Equivalent mutations in the eight subunits of the chaperonin CCT produce dramatically different cell phenotypes.
ABSTRACT: The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. Here, we decided to test the significance of the hetero-oligomeric nature of CCT for its function by introducing, in each of the eight subunits in turn, an identical mutation at a position involved in ATP binding and conserved in all the subunits, in order to establish the extent of ‘individuality’ of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, cells with the mutation in CCT2 have an excess of actin patches and are the only pseudo-diploid strain. By contrast, cells with the mutation in CCT7 are the only ones to accumulate juxta-nuclear protein aggregates that may reflect the absence of stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks including ribosome biogenesis and TOR2 that help to explain the phenotypic variability observed We used microarrays to reveal the differences in mRNA expression caused by the different mutations. Overall design: All yeast strains were grown at 30 °C to OD(600)=0.5. Their total RNA was extracted and reverse transcribed to cDNA and transcribed back to RNA in the presence of biotinylated nucleotide analog. The biotinylated RNA was fragmented and hybridized to GenCHip Yeast Genome 2.0 array.
Project description:The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins and other proteins in an ATP-dependent manner. Here, we decided to test the significance of the hetero-oligomeric nature of CCT for its function by introducing, in each of the eight subunits in turn, an identical mutation at a position involved in ATP binding and conserved in all the subunits, in order to establish the extent of ‘individuality’ of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, cells with the mutation in CCT2 have an excess of actin patches and are the only pseudo-diploid strain. By contrast, cells with the mutation in CCT7 are the only ones to accumulate juxta-nuclear protein aggregates that may reflect the absence of stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks including ribosome biogenesis and TOR2 that help to explain the phenotypic variability observed We used microarrays to reveal the differences in mRNA expression caused by the different mutations. All yeast strains were grown at 30 °C to OD(600)=0.5. Their total RNA was extracted and reverse transcribed to cDNA and transcribed back to RNA in the presence of biotinylated nucleotide analog. The biotinylated RNA was fragmented and hybridized to GenCHip Yeast Genome 2.0 array.
Project description:NF-kB plays a crucial role in immunity to infection. This transcription factor consists in different combinatory homo- and hetero-dimers of the five members of the Rel family. cRel/p50-containing dimers have been involved in the development and function of the immune cells. However, the transcriptional roles of these two subunits still remain poorly explored in innate immune cells. By a multiple approach integrating ex vivo genomic analysis and an in vivo experimental study, we have investigated the consequences of the combined absence of cRel and p50 subunits of NF-kB in the innate response to infection. We have performed gene profiling of cRel-/-p50-/- (DKO) and wild type (WT) peritoneal macrophages stimulated with endotoxin for 2, 6 or 18 hours.
Project description:Three types of phenotypic expression of ß-lactam resistance has been reported in MRSA: heterogeneous-, homogeneous-, and Eagle-type resistance. Heterogeneous to homogeneous (hetero-to-homo) conversion of ß-lactam resistance is postulated to be caused by a chromosomal mutation (chr*) together with mecA-gene expression. The Eagle-type resistance is a special pattern of chr* expression in the pre-MRSA strain N315 under the strong mecI-gene mediated repression of mecA gene transcription. Here, for the identification of chr*, experiments were conducted using an in-vitro derived homogeneously imipenem-resistant MRSA strain N315∆IPH5 (∆IPH5). The strain was selected with imipenem 8 mg/L from the heterogeneously imipenem-resistant MRSA strain N315∆IP (∆IP). The whole genome sequencing of ∆IPH5 revealed the presence of a unique mutation in the rpoB gene, rpoB(N967I), causing the amino-acid (AA) substitution of Asp by Ile at the 967th AA position of the RNA polymerase ß subunit. The effect of the mutation on the phenotypic change was confirmed by constructing and studying the phenotype of the strains H5rpoB(I967N), a ∆IPH5-derived strain cured of the rpoB mutation, and N315rpoB(N967I), a N315-derived strain introduced with the mutated rpoB gene. H5rpoB(I967N) regained the hetero-MRSA phenotype, and the mutant strain N315rpoB(N967I) showed an Eagle-type phenotype similar to that of N315h4. Furthermore, subsequent whole genome sequencing revealed that N315h4 also had a missense mutation in the rpoB gene rpoB(R644H). The rpoB mutations caused decreased autolysis, prolonged doubling-time, and tolerance to bactericidal concentrations of methicillin. We concluded that the certain rpoB mutations are chr* responsible for the hetero-to-homo phenotypic conversion of MRSA. We compared the gene expression profiles of the wild-type strain and rpoB mutant (N967I) using a 60mer oligo array.
Project description:ATP-dependent chromatin remodelers regulate chromatin structure during multiple stages of transcription. We report that RSC, an essential chromatin remodeler, is recruited to the open reading frames (ORFs) of actively transcribed genes genome-wide, suggesting a role for RSC in regulating transcription elongation. Consistent with such a role, Pol II occupancy in the ORFs of weakly transcribed genes is drastically reduced upon depletion of the RSC catalytic subunit Sth1. RSC inactivation also reduced histone H3 occupancy across transcribed regions. Remarkably, the strongest effects on Pol II and H3 occupancy were confined to the genes displaying the greatest RSC ORF enrichment. Additionally, RSC recruitment to the ORF requires the activities of the SAGA and NuA4 HAT complexes and is aided by the activities of the Pol II CTD Ser2 kinases Bur1 and Ctk1. Overall, our findings strongly implicate ORF-associated RSC in governing Pol II function and in maintaining chromatin structure over transcribed regions. In these experiments, we have analyzed Sth1 (catalytic subunit of the RSC chromatin remodeling complex) enrichment to the transcribing genes. The cells (WT and gcn4Δ) harboring STH1-MYC allele were treated by SM for 20 minutes to induce Gcn4 regulated genes. The chromatin extracts were prepared and subjected to chromatin immunoprecipitation using anti-Myc antibodies. The ChIP DNA as well the corresponding input DNA were biotinylated and hybridized to the Affymetrix tiling Arrays. Chromatin samples from two different cultures were used in this analysis.
Project description:Wang2007 - ATP induced intracellular Calicum Oscillation
The model simulate the ATP-induced intracellular Ca2+ oscillations and the quantitative effect of ATP concentration on the oscillation characteristics such as the duration, peak concentration of intracellular Ca2+ and average interval.
This model is described in the article:
A quantitative kinetic model for ATP-induced intracellular Ca2+ oscillations.
Wang J, Huang X, Huang W.
J. Theor. Biol. 2007 Apr; 245(3): 510-519
A quantitative kinetic model is proposed to simulate the ATP-induced intracellular Ca(2+) oscillations. The quantitative effect of ATP concentration upon the oscillations was successfully simulated. Our simulation results support previous experimental explanations that the Ca(2+) oscillations are mainly due to interaction of Ca(2+) release from the endoplasmic reticulum (ER) and the ATP-dependent Ca(2+) pump back into the ER, and the oscillations are prolonged by extracellular Ca(2+) entry that maintains the constant Ca(2+) supplies to its intracellular stores. The model is also able to simulate the sudden disappearance phenomenon of the Ca(2+) oscillations observed in some cell types by taking into account of the biphasic characteristic of the Ca(2+) release from the endoplasmic reticulum (ER). Moreover, the model simulation results for the Ca(2+) oscillations characteristics such as duration, peak [Ca(2+)](cyt), and average interval, etc., lead to prediction of some possible factors responsible for the variations of Ca(2+) oscillations in different types of cells.
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Project description:Temporal coordination of developmental programs is necessary for normal ontogeny, but the mechanism by which this is accomplished is poorly understood. We have previously shown that two components of the Mediator CDK8 module, CENTER CITY (CCT/MED12) and GRAND CENTRAL (GCT/MED13), are required for timing of pattern formation during embryogenesis in Arabidopsis. Here, we performed global gene expression analyses of wild-type, cct-1, and gct-2 seedlings (above-ground portions only) to help analyze their post-embryonic phenotypes. Our results suggest that MED12 and MED13 act as global regulators of developmental timing by fine-tuning expression of temporal regulatory genes. Seeds of wild-type Col-0, cct-1, and gct-2 were sown in Fafard #2 soil. Seedlings (above-ground portions only) were harvested when the first two leaf primordia were 1 mm in length, which was at day 7 for wild-type seedlings and day 9 for the two mutants. 75-100 seedlings were used for each of three biological replicates.
Project description:Promoter recognition by bacterial RNA polymerase is mediated by σ subunits, which assemble transiently to RNA polymerase core enzyme (E) during transcription initiation. σ subunits drive transcription of specific sets of genes by allowing RNA polymerase to interact with different promoter sequences. However, σ70, the housekeeping σ subunit, and σS, an alternative σ subunit mainly active during slow growth and in response to cellular stresses, appear to recognize almost identical promoter sequences, raising the question of how promoter selectivity is achieved in the bacterial cell. To identify sequence determinants for selective promoter recognition, we performed a run-off/microarray experiment (ROMA): in vitro transcription experiments were carried out with RNA polymerase saturated either with σ70 (Eσ70) or with σS (EσS) using the whole Escherichia coli genome as DNA template, and transcript levels were determined by microarray analysis. We found that several genes associated with bacterial growth (e.g., ribosomal operons) were transcribed more efficiently by Eσ70. In contrast, EσS transcribed preferentially genes involved in stress responses, secondary metabolism, as well as regulatory RNAs and intergenic regions with yet unknown function. Genes preferentially recognized in vitro by EσS showed reduced expression in EσS -deficient mutant strain of E. coli. Sequence comparison of Eσ70- versus EσS –dependent promoters confirms that the presence of a -35 sequence and the relative location of UP elements affect promoter interaction with either form of RNA polymerase, and suggests that a G/C bias in the -2/+1 nucleotides would favour efficient promoter recognition by Eσ70. Overall design: We have performed in vitro transcription experiments with either Eσ70 or EσS, using the whole E. coli genome as template, to identify promoter regions selectively recognized by two forms of RNA polymerase by using E.coli genome 2.0 GeneChips.
Project description:The mitochondrial ATP synthase is a macromolecular motor that uses the proton gradient to generate ATP. Proper ATP synthase function requires a stator linking the catalytic and rotary portions of the complex. However, sequence-based searches fail to identify genes encoding stator subunits in apicomplexan parasites like Toxoplasma gondii or the related organisms that cause malaria. Here, we identify 11 previously unknown subunits from the Toxoplasma ATP synthase, which lack homologs outside the phylum. Hidden Markov modeling suggests that two of them—ICAP2 and ICAP18—share distant homology with mammalian stator subunits. Our mass spcetrometry analysis indicates that both proteins form part of the ATP synthase complex.
Project description:Extracellular adenosine 5’-triphosphate (ATP) is a signaling molecule. To define the global transcriptional response to ATP, we performed DNA microarray analysis using RNA derived from wild-type (Col-0) Arabidopsis and the dorn1-1 mutant, Lectin receptor kinase I.9 (At5g60300) carrying a EMS point mutation, seedlings after 100 μM ATP. 12 samples ( 2 genotypes, 2 treatments, 3 biological replicates)