Comparative genomic hybridization of wild type and myo1 knock out yeast to identify aneuploidies and CNVs in evolved mutants that can successfully perform cytokinesis
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
SUBMITTER: Ariel Paulson
PROVIDER: E-TABM-557 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:In general, yeast strains with myo1 knocked out cannot perform cytokinesis, but some evolve new ways of accomplishing the task. This is an examination of differential gene expression patterns in a selection of evolved mutants that can successfully perform cytokinesis. The purpose is to attempt a molecular characterization of the evolved responses.
2008-11-24 | E-TABM-559 | biostudies-arrayexpress
Project description:Aneuploidy, the condition of having an abnormal number of chromosomes, is a hallmark of cancer cells. Recent work performed in yeast and mouse models suggests that aneuploidy interferes with cell proliferation and organism development. However, other recent experiments suggested that aneuploidy might play a positive role in tumor initiation and/or progression and in the emergence of chemotherapy as well as antibiotic resistances. Given its general deleterious effects on cellular fitness, how could aneuploidy be beneficial? In this set of experiments we plan to provide evidence that aneuploidy induces phenotypic variation through direct and indirect changes not only at the transcriptome but also at the proteome level.
2010-10-01 | E-MTAB-318 | biostudies-arrayexpress
Project description:During cytokinesis in the budding yeast Saccharomyces cerevisiae, contraction of the cytokinetic ring and primary septum synthesis by chitin synthase II (Chs2p) are coupled processes. Myosin II (Myo1p), is involved in the actomyosin ring formation, required for proper cytokinesis, while Chs2p is responsible for the chitin primary septum formation which is necessary to stabilize the cytokinetic ring during its contraction. Morphological phenotypes of myo1∆ and chs2∆ mutants are therefore similar particularly in that both are unable to complete normal cytokinesis. A comparison between the global mRNA transcription profiles of myo1∆ and chs2∆ strains was conducted using oligonucleotide microarrays to establish if these cytokinesis mutants exhibited similar mRNA expression patterns and signature profiles were later generated from gene set enrichment analysis (GSEA). Genetic experiments were conducted to further test predictions based on the GSEA results. As reported previously in myo1∆ strains, a significant number of protein biosynthesis and RNA processing genes that may be attributed to regulation by cell integrity pathway(s) were also down-regulated in the chs2∆ strain. Genes coding for proteins involved in autophagy were coordinately upregulated only in the chs2∆ strain yet an ATG9 genetic knockout that completely blocks autophagy was viable in chs2∆ and myo1∆ genetic backgrounds.. However, the chs2∆ strain was significantly more susceptible to lysis by B-1,3-glucanase than myo1∆ and fks1 control strains. We interpret this result to indicate that these two cytokinesis mutant strains possess different cell wall properties. This interpretation was further supported by the observation that Slt2p hyperphosphorylation was detected in the chs2∆ strain yet expression of SLT2 was not required for cell viability. We conclude that in contrast to myo1∆ strains, chs2∆ strains do not activate the cell integrity pathway.
2009-05-21 | GSE12994 | GEO
Project description:The Saccharomyces cerevisiae MYO1 gene encodes the myosin type II heavy chain (Myo1p), a protein required for normal cytokinesis in budding yeast. Deletion of the MYO1 gene prevents actomyosin-driven cytokinesis thereby activating an alternative mechanism that involves the synthesis of a remedial septum. Myo1p deficiency in yeast (myo1) also causes the formation of attached cells, abnormal budding patterns, formation of enlarged and elongated cells, increased osmotic sensitivity, delocalized chitin deposition, and increased chitin synthesis. To determine how the differential expression of genes is related to these diverse phenotypes, we analyzed the global mRNA expression profile of myo1 strains. Global mRNA expression profiles of myo1 strains and their corresponding wild type controls were obtained by hybridization to yeast oligonucleotide microarrays. Results for selected genes were confirmed by real time RT-PCR. A total of 547 differentially expressed genes were identified with 263 up-regulated and 284 down regulated genes in the myo1 strains. Gene set enrichment analysis revealed the significant over-representation of genes in the protein biosynthesis and stress response categories. Genes involved in cell wall assembly (GAS1, PSA1, CIS3, FIT1, WSC2), MAP kinase activity (SLT2), Rho1p Guanine Exchange Factor (ROM1), and regulation of cell proliferation (RAS1) were also differentially expressed in myo1 strains. Conclusions: We have presented a global mRNA expression analysis of yeast myo1 strains and hypothesized about possible correlations with morphological and biochemical phenotypes observed in these strains. We report 547 differentially regulated genes in the myo1 mutant strains. Genes involved in the control of cell proliferation, protein synthesis and maturation, DNA replication, and cell division processes were largely down regulated, suggesting a mechanism for delayed cell cycle progression and growth that involves coordinated regulation of these processes in myo1 strains. Other genes involved in the cellular response to cell wall stress and cell wall organization were largely up-regulated suggesting that cell wall biogenesis is important for the completion of cytokinesis and cell wall morphogenetic processes that may also be affected by myosin II deficiency. Gene set enrichment analysis indicates that stress response and protein biosynthesis gene categories are inversely correlated in this mutant. Keywords: Comparative genomic hybridization
2008-09-18 | GSE5931 | GEO
Project description:The microarrays experiments of three biological and one technical replicates were performed in YJR12 (wt) and YJR13 (myo1?) strains. YJR12 (wild type) and YJR13 (myo1?) strains were obtained as haploid segregants from a cross between YJR6 (myo1::HIS5 strain) and BY4741 (obtained from ATTC). Cultures were grown overnight at 26ºC to an optical density between 0.5-0.8 (OD600) in complete synthetic media (CSM, 2% glucose, 1X Nitrogen base) with continuous shaking at 200 rpm. RNA was extracted from ribosomal pellets using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s instructions. RNA concentrations were determined by measuring absorbance at 260nm using a Nanodrop spectrophotometer (Nanodrop Technologies). The purity and integrity of the RNA was monitored using an Agilent Bioanalyzer (Agilent Technologies) following the manufacturer’s instructions. 1.0 µg of RNA extracted form ribosomal pellets from each sample was amplified using the Low RNA Input Fluorescent Linear Amplification kit (Agilent Technologies). The amplified cRNA was labeled with 10mM Cyanine 5-CTP (Cy5) or Cyanine 3-CTP (Cy3) (Perkin Elmer Life Sciences). Labeled cRNA’s were purified with Qiagen RNeasy mini spin columns and dye incorporation was monitored on an Agilent Bioanalyzer. Hybridization of Cy5 and Cy3 labeled cRNA’s were performed using Yeast Oligo Microarray slides and hybridization kit from Agilent Technologies (Sheldon Manufacturing) at 60ºC for 17 hours. Slides were washed and scanned with a VersArray Chip Reader system (BioRad, Hercules, CA) at a resolution of 5mm with detector sensitivity values between 704-800 and laser power at 85%. Scanned images were transferred to the Imagene 3.0 software (Biodiscovery) for further analysis to locate spots, adjust the appropriate grid, and obtain the Cy3 and Cy5 TIFF files. The microarrays raw data generated with Imagene 3.0 were analyzed using Limma software (Bioconductor Package 1.7). The data was prepared for analysis by correcting for background intensity. The individual data sets were normalized using the locally weighted linear regression (Lowess) within each array. After normalization, the difference between the experimental and control signal was calculated, replicates were combined, and their averages were calculated. The fold change in gene expression was calculated by 2^(M), where M is the log2-fold change after background correction and normalization. An Empirical Bayes Statistics for differential expression analysis (eBayes statistics) was performed by Limma. Genes with a p-value ? 0.018 were established as a cutoff for differential expression. In addition, a false discovery rate (FDR) test was performed by Limma program. Previous analysis of global mRNA expression in Saccharomyces cerevisiae myosin type II deficient strains (myo1?) revealed 547 genes related to the stress response that were proposed to be regulated at the transcriptional level. The objective of this study is to explore the post-transcriptional regulation of the stress response in these strains. We have identified 1,271 differentially regulated mRNAs bound to ribosomes extracted from myo1? strains compared to wild type controls. To assess the mode of regulation of these putative translationally regulated genes, ribosomal protein (RP) mRNAs were analyzed for their polysomal distribution in sucrose gradient fractions of myo1? and wild-type (wt) cells. Our analysis of three representative RP mRNAs showed that RPS8A, RPL7B and RPL3 were recruited from heavy to lighter polyribosome fractions in myo1?, suggesting that these RP mRNAs were less efficiently translated. Western blot analysis revealed accumulation of the phosphorylated form of eukaryotic translation initiation factor 2 (eIF2?-P) in myo1? strains and RPS8A, RPL7B and RPL3 mRNAs were found co-precipitated with immunoprecipitated eIF2?-P, suggesting a direct association between eIF2?-P and translationally regulated RP mRNAs. In yeast, GCN2 codes for the only eIF2? kinase that is directly regulated by TOR (target of rapamycin) pathway. Repression of TOR by rapamycin in a myo1? strain did not increase levels of eIF2?-P yet, a gcn2?myo1? strain exhibited a severe synthetic growth defect suggesting an important survival role for TOR mediated translational regulation in these strains. Reduced steady state levels of the translation initiation factor eIF4G, were also observed in myo1? further supporting the regulation of translation by the TOR pathway. These findings support the conclusion that post-transcriptional control specifically by translation inhibition is a key component of the stress response in yeast.
2010-12-08 | E-GEOD-20203 | biostudies-arrayexpress
Project description:In fission yeast the SET domain protein, Set3p is required for the reliable execution of cytokinesis. To address whether the deletion of the set3 gene might selectively alter expression of cytokinesis genes, expression profiling of wildtype and set3D strains was performed. Wild-type or set3D strains were grown to mid-log phase in YES media and treated with 0.5 mM LatA (or the solvent control, DMSO) for three hours at 30 degrees C. Three biological replicates were performed.
2012-01-01 | E-GEOD-33228 | biostudies-arrayexpress
Project description:In fission yeast, the nuclear-localized Lsk1p-Lsc1p-Lsg1p cyclin dependent kinase complex is required for the reliable execution of cytokinesis and is also required for Ser-2 phosphorylation RNA pol II carboxy terminal domain. To address whether alterations in CTD phosphorylation might selectively alter expression of cytokinesis genes, expression profiling of site-directed CTD mutants was performed. Strains bearing the rpb1-12XCTD and rpb1-12XS2ACTD mutations were grown to mid-log phase in YES media and treated with 0.5uM LatA (or the solvent control, DMSO) for three hours at 30C. Three biological replicates were performed.
2011-08-31 | E-GEOD-31369 | biostudies-arrayexpress
Project description:Aim of this project is to identify biomarkers associated with fungal persistence in the host and genomic variability among strains isolated from different environments.
2016-04-30 | E-SYBR-8 | biostudies-arrayexpress
Project description:Prolonged cultivation of Saccharomyces cerevisiae in aerobic, glucose-limited chemostat cultures (dilution rate, 0·10 hâ1) resulted in a progressive decrease of the residual glucose concentration (from 20 to 8 mg lâ1 after 200 generations). This increase in the affinity for glucose was accompanied by a fivefold decrease of fermentative capacity, and changes in cellular morphology. These phenotypic changes were retained when single-cell isolates from prolonged cultures were used to inoculate fresh chemostat cultures, indicating that genetic changes were involved. Kinetic analysis of glucose transport in an âevolvedâ strain revealed a decreased Km, while Vmax was slightly increased relative to the parental strain. Apparently, fermentative capacity in the evolved strain was not controlled by glucose uptake. Instead, enzyme assays in cell extracts of the evolved strain revealed strongly decreased capacities of enzymes in the lower part of glycolysis. This decrease was corroborated by genome-wide transcriptome analysis using DNA microarrays. In aerobic batch cultures on 20 g glucose lâ1, the specific growth rate of the evolved strain was lower than that of the parental strain (0·28 and 0·37 hâ1, respectively). Instead of the characteristic instantaneous production of ethanol that is observed when aerobic, glucose-limited cultures of wild-type S. cerevisiae are exposed to excess glucose, the evolved strain exhibited a delay of 90 min before aerobic ethanol formation set in. This study demonstrates that the effects of selection in glucose-limited chemostat cultures extend beyond glucose-transport kinetics. Although extensive physiological analysis offered insight into the underlying cellular processes, the evolutionary âdriving forceâ for several of the observed changes remains to be elucidated Experiment Overall Design: A crucial feature of bakers' yeast is its capacity to produce CO2, referred to as fermentative capacity (van Hoek et al., 1998). After prolonged glucose-limited cultivation of S. cerevisiae, in addition to an increased affinity for glucose, we observed a dramatic decrease in fermentative capacity. Consequently, the aim of the present study was to perform an integral analysis of the long-term adaptation of S. cerevisiae during prolonged glucose-limited, aerobic cultivation in chemostat cultures, with special emphasis on the regulation of glucose transport and glycolytic capacity. To this end, we applied an integrated approach that combined transcriptome analysis, measurement of fermentative capacity and activities of glucose transport and glycolytic enzymes, and characterization of cellular morphology.
2008-06-16 | E-GEOD-8898 | biostudies-arrayexpress
Project description:mRNA sequencing was performed on E. coli evolved at 37C on M9 Minimal Media with Glucose Growth of evolved E. coli was characterized under the same conditions as the evolution and compared to the wild type parent strain
2014-12-03 | E-GEOD-61327 | biostudies-arrayexpress