cerevisiae was reconstructed using currently available genomic, biochemical, and physiological information. The metabolic reactions were compartmentalized between the cytosol and the mitochondria, and transport steps between the compartments and the...... containing 1175 metabolic reactions and 584 metabolites. The number of gene functions included in the reconstructed network corresponds to similar to16% of all characterized ORFs in S. cerevisiae. Using the reconstructed network, the metabolic capabilities of S. cerevisiae were calculated and compared with...

  17. Phosphate transport and sensing in Saccharomyces cerevisiae.

    OpenAIRE

    Wykoff, D D; O'Shea, E K

    2001-01-01

    Cellular metabolism depends on the appropriate concentration of intracellular inorganic phosphate; however, little is known about how phosphate concentrations are sensed. The similarity of Pho84p, a high-affinity phosphate transporter in Saccharomyces cerevisiae, to the glucose sensors Snf3p and Rgt2p has led to the hypothesis that Pho84p is an inorganic phosphate sensor. Furthermore, pho84Delta strains have defects in phosphate signaling; they constitutively express PHO5, a phosphate starvat...

  18. Viruses and prions of Saccharomyces cerevisiae

    OpenAIRE

    Wickner, Reed B.; Fujimura, Tsutomu; Esteban, Rosa

    2013-01-01

    Saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including dsRNA viruses, ssRNA viruses, and prions. Studies of the mechanisms of virus and prion replication, virus structure, and structure of the amyloid filaments that are the basis of yeast prions have been at the forefront of such studies in these classes of infectious entities. Yeast has been particularly useful in defining the interactions of the infectious elements with cellular compone...

  19. Identification of coated vesicles in Saccharomyces cerevisiae

    OpenAIRE

    1984-01-01

    Clathrin-coated vesicles were found in yeast, Saccharomyces cerevisiae, and enriched from spheroplasts by a rapid procedure utilizing gel filtration on Sephacryl S-1000. The coated vesicles (62-nm diam) were visualized by negative stain electron microscopy and clathrin triskelions were observed by rotary shadowing. The contour length of a triskelion leg was 490 nm. Coated vesicle fractions contain a prominent band with molecular weight of approximately 185,000 when analyzed by SDS PAGE. The p...

  20. Cell Wall Assembly in Saccharomyces cerevisiae

    OpenAIRE

    Lesage, Guillaume; Bussey, Howard

    2006-01-01

    An extracellular matrix composed of a layered meshwork of β-glucans, chitin, and mannoproteins encapsulates cells of the yeast Saccharomyces cerevisiae. This organelle determines cellular morphology and plays a critical role in maintaining cell integrity during cell growth and division, under stress conditions, upon cell fusion in mating, and in the durable ascospore cell wall. Here we assess recent progress in understanding the molecular biology and biochemistry of cell wall synthesis and it...

  1. Impairment of cobalt-induced riboflavin biosynthesis in a Debaryomyces hansenii mutant.

    Science.gov (United States)

    Seda-Miró, Jasmine M; Arroyo-González, Nancy; Pérez-Matos, Ana; Govind, Nadathur S

    2007-11-01

    Flavinogenic yeasts such as Debaryomyces hansenii overproduce riboflavin (RF) in the presence of heavy metals. Growth and RF production were compared between wild-type D. hansenii and a RF production-impaired metal-tolerant ura3 mutant in the presence of sublethal cobalt(II) concentrations. Debaryomyces hansenii (wild type) exhibits an extended lag phase with an increase in RF synthesis. Supplementation of exogenous uracil shortened the lag phase at the highest concentration of cobalt(II) used, suggesting that uracil has a possible role in metal acclimation. The D. hansenii ura3 mutant isolated by chemical mutagenesis exhibited a higher level of metal tolerance, no extended lag phase, and no marked increase in RF synthesis. Transformation of the mutant with the URA3 gene isolated from Saccharyomyces cerevisiae or D. hansenii did not restore wild-type characteristics, suggesting a second mutation that impairs RF oversynthesis. Our results demonstrate that growth, metal sensitivity, and RF biosynthesis are linked. PMID:18026221

  2. CaMac1, a Candida albicans Copper Ion-sensing Transcription Factor, Pro- motes Filamentous and Invasive Growth in Saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    Guang-Hua HUANG; Xin-Yi NIE; Jiang-Ye CHEN

    2006-01-01

    Molecular mechanisms of morphogenesis share many common components between Candida albicans and Saccharomyces cerevisiae. The Kssl-associated MAPK cascade and the cAMP/PKA pathway are two important signal transduction pathways that control morphogenesis in S. cerevisiae. A C. albicans copper ion-sensing transcription factor gene, CaMAC1, was cloned from C. albicans SC5314. Ectopic expression of CaMAC1 in S. cerevisiae promoted filamentous and invasive growth. In diploid cells, CaMacl could suppress the filamentous growth defect of mutants in the Kss 1-associated MAPK pathway and the cAMP/PKA pathway. In haploid strains, ectopic expression of CaMAC1 suppressed the invasive growth defect of mutants in the MAPK pathway (ste7, stel2 and tecl), but failed to suppress the invasive growth defect of thefio8 mutant. Our results suggest that the activation of CaMacl is independent of the MAPK and cAMP/PKA pathways in filament formation, but requires Flo8 factor for invasive growth. In the media containing a high concentration of CuSO4, the yeast filamentous and invasive growth was blocked. The activating effect of CaMacl is inhibited by copper ions.

  3. The meiotic recombination checkpoint suppresses NHK-1 kinase to prevent reorganisation of the oocyte nucleus in Drosophila.

    Directory of Open Access Journals (Sweden)

    Oscar M Lancaster

    2010-10-01

    Full Text Available The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1 is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired.

  4. Pronounced and Extensive Microtubule Defects in a Saccharomyces cerevisiae DIS3 Mutant

    OpenAIRE

    Smith, Sarah B.; Kiss, Daniel L.; Turk, Edward; Tartakoff, Alan M.; Andrulis, Erik D

    2011-01-01

    Subunits of the RNA processing exosome assemble into structurally distinct protein complexes that function in disparate cellular compartments and RNA metabolic pathways. Here, in a genetic, cell biological, and transcriptomic analysis, we examine the role of Dis3 – an essential polypeptide with endo- and 3’ to 5’ exo-ribonuclease activity – in cell cycle progression. We present several lines of evidence that perturbation of DIS3 affects microtubule (MT) localization and structure in Saccharom...

  5. Phenotypic characterization of glucose repression mutants of Saccharomyce cerevisiae usinge experiments with C-13-labelled glucose

    DEFF Research Database (Denmark)

    Vijayendran, Raghevendran; Gombert, A.K.; Christensen, B.;

    2004-01-01

    In the field of metabolic engineering and functional genomics, methods for analysis of metabolic fluxes in the cell are attractive as they give an overview of the phenotypic response of the cells at the level of the active metabolic network. This is unlike several other high-throughput experiment...

  6. Genetic analysis of the spindle checkpoint genes san-1, mdf-2, bub-3 and the CENP-F homologues hcp-1 and hcp-2 in Caenorhabditis elegans

    Directory of Open Access Journals (Sweden)

    Moore Landon L

    2008-02-01

    Full Text Available Abstract Background The spindle checkpoint delays the onset of anaphase until all sister chromatids are aligned properly at the metaphase plate. To investigate the role san-1, the MAD3 homologue, has in Caenorhabditis elegans embryos we used RNA interference (RNAi to identify genes synthetic lethal with the viable san-1(ok1580 deletion mutant. Results The san-1(ok1580 animal has low penetrating phenotypes including an increased incidence of males, larvae arrest, slow growth, protruding vulva, and defects in vulva morphogenesis. We found that the viability of san-1(ok1580 embryos is significantly reduced when HCP-1 (CENP-F homologue, MDF-1 (MAD-1 homologue, MDF-2 (MAD-2 homologue or BUB-3 (predicted BUB-3 homologue are reduced by RNAi. Interestingly, the viability of san-1(ok1580 embryos is not significantly reduced when the paralog of HCP-1, HCP-2, is reduced. The phenotype of san-1(ok1580;hcp-1(RNAi embryos includes embryonic and larval lethality, abnormal organ development, and an increase in abnormal chromosome segregation (aberrant mitotic nuclei, anaphase bridging. Several of the san-1(ok1580;hcp-1(RNAi animals displayed abnormal kinetochore (detected by MPM-2 and microtubule structure. The survival of mdf-2(RNAi;hcp-1(RNAi embryos but not bub-3(RNAi;hcp-1(RNAi embryos was also compromised. Finally, we found that san-1(ok1580 and bub-3(RNAi, but not hcp-1(RNAi embryos, were sensitive to anoxia, suggesting that like SAN-1, BUB-3 has a functional role as a spindle checkpoint protein. Conclusion Together, these data suggest that in the C. elegans embryo, HCP-1 interacts with a subset of the spindle checkpoint pathway. Furthermore, the fact that san-1(ok1580;hcp-1(RNAi animals had a severe viability defect whereas in the san-1(ok1580;hcp-2(RNAi and san-1(ok1580;hcp-2(ok1757 animals the viability defect was not as severe suggesting that hcp-1 and hcp-2 are not completely redundant.

  7. The Saccharomyces cerevisiae RAD9, RAD17, RAD24 and MEC3 genes are required for tolerating irreparable, ultraviolet-induced DNA damage

    International Nuclear Information System (INIS)

    In wild-type Saccharomyces cerevisiae, a checkpoint slows the rate of progression of an ongoing S phase in response to exposure to a DNA-alkylating agent. Mutations that eliminate S phase regulation also confer sensitivity to alkylating agents, leading us to suggest that, by regulating the S phase rate, cells are either better able to repair or better able to replicate damaged DNA. In this study, we determine the effects of mutations that impair S phase regulation on the ability of excision repair-defective cells to replicate irreparably UV-damaged DNA. We assay survival after UV irradiation, as well as the genetic consequences of replicating a damaged template, namely mutation and sister chromatid exchange induction. We find that RAD9, RAD17, RAD24, and MEC3 are required for UV-induced (although not spontaneous) mutagenesis, and that RAD9 and RAD17 (but not REV3, RAD24, and MEC3) are required for maximal induction of replication-dependent sister chromatid exchange. Therefore, checkpoint genes not only control cell cycle progression in response to damage, but also play a role in accommodating DNA damage during replication. (author)

  8. A yeast tRNA mutant that causes pseudohyphal growth exhibits reduced rates of CAG codon translation

    OpenAIRE

    Kemp, Alain J; Betney, Russell; Ciandrini, Luca; Schwenger, Alexandra C M; Romano, M. Carmen; Stansfield, Ian

    2012-01-01

    In Saccharomyces cerevisiae, the SUP70 gene encodes the CAG-decoding tRNAGln CUG. A mutant allele, sup70-65 , induces pseudohyphal growth on rich medium, an inappropriate nitrogen starvation response. This mutant tRNA is also a UAG nonsense suppressor via first base wobble. To investigate the basis of the pseudohyphal phenotype, 10 novel sup70 UAG suppressor alleles were identified, defining positions in the tRNAGln CUG anticodon stem that restrict first base wobble. However, none conferred p...

  9. Time dependent physiological characterization of yeast oxidative stress response and growth modulation of protein kinase/phosphatase mutants

    DEFF Research Database (Denmark)

    Altintas, Ali; Workman, Christopher

    mutants were selected for their known activities in various stress response pathways, including oxidative stress, and were investigated for their response to oxidative stress. Hydrogen peroxide was us ed as the oxidizing agent at a number of different concentrations ranging from mild to moderate stress (0......The objective of the project was to investigate the time-dependent batch growth effects of oxidative environmental conditions on protein ki nase (PK) and phosphatase (PP) deletion mutants and relevant wild type strains of Saccharomyces cerevisiae . To achieve this goal, 44 different PK and PP...

  10. Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants

    International Nuclear Information System (INIS)

    A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms

  11. The fate of linear DNA in Saccharomyces cerevisiae and Candida glabrata: the role of homologous and non-homologous end joining.

    Directory of Open Access Journals (Sweden)

    Mary W Corrigan

    Full Text Available In vivo assembly of plasmids has become an increasingly used process, as high throughput studies in molecular biology seek to examine gene function. In this study, we investigated the plasmid construction technique called gap repair cloning (GRC in two closely related species of yeast - Saccharomyces cerevisiae and Candida glabrata. GRC utilizes homologous recombination (HR activity to join a linear vector and a linear piece of DNA that contains base pair homology. We demonstrate that a minimum of 20 bp of homology on each side of the linear DNA is required for GRC to occur with at least 10% efficiency. Between the two species, we determine that S. cerevisiae is slightly more efficient at performing GRC. GRC is less efficient in rad52 deletion mutants, which are defective in HR in both species. In dnl4 deletion mutants, which perform less non-homologous end joining (NHEJ, the frequency of GRC increases in C. glabrata, whereas GRC frequency only minimally increases in S. cerevisiae, suggesting that NHEJ is more prevalent in C. glabrata. Our studies allow for a model of the fate of linear DNA when transformed into yeast cells. This model is not the same for both species. Most significantly, during GRC, C. glabrata performs NHEJ activity at a detectable rate (>5%, while S. cerevisiae does not. Our model suggests that S. cerevisiae is more efficient at HR because NHEJ is less prevalent than in C. glabrata. This work demonstrates the determinants for GRC and that while C. glabrata has a lower efficiency of GRC, this species still provides a viable option for GRC.

  12. Optimization of feeding strategy for the ergosterol production by yeasts Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Mojmir Rychtera

    2010-08-01

    Full Text Available Objective of this study was to optimize ergosterol production by yeast strain Saccharomyces cerevisiae with the use of computer controlled feeding of cultivation medium. Baker´s yeasts strain of Saccharomyces cerevisiae originally modified and selected as mutant D7 was further applied in an industrial scale and also in this investigation. Composition of cultivation medium was optimized with the use of a modified Rosenbrock´s method with regard to following components: glucose, yeast extract, ammonium sulphate, potassium dihydrogen phosphate, magnesium sulphate and calcium chloride. Cultivation of yeast culture was performed in 7 L laboratory bioreactor with a working volume of 5 L equipped with a control unit and linked to a computer, with dissolved oxygen tension measurement, oxygen and carbon dioxide analyzers. BIOGENES prototype software was created from the commercial control system Genesis for Windows 3.0 (GFW, from Iconics and CLIPS 6.04 for the PC-Windows platform. From various factors affecting sterol biosynthesis a specific growth rate was chosen. Feed rate was controlled according to mathematical model. In this case it dealt with a design of optimal profile of specific growth rate with consequent calculation of carbon dioxide profile. Sterol concentration in the dry biomass increased from 1.0 % up to 3 %. Key words: Saccharomyces cerevisiae yeasts, ergosterol, fed-batch cultivation control, effect of the specific growth rate. Resumen: El objetivo de este estudio fue optimizar la producción de ergosterol por una cepa de levadura Saccharomyces cerevisiae, controlando la alimentación de medio de cultivo por computadora. La cepa de levadura panadera Saccharomyces cerevisiae originalmente modificada y seleccionada como mutante D7 fue posteriormente utilizada a escala industrial y también para esta investigación. La composición del medio de cultivo fue optimizada usando el método modificado de Rosenbrock respecto a los siguientes

  13. Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Christopher L Brett

    Full Text Available Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pH(v in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pH(v (5.27±0.13 was resistant to acid stress (5.28±0.14 but shifted significantly in response to alkali stress (5.83±0.13. Of 107 mutants that displayed aberrant pH(v under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pH(v dysregulation in a neo1(ts mutant restored viability whereas cholesterol accumulation in human NPC1(-/- fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation.

  14. DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization

    DEFF Research Database (Denmark)

    Reinhardt, H Christian; Hasskamp, Pia; Schmedding, Ingolf;

    2010-01-01

    Following genotoxic stress, cells activate a complex kinase-based signaling network to arrest the cell cycle and initiate DNA repair. p53-defective tumor cells rewire their checkpoint response and become dependent on the p38/MK2 pathway for survival after DNA damage, despite a functional ATR-Chk1...... pathway. We used functional genetics to dissect the contributions of Chk1 and MK2 to checkpoint control. We show that nuclear Chk1 activity is essential to establish a G(2)/M checkpoint, while cytoplasmic MK2 activity is critical for prolonged checkpoint maintenance through a process of...... posttranscriptional mRNA stabilization. Following DNA damage, the p38/MK2 complex relocalizes from nucleus to cytoplasm where MK2 phosphorylates hnRNPA0, to stabilize Gadd45α mRNA, while p38 phosphorylates and releases the translational inhibitor TIAR. In addition, MK2 phosphorylates PARN, blocking Gadd45α m...

  15. Investigation of autonomous cell cycle oscillation in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hansen, Morten Skov

    2007-01-01

    Autonome Oscillationer i kontinuert kultivering af Saccharomyces cerevisiae Udgangspunktet for dette Ph.d. projekt var at søge at forstå, hvad der gør det muligt at opnå multiple statiske tilstande ved kontinuert kultivering af Saccharomyces cerevisiae med glukose som begrænsende substrat...

  16. Kluyveromyces lactis maintains Saccharomyces cerevisiae intron-encoded splicing signals.

    OpenAIRE

    Deshler, J O; Larson, G P; Rossi, J J

    1989-01-01

    The actin (ACT) gene from the budding yeast Kluyveromyces lactis was cloned, and the nucleotide sequence was determined. The gene had a single intron 778 nucleotides in length which possessed the highly conserved splicing signals found in Saccharomyces cerevisiae introns. We demonstrated splicing of heterologous ACT transcripts in both K. lactis and S. cerevisiae.

  17. Nucleosome alterations caused by mutations at modifiable histone residues in Saccharomyces cerevisiae

    Science.gov (United States)

    Liu, Hongde; Wang, Pingyan; Liu, Lingjie; Min, Zhu; Luo, Kun; Wan, Yakun

    2015-01-01

    Nucleosome organization exhibits dynamic properties depending on the cell state and environment. Histone proteins, fundamental components of nucleosomes, are subject to chemical modifications on particular residues. We examined the effect of substituting modifiable residues of four core histones with the non-modifiable residue alanine on nucleosome dynamics. We mapped the genome-wide nucleosomes in 22 histone mutants of Saccharomyces cerevisiae and compared the nucleosome alterations relative to the wild-type strain. Our results indicated that different types of histone mutation resulted in different phenotypes and a distinct reorganization of nucleosomes. Nucleosome occupancy was altered at telomeres, but not at centromeres. The first nucleosomes upstream (−1) and downstream (+1) of the transcription start site (TSS) were more dynamic than other nucleosomes. Mutations in histones affected the nucleosome array downstream of the TSS. Highly expressed genes, such as ribosome genes and genes involved in glycolysis, showed increased nucleosome occupancy in many types of histone mutant. In particular, the H3K56A mutant exhibited a high percentage of dynamic genomic regions, decreased nucleosome occupancy at telomeres, increased occupancy at the +1 and −1 nucleosomes, and a slow growth phenotype under stress conditions. Our findings provide insight into the influence of histone mutations on nucleosome dynamics. PMID:26498326

  18. Biogenesis of RNA Polymerases II and III Requires the Conserved GPN Small GTPases in Saccharomyces cerevisiae

    Science.gov (United States)

    Minaker, Sean W.; Filiatrault, Megan C.; Ben-Aroya, Shay; Hieter, Philip; Stirling, Peter C.

    2013-01-01

    The GPN proteins are a poorly characterized and deeply evolutionarily conserved family of three paralogous small GTPases, Gpn1, 2, and 3. The founding member, GPN1/NPA3/XAB1, is proposed to function in nuclear import of RNA polymerase II along with a recently described protein called Iwr1. Here we show that the previously uncharacterized protein Gpn2 binds both Gpn3 and Npa3/Gpn1 and that temperature-sensitive alleles of Saccharomyces cerevisiae GPN2 and GPN3 exhibit genetic interactions with RNA polymerase II mutants, hypersensitivity to transcription inhibition, and defects in RNA polymerase II nuclear localization. Importantly, we identify previously unrecognized RNA polymerase III localization defects in GPN2, GPN3, and IWR1 mutant backgrounds but find no localization defects of unrelated nuclear proteins or of RNA polymerase I. Previously, it was unclear whether the GPN proteins and Iwr1 had overlapping function in RNA polymerase II assembly or import. In this study, we show that the nuclear import defect of iwr1Δ, but not the GPN2 or GPN3 mutant defects, is partially suppressed by fusion of a nuclear localization signal to the RNA polymerase II subunit Rpb3. These data, combined with strong genetic interactions between GPN2 and IWR1, suggest that the GPN proteins function upstream of Iwr1 in RNA polymerase II and III biogenesis. We propose that the three GPN proteins execute a common, and likely essential, function in RNA polymerase assembly and transport. PMID:23267056

  19. The neglected nano-specific toxicity of ZnO nanoparticles in the yeast Saccharomyces cerevisiae

    Science.gov (United States)

    Zhang, Weicheng; Bao, Shaopan; Fang, Tao

    2016-04-01

    Nanoparticles (NPs) with unique physicochemical properties induce nano-specific (excess) toxicity in organisms compared with their bulk counterparts. Evaluation and consideration of nano-specific toxicity are meaningful for the safe design and environmental risk assessment of NPs. However, ZnO NPs have been reported to lack excess toxicity for diverse organisms. In the present study, the nano-specific toxicity of ZnO NPs was evaluated in the yeast Saccharomyces cerevisiae. Nano-specific toxicity of ZnO NPs was not observed in the wild type yeast. However, the ZnO NPs induced very similar nano-specific toxicities in the three mutants with comparable log Te (particle) values (0.64 vs 0.65 vs 0.62), suggesting that the mutants were more sensitive and specific for the NPs’ nano-specific toxicity. The toxic effects in the yeast were slightly attributable to dissolved zinc ions from the ZnO (nano or bulk) particles. Oxidative damage and mechanical damage contributed to the toxic effect of the ZnO particles. The mechanism of mechanical damage is proposed to be an inherent characteristic underlying the nano-specific toxicity in the mutants. The log Te (particle) was a useful parameter for evaluation of NPs nano-specific toxicity, whereas log Te (ion) efficiently determined the NPs toxicity associated with released ions.

  20. Production of 2,3-butanediol in Saccharomyces cerevisiae by in silico aided metabolic engineering

    Directory of Open Access Journals (Sweden)

    Ng Chiam Yu

    2012-05-01

    Full Text Available Abstract Background 2,3-Butanediol is a chemical compound of increasing interest due to its wide applications. It can be synthesized via mixed acid fermentation of pathogenic bacteria such as Enterobacter aerogenes and Klebsiella oxytoca. The non-pathogenic Saccharomyces cerevisiae possesses three different 2,3-butanediol biosynthetic pathways, but produces minute amount of 2,3-butanediol. Hence, we attempted to engineer S. cerevisiae strain to enhance 2,3-butanediol production. Results We first identified gene deletion strategy by performing in silico genome-scale metabolic analysis. Based on the best in silico strategy, in which disruption of alcohol dehydrogenase (ADH pathway is required, we then constructed gene deletion mutant strains and performed batch cultivation of the strains. Deletion of three ADH genes, ADH1, ADH3 and ADH5, increased 2,3-butanediol production by 55-fold under microaerobic condition. However, overproduction of glycerol was observed in this triple deletion strain. Additional rational design to reduce glycerol production by GPD2 deletion altered the carbon fluxes back to ethanol and significantly reduced 2,3-butanediol production. Deletion of ALD6 reduced acetate production in strains lacking major ADH isozymes, but it did not favor 2,3-butanediol production. Finally, we introduced 2,3-butanediol biosynthetic pathway from Bacillus subtilis and E. aerogenes to the engineered strain and successfully increased titer and yield. Highest 2,3-butanediol titer (2.29 g·l-1 and yield (0.113 g·g-1 were achieved by Δadh1 Δadh3 Δadh5 strain under anaerobic condition. Conclusions With the aid of in silico metabolic engineering, we have successfully designed and constructed S. cerevisiae strains with improved 2,3-butanediol production.

  1. Mechanisms of DNA repair, recombination and mutagenesis in Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Full text. 1. It was confirmed that from the six DNA polymerases discovered in yeast cells, only DNA polymerases δ, ε and ζ are engaged in dark repair of lesions caused by UV-light and MMS. DNA polymerase δ is involved in the repair of both types of lesions, while DNA polymerase ε and ζ only in lesions caused by UV and MMS, respectively. Other polymerases are not involved or play only a minor role in repair. The results obtained are being prepared for publication. 2. Studies on the involvement of the three replicative DNA polymerases in mitotic gene conversion induced by mono- and bifunctional psoralens (and also by UV- light or MMS) revealed that DNA polymerases α and δ are the main polymerases responsible for induced intragenic conversion. DNA polymerase ε seems to play minor role in this process. It is possible that DNA polymerase α may also be involved in DNA repair synthesis but only in cases when the opening of new replication forks is necessary for repair. 3. Studies on the influence of mutations in the replicative and nonreplicative DNA polymerases on adaptive mutations in the cells of Saccharomyces cerevisiae were continued. We found that thermosensitive mutation in the POL2 gene encoding DNA polymerase ε increased the frequency of adaptive mutation in a similar manner as found earlier for DNA polymerase δ. A similar effect was observed also in strains with deletions in the MSH3 gene responsible for mismatch repair. Mutations in other DNA polymerases, including the essential DNA polymerase α and the inessential DNA polymerases β and ζ revealed no effect on this process. Analysis of DNA sequences in the revertants showed that in all cases the obtained reversions resulted from a single nucleotide deletion most often in sequences having short homopolymer tracts. The results obtained suggest that errors arising during DNA elongation and their persistence in mutants deficient in mismatch repair activity seem to be the source of the adaptive

  2. Ghost Process: a Sound Basis to Implement Process Duplication, Migration and Checkpoint-Restart in Linux Clusters

    OpenAIRE

    Vallée, Geoffroy; Lottiaux, Renaud; Margery, David; Morin, Christine; Berthou, Jean-Yves

    2005-01-01

    Process management mechanisms (process duplication, migration and checkpoint-restart) are very useful for high performance and high availability in clustering systems. The single system image approach aims at providing a global process management service with mechanisms for process checkpoint, process migration and process duplication. In this context, a common mechanism for process virtualization is highly desirable but traditional operating systems do not provide such a mechanism. This pape...

  3. DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age

    OpenAIRE

    Marangos, P; Stevense, M.; Niaka, K.; Lagoudaki, M.; Nabti, I.; Jessberger, R.; Carroll, J.

    2015-01-01

    In mammalian oocytes DNA damage can cause chromosomal abnormalities that potentially lead to infertility and developmental disorders. However, there is little known about the response of oocytes to DNA damage. Here we find that oocytes with DNA damage arrest at metaphase of the first meiosis (MI). The MI arrest is induced by the spindle assembly checkpoint (SAC) because inhibiting the SAC overrides the DNA damage-induced MI arrest. Furthermore, this MI checkpoint is compromised in oocytes fro...

  4. Telomerase expression is sufficient for chromosomal integrity in cells lacking p53 dependent G1 checkpoint function

    Directory of Open Access Journals (Sweden)

    Simpson Dennis

    2005-01-01

    Full Text Available Abstract Background Secondary cultures of human fibroblasts display a finite lifespan ending at senescence. Loss of p53 function by mutation or viral oncogene expression bypasses senescence, allowing cell division to continue for an additional 10 – 20 doublings. During this time chromosomal aberrations seen in mitotic cells increase while DNA damage and decatenation checkpoint functions in G2 cells decrease. Methods To explore this complex interplay between chromosomal instability and checkpoint dysfunction, human fibroblast lines were derived that expressed HPV16E6 oncoprotein or dominant-negative alleles of p53 (A143V and H179Q with or without the catalytic subunit of telomerase. Results Cells with normal p53 function displayed 86 – 93% G1 arrest after exposure to 1.5 Gy ionizing radiation (IR. Expression of HPV16E6 or p53-H179Q severely attenuated G1 checkpoint function (3 – 20% arrest while p53-A143V expression induced intermediate attenuation (55 – 57% arrest irrespective of telomerase expression. All cell lines, regardless of telomerase expression or p53 status, exhibited a normal DNA damage G2 checkpoint response following exposure to 1.5 Gy IR prior to the senescence checkpoint. As telomerase-negative cells bypassed senescence, the frequencies of chromosomal aberrations increased generally congruent with attenuation of G2 checkpoint function. Telomerase expression allowed cells with defective p53 function to grow >175 doublings without chromosomal aberrations or attenuation of G2 checkpoint function. Conclusion Thus, chromosomal instability in cells with defective p53 function appears to depend upon telomere erosion not loss of the DNA damage induced G1 checkpoint.

  5. Intestinal microbiome analyses identify melanoma patients at risk for checkpoint-blockade-induced colitis.

    Science.gov (United States)

    Dubin, Krista; Callahan, Margaret K; Ren, Boyu; Khanin, Raya; Viale, Agnes; Ling, Lilan; No, Daniel; Gobourne, Asia; Littmann, Eric; Huttenhower, Curtis; Pamer, Eric G; Wolchok, Jedd D

    2016-01-01

    The composition of the intestinal microbiota influences the development of inflammatory disorders. However, associating inflammatory diseases with specific microbial members of the microbiota is challenging, because clinically detectable inflammation and its treatment can alter the microbiota's composition. Immunologic checkpoint blockade with ipilimumab, a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) signalling, is associated with new-onset, immune-mediated colitis. Here we conduct a prospective study of patients with metastatic melanoma undergoing ipilimumab treatment and correlate the pre-inflammation faecal microbiota and microbiome composition with subsequent colitis development. We demonstrate that increased representation of bacteria belonging to the Bacteroidetes phylum is correlated with resistance to the development of checkpoint-blockade-induced colitis. Furthermore, a paucity of genetic pathways involved in polyamine transport and B vitamin biosynthesis is associated with an increased risk of colitis. Identification of these biomarkers may enable interventions to reduce the risk of inflammatory complications following cancer immunotherapy. PMID:26837003

  6. Social and ethical checkpoints for bottom-up synthetic biology, or protocells.

    Science.gov (United States)

    Bedau, Mark A; Parke, Emily C; Tangen, Uwe; Hantsche-Tangen, Brigitte

    2009-12-01

    An alternative to creating novel organisms through the traditional "top-down" approach to synthetic biology involves creating them from the "bottom up" by assembling them from non-living components; the products of this approach are called "protocells." In this paper we describe how bottom-up and top-down synthetic biology differ, review the current state of protocell research and development, and examine the unique ethical, social, and regulatory issues raised by bottom-up synthetic biology. Protocells have not yet been developed, but many expect this to happen within the next five to ten years. Accordingly, we identify six key checkpoints in protocell development at which particular attention should be given to specific ethical, social and regulatory issues concerning bottom-up synthetic biology, and make ten recommendations for responsible protocell science that are tied to the achievement of these checkpoints. PMID:19816801

  7. An Overview of the Spindle Assembly Checkpoint Status in Oral Cancer

    Science.gov (United States)

    Teixeira, José Henrique; Silva, Patrícia Manuela; Reis, Rita Margarida; Moura, Inês Moranguinho; Marques, Sandra; Fonseca, Joana; Monteiro, Luís Silva; Bousbaa, Hassan

    2014-01-01

    Abnormal chromosome number, or aneuploidy, is a common feature of human solid tumors, including oral cancer. Deregulated spindle assembly checkpoint (SAC) is thought as one of the mechanisms that drive aneuploidy. In normal cells, SAC prevents anaphase onset until all chromosomes are correctly aligned at the metaphase plate thereby ensuring genomic stability. Significantly, the activity of this checkpoint is compromised in many cancers. While mutations are rather rare, many tumors show altered expression levels of SAC components. Genomic alterations such as aneuploidy indicate a high risk of oral cancer and cancer-related mortality, and the molecular basis of these alterations is largely unknown. Yet, our knowledge on the status of SAC components in oral cancer remains sparse. In this review, we address the state of our knowledge regarding the SAC defects and the underlying molecular mechanisms in oral cancer, and discuss their therapeutic relevance, focusing our analysis on the core components of SAC and its target Cdc20. PMID:24995269

  8. Cloud object store for checkpoints of high performance computing applications using decoupling middleware

    Energy Technology Data Exchange (ETDEWEB)

    Bent, John M.; Faibish, Sorin; Grider, Gary

    2016-04-19

    Cloud object storage is enabled for checkpoints of high performance computing applications using a middleware process. A plurality of files, such as checkpoint files, generated by a plurality of processes in a parallel computing system are stored by obtaining said plurality of files from said parallel computing system; converting said plurality of files to objects using a log structured file system middleware process; and providing said objects for storage in a cloud object storage system. The plurality of processes may run, for example, on a plurality of compute nodes. The log structured file system middleware process may be embodied, for example, as a Parallel Log-Structured File System (PLFS). The log structured file system middleware process optionally executes on a burst buffer node.

  9. Metabolic alterations during ascosporogenesis of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Sporulation of S. cerevisiae has been shown to alter the profiles of enzymes involved in gluconeogenesis and glycolysis. The enhancement in the levels of total cellular carbohydrates could be correlated with the enhancement in fructose 1,6-diphosphatase and trehalose-phosphate synthetase. The latter activity could account for the 15-fold increase in trehalose levels in sporulating cells. Glucose-6-phosphatase, pyruvate kinase and phosphofructokinase showed continuous decline during ascosporogenesis. The relative incorporation of radioactivity from possible precursors of gluconeogenesis indicated that acetate-2-14C alone could contribute to carbohydrate synthesis. (author)

  10. Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint.

    Directory of Open Access Journals (Sweden)

    Sagar P Mahale

    Full Text Available The multi-functional molecular motor cytoplasmic dynein performs diverse essential roles during mitosis. The mechanistic importance of the dynein Light Intermediate Chain homologs, LIC1 and LIC2 is unappreciated, especially in the context of mitosis. LIC1 and LIC2 are believed to exist in distinct cytoplasmic dynein complexes as obligate subunits. LIC1 had earlier been reported to be required for metaphase to anaphase progression by inactivating the kinetochore-microtubule attachment-sensing arm of the spindle assembly checkpoint (SAC. However, the functional importance of LIC2 during mitosis remains elusive. Here we report prominent novel roles for the LIC2 subunit of cytoplasmic dynein in regulating the spindle assembly checkpoint. LIC2 depletion in mammalian cells led to prolonged metaphase arrest in the presence of an active SAC and also to stretched kinetochores, thus implicating it in SAC inactivation. Quantitative fluorescence microscopy of SAC components revealed accumulation of both attachment- and tension-sensing checkpoint proteins at metaphase kinetochores upon LIC2 depletion. These observations support a stronger and more diverse role in checkpoint inactivation for LIC2 in comparison to its close homolog LIC1. Our study uncovers a novel functional hierarchy during mitotic checkpoint inactivation between the closely related but homologous LIC subunits of cytoplasmic dynein. These subtle functional distinctions between dynein subpopulations could be exploited to study specific aspects of the spindle assembly checkpoint, which is a key mediator of fidelity in eukaryotic cell division.

  11. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    International Nuclear Information System (INIS)

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint

  12. Differential activation of intra-S-phase checkpoint in response to tripchlorolide and its effects on DNA replication

    Institute of Scientific and Technical Information of China (English)

    Yan REN; Jia Rui WU

    2004-01-01

    DNA replication is tightly regulated during the S phase of the cell cycle, and the activation of the intra-S-phase checkpoint due to DNA damage usually results in arrest of DNA synthesis. However, the molecular details about the correlation between the checkpoint and regulation of DNA replication are still unclear. To investigate the connections between DNA replication and DNA damage checkpoint, a DNA-damage reagent, tripchlorolide, was applied to CHO (Chinese ovary hamster) cells at early- or middle-stages of the S phase. The early-S-phase treatment with TC significantly delayed the progression of the S phase and caused the phosphorylation of the Chk1 checkpoint protein, whereas the middle-S-phase treatment only slightly slowed down the progression of the S phase. Furthermore, the analysis of DNA replication patterns revealed that replication pattern Ⅱ was greatly prolonged in the cells treated with the drug during the early-S phase, whereas the late-replication patterns of these cells were hardly detected, suggesting that the activation of the intra-S-phase checkpoint inhibits the late-origin firing of DNA replication. We conclude that cells at different stages of the S phase are differentially sensitive to the DNA-damage reagent, and the activation of the intra-Sphase checkpoint blocks the DNA replication progression in the late stage of S phase.

  13. Dynein Light Intermediate Chain 2 Facilitates the Metaphase to Anaphase Transition by Inactivating the Spindle Assembly Checkpoint

    Science.gov (United States)

    Mahale, Sagar P.; Sharma, Amit; Mylavarapu, Sivaram V. S.

    2016-01-01

    The multi-functional molecular motor cytoplasmic dynein performs diverse essential roles during mitosis. The mechanistic importance of the dynein Light Intermediate Chain homologs, LIC1 and LIC2 is unappreciated, especially in the context of mitosis. LIC1 and LIC2 are believed to exist in distinct cytoplasmic dynein complexes as obligate subunits. LIC1 had earlier been reported to be required for metaphase to anaphase progression by inactivating the kinetochore-microtubule attachment-sensing arm of the spindle assembly checkpoint (SAC). However, the functional importance of LIC2 during mitosis remains elusive. Here we report prominent novel roles for the LIC2 subunit of cytoplasmic dynein in regulating the spindle assembly checkpoint. LIC2 depletion in mammalian cells led to prolonged metaphase arrest in the presence of an active SAC and also to stretched kinetochores, thus implicating it in SAC inactivation. Quantitative fluorescence microscopy of SAC components revealed accumulation of both attachment- and tension-sensing checkpoint proteins at metaphase kinetochores upon LIC2 depletion. These observations support a stronger and more diverse role in checkpoint inactivation for LIC2 in comparison to its close homolog LIC1. Our study uncovers a novel functional hierarchy during mitotic checkpoint inactivation between the closely related but homologous LIC subunits of cytoplasmic dynein. These subtle functional distinctions between dynein subpopulations could be exploited to study specific aspects of the spindle assembly checkpoint, which is a key mediator of fidelity in eukaryotic cell division. PMID:27441562

  14. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    Energy Technology Data Exchange (ETDEWEB)

    Fukumoto, Yasunori, E-mail: fukumoto@faculty.chiba-u.jp; Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto, E-mail: nyama@faculty.chiba-u.jp

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  15. Evolutionary engineering of Saccharomyces cerevisiae for efficient conversion of red algal biosugars to bioethanol.

    Science.gov (United States)

    Lee, Hye-Jin; Kim, Soo-Jung; Yoon, Jeong-Jun; Kim, Kyoung Heon; Seo, Jin-Ho; Park, Yong-Cheol

    2015-09-01

    The aim of this work was to apply the evolutionary engineering to construct a mutant Saccharomyces cerevisiae HJ7-14 resistant on 2-deoxy-D-glucose and with an enhanced ability of bioethanol production from galactose, a mono-sugar in red algae. In batch and repeated-batch fermentations, HJ7-14 metabolized 5-fold more galactose and produced ethanol 2.1-fold faster than the parental D452-2 strain. Transcriptional analysis of genes involved in the galactose metabolism revealed that moderate relief from the glucose-mediated repression of the transcription of the GAL genes might enable HJ7-14 to metabolize galactose rapidly. HJ7-14 produced 7.4 g/L ethanol from hydrolysates of the red alga Gelidium amansii within 12 h, which was 1.5-times faster than that observed with D452-2. We demonstrate conclusively that evolutionary engineering is a promising tool to manipulate the complex galactose metabolism in S. cerevisiae to produce bioethanol from red alga. PMID:25804535

  16. Design and engineering of intracellular-metabolite-sensing/regulation gene circuits in Saccharomyces cerevisiae.

    Science.gov (United States)

    Wang, Meng; Li, Sijin; Zhao, Huimin

    2016-01-01

    The development of high-throughput phenotyping tools is lagging far behind the rapid advances of genotype generation methods. To bridge this gap, we report a new strategy for design, construction, and fine-tuning of intracellular-metabolite-sensing/regulation gene circuits by repurposing bacterial transcription factors and eukaryotic promoters. As proof of concept, we systematically investigated the design and engineering of bacterial repressor-based xylose-sensing/regulation gene circuits in Saccharomyces cerevisiae. We demonstrated that numerous properties, such as induction ratio and dose-response curve, can be fine-tuned at three different nodes, including repressor expression level, operator position, and operator sequence. By applying these gene circuits, we developed a cell sorting based, rapid and robust high-throughput screening method for xylose transporter engineering and obtained a sugar transporter HXT14 mutant with 6.5-fold improvement in xylose transportation capacity. This strategy should be generally applicable and highly useful for evolutionary engineering of proteins, pathways, and genomes in S. cerevisiae. PMID:26059511

  17. Comparative genomic analysis reveals a critical role of de novo nucleotide biosynthesis for Saccharomyces cerevisiae virulence.

    Directory of Open Access Journals (Sweden)

    Roberto Pérez-Torrado

    Full Text Available In recent years, the number of human infection cases produced by the food related species Saccharomyces cerevisiae has increased. Whereas many strains of this species are considered safe, other 'opportunistic' strains show a high degree of potential virulence attributes and can cause infections in immunocompromised patients. Here we studied the genetic characteristics of selected opportunistic strains isolated from dietary supplements and also from patients by array comparative genomic hybridization. Our results show increased copy numbers of IMD genes in opportunistic strains, which are implicated in the de novo biosynthesis of the purine nucleotides pathway. The importance of this pathway for virulence of S. cerevisiae was confirmed by infections in immunodeficient murine models using a GUA1 mutant, a key gene of this pathway. We show that exogenous guanine, an end product of this pathway in its triphosphorylated form, increases the survival of yeast strains in ex vivo blood infections. Finally, we show the importance of the DNA damage response that activates dNTP biosynthesis in yeast cells during ex vivo blood infections. We conclude that opportunistic yeasts may use an enhanced de novo biosynthesis of the purine nucleotides pathway to increase survival and favor infections in the host.

  18. Requirement of copper for 1st-log growth of the yeast Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Como, S.A.; Valerio, V.; Nickless, S.; Connelly, J.L.

    1986-05-01

    Routine evaluation of the role of copper (Cu) in the growth of various mutants of the yeast Saccharomyces Cerevisiae disclosed an unexpected effect of Cu on the fermentative first-log growth. The authors subsequent studies are attempting to ascertain the nature and significance of this observation. Cells are grown on glucose in a supplemented minimal media at 29/sup 0/C for 48-72 hrs. using New Brunswick incubator shaking at 200 rpm. Cu concentration was varied by addition of Cu salts or bathocuproine disulfonate (BC), a highly specific Cu chelator. Samples were removed periodically from flasks and dry weights were determined. Growth curve plots of normal yeasts grown in the presence of 1mM to 38mM Cu showed little variation in the expected 1st log; diauxi; 2nd log; stationary phase picture. However, in the presence of BC growth rate in the 1st log was significantly slowed and as expected 2nd log growth was essentially stopped. The low 1st log growth rate could be titrated to normal (+Cu) levels by increments of added Cu but not by added iron. The effect was not seen when Rho-minus strains were used nor when growth was followed under anaerobic conditions. Results to date implicate a mitochondrial protein, oxygen and copper in the 1st log growth of S Cerevisiae. The character of the protein agent and the possible contribution of cytochrome oxidase activity to the lst log growth are being evaluated.

  19. Evaluation of Brachypodium distachyon L-Tyrosine Decarboxylase Using L-Tyrosine Over-Producing Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Shuhei Noda

    Full Text Available To demonstrate that herbaceous biomass is a versatile gene resource, we focused on the model plant Brachypodium distachyon, and screened the B. distachyon for homologs of tyrosine decarboxylase (TDC, which is involved in the modification of aromatic compounds. A total of 5 candidate genes were identified in cDNA libraries of B. distachyon and were introduced into Saccharomyces cerevisiae to evaluate TDC expression and tyramine production. It is suggested that two TDCs encoded in the transcripts Bradi2g51120.1 and Bradi2g51170.1 have L-tyrosine decarboxylation activity. Bradi2g51170.1 was introduced into the L-tyrosine over-producing strain of S. cerevisiae that was constructed by the introduction of mutant genes that promote deregulated feedback inhibition. The amount of tyramine produced by the resulting transformant was 6.6-fold higher (approximately 200 mg/L than the control strain, indicating that B. distachyon TDC effectively converts L-tyrosine to tyramine. Our results suggest that B. distachyon possesses enzymes that are capable of modifying aromatic residues, and that S. cerevisiae is a suitable host for the production of L-tyrosine derivatives.

  20. Intestinal microbiome analyses identify melanoma patients at risk for checkpoint-blockade-induced colitis

    OpenAIRE

    Dubin, Krista; Callahan, Margaret K.; Ren, Boyu; Khanin, Raya; Viale, Agnes; Ling, Lilan; No, Daniel; Gobourne, Asia; Littmann, Eric; Huttenhower, Curtis; Pamer, Eric G.; Wolchok, Jedd D.

    2016-01-01

    The composition of the intestinal microbiota influences the development of inflammatory disorders. However, associating inflammatory diseases with specific microbial members of the microbiota is challenging, because clinically detectable inflammation and its treatment can alter the microbiota's composition. Immunologic checkpoint blockade with ipilimumab, a monoclonal antibody that blocks cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) signalling, is associated with new-onset, immune-med...

  1. Handoff Based Secure Checkpointing and Log Based Rollback Recovery for Mobile Hosts

    Directory of Open Access Journals (Sweden)

    Priyanka Dey and Suparna Biswas

    2012-10-01

    Full Text Available An efficient fault tolerant algorithm based on movement-based secure checkpointing and logging formobile computing system is proposed here. The recovery scheme proposed here combines independentcheckpointing and message logging. Here we consider mobility rate of the user in checkpointing so thatmobile host can manage recovery information such as checkpoints and logs properly so that a mobilehost takes less recovery time after failure. Mobile hosts save checkpoints when number of hand-offexceeds a predefined hand-off threshold value. Current approaches save logs in base station. But thisapproach maximizes recovery time if message passing frequency is large. If a mobile host saves log in itsown memory, recovery cost will be less because log retrieval time will be small after failure. But there isa probability of memory crash of a mobile host. In that case logs can not be retrieved if it is saved only inmobile node. If the failure is transient then logs can be retrieved from the memory of mobile node.Hence in this algorithm mobile hosts also save log in own memory and base station. In case of crashrecovery, log will be retrieved from base station and in case of transient failure recovery logs will beretrieved from mobile host. In this algorithm recovery probability is optimized and total recovery time isreduced in comparison to existing works. Logs are very small in size. Hence saving logs in mobile hostsdoes not cause much memory overhead. Hand-off threshold is a function of mobility rate, messagepassing frequency and failure rate of mobile hosts. This algorithm describes a secure check pointingtechnique as a method for providing fault tolerance while preventing information leakage through thecheckpoint data.

  2. In vivo role of checkpoint kinase 2 in signaling telomere dysfunction

    OpenAIRE

    García-Beccaria, María; Martínez, Paula; Flores, Juana M.; Blasco, Maria A.

    2014-01-01

    Checkpoint kinase 2 (CHK2) is a downstream effector of the DNA damage response (DDR). Dysfunctional telomeres, either owing to critical shortening or disruption of the shelterin complex, activate a DDR, which eventually results in cell cycle arrest, senescence and/or apoptosis. Successive generations of telomerase-deficient (Terc) mice show accelerated aging and shorter lifespan due to tissue atrophy and impaired organ regeneration associated to progressive telomere shortening. In contrast, m...

  3. Cdk2 is required for p53-independent G2/M checkpoint control.

    Directory of Open Access Journals (Sweden)

    Jon H Chung

    2010-02-01

    Full Text Available The activation of phase-specific cyclin-dependent kinases (Cdks is associated with ordered cell cycle transitions. Among the mammalian Cdks, only Cdk1 is essential for somatic cell proliferation. Cdk1 can apparently substitute for Cdk2, Cdk4, and Cdk6, which are individually dispensable in mice. It is unclear if all functions of non-essential Cdks are fully redundant with Cdk1. Using a genetic approach, we show that Cdk2, the S-phase Cdk, uniquely controls the G(2/M checkpoint that prevents cells with damaged DNA from initiating mitosis. CDK2-nullizygous human cells exposed to ionizing radiation failed to exclude Cdk1 from the nucleus and exhibited a marked defect in G(2/M arrest that was unmasked by the disruption of P53. The DNA replication licensing protein Cdc6, which is normally stabilized by Cdk2, was physically associated with the checkpoint regulator ATR and was required for efficient ATR-Chk1-Cdc25A signaling. These findings demonstrate that Cdk2 maintains a balance of S-phase regulatory proteins and thereby coordinates subsequent p53-independent G(2/M checkpoint activation.

  4. Biological significance of the focus on DNA damage checkpoint factors remained after irradiation of ionizing radiation

    International Nuclear Information System (INIS)

    This paper reviews recent reports on the focus formation and participation to checkpoint of (such phosphorylated (P-d) as below) ATM and H2AX, MDC1, 53BP1 and NBS1, and discusses their role in DNA damage checkpoint induction mainly around authors' studies. When the cell is irradiated by ionizing radiation, the subtype histone like H2AX is P-d and the formed focus', seen in the nucleus on immuno-fluorographic observation, represents the P-d H2AX at the damaged site of DNA. The role of P-d ATM (the product of causative gene of ataxia-telangiectasia mutation, a protein kinase) has been first shown by laser beam irradiation. Described are discussions on the roles and functions after irradiation in focus formation and DNA damage checkpoint of P-d H2AX (a specific histone product by the radiation like γ-ray as above), P-d ATM, MDC1 (a mediator of DNA damage check point protein 1), 53BP1, (a p53 binding protein) and NBS1 (the product of the causative gene of Nijmegen Breakage Syndrome). Authors have come to point out the remained focal size increase as implications of the efficient repair of damaged DNA, and the second cycled p53 accumulation, of tumor suppression. Thus evaluation of biological significance of these aspects, scarcely noted hitherto, is concluded important. (S.I.)

  5. Efficient checkpointing schemes for depletion perturbation solutions on memory-limited architectures

    International Nuclear Information System (INIS)

    We describe a methodology for decreasing the memory footprint and machine I/O load associated with the need to access a forward solution during an adjoint solve. Specifically, we are interested in the depletion perturbation equations, where terms in the adjoint Bateman and transport equations depend on the forward flux solution. Checkpointing is the procedure of storing snapshots of the forward solution to disk and using these snapshots to recompute the parts of the forward solution that are necessary for the adjoint solve. For large problems, however, the storage cost of just a few copies of an angular flux vector can exceed the available RAM on the host machine. We propose a methodology that does not checkpoint the angular flux vector; instead, we write and store converged source moments, which are typically of a much lower dimension than the angular flux solution. This reduces the memory footprint and I/O load of the problem, but requires that we perform single sweeps to reconstruct flux vectors on demand. We argue that this trade-off is exactly the kind of algorithm that will scale on advanced, memory-limited architectures. We analyze the cost, in terms of FLOPS and memory footprint, of five checkpointing schemes. We also provide computational results that support the analysis and show that the memory-for-work trade off does improve time to solution. (authors)

  6. Efficient checkpointing schemes for depletion perturbation solutions on memory-limited architectures

    Energy Technology Data Exchange (ETDEWEB)

    Stripling, H. F.; Adams, M. L.; Hawkins, W. D. [Texas A and M University, Department of Nuclear Engineering, 3133 TAMU, College Station, TX 77843-3133 (United States)

    2013-07-01

    We describe a methodology for decreasing the memory footprint and machine I/O load associated with the need to access a forward solution during an adjoint solve. Specifically, we are interested in the depletion perturbation equations, where terms in the adjoint Bateman and transport equations depend on the forward flux solution. Checkpointing is the procedure of storing snapshots of the forward solution to disk and using these snapshots to recompute the parts of the forward solution that are necessary for the adjoint solve. For large problems, however, the storage cost of just a few copies of an angular flux vector can exceed the available RAM on the host machine. We propose a methodology that does not checkpoint the angular flux vector; instead, we write and store converged source moments, which are typically of a much lower dimension than the angular flux solution. This reduces the memory footprint and I/O load of the problem, but requires that we perform single sweeps to reconstruct flux vectors on demand. We argue that this trade-off is exactly the kind of algorithm that will scale on advanced, memory-limited architectures. We analyze the cost, in terms of FLOPS and memory footprint, of five checkpointing schemes. We also provide computational results that support the analysis and show that the memory-for-work trade off does improve time to solution. (authors)

  7. Immune checkpoint blockade therapy: The 2014 Tang prize in biopharmaceutical science

    Directory of Open Access Journals (Sweden)

    Ya-Shan Chen

    2015-02-01

    Full Text Available The first Tang Prize for Biopharmaceutical Science has been awarded to Prof. James P. Allison and Prof. Tasuku Honjo for their contributions leading to an entirely new way to treat cancer by blocking the molecules cytotoxic T lymphocyte-associated antigen 4 (CTLA-4 and programmed cell death protein 1 (PD-1 that turn off immune response. The treatment, called "immune checkpoint blockade therapy," has opened a new therapeutic era. Here the discoveries of the immune checkpoints and how they contribute to the maintenance of self-tolerance, as well as how to protect tissues from the excess immune responses causing damage are reviewed. The efforts made by Prof. Allison and Prof. Honjo for developing the most promising approaches to activate therapeutic antitumor immunity are also summarized. Since these certain immune checkpoint pathways appear to be one of the major mechanisms resulting in immune escape of tumors, the presence of anti-CTLA-4 and/or anti-PD-1 should contribute to removal of the inhibition signals for T cell activation. Subsequently, it will enhance specific T cell activation and, therefore, strengthen antitumor immunity.

  8. Immunologic checkpoints in cancer therapy: focus on the programmed death-1 (PD-1 receptor pathway

    Directory of Open Access Journals (Sweden)

    Momtaz P

    2014-11-01

    Full Text Available Parisa Momtaz,1,2 Michael A Postow1,2 1Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA; 2Weill Cornell Medical College, New York, NY, USA Abstract: T-lymphocytes have the potential to recognize cancer antigens as foreign and therefore eliminate them. However, immune checkpoints such as cytotoxic T-lymphocyte-associated antigen (CTLA-4 and programmed cell death (PD-1 receptor and its ligands (PD-L1, PD-L2 suppress the activity of T-lymphocytes. Advances in the understanding of immunology and its role in cancer have led to the development of immune checkpoint inhibitors that block CTLA-4 and PD-1 and result in durable responses in patients with a wide range of cancers. PD-1 and PD-L1 inhibitors are currently in many stages of clinical investigation, and the anti-PD-1 antibody, pembrolizumab, was recently approved by the US Food and Drug Administration. Many questions remain to be answered, such as the optimal administration schedule, biomarkers that associate with benefit, and potential for use of PD-1 agents in combination approaches. Nonetheless, immunotherapy with PD-1 blocking antibodies is now becoming an integral part in the management of cancer. Keyword: immune checkpoints, immunotherapy, programmed cell death protein-1, cytotoxic T-lymphocyte antigen 4

  9. Eavesdropping on the cytoskeleton: progress and controversy in the yeast morphogenesis checkpoint.

    Science.gov (United States)

    Keaton, Mignon A; Lew, Daniel J

    2006-12-01

    The morphogenesis checkpoint provides a link between bud formation and mitosis in yeast. In this pathway, insults affecting the actin or septin cytoskeleton trigger a cell cycle arrest, mediated by the Wee1 homolog Swe1p, which catalyzes the inhibitory phosphorylation of the mitosis-promoting cyclin-dependent kinase (CDK) on a conserved tyrosine residue. Analyses of Swe1p phosphorylation have mapped 61 sites targeted by CDKs and Polo-related kinases, which control both Swe1p activity and Swe1p degradation. Although the sites themselves are not evolutionarily conserved, the control of Swe1p degradation exhibits many conserved features, and is linked to DNA-responsive checkpoints in vertebrate cells. At the 'sensing' end of the checkpoint, recent work has begun to shed light on how septins are organized and how they impact Swe1p regulators. However, the means by which Swe1p responds to actin perturbations once a bud has formed remains controversial. PMID:17055334

  10. Multicellular tumor spheroid models to explore cell cycle checkpoints in 3D

    International Nuclear Information System (INIS)

    MultiCellular Tumor Spheroid (MCTS) mimics the organization of a tumor and is considered as an invaluable model to study cancer cell biology and to evaluate new antiproliferative drugs. Here we report how the characteristics of MCTS in association with new technological developments can be used to explore the regionalization and the activation of cell cycle checkpoints in 3D. Cell cycle and proliferation parameters were investigated in Capan-2 spheroids by immunofluorescence staining, EdU incorporation and using cells engineered to express Fucci-red and -green reporters. We describe in details the changes in proliferation and cell cycle parameters during spheroid growth and regionalization. We report the kinetics and regionalized aspects of cell cycle arrest in response to checkpoint activation induced by EGF starvation, lovastatin treatment and etoposide-induced DNA damage. Our data present the power and the limitation of spheroids made of genetically modified cells to explore cell cycle checkpoints. This study paves the way for the investigation of molecular aspects and dynamic studies of the response to novel antiproliferative agents in 3D models

  11. RNF8 Transduces the DNA-Damage Signal Via Histone Ubiquitylation And Checkpoint Protein Assembly

    Energy Technology Data Exchange (ETDEWEB)

    Huen, M.S.Y.; Grant, R.; Manke, I.; Minn, K.; Yu, X.; Yaffe, M.B.; Chen, J.

    2009-06-01

    DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 {angstrom}. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress.

  12. Vanillin causes the activation of Yap1 and mitochondrial fragmentation in Saccharomyces cerevisiae.

    Science.gov (United States)

    Nguyen, Trinh Thi My; Iwaki, Aya; Ohya, Yoshikazu; Izawa, Shingo

    2014-01-01

    Vanillin and furfural are derived from lignocellulosic biomass and inhibit yeast growth and fermentation as biomass conversion inhibitors. Furfural has been shown to induce oxidative stress in Saccharomyces cerevisiae. Since there has been no report on the relationship between vanillin and oxidative stress, we investigated whether vanillin caused oxidative stress in yeast cells. We showed that vanillin caused the nuclear accumulation of Yap1, an oxidative stress responsive transcription factor, and subsequent transcriptional activation of Yap1-target genes. The growth of the null mutant of the YAP1 gene (yap1Δ) was delayed in the presence of vanillin, which indicated that Yap1 plays a role in the acquisition of tolerance to vanillin. We also demonstrated that vanillin facilitated the fragmentation of mitochondria. These findings suggest that the toxicity of vanillin involves damage induced by oxidative stress. PMID:23850265

  13. Fermentation of grain sorghum starch by co-cultivation of Schwanniomyces occidentalis and Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Horn, C.H.; Preez, J.C. du; Kilian, S.G. (Orange Free State Univ., Bloemfontein (ZA). Dept. of Microbiology and Biochemistry)

    1992-01-01

    The amylases of a Schwanniomyces occidentalis mutant hydrolysed grain sorghum starch completely. Heat pretreatment to effect gelatinization was essential for total starch hydrolysis. Ethanol fermentations were conducted by using a stationary-phase culture of S. occidentalis in conjunction with Saccharomyces cerevisiae to ferment the sugars liberated from the grain sorghum starch by the amylases of S. occidentalis. Increasing the grain sorghum concentration from 8 to 28% (m/v) did not affect the final ethanol yield of 0.45 g ethanol/g glucose equivalents, although the fermentation rate decreased considerably at the higher slurry concentration, requiring 8 days for completion of the fermentation. A 28% grain sorghum slurry yielded 12.5% (w/v) ethanol, indicating that nearly 390 1 of ethanol could be produced per tonne grain sorghum, with about 430 kg residual biomass containing 43% crude protein. (Author).

  14. MGL2/YMR210w encodes a monoacylglycerol lipase in Saccharomyces cerevisiae.

    Science.gov (United States)

    Selvaraju, Kandasamy; Gowsalya, Ramachandran; Vijayakumar, Rajendran; Nachiappan, Vasanthi

    2016-04-01

    In silico analysis of the uncharacterized open reading frame YMR210w in Saccharomyces cerevisiae revealed that it possesses both an α/β hydrolase domain (ABHD) and a typical lipase (GXSXG) motif. The purified protein displayed monoacylglycerol (MAG) lipase activity and preferred palmitoyl-MAG. Overexpression of YMR210w in the known MAG lipase mutant yju3Δ clearly revealed that the protein had MAG lipase activity, hence we named the ORF MGL2. Overexpression of YMR210w decreased the cellular triacylglycerol levels. Analysis of the overexpressed strains showed reduction in the lipid droplets number and size. Phenotype studies revealed that the double deletion yju3Δmgl2Δ displayed a growth defect that was partially restored by MGL2 overexpression. PMID:26991558

  15. KONSTRUKSI MUTAN PROTEIN FOSFATASE ptc2D Saccharomyces cerevisiae DENGAN METODE PENGGANTIAN GEN TARGET DENGAN POLYMERASE CHAIN REACTION (PCR

    Directory of Open Access Journals (Sweden)

    Hermansyah

    2011-05-01

    Full Text Available Yeast Saccharomyces cerevisiae is an excellent model to studi genes function of eukarotic cells such as study of gene encoding protein phosphatase PTC2. Novel phenotypic caused by mutated gene is an important step to study function of gene. In this study constructed mutant of PTC2 gene encoding protein phosphatase. Method that used in this construction was replacement of target gene (PTC2 with auxotroph marker Candida albicans HIS3 by Polymer Chain Reaction (PCR or called by PCR-mediated disruption. Mutant colonies which grew in selective medium SC without histidine were confirmed by PCR amplification. By using 1% Agarose gel electrophoresis the result showed that size of ptc2D::CgHIS3 transformant was 3.52 kb while wild type strain was 2.9 kb, indicated that ptc2D::CgHIS3 has integrated on chromosome V replacing PTC2 wild type.

  16. Regulation of Saccharomyces cerevisiae maltose fermentation by cold temperature and CSF1 Regulação da fermentação de maltose em Saccharomyces cerevisiae por baixas temperaturas e CSF1

    Directory of Open Access Journals (Sweden)

    Claudia Hollatz

    2003-11-01

    Full Text Available We studied the influence of cold temperature (10ºC on the fermentation of maltose by a S. cerevisiae wild-type strain, and a csf1delta mutant impaired in glucose and leucine uptake at low temperatures. Cold temperature affected the fermentation kinetics by decreasing the growth rate and the final cell yield, with almost no ethanol been produced from maltose by the wild-type cells at 10ºC. The csf1delta strain did not grew on maltose when cultured at 10ºC, indicating that the CSF1 gene is also required for maltose consumption at low temperatures. However, this mutant also showed increased inhibition of glucose and maltose fermentation under salt stress, indicating that CSF1 is probably involved in the regulation of other physiological processes, including ion homeostasis.Foi estudado o efeito da baixa temperatura (10ºC na fermentação de maltose por uma cepa de S. cerevisiae selvagem, e uma cepa csf1delta mutante incapaz de transportar glicose e leucina a baixas temperaturas. A baixa temperatura afeta a cinética da fermentação por diminuir a velocidade de crescimento e rendimento celular final, com quase nenhum etanol produzido a partir de maltose pelas células selvagems a 10ºC. A cepa csf1delta foi incapaz de crescer em maltose a 10ºC, indicando que o gene CSF1 é também necessário para a utilização de maltose a baixas temperaturas. Entretanto, o mutante também mostrou inibição acentuada da fermentação de glicose e maltose por estresse salino, indicando que CSF1 também estaria envolvido na regulação de outros processos fisiológicos, incluindo a homeostase iónica.

  17. Esc2 and Sgs1 act in functionally distinct branches of the homologous recombination repair pathway in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Ngo, Hien-Ping; Hickson, Ian D

    2009-01-01

    homologous recombination repair (HRR) intermediates. These roles are qualitatively similar to those of Sgs1, the yeast ortholog of the human Bloom's syndrome protein, BLM. However, whereas mutation of either ESC2 or SGS1 leads to the accumulation of unprocessed HRR intermediates in the presence of MMS, the...... accumulation of these structures in esc2 (but not sgs1) mutants is entirely dependent on Mph1, a protein that shows structural similarity to the Fanconi anemia group M protein (FANCM). In the absence of both Esc2 and Sgs1, the intra-S-phase DNA damage checkpoint response is compromised after exposure to MMS......, and sgs1esc2 cells attempt to undergo mitosis with unprocessed HRR intermediates. We propose a model whereby Esc2 acts in an Mph1-dependent process, separately from Sgs1, to influence the repair/tolerance of MMS-induced lesions during S-phase....

  18. Isolation and Characterization of Chromosome-Gain and Increase-in-Ploidy Mutants in Yeast

    OpenAIRE

    Chan, CSM.; Botstein, D

    1993-01-01

    We have developed a colony papillation assay for monitoring the copy number of genetically marked chromosomes II and III in Saccharomyces cerevisiae. The unique feature of this assay is that it allows detection of a gain of the marked chromosomes even if there is a gain of the entire set of chromosomes (increase-in-ploidy). This assay was used to screen for chromosome-gain or increase-in-ploidy mutants. Five complementation groups have been defined for recessive mutations that confer an incre...

  19. Revertants of a Transcription Termination Mutant of Yeast Contain Diverse Genetic Alterations

    OpenAIRE

    Kotval, Jeroo; Zaret, Kenneth S.; Consaul, Sandra; Sherman, Fred

    1983-01-01

    Revertants of the cyc1-512 transcription termination mutant of the yeast Saccharomyces cerevisiae were isolated and subjected to a detailed genetic analysis. The cyc1-512 mutation previously was shown to be a 38-base pair deletion that causes only 10% of the normal steady-state levels of CYC1 mRNA and of the CYC1 gene product, iso-1-cytochrome c. Forty-one cyc1-512 revertants were classified by their content of iso-1-cytochrome c and by their genetic properties in meiotic crosses. Many of the...

  20. Isolation of yeast mutants defective for localization of vacuolar vital dyes

    OpenAIRE

    Zheng, Bing; Wu, Jennifer N.; Schober, Wendy; Dorothy E Lewis; Vida, Thomas

    1998-01-01

    An application of flow cytometric sorting is used for isolation of Saccharomyces cerevisiae mutants that mislocalize vacuolar vital dyes. This screen is based on the ability of a lipophilic styryl compound, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM4–64), to label endocytic intermediates from the plasma membrane to the vacuole membrane at 15°C. Cells stained at 15°C for both FM4–64 and carboxydichlorofluorescein diacetate (a vacuolar luminal...

  1. Overproduction and secretion of free fatty acids through disrupted neutral lipid recycle in Saccharomyces cerevisiae.

    Science.gov (United States)

    Leber, Christopher; Polson, Brian; Fernandez-Moya, Ruben; Da Silva, Nancy A

    2015-03-01

    The production of fuels and chemicals from biorenewable resources is important to alleviate the environmental concerns, costs, and foreign dependency associated with the use of petroleum feedstock. Fatty acids are attractive biomolecules due to the flexibility of their iterative biosynthetic pathway, high energy content, and suitability for conversion into other secondary chemicals. Free fatty acids (FFAs) that can be secreted from the cell are particularly appealing due to their lower harvest costs and straightforward conversion into a broad range of biofuel and biochemical products. Saccharomyces cerevisiae was engineered to overproduce extracellular FFAs by targeting three native intracellular processes. β-oxidation was disrupted by gene knockouts in FAA2, PXA1 and POX1, increasing intracellular fatty acids levels up to 55%. Disruptions in the acyl-CoA synthetase genes FAA1, FAA4 and FAT1 allowed the extracellular detection of free fatty acids up to 490mg/L. Combining these two disrupted pathways, a sextuple mutant (Δfaa1 Δfaa4 Δfat1 Δfaa2 Δpxa1 Δpox1) was able to produce 1.3g/L extracellular free fatty acids. Further diversion of carbon flux into neutral lipid droplet formation was investigated by the overexpression of DGA1 or ARE1 and by the co-overexpression of a compatible lipase, TGL1, TGL3 or TGL5. The sextuple mutant overexpressing the diacylglycerol acyltransferase, DGA1, and the triacylglycerol lipase, TGL3, yielded 2.2g/L extracellular free fatty acids. This novel combination of pathway interventions led to 4.2-fold higher extracellular free fatty acid levels than previously reported for S. cerevisiae. PMID:25461829

  2. The Swedish mutant barley collection

    International Nuclear Information System (INIS)

    Full text: The Swedish mutation research programme in barley began about 50 years ago and has mainly been carried out at Svaloev in co-operation with the institute of Genetics at the University of Lund. The collection has been produced from different Swedish high-yielding spring barley varieties, using the following mutagens: X-rays, neutrons, several organic chemical compounds such as ethyleneimine, several sulfonate derivatives and the inorganic chemical mutagen sodium azide. Nearly 10,000 barley mutants are stored in the Nordic Gene Bank and documented in databases developed by Udda Lundquist, Svaloev AB. The collection consists of the following nine categories with 94 different types of mutants: 1. Mutants with changes in the spike and spikelets; 2. Changes in culm length and culm composition; 3. Changes in growth types; 4. Physiological mutants; 5. Changes in awns; 6. Changes in seed size and shape; 7. Changes in leaf blades; 8. Changes in anthocyanin and colour; 9. Resistance to barley powdery mildew. Barley is one of the most thoroughly investigated crops in terms of induction of mutations and mutation genetics. So far, about half of the mutants stored at the Nordic Gene Bank, have been analysed genetically; They constitute, however, only a minority of the 94 different mutant types. The genetic analyses have given valuable insights into the mutation process but also into the genetic architecture of various characters. A number of mutants of two-row barley have been registered and commercially released. One of the earliest released, Mari, an early maturing, daylength neutral, straw stiff mutant, is still grown in Iceland. The Swedish mutation material has been used in Sweden, but also in other countries, such as Denmark, Germany, and USA, for various studies providing a better understanding of the barley genome. The collection will be immensely valuable for future molecular genetical analyses of clone mutant genes. (author)

  3. Biosorption of cesium by saccharomyces cerevisia

    International Nuclear Information System (INIS)

    The characteristics of Cs+ biosorption by Saccharornyces cerevisia was investigated, including the biosorption kinetics, biosorption equilibrium, isotherm as well as the IR spectrum of biomass pre- and post-biosorption. The experimental results show that the process of Cs+ biosorption onto the biomass of Saccharornyces cerevisia can be devided into two stages, the first stage is physical sorption and the sorption equilibrium is very quickly reached (within 20 min). The biosorption kinetics can be described by the pseudo second-order equation quite well (R2=0.989), the kinetic parameters k2 and qe are 3.56 x 10-3 g/(mg·min) and 7.18 mg/g, respectively. The equilibrium isotherm data can be fitted with Langmuir and Freundlich models, with the maximum biosorptive capacity of 10.13 mg/g. Both the IR spectra of the biomass pre- and post-biosorption almost are same, and it indicates that the biosorption of Cs+ does not change the structure of the biomass, however, some adsorptive peaks shift. (authors)

  4. Heterooligomeric phosphoribosyl diphosphate synthase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Hove-Jensen, Bjarne

    2004-01-01

    The yeast Saccharomyces cerevisiae contains five phosphoribosyl diphosphate (PRPP) synthase-homologous genes (PRS1-5), which specify PRPP synthase subunits 1-5. Expression of the five S. cerevisiae PRS genes individually in an Escherichia coli PRPP-less strain (Deltaprs) showed that a single PRS...... gene product had no PRPP synthase activity. In contrast, expression of five pairwise combinations of PRS genes resulted in the formation of active PRPP synthase. These combinations were PRS1 PRS2, PRS1 PRS3, and PRS1 PRS4, as well as PRS5 PRS2 and PRS5 PRS4. None of the remaining five possible pairwise...... combinations of PRS genes appeared to produce active enzyme. Extract of an E. coli strain containing a plasmid-borne PRS1 gene and a chromosome-borne PRS3 gene contained detectable PRPP synthase activity, whereas extracts of strains containing PRS1 PRS2, PRS1 PRS4, PRS5 PRS2, or PRS5 PRS4 contained no...

  5. Response of Saccharomyces cerevisiae to cadmium stress

    Energy Technology Data Exchange (ETDEWEB)

    Moreira, Luciana Mara Costa; Ribeiro, Frederico Haddad; Neves, Maria Jose [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Lab. de Radiobiologia], e-mail: luamatu@uol.com.br; Porto, Barbara Abranches Araujo; Amaral, Angela M.; Menezes, Maria Angela B.C. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Lab. de Ativacao Neutronica], e-mail: menezes@cdtn.br; Rosa, Carlos Augusto [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Microbiologia], e-mail: carlrosa@icb.ufmg

    2009-07-01

    The intensification of industrial activity has been greatly contributing with the increase of heavy metals in the environment. Among these heavy metals, cadmium becomes a serious pervasive environmental pollutant. The cadmium is a heavy metal with no biological function, very toxic and carcinogenic at low concentrations. The toxicity of cadmium and several other metals can be mainly attributed to the multiplicity of coordination complexes and clusters that they can form. Some aspects of the cellular response to cadmium were extensively investigated in the yeast Saccharomyces cerevisiae. The primary site of interaction between many toxic metals and microbial cells is the plasma membrane. Plasma-membrane permeabilisation has been reported in a variety of microorganisms following cadmium exposure, and is considered one mechanism of cadmium toxicity in the yeast. In this work, using the yeast strain S. cerevisiae W303-WT, we have investigated the relationships between Cd uptake and release of cellular metal ions (K{sup +} and Na{sup +}) using neutron activation technique. The neutron activation was an easy, rapid and suitable technique for doing these metal determinations on yeast cells; was observed the change in morphology of the strains during the process of Cd accumulation, these alterations were observed by Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) during incorporation of cadmium. (author)

  6. Boolean network model predicts knockout mutant phenotypes of fission yeast.

    Directory of Open Access Journals (Sweden)

    Maria I Davidich

    Full Text Available BOOLEAN NETWORKS (OR: networks of switches are extremely simple mathematical models of biochemical signaling networks. Under certain circumstances, Boolean networks, despite their simplicity, are capable of predicting dynamical activation patterns of gene regulatory networks in living cells. For example, the temporal sequence of cell cycle activation patterns in yeasts S. pombe and S. cerevisiae are faithfully reproduced by Boolean network models. An interesting question is whether this simple model class could also predict a more complex cellular phenomenology as, for example, the cell cycle dynamics under various knockout mutants instead of the wild type dynamics, only. Here we show that a Boolean network model for the cell cycle control network of yeast S. pombe correctly predicts viability of a large number of known mutants. So far this had been left to the more detailed differential equation models of the biochemical kinetics of the yeast cell cycle network and was commonly thought to be out of reach for models as simplistic as Boolean networks. The new results support our vision that Boolean networks may complement other mathematical models in systems biology to a larger extent than expected so far, and may fill a gap where simplicity of the model and a preference for an overall dynamical blueprint of cellular regulation, instead of biochemical details, are in the focus.

  7. Dynamic model-based analysis of furfural and HMF detoxification by pure and mixed batch cultures of S. cerevisiae and S. stipitis.

    Science.gov (United States)

    Hanly, Timothy J; Henson, Michael A

    2014-02-01

    Inhibitory compounds that result from biomass hydrolysis are an obstacle to the efficient production of second-generation biofuels. Fermentative microorganisms can reduce compounds such as furfural and 5-hydroxymethyl furfural (HMF), but detoxification is accompanied by reduced growth rates and ethanol yields. In this study, we assess the effects of these furan aldehydes on pure and mixed yeast cultures consisting of a respiratory deficient mutant of Saccharomyces cerevisiae and wild-type Scheffersomyces stipitis using dynamic flux balance analysis. Uptake kinetics and stoichiometric equations for the intracellular reduction reactions associated with each inhibitor were added to genome-scale metabolic reconstructions of the two yeasts. Further modification of the S. cerevisiae metabolic network was necessary to satisfactorily predict the amount of acetate synthesized during HMF reduction. Inhibitory terms that captured the adverse effects of the furan aldehydes and their corresponding alcohols on cell growth and ethanol production were added to attain qualitative agreement with batch experiments conducted for model development and validation. When the two yeasts were co-cultured in the presence of the furan aldehydes, inoculums that reduced the synthesis of highly toxic acetate produced by S. cerevisiae yielded the highest ethanol productivities. The model described here can be used to generate optimal fermentation strategies for the simultaneous detoxification and fermentation of lignocellulosic hydrolysates by S. cerevisiae and/or S. stipitis. PMID:23983023

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

    Science.gov (United States)

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

    2016-03-18

    The d or l form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high d-2-hydroxyglutarate (d-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the d enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human d-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3Δ knock-out strain accumulates millimolar levels of d-2HG, whereas a dld2Δ knock-out strain displayed only very moderate increases in d-2HG. Recombinant Dld2 and Dld3, both currently annotated as d-lactate dehydrogenases, efficiently oxidized d-2HG to α-ketoglutarate. Depletion of d-lactate levels in the dld3Δ, but not in the dld2Δ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert d-2HG to α-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce α-ketoglutarate to d-2HG using NADH and represent major intracellular sources of d-2HG in yeast. Based on our observations, we propose that d-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples d-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the d-lactate dehydrogenase Dld1. PMID:26774271

  9. Evidence for Domesticated and Wild Populations of Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    2005-07-01

    Full Text Available Saccharomyces cerevisiae is predominantly found in association with human activities, particularly the production of alcoholic beverages. S. paradoxus, the closest known relative of S. cerevisiae, is commonly found on exudates and bark of deciduous trees and in associated soils. This has lead to the idea that S. cerevisiae is a domesticated species, specialized for the fermentation of alcoholic beverages, and isolates of S. cerevisiae from other sources simply represent migrants from these fermentations. We have surveyed DNA sequence diversity at five loci in 81 strains of S. cerevisiae that were isolated from a variety of human and natural fermentations as well as sources unrelated to alcoholic beverage production, such as tree exudates and immunocompromised patients. Diversity within vineyard strains and within saké strains is low, consistent with their status as domesticated stocks. The oldest lineages and the majority of variation are found in strains from sources unrelated to wine production. We propose a model whereby two specialized breeds of S. cerevisiae have been created, one for the production of grape wine and one for the production of saké wine. We estimate that these two breeds have remained isolated from one another for thousands of years, consistent with the earliest archeological evidence for winemaking. We conclude that although there are clearly strains of S. cerevisiae specialized for the production of alcoholic beverages, these have been derived from natural populations unassociated with alcoholic beverage production, rather than the opposite.

  10. Industrial systems biology of Saccharomyces cerevisiae enables novel succinic acid cell factory.

    Directory of Open Access Journals (Sweden)

    José Manuel Otero

    Full Text Available Saccharomyces cerevisiae is the most well characterized eukaryote, the preferred microbial cell factory for the largest industrial biotechnology product (bioethanol, and a robust commerically compatible scaffold to be exploitted for diverse chemical production. Succinic acid is a highly sought after added-value chemical for which there is no native pre-disposition for production and accmulation in S. cerevisiae. The genome-scale metabolic network reconstruction of S. cerevisiae enabled in silico gene deletion predictions using an evolutionary programming method to couple biomass and succinate production. Glycine and serine, both essential amino acids required for biomass formation, are formed from both glycolytic and TCA cycle intermediates. Succinate formation results from the isocitrate lyase catalyzed conversion of isocitrate, and from the α-keto-glutarate dehydrogenase catalyzed conversion of α-keto-glutarate. Succinate is subsequently depleted by the succinate dehydrogenase complex. The metabolic engineering strategy identified included deletion of the primary succinate consuming reaction, Sdh3p, and interruption of glycolysis derived serine by deletion of 3-phosphoglycerate dehydrogenase, Ser3p/Ser33p. Pursuing these targets, a multi-gene deletion strain was constructed, and directed evolution with selection used to identify a succinate producing mutant. Physiological characterization coupled with integrated data analysis of transcriptome data in the metabolically engineered strain were used to identify 2(nd-round metabolic engineering targets. The resulting strain represents a 30-fold improvement in succinate titer, and a 43-fold improvement in succinate yield on biomass, with only a 2.8-fold decrease in the specific growth rate compared to the reference strain. Intuitive genetic targets for either over-expression or interruption of succinate producing or consuming pathways, respectively, do not lead to increased succinate. Rather, we

  11. Indicadores fisiológicos de la respuesta al estrés en cepas Saccharomyces cerevisiae alcholeras

    Directory of Open Access Journals (Sweden)

    Roxana García

    2009-01-01

    Full Text Available El fenómeno de tolerancia cruzada en levaduras se ha propuesto como indicador fisiológico útil en estudios de selección de cepas S. cerevisiae para la obtención de etanol. Se evalúa para cinco cepas de esta especie la relación entre resistencia a H2O2 3,2 M y a presiones hidrostáticas de 200 MPa durante 30 minutos y la actividad enzimática alcohol deshidrogenasa (ADH en presencia de etanol y glucosa. Solo el mutante auxótrofo XA3-1 no muestra un crecimiento apreciable ante el estrés oxidativo. Su cepa salvaje A3 es la única capaz de crecer hasta la mayor dilución a altas presiones hidrostáticas. Las cepas CM y 373 muestran patrones similares de actividad ADH en presencia de glucosa o etanol, mientras que las cepas A3 y su mutante presentan niveles de ADH superiores en presencia de 5% (v/v de etanol. Los resultados indican una estrecha vinculación entre la resistencia a estreses no necesariamente propios de la fermentación alcohólica y el comportamiento fermentativo en cepas S. cerevisiae, lo cual abre la posibilidad de emplear estos criterios para estudios de selección de cepas alcoholeras dirigidos a futuras estrategias de mejoramiento genético.

  12. Saccharomyces cerevisiae Bat1 and Bat2 aminotransferases have functionally diverged from the ancestral-like Kluyveromyces lactis orthologous enzyme.

    Directory of Open Access Journals (Sweden)

    Maritrini Colón

    Full Text Available BACKGROUND: Gene duplication is a key evolutionary mechanism providing material for the generation of genes with new or modified functions. The fate of duplicated gene copies has been amply discussed and several models have been put forward to account for duplicate conservation. The specialization model considers that duplication of a bifunctional ancestral gene could result in the preservation of both copies through subfunctionalization, resulting in the distribution of the two ancestral functions between the gene duplicates. Here we investigate whether the presumed bifunctional character displayed by the single branched chain amino acid aminotransferase present in K. lactis has been distributed in the two paralogous genes present in S. cerevisiae, and whether this conservation has impacted S. cerevisiae metabolism. PRINCIPAL FINDINGS: Our results show that the KlBat1 orthologous BCAT is a bifunctional enzyme, which participates in the biosynthesis and catabolism of branched chain aminoacids (BCAAs. This dual role has been distributed in S. cerevisiae Bat1 and Bat2 paralogous proteins, supporting the specialization model posed to explain the evolution of gene duplications. BAT1 is highly expressed under biosynthetic conditions, while BAT2 expression is highest under catabolic conditions. Bat1 and Bat2 differential relocalization has favored their physiological function, since biosynthetic precursors are generated in the mitochondria (Bat1, while catabolic substrates are accumulated in the cytosol (Bat2. Under respiratory conditions, in the presence of ammonium and BCAAs the bat1Δ bat2Δ double mutant shows impaired growth, indicating that Bat1 and Bat2 could play redundant roles. In K. lactis wild type growth is independent of BCAA degradation, since a Klbat1Δ mutant grows under this condition. CONCLUSIONS: Our study shows that BAT1 and BAT2 differential expression and subcellular relocalization has resulted in the distribution of the

  13. Root hair mutants of barley

    International Nuclear Information System (INIS)

    Barley mutants without root hairs or with short or reduced root hairs were isolated among M2 seeds of 'Lux' barley (Hordeum vulgare L.) after acidified sodium azide mutagenesis. Root hair mutants are investigated intensively in Arabidopsis where about 40 genes are known. A few root hair mutants are known in maize, rice, barley and tomato. Many plants without root hairs grow quite well with good plant nutrition, and mutants have been used for investigations of uptake of strongly bound nutrients like phosphorus, iron, zinc and silicon. Seed of 'Lux' barley (Sejet Plant Breeding, Denmark) were soaked overnight, and then treated with 1.5-millimolarsodium azide in 0.1 molar sodium phosphate buffer, pH 3, for 2.5 hours according to the IAEA Manual on Mutation Breeding (2nd Ed.). After rinsing in tap water and air-drying, the M2 seeds were sown in the field the same day. Spikes, 4-6 per M1 plant, were harvested. The mutation frequency was similar to that obtained with other barley cultivars from which low-phytate mutants were isolated [5]. Seeds were germinated on black filter paper in tap water for 3 or 4 days before scoring for root hair mutants

  14. Mutants of alfalfa mosaic virus

    International Nuclear Information System (INIS)

    In this thesis the isolation and characterization of a number of mutants of alfalfa mosaic virus, a plant virus with a coat protein dependent genome, is described. Thermo-sensitive (ts) mutants were selected since, at least theoretically, ts mutations can be present in all virus coded functions. It was found that a high percentage of spontaneous mutants, isolated because of their aberrant symptoms, were ts. The majority of these isolates could grow at the non-permissive temperature in the presence of a single wild type (wt) component. To increase the mutation rate virus preparations were treated with several mutagens. After nitrous acid treatment or irradiation with ultraviolet light, an increase in the level of mutations was observed. UV irradiation was preferred since it did not require large amounts of purified viral components. During the preliminary characterization of potential ts mutants the author also obtained one structural and several symptom mutants which were analysed further (chapter 7, 8 and 9). The properties of the ts mutants are described in chapter 3-7. (Auth.)

  15. Mutant strains of Pichia pastoris with enhanced secretion of recombinant proteins.

    Science.gov (United States)

    Larsen, Sasha; Weaver, Jun; de Sa Campos, Katherine; Bulahan, Rhobe; Nguyen, Jackson; Grove, Heather; Huang, Amy; Low, Lauren; Tran, Namphuong; Gomez, Seth; Yau, Jennifer; Ilustrisimo, Thomas; Kawilarang, Jessica; Lau, Jonathan; Tranphung, Maivi; Chen, Irene; Tran, Christina; Fox, Marcia; Lin-Cereghino, Joan; Lin-Cereghino, Geoff P

    2013-11-01

    Although Pichia pastoris is a popular protein expression system, it exhibits limitations in its ability to secrete heterologous proteins. Therefore, a REMI (restriction enzyme mediated insertion) strategy was utilized to select mutant beta-g alactosidase s upersecretion (bgs) strains that secreted increased levels of a β-galactosidase reporter. Many of the twelve BGS genes may have functions in intracellular signaling or vesicle transport. Several of these strains also appeared to contain a more permeable cell wall. Preliminary characterization of four bgs mutants showed that they differed in the ability to enhance the export of other reporter proteins. bgs13, which has a disruption in a gene homologous to Saccharomyces cerevisiae protein kinase C (PKC1), gave enhanced secretion of most recombinant proteins that were tested, raising the possibility that it has the universal super-secreter phenotype needed in an industrial production strain of P. pastoris. PMID:23881328

  16. Improved ethanol fermentation of a yeast mutant by C-12 ion beam irradiation

    International Nuclear Information System (INIS)

    The yeast Saccharomyces cerevisiae YY was irradiated with 100 MeV/u 12C6+ ion beams. After screening,we obtained the mutant strain C03A of high ethanol yield. The influence of fermentation temperature, pH and concentration of sugar on ethanol fermentation were studied. The range analysis and analysis of variance were applied for the result of orthogonal experiments. The optimal ethanol fermentation conditions are: fermentation temperature 35 degree C, pH value 5.0, and sugar concentration 24%. The results of fermentation in the 10 L bioreactor showed that the ethanol fermentation of the mutant strain could be completed in 36 hours, the production of ethanol was to 13.2%(V/V), which means 12 hours faster and 1.6%(V /V) ethanol yield higher than original strain. (authors)

  17. Genes regulation encoding ADP/ATP carrier in yeasts Saccharomyces cerevisiae and Candida parapsilosis

    International Nuclear Information System (INIS)

    Genes encoding a mitochondrial ADP/ATP carrier (AAC) in yeast Saccharomyces cerevisiae and Candida parapsilosis were investigated. AAC2 is coding for the major AAC isoform in S. cerevisiae. We suggest that AAC2 is a member of a syn-expression group of genes encoding oxidative phosphorylation proteins. Within our previous studies on the regulation of the AAC2 transcription an UAS (-393/-268) was identified that is essential for the expression of this gene. Two functional regulatory cis-elements are located within this UAS -binding sites for an ABFl factor and for HAP2/3/4/5 heteromeric complex. We examined relative contributions and mutual interactions of the ABFl and HAP2/3/4/5 factors in the activation of transcription from the UAS of the AAC2 gene. The whole UAS was dissected into smaller sub-fragments and tested for (i) the ability to form DNA-protein complexes with cellular proteins in vitro, (ii) the ability to confer heterologous expression using AAC3 gene lacking its own promoter, and (iii) the expression of AAC3-lacZ fusion instead of intact AAC3 gene. The obtained results demonstrated that: a) The whole UAS as well as sub-fragment containing only ABF1-binding site are able to form DNA-protein complexes with cellular proteins in oxygen- and heme- dependent manner. The experiments with antibody against the ABF1 showed that the ABF1 factor is one of the proteins binding to AAC2 promoter. We have been unsuccessful to prove the binding of cellular proteins to the HAP2/3/4/5-binding site. However, the presence of HAP2/3/4/5-binding site is necessary to drive a binding of cellular proteins to the ABF1-binding site in carbon source-dependent manner. b) The presence of both ABF1- and HAP2/3/4/5-binding sites and original spacing between them is necessary to confer the growth of Aaac2 mutant strain on non- fermentable carbon source when put in front of AAC3 gene introduced on centromeric vector to Aaac2 mutant strain. c) For the activation of AAC3-lacZ expression on

  18. A genome-wide imaging-based screening to identify genes involved in synphilin-1 inclusion formation in Saccharomyces cerevisiae

    Science.gov (United States)

    Zhao, Lei; Yang, Qian; Zheng, Ju; Zhu, Xuefeng; Hao, Xinxin; Song, Jia; Lebacq, Tom; Franssens, Vanessa; Winderickx, Joris; Nystrom, Thomas; Liu, Beidong

    2016-01-01

    Synphilin-1 is a major component of Parkinson’s disease (PD) inclusion bodies implicated in PD pathogenesis. However, the machinery controlling synphilin-1 inclusion formation remains unclear. Here, we investigated synphilin-1 inclusion formation using a systematic genome-wide, high-content imaging based screening approach (HCI) in the yeast Saccharomyces cerevisiae. By combining with a secondary screening for mutants showing significant changes on fluorescence signal intensity, we filtered out hits that significantly decreased the expression level of synphilin-1. We found 133 yeast genes that didn’t affect synphilin-1 expression but that were required for the formation of synphilin-1 inclusions. Functional enrichment and physical interaction network analysis revealed these genes to encode for functions involved in cytoskeleton organization, histone modification, sister chromatid segregation, glycolipid biosynthetic process, DNA repair and replication. All hits were confirmed by conventional microscopy. Complementation assays were performed with a selected group of mutants, results indicated that the observed phenotypic changes in synphilin-1 inclusion formation were directly caused by the loss of corresponding genes of the deletion mutants. Further growth assays of these mutants showed a significant synthetic sick effect upon synphilin-1 expression, which supports the hypothesis that matured inclusions represent an end stage of several events meant to protect cells against the synphilin-1 cytotoxicity. PMID:27440388

  19. Probing glycolytic and membrane potential oscillations in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Poulsen, Allan K.; Andersen, Ann Zahle; Brasen, Jens Christian; Scharff-Poulsen, Anne Marie; Olsen, Lars Folke

    2008-01-01

    We have investigated glycolytic oscillations under semi-anaerobic conditions in Saccharomyces cerevisiae by means of NADH fluorescence, measurements of intracellular glucose concentration, and mitochondrial membrane potential. The glucose concentration was measured using an optical nanosensor...

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

    DEFF Research Database (Denmark)

    Scalcinati, Gionata; Otero, José Manuel; Van Vleet, Jennifer R. H.;

    2012-01-01

    flux to biomass production. Such a platform may then be enhanced with complementary metabolic engineering strategies that couple biomass production with high value-added chemical. Saccharomyces cerevisiae, expressing xylose reductase, xylitol dehydrogenase and xylulose kinase, from the native xylose...

  1. Biosorption of 241Am by immobilized Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Americium-241 is one of the most serious radioactive contaminating nuclides due to its high toxicity and long half-life. The encouraging biosorption of 241Am from aqueous solutions by free Saccharomyces cerevisiae (S. cerevisiae) has been observed in our previous experiments. 241Am biosorption by immobilized S. cerevisiae and the effect of the various experimental conditions on the adsorption were investigated. The results indicated that the 241Am biosorption by immobilized S. cerevisiae is still very efficient, and immobilized S. cerevisiae can be used repeatedly or continuously. The biosorption equilibrium was achieved within 2 hours, and more than 92% of 241Am was removed by immobilized S. cerevisiae in the pH 1-4 range. No significant differences in 241Am biosorption were observed at 15-45 deg C. The immobilized S. cerevisiae, even after used repeatedly for 6 times, still could adsorb more than 90% of 241Am in solutions of 1.08 MBq/l (8.5 μg/l). At this moment, the total adsorption capacity for 241Am was more than 63.3 KBq/g globe (0.5 μg/g), but has not reached saturation yet. The 241Am left in solutions with initial concentration of 1.08 MBq/l (8.5 μg/l) was noted as low as ∼10 Bq/l (∼8.0 x 10-5 μg/l) after adsorption by the immobilized S. cerevisiae for 3 times. (author)

  2. Research on biosorption of uranium by saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The effects of pH and the granularity of S. cerevisiae on the biosorption capacity were examined in order to study the properties of the biosorption of uranium from effluent by Saccharomyces cerevisiae. The isotherm was drawn. From the isotherm, the equations of Langmuir and Freundlich were achieved. The results showed the highest biosorption capacity was obtained when the pH value was about 6 and the granularity was 0.15-0.13 mm

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

    DEFF Research Database (Denmark)

    Rødkaer, Steven V; Færgeman, Nils J.

    2014-01-01

    steps and by numerous different regulators. As numerous of these regulating proteins, biochemical mechanisms, and cellular pathways are evolutionary conserved, complex biochemical information relevant to humans can be obtained by studying simple organisms. Thus, the yeast Saccharomyces cerevisiae has...... been recognized as a powerful model system to study fundamental biochemical processes. In the present review, we highlight central signaling pathways and molecular circuits conferring nitrogen- and glucose sensing in S. cerevisiae....

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

    OpenAIRE

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

    1985-01-01

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

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

    OpenAIRE

    Hana Šuranská; Dana Vránová; Jiřina Omelková

    2016-01-01

    Abstract In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typ...

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

    OpenAIRE

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

    In the present work we isolated and identified various indigenous Saccharomyces cerevisiae strains and screened them for the selected oenological properties. These S. cerevisiae strains were isolated from berries and spontaneously fermented musts. The grape berries (Sauvignon blanc and Pinot noir) were grown under the integrated and organic mode of farming in the South Moravia (Czech Republic) wine region. Modern genotyping techniques such as PCR-fingerprinting and interdelta PCR typing were ...

  7. The effect of heavy ion on DNA damage checkpoint, and the discovery of its sensitizing compounds

    International Nuclear Information System (INIS)

    We demonstrated that Schisandrin B (SchB) and persimmon leaf extract (PLE) inhibited activation of checkpoint signaling molecule of cancer cells by heavy ion radiation. Therefore, SchB and PLE raise the possibility of sensitizing agents for heavy ion cancer therapy. In the present study, we examined whether SchB and PLE can sensitize the effect of heavy ion on tumor growth by using xenograft study. The oral administration of SchB and PLE for 31 days did not clearly reduce a volume of tumor in Scid mice transplanted A549 human adenocarcinoma cells. Tumor growth was obviously inhibited by the irradiation of heavy ion (C290 MeV/u, spread-out Bragg peak (SOBP)). SchB and PLE reduced the tumor volume in 3 Gy heavy ion exposed mice, respectively. Similarly, we examined whether SchB and PLE can sensitize the effect of X-ray also. SchB did not sensitize the effect of X-ray. However, PLE reduced the tumor volume in 6 Gy X-ray exposed mice. These results indicated that tumor volume in heavy ion exposed mice was inhibited by SchB and PLE treatment through the inhibition of DNA damage checkpoint signaling pathway. On the other hand, SchB could not sensitize the effect of X-ray on tumor growth. These results suggest that the modulating effects of SchB and PLE on DNA damage checkpoint are targeting independent molecules. These mechanisms should be clarified in further study. (author)

  8. Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

    Directory of Open Access Journals (Sweden)

    Chen Bo-Ruei

    2012-05-01

    Full Text Available Abstract Background Barcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques. Results An insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites. Conclusions This collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning

  9. Acute renal allograft rejection after immune checkpoint inhibitor therapy for metastatic melanoma.

    Science.gov (United States)

    Spain, L; Higgins, R; Gopalakrishnan, K; Turajlic, S; Gore, M; Larkin, J

    2016-06-01

    Immune checkpoint inhibitors such as ipilimumab and nivolumab improve survival in patients with advanced melanoma and are increasingly available to clinicians for use in the clinic. Their safety in organ transplant recipients is not well defined but published case reports describing treatment with ipilimumab have not been complicated by graft rejection. No cases of anti-programmed cell death protein 1 administration are reported in this group. We describe a case of acute graft rejection in a kidney transplant recipient after treatment with nivolumab, after progression on ipilimumab. Potential factors increasing the risk of graft rejection in this case are discussed, in particular the contribution of nivolumab. PMID:26951628

  10. Human Embryonic Stem Cells Are Capable of Executing G1/S Checkpoint Activation

    Czech Academy of Sciences Publication Activity Database

    Bárta, Tomáš; Vinarský, Vladimír; Holubcová, Z.; Doležalová, Dáša; Verner, J.; Pospíšilová, Š.; Dvořák, Petr; Hampl, Aleš

    2010-01-01

    Roč. 28, č. 7 (2010), s. 1143-1152. ISSN 1066-5099 Grant ostatní: GA MŠk(CZ) MSM0021622430; GA MŠk(CZ) 1M0538; GA MZd(CZ) NS10439; GA MŠk(CZ) MUNIE/E/0118/2009; EC FP6(XE) LSHG-CT-2006-018739 Institutional research plan: CEZ:AV0Z50390512; CEZ:AV0Z50390703 Keywords : human embryonic stem cells * DNA damage * checkpoint activation Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.871, year: 2010

  11. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair

    DEFF Research Database (Denmark)

    Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw;

    2005-01-01

    The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination......, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51...

  12. Transparent Checkpoint-Restart for Hardware-Accelerated 3D Graphics

    OpenAIRE

    Nafchi, Samaneh Kazemi; Garg, Rohan; Cooperman, Gene

    2013-01-01

    Providing fault-tolerance for long-running GPU-intensive jobs requires application-specific solutions, and often involves saving the state of complex data structures spread among many graphics libraries. This work describes a mechanism for transparent GPU-independent checkpoint-restart of 3D graphics. The approach is based on a record-prune-replay paradigm: all OpenGL calls relevant to the graphics driver state are recorded; calls not relevant to the internal driver state as of the last graph...

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

    Science.gov (United States)

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

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

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

    Science.gov (United States)

    Šuranská, Hana; Vránová, Dana; Omelková, Jiřina

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hana Šuranská

    2016-03-01

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

  16. Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration

    International Nuclear Information System (INIS)

    Research highlights: → COQ10 deletion elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q2, a synthetic diffusible ubiquinone. → The significance that purified Coq10p contains bound Q6 was examined by testing over-expression of Coq10p on respiration. → Inhibition of CoQ function due to Coq10p excess strength our hypothesis of Coq10p function in CoQ delivery. → Respiratory deficiency caused by more Coq10p was specific and restored by Q2 in mitochondria or by Coq8p in cells. → Coq8p over-production on other coq mutants revealed a surprisingly higher stability of other Coq proteins. -- Abstract: COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q2. Rescue of respiration by Q2 is a characteristic of mutants blocked in coenzyme Q6 synthesis. Unlike Q6 deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q6. The physiological significance of earlier observations that purified Coq10p contains bound Q6 was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q2. This suggests that in vivo binding of Q6 by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains over-producing Coq10p.

  17. Over-expression of COQ10 in Saccharomyces cerevisiae inhibits mitochondrial respiration

    Energy Technology Data Exchange (ETDEWEB)

    Zampol, Mariana A.; Busso, Cleverson; Gomes, Fernando [Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo (Brazil); Ferreira-Junior, Jose Ribamar [Escola de Artes, Ciencias e Humanidades, Universidade de Sao Paulo, Sao Paulo (Brazil); Tzagoloff, Alexander [Department of Biological Sciences, Columbia University, NY (United States); Barros, Mario H., E-mail: mariohb@usp.br [Departamento de Microbiologia, Instituto de Ciencias Biomedicas, Universidade de Sao Paulo, Sao Paulo (Brazil)

    2010-11-05

    Research highlights: {yields} COQ10 deletion elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}, a synthetic diffusible ubiquinone. {yields} The significance that purified Coq10p contains bound Q{sub 6} was examined by testing over-expression of Coq10p on respiration. {yields} Inhibition of CoQ function due to Coq10p excess strength our hypothesis of Coq10p function in CoQ delivery. {yields} Respiratory deficiency caused by more Coq10p was specific and restored by Q{sub 2} in mitochondria or by Coq8p in cells. {yields} Coq8p over-production on other coq mutants revealed a surprisingly higher stability of other Coq proteins. -- Abstract: COQ10 deletion in Saccharomyces cerevisiae elicits a defect in mitochondrial respiration correctable by addition of coenzyme Q{sub 2}. Rescue of respiration by Q{sub 2} is a characteristic of mutants blocked in coenzyme Q{sub 6} synthesis. Unlike Q{sub 6} deficient mutants, mitochondria of the coq10 null mutant have wild-type concentrations of Q{sub 6}. The physiological significance of earlier observations that purified Coq10p contains bound Q{sub 6} was examined in the present study by testing the in vivo effect of over-expression of Coq10p on respiration. Mitochondria with elevated levels of Coq10p display reduced respiration in the bc1 span of the electron transport chain, which can be restored with exogenous Q{sub 2}. This suggests that in vivo binding of Q{sub 6} by excess Coq10p reduces the pool of this redox carrier available for its normal function in providing electrons to the bc1 complex. This is confirmed by observing that extra Coq8p relieves the inhibitory effect of excess Coq10p. Coq8p is a putative kinase, and a high-copy suppressor of the coq10 null mutant. As shown here, when over-produced in coq mutants, Coq8p counteracts turnover of Coq3p and Coq4p subunits of the Q-biosynthetic complex. This can account for the observed rescue by COQ8 of the respiratory defect in strains

  18. Expression and purification of correctly processed, active human TACE catalytic domain in Saccharomyces cerevisiae.

    Science.gov (United States)

    Clarke, H R; Wolfson, M F; Rauch, C T; Castner, B J; Huang, C P; Gerhart, M J; Johnson, R S; Cerretti, D P; Paxton, R J; Price, V L; Black, R A

    1998-06-01

    Human tumor necrosis factor-alpha (TNF alpha) converting enzyme (TACE) releases soluble TNF alpha from cells. It is a member of the adamalysin family of metalloproteases. A truncated form of TACE cDNA was expressed in Saccharomyces cerevisiae and purified to homogeneity in order to study TACE structure and function. Recombinant TACE was expressed as a preproprotein including the pro- and catalytic (PROCAT) domains fused to the yeast alpha-factor leader. A C-terminal immunoreactive FLAG peptide was added for Western blot detection and anti-FLAG antibody column purification. We constructed two glycosylation mutant PROCAT TACE isoforms to facilitate purification. A PROCAT isoform, mutated to eliminate two N-linked glycosylation sites, was buffer exchanged and purified to homogeneity by ion exchange chromatography and an anti-FLAG antibody affinity step. N-terminal sequence analysis showed that the mutant preproprotein was processed in yeast at the furin protease cleavage site and yielded an active catalytic domain which has TNF alpha peptide-specific protease activity. Mass spectrometry of the purified catalytic domain showed that removal of both N-linked sites results in a homogeneous sized polypeptide lacking further posttranslational modifications. PMID:9631522

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

    Science.gov (United States)

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

    2016-06-01

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

  20. N-acetyltransferase Mpr1 confers ethanol tolerance on Saccharomyces cerevisiae by reducing reactive oxygen species

    Energy Technology Data Exchange (ETDEWEB)

    Du, Xiaoyi [Fukui Prefectural Univ., Fukui (Japan). Dept. of Bioscience; Takagi, Hiroshi [Nara Inst. of Science and Technology, Ikoma, Nara (Japan). Graduate School of Biological Sciences

    2007-07-15

    N-Acetyltransferase Mpr1 of Saccharomyces cerevisiae can reduce intracellular oxidation levels and protect yeast cells under oxidative stress, including H{sub 2}O{sub 2}, heat-shock, or freeze-thaw treatment. Unlike many antioxidant enzyme genes induced in response to oxidative stress, the MPR1 gene seems to be constitutively expressed in yeast cells. Based on a recent report that ethanol toxicity is correlated with the production of reactive oxygen species (ROS), we examined here the role of Mpr1 under ethanol stress conditions. The null mutant of the MPR1 and MPR2 genes showed hypersensitivity to ethanol stress, and the expression of the MPR1 gene conferred stress tolerance. We also found that yeast cells exhibited increased ROS levels during exposure to ethanol stress, and that Mpr1 protects yeast cells from ethanol stress by reducing intracellular ROS levels. When the MPR1 gene was overexpressed in antioxidant enzyme-deficient mutants, increased resistance to H{sub 2}O{sub 2} or heat shock was observed in cells lacking the CTA1, CTT1, or GPX1 gene encoding catalase A, catalase T, or glutathione peroxidase, respectively. These results suggest that Mpr1 might compensate the function of enzymes that detoxify H{sub 2}O{sub 2}. Hence, Mpr1 has promising potential for the breeding of novel ethanol-tolerant yeast strains. (orig.)

  1. Histone H3K36 methylation regulates pre-mRNA splicing in Saccharomyces cerevisiae.

    Science.gov (United States)

    Sorenson, Matthew R; Jha, Deepak K; Ucles, Stefanie A; Flood, Danielle M; Strahl, Brian D; Stevens, Scott W; Kress, Tracy L

    2016-04-01

    Co-transcriptional splicing takes place in the context of a highly dynamic chromatin architecture, yet the role of chromatin restructuring in coordinating transcription with RNA splicing has not been fully resolved. To further define the contribution of histone modifications to pre-mRNA splicing in Saccharomyces cerevisiae, we probed a library of histone point mutants using a reporter to monitor pre-mRNA splicing. We found that mutation of H3 lysine 36 (H3K36) - a residue methylated by Set2 during transcription elongation - exhibited phenotypes similar to those of pre-mRNA splicing mutants. We identified genetic interactions between genes encoding RNA splicing factors and genes encoding the H3K36 methyltransferase Set2 and the demethylase Jhd1 as well as point mutations of H3K36 that block methylation. Consistent with the genetic interactions, deletion of SET2, mutations modifying the catalytic activity of Set2 or H3K36 point mutations significantly altered expression of our reporter and reduced splicing of endogenous introns. These effects were dependent on the association of Set2 with RNA polymerase II and H3K36 dimethylation. Additionally, we found that deletion of SET2 reduces the association of the U2 and U5 snRNPs with chromatin. Thus, our study provides the first evidence that H3K36 methylation plays a role in co-transcriptional RNA splicing in yeast. PMID:26821844

  2. Molecular mechanisms of pyrimidine dimer excision in Saccharomyces cerevisiae: incision of ultraviolet-irradiated deoxyribonucleic acid in vivo

    International Nuclear Information System (INIS)

    A group of genetically related ultraviolet (uv)-sensitive mutants of Saccharomyces cerevisiae has been examined in terms of their survival after exposure to uv radiation, their ability to carry out excision repair or pyrimidine dimers as measured by the loss of sites (pyrimidine dimers) sensitive to a dimer-specific enzyme probe, and in terms of their ability to effect incision of their deoxyribonucleic acid (DNA) during post-uv incubation in vivo (as measured by the detection of single-strand breaks in nuclear DNA). In addition to a haploid RAD+ strain (S288C), 11 different mutants representing six RAD loci (RAD1, RAD2, RAD3, RAD4, RAD14, and RAD18) were examined. Quantitative analysis of excision repair capacity, as determined by the loss of sites in DNA sensitive to an enzyme preparation from M. luteus which is specific for pyrimidine dimers, revealed a profound defect in this parameter in all but three of the strains examined. The rad14-1 mutant showed reduced but significant residual capacity to remove enzyme-sensitive sites as did the rad2-4 mutant. The latter was the only one of three different rad2 alleles examined which was leaky in this respect. The uv-sensitive strain carrying the mutant allele rad18-1 exhibited normal loss of enzyme-sensitive sites consistent with its assignment to the RAD6 rather than the RAD3 epistatic group. All strains having mutant alleles of the RAD1, RAD2, RAD3, RAD4, and RAD14 loci showed no detectable incubation-dependent strand breaks in nuclear DNA after exposure to uv radiation. These experiments suggest that the RAD1, RAD2, RAD3, RAD4 (and probably RAD14) genes are all required for the incision of uv-irradiated DNA during pyrimidine dimer excision in vivo

  3. DNA damage-induced metaphase I arrest is mediated by the spindle assembly checkpoint and maternal age.

    Science.gov (United States)

    Marangos, Petros; Stevense, Michelle; Niaka, Konstantina; Lagoudaki, Michaela; Nabti, Ibtissem; Jessberger, Rolf; Carroll, John

    2015-01-01

    In mammalian oocytes DNA damage can cause chromosomal abnormalities that potentially lead to infertility and developmental disorders. However, there is little known about the response of oocytes to DNA damage. Here we find that oocytes with DNA damage arrest at metaphase of the first meiosis (MI). The MI arrest is induced by the spindle assembly checkpoint (SAC) because inhibiting the SAC overrides the DNA damage-induced MI arrest. Furthermore, this MI checkpoint is compromised in oocytes from aged mice. These data lead us to propose that the SAC is a major gatekeeper preventing the progression of oocytes harbouring DNA damage. The SAC therefore acts to integrate protection against both aneuploidy and DNA damage by preventing production of abnormal mature oocytes and subsequent embryos. Finally, we suggest escaping this DNA damage checkpoint in maternal ageing may be one of the causes of increased chromosome anomalies in oocytes and embryos from older mothers. PMID:26522734

  4. Mutant Varieties of Crop Plants

    International Nuclear Information System (INIS)

    Since 1969, the Joint PAO/IAEA Division undertakes to collect and publish information on varieties of crop plants that were developed directly from induced mutants or by using mutants in cross breeding (Micke 1972 and Sigurbjörnsson and Micke (1969, 1974). The purpose of this undertaking is to assess realistically the potential of induced mutation techniques to contribute towards progress in plant breeding. Varieties which have successfully passed official trials and were approved or recommended by national governmental authorities for cultivation, appear to be good indicators of practical success. By 1 October 1978, we know about 195 of such varieties in agricultural crop plants). They belong to 37 different plant species and come from 30 different countries. In addition, there are more than 120 mutant cultivars of ornamental plants known, which represent a considerable economic value for countries with developed horticulture (Broertjes and van Harten 1978)

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

    DEFF Research Database (Denmark)

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

    2005-01-01

    nontumorigenic porcine jejunal epithelial cell line (IPEC-J2) was investigated by incorporation of H-3-methionine into the yeast cells and use of liquid scintillation counting. Only few of the food-borne S. cerevisiae strains exhibited noteworthy adhesiveness with the strongest levels of adhesion (13...... effects hence indicating that food-borne strains of S. cerevisiae may possess probiotic properties in spite of low adhesiveness. © 2004 Elsevier B.V. All rights reserved....

  6. Crystal structure of Saccharomyces cerevisiae 6-phosphogluconate dehydrogenase Gnd1

    Directory of Open Access Journals (Sweden)

    Zhou Cong-Zhao

    2007-06-01

    Full Text Available Abstract Background As the third enzyme of the pentose phosphate pathway, 6-phosphogluconate dehydrogenase (6PGDH is the main generator of cellular NADPH. Both thioredoxin reductase and glutathione reductase require NADPH as the electron donor to reduce oxidized thioredoxin or glutathione (GSSG. Since thioredoxin and GSH are important antioxidants, it is not surprising that 6PGDH plays a critical role in protecting cells from oxidative stress. Furthermore the activity of 6PGDH is associated with several human disorders including cancer and Alzheimer's disease. The 3D structural investigation would be very valuable in designing small molecules that target this enzyme for potential therapeutic applications. Results The crystal structure of 6-phosphogluconate dehydrogenase (6PGDH/Gnd1 from Saccharomyces cerevisiae has been determined at 2.37 Å resolution by molecular replacement. The overall structure of Gnd1 is a homodimer with three domains for each monomer, a Rossmann fold NADP+ binding domain, an all-α helical domain contributing the majority to hydrophobic interaction between the two subunits and a small C-terminal domain penetrating the other subunit. In addition, two citrate molecules occupied the 6PG binding pocket of each monomer. The intact Gnd1 had a Km of 50 ± 9 μM for 6-phosphogluconate and of 35 ± 6 μM for NADP+ at pH 7.5. But the truncated mutants without the C-terminal 35, 39 or 53 residues of Gnd1 completely lost their 6PGDH activity, despite remaining the homodimer in solution. Conclusion The overall tertiary structure of Gnd1 is similar to those of 6PGDH from other species. The substrate and coenzyme binding sites are well conserved, either from the primary sequence alignment, or from the 3D structural superposition. Enzymatic activity assays suggest a sequential mechanism of catalysis, which is in agreement with previous studies. The C-terminal domain of Gnd1 functions as a hook to further tighten the dimer, but it is not

  7. Low doses effects of ionizing radiation on Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The exposure of living cells to low doses of ionizing radiation induce in response the activation of cellular protection mechanisms against subsequent larger doses of radiation. This cellular adaptive response may vary depending on radiation intensity and time of exposure, and also on the testing probes used whether they were mammalian cells, yeast, bacteria and other organisms or cell types. The mechanisms involved are the genome activation, followed by DNA repair enzymes synthesis. Due to the prompt cell response, the cell cycle can be delayed, and the secondary detoxification of free radicals and/or activation of membrane bound receptors may proceed. All these phenomena are submitted to intense scientific research nowadays, and their elucidation will depend on the complexity of the organism under study. In the present work, the effects of low doses of ionizing radiation (gamma rays) over a suspension of the yeast Saccharomyces cerevisiae (Baker's yeast) was studied, mainly in respect to survival rate and radio-adaptive response. At first, the yeast surviving curve was assessed towards increasing doses, and an estimation of Lethal Dose 50 (LD50) was made. The irradiation tests were performed at LINAC (electrons Linear Accelerator) where electron energy reached approximately 2.65 MeV, and gamma-radiation was produced for bremsstrahlung process over an aluminium screen target. A series of experiments of conditioning doses was performed and an increment surviving fraction was observed when the dose was 2.3 Gy and a interval time between this and a higher dose (challenging dose) of 27 Gy was 90 minutes. A value of 58 ± 4 Gy was estimated for LD50, at a dose rate of 0.44 ± 0.03 Gy/min These quantities must be optimized. Besides data obtained over yeast survival, an unusual increasing amount of tiny yeast colonies appeared on the agar plates after incubation, and this number increased as increasing the time exposure. Preliminary results indicate these colonies as

  8. The F-box protein Rcy1p is involved in endocytic membrane traffic and recycling out of an early endosome in Saccharomyces cerevisiae.

    Science.gov (United States)

    Wiederkehr, A; Avaro, S; Prescianotto-Baschong, C; Haguenauer-Tsapis, R; Riezman, H

    2000-04-17

    In Saccharomyces cerevisiae, endocytic material is transported through different membrane-bound compartments before it reaches the vacuole. In a screen for mutants that affect membrane trafficking along the endocytic pathway, we have identified a novel mutant disrupted for the gene YJL204c that we have renamed RCY1 (recycling 1). Deletion of RCY1 leads to an early block in the endocytic pathway before the intersection with the vacuolar protein sorting pathway. Mutation of RCY1 leads to the accumulation of an enlarged compartment that contains the t-SNARE Tlg1p and lies close to areas of cell expansion. In addition, endocytic markers such as Ste2p and the fluorescent dyes, Lucifer yellow and FM4-64, were found in a similar enlarged compartment after their internalization. To determine whether rcy1Delta is defective for recycling, we have developed an assay that measures the recycling of previously internalized FM4-64. This method enables us to follow the recycling pathway in yeast in real time. Using this assay, it could be demonstrated that recycling of membranes is rapid in S. cerevisiae and that a major fraction of internalized FM4-64 is secreted back into the medium within a few minutes. The rcy1Delta mutant is strongly defective in recycling. PMID:10769031

  9. The Saccharomyces cerevisiae RAD30 gene, a homologue of Escherichia coli dinB and umuC, is DNA damage inducible and functions in a novel error-free postreplication repair mechanism

    International Nuclear Information System (INIS)

    Damage-inducible mutagenesis in prokaryotes is largely dependent upon the activity of the UmuD'C-like proteins. Since many DNA repair processes are structurally and/or functionally conserved between prokaryotes and eukaryotes, we investigated the role of RAD30, a previously uncharacterized Saccharomyces cerevisiae DNA repair gene related to the Escherichia coli dinB, umuC and S. cerevisiae REV1 genes, in UV resistance and UV-induced mutagenesis. Similar to its prokaryotic homologues, RAD30 was found to be damage inducible. Like many S. cerevisiae genes involved in error-prone DNA repair, epistasis analysis clearly places RAD30 in the RAD6 group and rad30 mutants display moderate UV sensitivity reminiscent of rev mutants. However, unlike rev mutants, no defect in UV-induced reversion was seen in rad30 strains. While rad6 and rad18 are both epistatic to rad30, no epistasis was observed with rev1, rev3, rev7 or rad5, all of which are members of the RAD6 epistasis group. These findings suggest that RD30 participates in a novel error-free repair pathway dependent on RAD6 and RAD18, but independent of REV1, REV3, REV7 and RAD5. (author)

  10. The Geography of Deterrence: Exploring the Small Area Effects of Sobriety Checkpoints on Alcohol-Impaired Collision Rates within a City

    Science.gov (United States)

    Nunn, Samuel; Newby, William

    2011-01-01

    This article examines alcohol-impaired collision metrics around nine sobriety checkpoint locations in Indianapolis, Indiana, before and after implementation of 22 checkpoints, using a pre/post examination, a pre/post nonequivalent comparison group analysis, and an interrupted time series approach. Traffic safety officials used geographical…

  11. Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1

    Science.gov (United States)

    Breit, Claudia; Bange, Tanja; Petrovic, Arsen; Weir, John R.; Müller, Franziska; Vogt, Doro; Musacchio, Andrea

    2015-01-01

    The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint. PMID:26658523

  12. Tumor-suppressor genes, cell cycle regulatory checkpoints, and the skin

    Directory of Open Access Journals (Sweden)

    Ana Maria Abreu Velez

    2015-01-01

    Full Text Available The cell cycle (or cell-division cycle is a series of events that take place in a cell, leading to its division and duplication. Cell division requires cell cycle checkpoints (CPs that are used by the cell to both monitor and regulate the progress of the cell cycle. Tumor-suppressor genes (TSGs or antioncogenes are genes that protect the cell from a single event or multiple events leading to cancer. When these genes mutate, the cell can progress to a cancerous state. We aimed to perform a narrative review, based on evaluation of the manuscripts published in MEDLINE-indexed journals using the Medical Subject Headings (MeSH terms "tumor suppressor′s genes," "skin," and "cell cycle regulatory checkpoints." We aimed to review the current concepts regarding TSGs, CPs, and their association with selected cutaneous diseases. It is important to take into account that in some cell cycle disorders, multiple genetic abnormalities may occur simultaneously. These abnormalities may include intrachromosomal insertions, unbalanced division products, recombinations, reciprocal deletions, and/or duplication of the inserted segments or genes; thus, these presentations usually involve several genes. Due to their complexity, these disorders require specialized expertise for proper diagnosis, counseling, personal and family support, and genetic studies. Alterations in the TSGs or CP regulators may occur in many benign skin proliferative disorders, neoplastic processes, and genodermatoses.

  13. Anti-TNF-refractory colitis after checkpoint inhibitor therapy: Possible role of CMV-mediated immunopathogenesis.

    Science.gov (United States)

    Lankes, Katharina; Hundorfean, Gheorghe; Harrer, Thomas; Pommer, Ansgar J; Agaimy, Abbas; Angelovska, Irena; Tajmir-Riahi, Azadeh; Göhl, Jonas; Schuler, Gerold; Neurath, Markus F; Hohenberger, Werner; Heinzerling, Lucie

    2016-06-01

    Immune-related adverse events (irAEs) induced by checkpoint inhibitors are well known. Since fatal outcomes have been reported early detection and adequate management are crucial. In particular, colitis is frequently observed and can result in intestinal perforation. This is the first report of an autoimmune colitis that was treated according to algorithms but became resistant due to a CMV reactivation. The 32-y-old male patient with metastatic melanoma treated within an anti-PD-1/ipilimumab combination study developed severe immune-mediated colitis (CTCAE grade 3) with up to 18 watery stools per day starting 2 weeks after treatment initiation. After improving upon therapy with immunosuppressive treatment (high dose steroids and infliximab) combined with parenteral nutrition diarrhea again exacerbated. Additionally, the patient had asymptomatic grade 3 CTCAE amylase and lipase elevation. Colitis was monitored by weekly endoscopies and colon biopsies were analyzed histologically with CMV staining, multi-epitope ligand cartography (MELC) and qRT-PCR for inflammatory genes. In the course, CMV reactivation was detected in the colon and treated with antiviral medication in parallel to a reduction of corticosteroids. Subsequently, symptoms improved. The patient showed a complete response for 2 y now including regression of bone metastases. CMV reactivation under checkpoint inhibitor therapy in combination with immunosuppressive treatment for autoimmune side effects has to be considered in these patients and if present treated. Potentially, CMV reactivation is underdiagnosed. Treatment algorithms should include CMV diagnostics. PMID:27471608

  14. A2aR antagonists: Next generation checkpoint blockade for cancer immunotherapy

    Directory of Open Access Journals (Sweden)

    Robert D. Leone

    2015-01-01

    Full Text Available The last several years have witnessed exciting progress in the development of immunotherapy for the treatment of cancer. This has been due in great part to the development of so-called checkpoint blockade. That is, antibodies that block inhibitory receptors such as CTLA-4 and PD-1 and thus unleash antigen-specific immune responses against tumors. It is clear that tumors evade the immune response by usurping pathways that play a role in negatively regulating normal immune responses. In this regard, adenosine in the immune microenvironment leading to the activation of the A2a receptor has been shown to represent one such negative feedback loop. Indeed, the tumor microenvironment has relatively high concentrations of adenosine. To this end, blocking A2a receptor activation has the potential to markedly enhance anti-tumor immunity in mouse models. This review will present data demonstrating the ability of A2a receptor blockade to enhance tumor vaccines, checkpoint blockade and adoptive T cell therapy. Also, as several recent studies have demonstrated that under certain conditions A2a receptor blockade can enhance tumor progression, we will also explore the complexities of adenosine signaling in the immune response. Despite important nuances to the A2a receptor pathway that require further elucidation, studies to date strongly support the development of A2a receptor antagonists (some of which have already been tested in phase III clinical trials for Parkinson Disease as novel modalities in the immunotherapy armamentarium.

  15. Estimated Interval-Based Checkpointing (EIC on Spot Instances in Cloud Computing

    Directory of Open Access Journals (Sweden)

    Daeyong Jung

    2014-01-01

    Full Text Available In cloud computing, users can rent computing resources from service providers according to their demand. Spot instances are unreliable resources provided by cloud computing services at low monetary cost. When users perform tasks on spot instances, there is an inevitable risk of failures that causes the delay of task execution time, resulting in a serious deterioration of quality of service (QoS. To deal with the problem on spot instances, we propose an estimated interval-based checkpointing (EIC using weighted moving average. Our scheme sets the thresholds of price and execution time based on history. Whenever the actual price and the execution time cross over the thresholds, the system saves the state of spot instances. The Bollinger Bands is adopted to inform the ranges of estimated cost and execution time for user's discretion. The simulation results reveal that, compared to the HBC and REC, the EIC reduces the number of checkpoints and the rollback time. Consequently, the task execution time is decreased with EIC by HBC and REC. The EIC also provides the benefit of the cost reduction by HBC and REC, on average. We also found that the actual cost and execution time fall within the estimated ranges suggested by the Bollinger Bands.

  16. The impact of aviation checkpoint queues on optimizing security screening effectiveness

    International Nuclear Information System (INIS)

    Passenger screening at aviation security checkpoints is a critical component in protecting airports and aircraft from terrorist threats. Recent developments in screening device technology have increased the ability to detect these threats; however, the average amount of time it takes to screen a passenger still remains a concern. This paper models the queueing process for a multi-level airport checkpoint security system, where multiple security classes are formed through subsets of specialized screening devices. An optimal static assignment policy is obtained which minimizes the steady-state expected amount of time a passenger spends in the security system. Then, an optimal dynamic assignment policy is obtained through a transient analysis that balances the expected number of true alarms with the expected amount of time a passenger spends in the security system. Performance of a two-class system is compared to that of a selective security system containing primary and secondary levels of screening. The key contribution is that the resulting optimal assignment policies increase security and passenger throughput by efficiently and effectively utilizing available screening resources.

  17. Systematic Triple-Mutant Analysis Uncovers Functional Connectivity between Pathways Involved in Chromosome Regulation

    Directory of Open Access Journals (Sweden)

    James E. Haber

    2013-06-01

    Full Text Available Genetic interactions reveal the functional relationships between pairs of genes. In this study, we describe a method for the systematic generation and quantitation of triple mutants, termed triple-mutant analysis (TMA. We have used this approach to interrogate partially redundant pairs of genes in S. cerevisiae, including ASF1 and CAC1, two histone chaperones. After subjecting asf1Δ cac1Δ to TMA, we found that the Swi/Snf Rdh54 protein compensates for the absence of Asf1 and Cac1. Rdh54 more strongly associates with the chromatin apparatus and the pericentromeric region in the double mutant. Moreover, Asf1 is responsible for the synthetic lethality observed in cac1Δ strains lacking the HIRA-like proteins. A similar TMA was carried out after deleting both CLB5 and CLB6, cyclins that regulate DNA replication, revealing a strong functional connection to chromosome segregation. This approach can reveal functional redundancies that cannot be uncovered through traditional double-mutant analyses.

  18. Wild Accessions and Mutant Resources

    DEFF Research Database (Denmark)

    Kawaguchi, Masayoshi; Sandal, Niels Nørgaard

    2014-01-01

    Lotus japonicus, Lotus burttii, and Lotus filicaulis are species of Lotus genus that are utilized for molecular genetic analysis such as the construction of a linkage map and QTL analysis. Among them, a number of mutants have been isolated from two wild accessions: L. japonicus Gifu B-129 and Miy...

  19. Genomewide screening for genes associated with gliotoxin resistance and sensitivity in Saccharomyces cerevisiae.

    Science.gov (United States)

    Chamilos, Georgios; Lewis, Russell E; Lamaris, Gregory A; Albert, Nathaniel D; Kontoyiannis, Dimitrios P

    2008-04-01

    Gliotoxin (GT) is a secondary fungal metabolite with pleiotropic immunosuppressive properties that have been implicated in Aspergillus virulence. However, the mechanisms of GT cytotoxicity and its molecular targets in eukaryotic cells have not been fully characterized. We screened a haploid library of Saccharomyces cerevisiae single-gene deletion mutants (4,787 strains in EUROSCARF) to identify nonessential genes associated with GT increased resistance (GT-IR) and increased sensitivity (GT-IS). The susceptibility of the wild-type parental strain BY4741 to GT was initially assessed by broth microdilution methods using different media. GT-IR and GT-IS were defined as a fourfold increase and decrease, respectively, in MIC, and this was additionally confirmed by susceptibility testing on agar yeast extract-peptone-glucose plates. The specificity of GT-IR and GT-IS mutants exhibiting normal growth compared with the wild-type strain was further tested in studies of their susceptibility to conventional antifungal agents, cycloheximide, and H2O2. GT-IR was associated with the disruption of genes acting in general metabolism (OPI1, SNF1, IFA38), mitochondrial function (RTG2), DNA damage repair (RAD18), and vesicular transport (APL2) and genes of unknown function (YGL235W, YOR345C, YLR456W, YGL072C). The disruption of three genes encoding transsulfuration (CYS3), mitochondrial function (MEF2), and an unknown function (YKL037W) led to GT-IS. Specificity for GT-IR and GT-IS was observed in all mutants. Importantly, the majority (69%) of genes implicated in GT-IR (6/10) and GT-IS (2/3) have human homologs. We identified novel Saccharomyces genes specifically implicated in GT-IR or GT-IS. Because most of these genes are evolutionarily conserved, further characterization of their function could improve our understanding of GT cytotoxicity mechanisms in humans. PMID:18212113

  20. Repair of DNA double-strand breaks induced in Saccharomyces cerevisiae using different γ-ray dose rates: a pulsed-field gel electrophoresis analysis

    International Nuclear Information System (INIS)

    We investigated the effects of γ-ray exposures at high dose-rate (HDR, 23·2 Gy/min) and low dose-rate (LDR, 0·47 Gy/min) on survival and the induction of DNA double-strand breaks (dsb) in a diploid wild-type (D7) and the repair-deficient mutant strain rad52/rad52 of Saccharomyces cerevisiae. The relationship observed between γ-ray survival and dsb repair clearly indicates that increases in survival of wild-type cells, during LDR as compared with HDR exposures and after LHR, are strongly related to the repair of dsb. (author)

  1. MGA2 or SPT23 is required for transcription of the delta9 fatty acid desaturase gene, OLE1, and nuclear membrane integrity in Saccharomyces cerevisiae.

    OpenAIRE

    S. Zhang; Skalsky, Y; Garfinkel, D J

    1999-01-01

    MGA2 and SPT23 are functionally and genetically redundant homologs in Saccharomyces cerevisiae. Both genes are implicated in the transcription of a subset of genes, including Ty retrotransposons and Ty-induced mutations. Neither gene is essential for growth, but mga2 spt23 double mutants are inviable. We have isolated a gene-specific activator, SWI5, and the Delta9 fatty acid desaturase of yeast, OLE1, as multicopy suppressors of an mga2Delta spt23 temperature-sensitive mutation (spt23-ts). T...

  2. ATP25, a New Nuclear Gene of Saccharomyces cerevisiae Required for Expression and Assembly of the Atp9p Subunit of Mitochondrial ATPase

    OpenAIRE

    Zeng, Xiaomei; Barros, Mario H.; Shulman, Theodore; Tzagoloff, Alexander

    2008-01-01

    We report a new nuclear gene, designated ATP25 (reading frame YMR098C on chromosome XIII), required for expression of Atp9p (subunit 9) of the Saccharomyces cerevisiae mitochondrial proton translocating ATPase. Mutations in ATP25 elicit a deficit of ATP9 mRNA and of its translation product, thereby preventing assembly of functional F0. Unlike Atp9p, the other mitochondrial gene products, including ATPase subunits Atp6p and Atp8p, are synthesized normally in atp25 mutants. Northern analysis of...

  3. The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

    OpenAIRE

    Dang, V D; Bohn, C.; Bolotin-Fukuhara, M.; Daignan-Fornier, B

    1996-01-01

    In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on a...

  4. Biosynthesis of levan, a bacterial extracellular polysaccharide, in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Franken, Jaco; Brandt, Bianca A; Tai, Siew L; Bauer, Florian F

    2013-01-01

    Levans are fructose polymers synthesized by a broad range of micro-organisms and a limited number of plant species as non-structural storage carbohydrates. In microbes, these polymers contribute to the formation of the extracellular polysaccharide (EPS) matrix and play a role in microbial biofilm formation. Levans belong to a larger group of commercially important polymers, referred to as fructans, which are used as a source of prebiotic fibre. For levan, specifically, this market remains untapped, since no viable production strategy has been established. Synthesis of levan is catalysed by a group of enzymes, referred to as levansucrases, using sucrose as substrate. Heterologous expression of levansucrases has been notoriously difficult to achieve in Saccharomyces cerevisiae. As a strategy, this study used an invertase (Δsuc2) null mutant and two separate, engineered, sucrose accumulating yeast strains as hosts for the expression of the levansucrase M1FT, previously cloned from Leuconostoc mesenteroides. Intracellular sucrose accumulation was achieved either by expression of a sucrose synthase (Susy) from potato or the spinach sucrose transporter (SUT). The data indicate that in both Δsuc2 and the sucrose accumulating strains, the M1FT was able to catalyse fructose polymerisation. In the absence of the predicted M1FT secretion signal, intracellular levan accumulation was significantly enhanced for both sucrose accumulation strains, when grown on minimal media. Interestingly, co-expression of M1FT and SUT resulted in hyper-production and extracellular build-up of levan when grown in rich medium containing sucrose. This study presents the first report of levan production in S. cerevisiae and opens potential avenues for the production of levan using this well established industrial microbe. Furthermore, the work provides interesting perspectives when considering the heterologous expression of sugar polymerizing enzymes in yeast. PMID:24147008

  5. Biosynthesis of levan, a bacterial extracellular polysaccharide, in the yeast Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    Jaco Franken

    Full Text Available Levans are fructose polymers synthesized by a broad range of micro-organisms and a limited number of plant species as non-structural storage carbohydrates. In microbes, these polymers contribute to the formation of the extracellular polysaccharide (EPS matrix and play a role in microbial biofilm formation. Levans belong to a larger group of commercially important polymers, referred to as fructans, which are used as a source of prebiotic fibre. For levan, specifically, this market remains untapped, since no viable production strategy has been established. Synthesis of levan is catalysed by a group of enzymes, referred to as levansucrases, using sucrose as substrate. Heterologous expression of levansucrases has been notoriously difficult to achieve in Saccharomyces cerevisiae. As a strategy, this study used an invertase (Δsuc2 null mutant and two separate, engineered, sucrose accumulating yeast strains as hosts for the expression of the levansucrase M1FT, previously cloned from Leuconostoc mesenteroides. Intracellular sucrose accumulation was achieved either by expression of a sucrose synthase (Susy from potato or the spinach sucrose transporter (SUT. The data indicate that in both Δsuc2 and the sucrose accumulating strains, the M1FT was able to catalyse fructose polymerisation. In the absence of the predicted M1FT secretion signal, intracellular levan accumulation was significantly enhanced for both sucrose accumulation strains, when grown on minimal media. Interestingly, co-expression of M1FT and SUT resulted in hyper-production and extracellular build-up of levan when grown in rich medium containing sucrose. This study presents the first report of levan production in S. cerevisiae and opens potential avenues for the production of levan using this well established industrial microbe. Furthermore, the work provides interesting perspectives when considering the heterologous expression of sugar polymerizing enzymes in yeast.

  6. The transcriptional response of Saccharomyces cerevisiae to proapoptotic concentrations of Pichia membranifaciens killer toxin.

    Science.gov (United States)

    Santos, A; Marquina, D

    2011-10-01

    PMKT (Pichia membranifaciens killer toxin) reportedly has antimicrobial activity against yeasts and filamentous fungi. In previous research we posited that high PMKT concentrations pose a serious challenge for cell survival by disrupting plasma membrane electrochemical gradients, inducing a transcriptional response similar to that of certain stimuli such as hyperosmotic shock. This response was related to the HOG-pathway with Hog1p phosphorylation and a transitional increase in intracellular glycerol accumulation. Such a response was consistent with the notion that the effect induced by high PMKT concentrations lies in an alteration to the ionic homeostasis of the sensitive cell. By contrast, the evidence presented here shows that low PMKT doses lead to a cell death process in Saccharomyces cerevisiae accompanied by cytological and biochemical indicators of apoptotic programmed cell death, namely, the production of reactive oxygen species, DNA strand breaks, metacaspase activation and cytochrome c release. Furthermore, dying cells progressed from an apoptotic state to a secondary necrotic state, and the rate at which this change occurred was proportional to the intensity of the stimulus. We have explored the global gene expression response of S. cerevisiae during that stimulus. The results obtained from DNA microarrays indicate that genes related with an oxidative stress response were induced in response to proapoptotic concentrations of PMKT, showing that the coordinated transcriptional response is not coincident with that obtained when ionophoric concentrations of PMKT are used. By contrast, cwp2Δ mutants showed no signs of apoptosis, indicating that the initial steps of the killer mechanism coincide when proapoptotic (low) or ionophoric (high) PMKT concentrations are used. Additionally, low dosages of PMKT promoted Hog1p phosphorylation and glycerol accumulation. PMID:21801845

  7. Removing cadmium from electroplating wastewater by waste saccharomyces cerevisiae

    Institute of Scientific and Technical Information of China (English)

    DAI Shu-juan; WEI De-zhou; ZHOU Dong-qin; JIA Chun-yun; WANG Yu-juan; LIU Wen-gang

    2008-01-01

    The appropriate condition and scheme of removing cadmium from electroplating wastewater were investigated by adsorption-precipitation method using waste saccharomyces cerevisiae(WSC) as sorbent. Effect factors on biosorption of cadmium in cadmium-containing electroplating wastewater by waste saccharomyces cerevisiae and precipitation process of waste saccharomyces cerevisiae after adsorbing cadmium were studied. The results show that removal rate of cadmium is over 88% after 30 min adsorbing under the condition of cadmium concentration 26 mg/L, the dosage of waste saccharomyces cerevisiae 16.25 g/L, temperature 18 ℃, pH 6.0 and precipitation time 4 h. Biosorption-precipitation method is effective to remove cadmium in cadmium-containing electroplating wastewater by waste saccharomyces cerevisiae. The SEM, infrared spectroscopy and Zeta-potential of the cells show that chemical chelating is the main adsorption form; electrostatic attraction, hydrogen bonding and van der Waals force all function in adsorption process; and ―NH2―,―C=O―,―C=O―NH―,―CH3, ―OH are the main adsorption groups.

  8. Analysis of HIV-1 Vpr determinants responsible for cell growth arrest in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Yao Xiao-Jian

    2004-08-01

    Full Text Available Abstract Background The HIV-1 genome encodes a well-conserved accessory gene product, Vpr, that serves multiple functions in the retroviral life cycle, including the enhancement of viral replication in nondividing macrophages, the induction of G2 cell-cycle arrest, and the modulation of HIV-1-induced apoptosis. We previously reported the genetic selection of a panel of di-tryptophan (W-containing peptides capable of interacting with HIV-1 Vpr and inhibiting its cytostatic activity in Saccharomyces cerevisiae (Yao, X.-J., J. Lemay, N. Rougeau, M. Clément, S. Kurtz, P. Belhumeur, and E. A. Cohen, J. Biol. Chem. v. 277, p. 48816–48826, 2002. In this study, we performed a mutagenic analysis of Vpr to identify sequence and/or structural determinants implicated in the interaction with di-W-containing peptides and assessed the effect of mutations on Vpr-induced cytostatic activity in S. cerevisiae. Results Our data clearly shows that integrity of N-terminal α-helix I (17–33 and α-helix III (53–83 is crucial for Vpr interaction with di-W-containing peptides as well as for the protein-induced cytostatic effect in budding yeast. Interestingly, several Vpr mutants, mainly in the N- and C-terminal domains, which were previously reported to be defective for cell-cycle arrest or apoptosis in human cells, still displayed a cytostatic activity in S. cerevisiae and remained sensitive to the inhibitory effect of di-W-containing peptides. Conclusions Vpr-induced growth arrest in budding yeast can be effectively inhibited by GST-fused di-W peptide through a specific interaction of di-W peptide with Vpr functional domain, which includes α-helix I (17–33 and α-helix III (53–83. Furthermore, the mechanism(s underlying Vpr-induced cytostatic effect in budding yeast are likely to be distinct from those implicated in cell-cycle alteration and apoptosis in human cells.

  9. Divergence in wine characteristics produced by wild and domesticated strains of Saccharomyces cerevisiae

    OpenAIRE

    Katie E Hyma; Saerens, Sofie M; Verstrepen, Kevin J.; Justin C Fay

    2011-01-01

    The budding yeast Saccharomyces cerevisiae is the primary species used by wine makers to convert sugar into alcohol during wine fermentation. Saccharomyces cerevisiae is found in vineyards, but is also found in association with oak trees and other natural sources. Although wild strains of S. cerevisiae as well as other Saccharomyces species are also capable of wine fermentation, a genetically distinct group of S. cerevisiae strains is primarily used to produce wine, consistent with the idea t...

  10. Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint

    DEFF Research Database (Denmark)

    Sironi, L; Melixetian, M; Faretta, M;

    2001-01-01

    Mad2 is a key component of the spindle checkpoint, a device that controls the fidelity of chromosome segregation in mitosis. The ability of Mad2 to form oligomers in vitro has been correlated with its ability to block the cell cycle upon injection into Xenopus embryos. Here we show that Mad2 forms...

  11. The E3 ubiquitin ligase EDD regulates S-phase and G(2)/M DNA damage checkpoints.

    Science.gov (United States)

    Munoz, Marcia A; Saunders, Darren N; Henderson, Michelle J; Clancy, Jennifer L; Russell, Amanda J; Lehrbach, Gillian; Musgrove, Elizabeth A; Watts, Colin K W; Sutherland, Robert L

    2007-12-15

    The cellular response to DNA damage is critical for maintenance of genomic integrity and inhibition of tumorigenesis. Mutations or aberrant expression of the E3 ubiquitin ligase EDD have been observed in a number of carcinomas and we recently reported that EDD modulates activity of the DNA damage checkpoint kinase, CHK2. Here, we demonstrate that EDD is necessary for G(1)/S and intra S phase DNA damage checkpoint activation and for the maintenance of G(2)/M arrest after double strand DNA breaks. Defective checkpoint activation in EDD-depleted cells led to radio-resistant DNA synthesis, premature entry into mitosis, accumulation of polyploid cells, and cell death via mitotic catastrophe. In addition to decreased CHK2 activation in EDD-depleted cells, the expression of several key cell cycle mediators including Cdc25A/C and E2F1 was altered, suggesting that these checkpoint defects may be both CHK2-dependent and -independent. These data support a role for EDD in the maintenance of genomic stability, emphasising the potential importance of dysregulated EDD expression and/or function in the evolution of cancer. PMID:18073532

  12. Wip1 phosphatase is associated with chromatin and dephosphorylates gammaH2AX to promote checkpoint inhibition

    Czech Academy of Sciences Publication Activity Database

    Macůrek, Libor; Lindqvist, A.; Voets, O.; Kool, J.; Vos, H.R.; Medema, R.H.

    2010-01-01

    Roč. 29, č. 15 (2010), s. 2281-2291. ISSN 0950-9232 R&D Projects: GA ČR GPP305/10/P420 Institutional research plan: CEZ:AV0Z50520514 Keywords : DNA damage * checkpoint * phosphatase Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 7.414, year: 2010

  13. Killing the umpire: cooperative defects in mitotic checkpoint and BRCA2 genes on the road to transformation

    International Nuclear Information System (INIS)

    Recent findings from mouse models of BRCA2 genetic lesions have provided intriguing insights and important questions concerning modes of tumor development in familial breast and ovarian cancers. Fibroblasts from mice homozygous for the BRCA2Tr allele grow poorly and display an array of chromosomal abnormalities that are consistent with a role for BRCA2 in DNA repair. This growth defect can be overcome and cellular transformation promoted by the expression of defective, dominant negative alleles of p53 and of the mitotic checkpoint gene Bub1, both of which are known to induce chromosome instability. These findings are mirrored in the genetic lesions sustained in tumors found in the rare BRCA2Tr/Trmice that survive to adulthood, which include defects in p53 as well as the mitotic checkpoint proteins Bub1 and Mad3L. Together, these data hint that tumors in these mice evolve from an unusually intense selective pressure to remove DNA damage checkpoints, which in turn might be facilitated by chromosomal abolition of mitotic checkpoints and the consequent increase in shuffling of genetic information. How these genetic lesions co-operate to yield transformed cells and how these data relate to BRCA1 and BRCA2 defects in the human population are important questions raised by this work

  14. Induced High Lysine Mutants in Barley

    DEFF Research Database (Denmark)

    Doll, Hans; Køie, B.; Eggum, B. O.

    1974-01-01

    Screening of mutagenically treated materials by combined Kjeldahl nitrogen and dye-binding capacity determinations disclosed fourteen barley mutants, which have from a few to about 40 per cent more lysine in the protein and one mutant with 10 per cent less lysine in the protein than the parent...... variety. Comparisons of six high lysine mutants with the parent variety showed that grain yield and seed size of the mutants are reduced between 10 and 30 per cent. However, the most promising mutant had the lowest reduction in grain yield, and the absolute lysine yield of this mutant was some 30 per cent...... above that of the parent variety. Feeding tests with rats revealed substantial increases in the biological value of the high lysine mutant protein. Also the net protein utilization was improved but less so because of a somewhat reduced digestibility of the mutant protein....

  15. Directed Evolution towards Increased Isoprenoid Production in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Carlsen, Simon; Nielsen, Michael Lynge; Kielland-Brandt, Morten;

    Due to declining drug discovery rates from organic synthetic libraries, pharmaceutical companies are turning their attention towards secondary metabolites. Isoprenoids, also known as terpenoids, constitute the largest known group of secondary metabolites isolated to date, encompassing more than 55...... pyrophosphate and geranylgeranyl pyrophosphate for large-scale microbial production of terpenoids. Saccharomyces cerevisiae was chosen as production platform due to its widespread use in industrial production and the waste number of molecular biology tools which is available for its manipulation. The effort...... for discovering new genetic perturbations, which would results in and increased production of isoprenoids by S. cerevisiae has been very limited. This project is focus on creating diversity within a lycopene producing S. cerevisiae strain by construction of gDNA-, cDNA-, and transposon-libraries. The diversified...

  16. Toxicities of the anti-PD-1 and anti-PD-L1 immune checkpoint antibodies.

    Science.gov (United States)

    Naidoo, J; Page, D B; Li, B T; Connell, L C; Schindler, K; Lacouture, M E; Postow, M A; Wolchok, J D

    2015-12-01

    Immune checkpoint antibodies that augment the programmed cell death protein 1 (PD-1)/PD-L1 pathway have demonstrated antitumor activity across multiple malignancies, and gained recent regulatory approval as single-agent therapy for the treatment of metastatic malignant melanoma and nonsmall-cell lung cancer. Knowledge of toxicities associated with PD-1/PD-L1 blockade, as well as effective management algorithms for these toxicities, is pivotal in order to optimize clinical efficacy and safety. In this article, we review selected published and presented clinical studies investigating single-agent anti-PD-1/PD-L1 therapy and trials of combination approaches with other standard anticancer therapies, in multiple tumor types. We summarize the key adverse events reported in these studies and their management algorithms. PMID:26371282

  17. The Aurora B kinase in chromosome biorientation and spindle checkpoint signalling

    Directory of Open Access Journals (Sweden)

    Veronica eKrenn

    2015-10-01

    Full Text Available Aurora B, a member of the Aurora family of serine/threonine protein kinases, is a key player in chromosome segregation. As part of a macromolecular complex known as the chromosome passenger complex, Aurora B concentrates early during mitosis in the proximity of centromeres and kinetochores, the sites of attachment of chromosomes to spindle microtubules. There, it contributes to a number of processes that impart fidelity to cell division, including kinetochore stabilization, kinetochore-microtubule attachment, and the regulation of a surveillance mechanism named the spindle assembly checkpoint. In the regulation of these processes, Aurora B is the fulcrum of a remarkably complex network of interactions that feed back on its localization and activation state. In this review we discuss the multiple roles of Aurora B during mitosis, focusing in particular on its role at centromeres and kinetochores. Many details of the network of interactions at these locations remain poorly understood, and we focus here on several crucial outstanding questions.

  18. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade.

    Science.gov (United States)

    McGranahan, Nicholas; Furness, Andrew J S; Rosenthal, Rachel; Ramskov, Sofie; Lyngaa, Rikke; Saini, Sunil Kumar; Jamal-Hanjani, Mariam; Wilson, Gareth A; Birkbak, Nicolai J; Hiley, Crispin T; Watkins, Thomas B K; Shafi, Seema; Murugaesu, Nirupa; Mitter, Richard; Akarca, Ayse U; Linares, Joseph; Marafioti, Teresa; Henry, Jake Y; Van Allen, Eliezer M; Miao, Diana; Schilling, Bastian; Schadendorf, Dirk; Garraway, Levi A; Makarov, Vladimir; Rizvi, Naiyer A; Snyder, Alexandra; Hellmann, Matthew D; Merghoub, Taha; Wolchok, Jedd D; Shukla, Sachet A; Wu, Catherine J; Peggs, Karl S; Chan, Timothy A; Hadrup, Sine R; Quezada, Sergio A; Swanton, Charles

    2016-03-25

    As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8(+)tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non-small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy-induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens. PMID:26940869

  19. Meiosis I in Xenopus oocytes is not error-prone despite lacking spindle assembly checkpoint.

    Science.gov (United States)

    Liu, Dandan; Shao, Hua; Wang, Hongmei; Liu, X Johné

    2014-01-01

    The spindle assembly checkpoint, SAC, is a surveillance mechanism to control the onset of anaphase during cell division. SAC prevents anaphase initiation until all chromosome pairs have achieved bipolar attachment and aligned at the metaphase plate of the spindle. In doing so, SAC is thought to be the key mechanism to prevent chromosome nondisjunction in mitosis and meiosis. We have recently demonstrated that Xenopus oocyte meiosis lacks SAC control. This prompted the question of whether Xenopus oocyte meiosis is particularly error-prone. In this study, we have karyotyped a total of 313 Xenopus eggs following in vitro oocyte maturation. We found no hyperploid egg, out of 204 metaphase II eggs with countable chromosome spreads. Therefore, chromosome nondisjunction is very rare during Xenopus oocyte meiosis I, despite the lack of SAC. PMID:24646611

  20. Clonal neoantigens elicit T cell immunoreactivity and sensitivity to immune checkpoint blockade

    Science.gov (United States)

    McGranahan, Nicholas; Furness, Andrew J. S.; Rosenthal, Rachel; Ramskov, Sofie; Lyngaa, Rikke; Saini, Sunil Kumar; Jamal-Hanjani, Mariam; Wilson, Gareth A.; Birkbak, Nicolai J.; Hiley, Crispin T.; Watkins, Thomas B. K.; Shafi, Seema; Murugaesu, Nirupa; Mitter, Richard; Akarca, Ayse U.; Linares, Joseph; Marafioti, Teresa; Henry, Jake Y.; Van Allen, Eliezer M.; Miao, Diana; Schilling, Bastian; Schadendorf, Dirk; Garraway, Levi A.; Makarov, Vladimir; Rizvi, Naiyer A.; Snyder, Alexandra; Hellmann, Matthew D.; Merghoub, Taha; Wolchok, Jedd D.; Shukla, Sachet A.; Wu, Catherine J.; Peggs, Karl S.; Chan, Timothy A.; Hadrup, Sine R.; Quezada, Sergio A.; Swanton, Charles

    2016-01-01

    As tumors grow, they acquire mutations, some of which create neoantigens that influence the response of patients to immune checkpoint inhibitors. We explored the impact of neoantigen intratumor heterogeneity (ITH) on antitumor immunity. Through integrated analysis of ITH and neoantigen burden, we demonstrate a relationship between clonal neoantigen burden and overall survival in primary lung adenocarcinomas. CD8+ tumor-infiltrating lymphocytes reactive to clonal neoantigens were identified in early-stage non–small cell lung cancer and expressed high levels of PD-1. Sensitivity to PD-1 and CTLA-4 blockade in patients with advanced NSCLC and melanoma was enhanced in tumors enriched for clonal neoantigens. T cells recognizing clonal neoantigens were detectable in patients with durable clinical benefit. Cytotoxic chemotherapy–induced subclonal neoantigens, contributing to an increased mutational load, were enriched in certain poor responders. These data suggest that neoantigen heterogeneity may influence immune surveillance and support therapeutic developments targeting clonal neoantigens. PMID:26940869

  1. Checkpoint and Replication Oriented Fault Tolerant Mechanism for MapReduce Framework

    Directory of Open Access Journals (Sweden)

    Yang Liu

    2013-09-01

    Full Text Available MapReduce is an emerging programming paradigm and an associated implementation for processing and generating big data which has been widely applied in data-intensive systems. In cloud environment, node and task failure is no longer accidental but a common feature of large-scale systems. In MapReduce framework, although the rescheduling based fault-tolerant method is simple to implement, it failed to fully consider the location of distributed data, the computation and storage overhead. Thus, a single node failure will increase the completion time dramatically. In this paper, a Checkpoint and Replication Oriented Fault Tolerant scheduling algorithm (CROFT is proposed, which takes both task and node failure into consideration. Preliminary experiments show that with less storage and network overhead. CROFT will significantly reduce the completion time at failure time, and the overall performance of MapReduce can be improved at least over 30% than original mechanism in Hadoop.  

  2. Chromosome 17p deletion in human medulloblastoma: a missing checkpoint in the Hedgehog pathway.

    Science.gov (United States)

    De Smaele, Enrico; Di Marcotullio, Lucia; Ferretti, Elisabetta; Screpanti, Isabella; Alesse, Edoardo; Gulino, Alberto

    2004-10-01

    Although deregulation of Hedgehog signalling is considered to play a crucial oncogenic role and commonly occurrs in medulloblastoma, genetic lesions in components of this pathway are observed in a minority of cases. The recent identification of a novel putative tumor suppressor (REN(KCTD11)) on chromosome 17p13.2, a region most frequently lost in human medulloblastoma, highlights the role of allelic deletion of the gene in this brain malignancy, leading to the loss of growth inhibitory activity via suppression of Gli-dependent activation of Hedgehog target genes. The presence on 17p13 of another tumor suppressor gene (p53) whose inactivation cooperates with Hedgehog pathway for medulloblastoma formation, suggests that 17p deletion unveils haploinsufficiency conditions leading to abrogation of either direct and indirect checkpoints of Hedgehog signalling in cancer. PMID:15467454

  3. An extra early mutant of pigeonpea

    International Nuclear Information System (INIS)

    The redgram (Cajanus cajan (L.) Huth) variety 'Prabhat DT' was gamma irradiated with 100, 200, 300 and 400 Gy doses. Several mutants have been identified viz., extra early mutants, monostem mutants, obcordifoliate mutants and bi-stigmatic mutants. The extra early mutant was obtained when treated with 100 Gy dose. The mutant was selfed and forwarded from M2 to M4 generation. In the M4 generation the mutant line was raised along with the parental variety. Normal cultural practices were followed and the biometrical observations were recorded. It was observed that for the characters viz., total number of branches per plant, number of pods per plants, seeds per pod, 100 seed weight and seed yield per plant there was no difference between the mutant and parent variety. Whereas, regarding the days to flowering and maturity the mutants were earlier than the parents. The observation was recorded from two hundred plants each. The mutant gives the same yield in 90 days as that of the parent variety in 107 days, which make it an economic mutant

  4. Problem-Solving Test: Tryptophan Operon Mutants

    Science.gov (United States)

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  5. Expression of protein engineered NADP{sup +}-dependent xylitol dehydrogenase increases ethanol production from xylose in recombinant Saccharomyces cerevisiae

    Energy Technology Data Exchange (ETDEWEB)

    Matsushika, Akinori; Inoue, Hiroyuki; Murakami, Katsuji; Takimura, Osamu; Sawayama, Shigeki [National Institute of Advanced Industrial Science and Technology, Hiroshima (Japan). Biomass Technology Research Center; Watanabe, Seiya; Kodaki, Tsutomu; Makino, Keisuke [Kyoto Univ. (Japan). Inst. of Advanced Energy

    2008-11-15

    A recombinant Saccharomyces cerevisiae strain transformed with xylose reductase (XR) and xylitol dehydrogenase (XDH) genes from Pichia stipitis has the ability to convert xylose to ethanol together with the unfavorable excretion of xylitol, which may be due to cofactor imbalance between NADPH-preferring XR and NAD{sup +}-dependent XDH. To reduce xylitol formation, we have already generated several XDH mutants with a reversal of coenzyme specificity toward NADP{sup +}. In this study, we constructed a set of recombinant S. cerevisiae strains with xylose-fermenting ability, including protein-engineered NADP{sup +}-dependent XDH-expressing strains. The most positive effect on xylose-to-ethanol fermentation was found by using a strain named MA-N5, constructed by chromosomal integration of the gene for NADP{sup +}-dependent XDH along with XR and endogenous xylulokinase genes. The MA-N5 strain had an increase in ethanol production and decrease in xylitol excretion compared with the reference strain expressing wild-type XDH when fermenting not only xylose but also mixed sugars containing glucose and xylose. Furthermore, the MA-N5 strain produced ethanol with a high yield of 0.49 g of ethanol/g of total consumed sugars in the nonsulfuric acid hydrolysate of wood chips. The results demonstrate that glucose and xylose present in the lignocellulosic hydrolysate can be efficiently fermented by this redox-engineered strain. (orig.)

  6. Transcriptomics in human blood incubation reveals the importance of oxidative stress response in Saccharomyces cerevisiae clinical strains

    Directory of Open Access Journals (Sweden)

    Llopis Silvia

    2012-08-01

    Full Text Available Abstract Background In recent years an increasing number of yeast infections in humans have been related to certain clinical isolates of Saccharomyces cerevisiae. Some clinical strains showed in vivo and in vitro virulence traits and were able to cause death in mice whereas other clinical strains were avirulent. Results In this work, we studied the transcriptional profiles of two S. cerevisiae clinical strains showing virulent traits and two control non-virulent strains during a blood incubation model and detected a specific transcriptional response of clinical strains. This response involves an mRNA levels increase of amino acid biosynthesis genes and especially oxidative stress related genes. We observed that the clinical strains were more resistant to reactive oxygen species in vitro. In addition, blood survival of clinical isolates was high, reaching similar levels to pathogenic Candida albicans strain. Furthermore, a virulent strain mutant in the transcription factor Yap1p, unable to grow in oxidative stress conditions, presented decreased survival levels in human blood compared with the wild type or YAP1 reconstituted strain. Conclusions Our data suggest that this enhanced oxidative stress response in virulent clinical isolates, presumably induced in response to oxidative burst from host defense cells, is important to increase survival in human blood and can help to infect and even produce death in mice models.

  7. KTI11 and KTI13, Saccharomyces cerevisiae genes controlling sensitivity to G1 arrest induced by Kluyveromyces lactis zymocin.

    Science.gov (United States)

    Fichtner, Lars; Schaffrath, Raffael

    2002-05-01

    The Kluyveromyces lactis zymocin and its gamma-toxin subunit inhibit cell cycle progression of Saccharomyces cerevisiae. To identify S. cerevisiae genes conferring zymocin sensitivity, we complemented the unclassified zymocin-resistant kti11 and kti13 mutations using a single-copy yeast library. Thus, we identified yeast open reading frames (ORFs) YBL071w-A and YAL020c/ATS1 as KTI11 and KTI13 respectively. Disruption of KTI11 and KTI13 results in the complex tot phenotype observed for the gamma-toxin target site mutants, tot1-7, and includes zymocin resistance, thermosensitivity, hypersensitivity to drugs and slow growth. Both loci, KTI11 and KTI13, are actively transcribed protein-encoding genes as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and in vivo HA epitope tagging. Kti11p is highly conserved from yeast to man, and Kti13p/Ats1p is related to yeast Prp20p and mammalian RCC1, components of the Ran-GTP/GDP cycle. Combining disruptions in KTI11 or KTI13 with a deletion in TOT3/ELP3 coding for the RNA polymerase II (RNAPII) Elongator histone acetyltransferase (HAT) yielded synthetic effects on slow growth phenotype expression. This suggests genetic interaction and possibly links KTI11 and KTI13 to Elongator function. PMID:11994165

  8. Cloning of a cDNA encoding ATP sulfurylase from Arabidopsis thaliana by functional expression in Saccharomyces cerevisiae.

    Science.gov (United States)

    Leustek, T; Murillo, M; Cervantes, M

    1994-01-01

    ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of a cDNA encoding ATP sulfurylase (APS1) from Arabidopsis thaliana. APS1 was isolated by its ability to alleviate the methionine requirement of an ATP sulfurylase mutant strain of Saccharomyces cerevisiae (yeast). Expression of APS1 correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1748-bp cDNA with an open reading frame predicted to encode a 463-amino acid, 51,372-D protein. The predicted amino acid sequence of APS1 is similar to ATP sulfurylase of S. cerevisiae, with which it is 25% identical. Two lines of evidence indicate that APS1 encodes a chloroplast form of ATP sulfurylase. Its predicted amino-terminal sequence resembles a chloroplast transit peptide; and the APS1 polypeptide, synthesized in vitro, is capable of entering isolated intact chloroplasts. Several genomic DNA fragments that hybridize with the APS1 probe were identified. The APS1 cDNA hybridizes to three species of mRNA in leaves (1.85, 1.60, and 1.20 kb) and to a single species of mRNA in roots (1.85 kb). PMID:8058839

  9. Effect of 905 MHz microwave radiation on colony growth of the yeast Saccharomyces cerevisiae strains FF18733, FF1481 and D7

    International Nuclear Information System (INIS)

    The aim of this study was to evaluate the effect of weak radiofrequency microwave (RF/MW) radiation emitted by mobile phones on colony growth of the yeast Saccharomyces cerevisiae. S. cerevisiae strains FF18733 (wild-type), FF1481 (rad1 mutant) and D7 (commonly used to detect reciprocal and nonreciprocal mitotic recombinations) were exposed to a 905 MHz electromagnetic field that closely matched the Global System for Mobile Communication (GSM) pulse modulation signals for mobile phones at a specific absorption rate (SAR) of 0.12 W/kg. Following 15-, 30- and 60-minutes exposure to RF/MW radiation, strain FF18733 did not show statistically significant changes in colony growth compared to the control sample. The irradiated strains FF1481 and D7 demonstrated statistically significant reduction of colony growth compared to non-irradiated strains after all exposure times. Furthermore, strain FF1481 was more sensitive to RF/MW radiation than strain D7. The findings indicate that pulsed RF/MW radiation at a low SAR level can affect the rate of colony growth of different S. cerevisiae strains

  10. Microaerobic conversion of xylose to ethanol in recombinant Saccharomyces cerevisiae SX6(MUT) expressing cofactor-balanced xylose metabolic enzymes and deficient in ALD6.

    Science.gov (United States)

    Jo, Sung-Eun; Seong, Yeong-Je; Lee, Hyun-Soo; Lee, Soo Min; Kim, Soo-Jung; Park, Kyungmoon; Park, Yong-Cheol

    2016-06-10

    Xylose is a major monosugar in cellulosic biomass and should be utilized for cost-effective ethanol production. In this study, xylose-converting ability of recombinant Saccharomyces cerevisiae SX6(MUT) expressing NADH-preferring xylose reductase mutant (R276H) and other xylose-metabolic enzymes, and deficient in aldehyde dehydrogenase 6 (Ald6p) were characterized at microaerobic conditions using various sugar mixtures. The reduction of air supply from 0.5vvm to 0.1vvm increased specific ethanol production rate by 75% and did not affect specific xylose consumption rate. In batch fermentations using various concentrations of xylose (50-104g/L), higher xylose concentration enhanced xylose consumption rate and ethanol productivity but reduced ethanol yield, owing to the accumulation of xylitol and glycerol from xylose. SX6(MUT) consumed monosugars in pitch pine hydrolysates and produced 23.1g/L ethanol from 58.7g/L sugars with 0.39g/g ethanol yield, which was 14% higher than the host strain of S. cerevisiae D452-2 without the xylose assimilating enzymes. In conclusion, S. cerevisiae SX6(MUT) was characterized to possess high xylose-consuming ability in microaerobic conditions and a potential for ethanol production from cellulosic biomass. PMID:27059482

  11. Overexpressing enzymes of the Ehrlich pathway and deleting genes of the competing pathway in Saccharomyces cerevisiae for increasing 2-phenylethanol production from glucose.

    Science.gov (United States)

    Shen, Li; Nishimura, Yuya; Matsuda, Fumio; Ishii, Jun; Kondo, Akihiko

    2016-07-01

    2-Phenylethanol (2-PE) is a higher aromatic alcohol that is used in the cosmetics and food industries. The budding yeast Saccharomyces cerevisiae is considered to be a suitable host for the industrial production of higher alcohols, including 2-PE. To produce 2-PE from glucose in S. cerevisiae, we searched for suitable 2-keto acid decarboxylase (KDC) and alcohol dehydrogenase (ADH) enzymes of the Ehrlich pathway for overexpression in strain YPH499, and found that overexpression of the ARO10 and/or ADH1 genes increased 2-PE production from glucose. Further, we screened ten BY4741 single-deletion mutants of genes involved in the competing pathways for 2-PE production, and found that strains aro8Δ and aat2Δ displayed increased 2-PE production. Based on these results, we engineered a BY4741 strain that overexpressed ARO10 and contained an aro8Δ deletion, and demonstrated that the strain produced 96 mg/L 2-PE from glucose as the sole carbon source. As this engineered S. cerevisiae strain showed a significant increase in 2-PE production from glucose without the addition of an intermediate carbon substrate, it is a promising candidate for the large-scale production of 2-PE. PMID:26975754

  12. Immune checkpoint inhibitors enhance cytotoxicity of cytokine-induced killer cells against human myeloid leukaemic blasts.

    Science.gov (United States)

    Poh, Su Li; Linn, Yeh Ching

    2016-05-01

    We studied whether blockade of inhibitory receptors on cytokine-induced killer (CIK) cells by immune checkpoint inhibitors could increase its anti-tumour potency against haematological malignancies. CIK cultures were generated from seven normal donors and nine patients with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) or multiple myeloma (MM). The inhibitory receptors B and T lymphocyte attenuator, CD200 receptor, lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin and mucin-domain-containing-3 (TIM-3) were present at variable percentages in most CIK cultures, while cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death-1 (PD-1) and killer cell immunoglobulin-like receptors (KIR2DL1/2/3) were expressed at low level in most cultures. Without blockade, myeloid leukaemia cells were susceptible to autologous and allogeneic CIK-mediated cytotoxicity. Blockade of KIR, LAG-3, PD-1 and TIM-3 but not CTLA-4 resulted in remarkable increase in killing against these targets, even in those with poor baseline cytotoxicity. ALL and MM targets were resistant to CIK-mediated cytotoxicity, and blockade of receptors did not increase cytotoxicity to a meaningful extent. Combination of inhibitors against two receptors did not further increase cytotoxicity. Interestingly, potentiation of CIK killing by blocking antibodies was not predicted by expression of receptors on CIK and their respective ligands on the targets. Compared to un-activated T and NK cells, blockade potentiated the cytotoxicity of CIK cells to a greater degree and at a lower E:T ratio, but without significant increase in cytotoxicity against normal white cell. Our findings provide the basis for clinical trial combining autologous CIK cells with checkpoint inhibitors for patients with AML. PMID:26961084

  13. Understanding the 3-hydroxypropionic acid tolerance mechanism in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Kildegaard, Kanchana Rueksomtawin; Juncker, Agnieszka; Hallstrom, Bjorn; Jensen, Niels Bjerg; Maury, Jerome; Nielsen, Jen; Förster, Jochen; Borodina, Irina

    sustainable alternative for production of acrylic acid from renewable feedstocks. We are establishing Saccharomyces cerevisiae as an alternative host for 3HP production. However, 3HP also inhibits yeast grow th at level well below what is desired for commercial applications. Therefore, we are aiming to...... improve 3HP tolerance in S. cerevisiae by applying adaptive evolution approach. We have generated yeast strains with sign ificantly improved capacity for tolerating 3HP when compared to the wild-type. We will present physiolo gical characterization, genome re-sequencing, and transcriptome analysis of the...

  14. Accumulation of gold using Baker's yeast, Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Authors have reported preconcentration of 152Eu, a long-lived fission product, by yeast cells, Saccharomyces cerevisiae. Gold being a precious metal is used in electroplating, hydrogenation catalyst, etc. Heterogeneous composition of samples and low concentration offers renewed interest in its selective extraction of gold using various extractants. Gold can be recovered from different solutions using various chemical reagents like amines, organophosphorus compounds, and extractants containing sulphur as donor atom, etc. In the present work, two different strains of baker's yeast, Saccharomyces cerevisiae have been used to study the preconcentration of gold at various experimental conditions

  15. Structure of Ynk1 from the yeast Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    The crystal structure of Ynk1, an NDPK from the yeast Saccharomyces cerevisiae, has been solved at 3.1 Å resolution. Nucleoside diphosphate kinase (NDPK) catalyzes the transfer of the γ-phosphate from nucleoside triphosphates to nucleoside diphosphates. In addition to biochemical studies, a number of crystal structures of NDPK from various organisms, including both native proteins and complexes with nucleotides or nucleotide analogues, have been determined. Here, the crystal structure of Ynk1, an NDPK from the yeast Saccharomyces cerevisiae, has been solved at 3.1 Å resolution. Structural analysis strongly supports the oligomerization state of this protein being hexameric rather than tetrameric

  16. Genetic Control of the Trigger for the G2/M Checkpoint

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Eric J. [Columbia University; Smilenov, Lubomir B. [Columbia University; Young, Erik F. [Columbia University

    2013-10-01

    The work undertaken in this project addressed two seminal areas of low dose radiation biology that are poorly understood and controversial. These areas are the challenge to the linear-no-threshold (LNT) paradigm at low doses of radiation and, the fundamental elements of radiation bystander effect biology Genetic contributions to low dose checkpoint engagement: The LNT paradigm is an extrapolation of known, measured cancer induction endpoints. Importantly, data for lower doses is often not available. Debatably, radiation protection standards have been introduced which are prudently contingent on the adherence of cancer risk to the established trend seen at higher doses. Intriguing findings from other labs have hinted at separate DNA damage response programs that engage at low or high levels of radiation. Individual radiation sensitivity commensurate with hemizygosity for a radiation sensitivity gene has been estimated at 1-2% in the U.S.. Careful interrogation of the DNA damage response at low doses of radiation became important and served as the basis for this grant. Several genes were tested in combinations to determine if combined haploinsufficiency for multiple radiosensitizing genes could render a cell more sensitive to lower levels of acute radiation exposure. We measured a classical radiation response endpoint, cell cycle arrest prior to mitosis. Mouse embryo fibroblasts were used and provided a uniform, rapidly dividing and genetically manipulable population of study. Our system did not report checkpoint engagement at acute doses of gamma rays below 100 mGy. The system did report checkpoint engagement reproducibly at 500 mGy establishing a threshold for activation between 100 and 500 mGy. Engagement of the checkpoint was ablated in cells nullizygous for ATM but was otherwise unperturbed in cells combinatorially haploinsufficient for ATM and Rad9, ATM and PTEN or PTEN and Rad9. Taken together, these experiments tell us that, in a sensitive fibroblast culture

  17. Genome-wide screening of Saccharomyces cerevisiae genes required to foster tolerance towards industrial wheat straw hydrolysates.

    Science.gov (United States)

    Pereira, Francisco B; Teixeira, Miguel C; Mira, Nuno P; Sá-Correia, Isabel; Domingues, Lucília

    2014-12-01

    The presence of toxic compounds derived from biomass pre-treatment in fermentation media represents an important drawback in second-generation bio-ethanol production technology and overcoming this inhibitory effect is one of the fundamental challenges to its industrial production. The aim of this study was to systematically identify, in industrial medium and at a genomic scale, the Saccharomyces cerevisiae genes required for simultaneous and maximal tolerance to key inhibitors of lignocellulosic fermentations. Based on the screening of EUROSCARF haploid mutant collection, 242 and 216 determinants of tolerance to inhibitory compounds present in industrial wheat straw hydrolysate (WSH) and in inhibitor-supplemented synthetic hydrolysate were identified, respectively. Genes associated to vitamin metabolism, mitochondrial and peroxisomal functions, ribosome biogenesis and microtubule biogenesis and dynamics are among the newly found determinants of WSH resistance. Moreover, PRS3, VMA8, ERG2, RAV1 and RPB4 were confirmed as key genes on yeast tolerance and fermentation of industrial WSH. PMID:25287021

  18. Amino acid residues important for substrate specificity of the amino acid permeases Can I p and Gnp I p in Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Regenberg, Birgitte; Kielland-Brandt, M.C.

    2001-01-01

    mutations affecting six predicted domains (helices III and X, and loops 1. 2, 6 and 7) of the permeases. Helix III and loop 7 are candidates for domains in direct contact with the transported amino acid. Helix III was affected in both CAN1 (Y173H, Y173D) and GNP1 (W239C) mutants and has previously been...... found to be important for substrate preference in other members of the family. Furthermore, the mutations affecting loop 7 (residue T354, S355, Y356) are close to a glutamate side chain (E367) potentially interacting with the positively charged substrate, a notion supported by conservation of the side......Deletion of the general amino acid permease gene GAP1 abolishes uptake of L-citrulline in Saccharomyces cerevisiae, resulting in the inability to grow on L-citrulline as sole nitrogen source. Selection for suppressor mutants that restored growth on L-citrulline led to isolation of 21 mutations in...

  19. Impaired Uptake and/or Utilization of Leucine by Saccharomyces cerevisiae Is Suppressed by the SPT15-300 Allele of the TATA-Binding Protein Gene

    DEFF Research Database (Denmark)

    Baerends, RJ; Qiu, Jin-Long; Rasmussen, Simon;

    2009-01-01

    mutant allele of the SPT15 gene (SPT15-300) corresponding to the three amino acid changes F177S, Y195H, and K218R has been reported (H. Alper, J. Moxley, E. Nevoigt, G. R. Fink, and G. Stephanopoulos, Science 314:1565-1568, 2006). The SPT15 gene codes for the TATA-binding protein. This finding prompted...... us to examine the effect of expression of the SPT15-300 allele in various yeast species of industrial importance. Expression of SPT15-300 in leucine-prototrophic strains of S. cerevisiae, Saccharomyces bayanus, or Saccharomyces pastorianus (lager brewing yeast), however, did not improve tolerance to...... ethanol on complex rich medium (yeast extract-peptone-dextrose). The enhanced growth of the laboratory yeast strain BY4741 expressing the SPT15-300 mutant allele was seen only on defined media with low concentrations of leucine, indicating that the apparent improved growth in the presence of ethanol was...

  20. Xenopus Cds1 Is Regulated by DNA-Dependent Protein Kinase and ATR during the Cell Cycle Checkpoint Response to Double-Stranded DNA Ends

    OpenAIRE

    McSherry, Troy D.; Mueller, Paul R.

    2004-01-01

    The checkpoint kinase Cds1 (Chk2) plays a key role in cell cycle checkpoint responses with functions in cell cycle arrest, DNA repair, and induction of apoptosis. Proper regulation of Cds1 is essential for appropriate cellular responses to checkpoint-inducing insults. While the kinase ATM has been shown to be important in the regulation of human Cds1 (hCds1), here we report that the kinases ATR and DNA-dependent protein kinase (DNA-PK) play more significant roles in the regulation of Xenopus ...