Aspergillus fumigatus
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PROVIDER: PRJNA115787 | ENA |
REPOSITORIES: ENA
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Project description:A. fumigatus wild type strain transcriptome analysis upon 200 mM CaCl2 treatment.
| PRJNA103317 | ENA
Project description:CRZ is a C2H2 zinc finger transcription factor activated by calcineurin, a protein phosphatase activated by calcium-calmodulin, in different stress conditions related with calcium cell accumulation. CRZ binds to a specific element on the promoter region of genes called CDRE (calcineurin-dependent response element) shown to be sufficient for calcium- and calcineurin-dependent gene expression. In order to investigate which pathways are involved under calcium stress conditions, we determined the transcriptional profile of A. fumigatus delta crzA mutant strain upon a time course 200 mM calcium chloride treatment. Conidia from the mutant and wild type strains were incubated at 37°C in complete medium for 16 hours and were challenged with 200 mM calcium chloride for 10 and 30 minutes. At each time point, the mycelia were harvested by centrifugation and used for competitive microarray hybridizations. We were able to observe a decreased mRNA expression of genes encoding inorganic ion transport proteins and lipid transport. In contrast, increased mRNA expression was observed for several genes involved in transcription, energy production and conversion, cell cycle control, cell division, chromosome partitioning, amino acid, lipid and carbohydrate transport and metabolism, nucleotide transport, cell wall and membrane biogenesis, posttranslation modifications, protein turnover, chaperones, inorganic ion transport, signal transduction mechanisms, intracellular trafficking, secretion and vesicular transport. Keywords: gene expression array-based (log2 ratio) For the time course microarray experiments, 5.0 x 108 conidia of A. fumigatus wild type [CEA17 delta akuB(KU80)] and delta crzA strains were inoculated in 300 ml of pre-warmed liquid cultures (YG) in 500-mL erlenmeyer flasks and allowed to grow for 16 hours in a reciprocal shaker (250 rpm) at 37°C. After this time, the cultures were added by calcium chloride (200 mM) and incubated for 10 and 30 more minutes before mycelia recovery and RNA extraction. Hybridization experiments were competitive using probes derived from calcium chloride shock cultures using the wild type strain exposed to calcium (10 or 30 minutes) as the reference RNA for every hybridizantion. Normalized signal intensities were used to generate relative hybridization ratios (query/reference). Following normalization, the values for each gene's in-slide replicates were averaged (median variance <0.01), and corresponding flip-dye replicates were averaged to compensate for dye-specific effects. These final, processed data are linked below as supplementary files.
2010-06-20 | E-GEOD-15432 | biostudies-arrayexpress
Project description:CRZ is a C2H2 zinc finger transcription factor activated by calcineurin, a protein phosphatase activated by calcium-calmodulin, in different stress conditions related with calcium cell accumulation. CRZ binds to a specific element on the promoter region of genes called CDRE (calcineurin-dependent response element) shown to be sufficient for calcium- and calcineurin-dependent gene expression. In order to investigate which pathways are involved under calcium stress conditions, we determined the transcriptional profile of A. fumigatus delta crzA mutant strain upon a time course 200 mM calcium chloride treatment. Conidia from the mutant and wild type strains were incubated at 37°C in complete medium for 16 hours and were challenged with 200 mM calcium chloride for 10 and 30 minutes. At each time point, the mycelia were harvested by centrifugation and used for competitive microarray hybridizations. We were able to observe a decreased mRNA expression of genes encoding inorganic ion transport proteins and lipid transport. In contrast, increased mRNA expression was observed for several genes involved in transcription, energy production and conversion, cell cycle control, cell division, chromosome partitioning, amino acid, lipid and carbohydrate transport and metabolism, nucleotide transport, cell wall and membrane biogenesis, posttranslation modifications, protein turnover, chaperones, inorganic ion transport, signal transduction mechanisms, intracellular trafficking, secretion and vesicular transport. Keywords: gene expression array-based (log2 ratio)
2009-04-02 | GSE15432 | GEO
Project description:Transcriptome of A. fumigatus shifted from ammoniumtartrate to different nitrogen sources and incubated for a defined time was compared. After 16h preculture, the fungus was transferred into fresh medium containing ammonium tartrate, sodium nitrate, proline or bsa as nitrogen source. After 1h, fungus was reisolated, RNA was prepared from fungus, transcriptome was assessed and used for further analysis. Ammoniumtartrate, sodiumnitrate, BSA and proline as nitrogen sources, 2 biological replicates for each source. A. fumigatus liquid media shifts were performed according to ref. (Narendja et al.,2002) with minor modifications: 200 ml minimal medium base with 5 mM ammonium tartrate was inoculated with 10^8 conidia freshly harvested. A. fumigatus ATCC 46645 conidia were grown at 37°C and 150 rpm for 16 hours. This pre-culture was then harvested, washed liberally with sterile saline and divided into mycelial masses of equal size on a sterile surface. The portions were then added to 100 ml of minimal medium base without nitrogen source. When a defined carbon source was needed, 1% glucose was added. Flasks were incubated for 20 min at 37°C. Thereafter, one of the following sterile nitrogen sources was added: ammonium tartrate to a final concentration of 5 mM, Sodiumnitrate to a final concentration of 10 mM, 1 g proline suspended in 2 ml minimal medium base, or 0.5 g BSA (Albumin Fraktion V, Roth), suspended in 15 ml minimal medium base. The cultures were incubated for another 60 minutes, harvested by filtering through Miracloth, snap frozen in liquid nitrogen, and ground using a cooled mortar to obtain a fine powder.
2011-10-29 | E-GEOD-30384 | biostudies-arrayexpress
Project description:A. fumigatus wild type and delta phoB(PHO80) strains transcriptome analysis for growth upon minimal medium 0.1 mM Pi
| PRJNA105183 | ENA
Project description:A. fumigatus wild type and delta phoB(PHO80) strains transcriptome analysis for growth upon minimal medium 11 mM Pi
| PRJNA105185 | ENA
Project description:Purpose: Investigation of DDX41 chromatin binding sites in U2OS cells Method: Expression of DDX41-GFP was induced for 48 hours before crosslinking using 1µg/ml docycycline. Control cells were not induced with doxycline. Cells were mildly crosslinked using 1% FA for 2 min at RT. Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2).1*10^6 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 1 ml Wash Buffer. Cells were permeabilized with 0.05% digitonin in Wash buffer for 4 min at RT. 1 µg Nanobody-GFP-MNase (in-house protein production, PMID:25362362) binding was performed at 4°C for 30 min in 0.05% digitonin-Wash buffer. After 2x washing, the MNase was activated by adding 3mM CaCl2 on ice for 30 min. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, )) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We succesfully mapped DDX41 binding sites on cromatin in U2OS cells. DDX41 preferentially binds in the promoter region of genes.
2021-11-17 | GSE183083 | GEO
Project description:Purpose: Investigation of genome-wide changes in R-loop levels after knockdown of DDX41 HCT116 cells in comparison to a control knockdown. Method: Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2). 1*10^6HCT116 cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 50 µl Wash Buffer containing 0.05% Digitonin. Either pA-MNase or RHΔ-MNase were added to the cells overnight at 4°C on a rotating wheel. After three washes with Wash Buffer containing 0.05% Digitonin, samples resuspended in 100 µl Dig-Wash-Buffer were equilibrated on ice. Activity of the MNase was triggered by adding 2mM CaCl2 to the samples for 30 minutes. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, 20 pg/ml spike-in Drosophila DNA (Active Motif)) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We were able to map R-loop dynamics genome-wide in HCT116 cells in biological triplicates. MapR signal was significantly increased around the transcription start site in the absence of DDX41
2021-11-17 | GSE183081 | GEO
Project description:We previously observed in mice that Bacteroides thetaiotaomicron (B. theta) significantly increased in abundance in the gut microbiome of mice when mice were fed egg and yeast dietary protein sources. We also observed that B. theta was expressing proteins previously connected to growth on mucin glycans when mice were fed an egg-white diet. To confirm that the bacterium were actually responding to the protein sources, we grew it in vitro using a defined medium, where the sole carbon source was dietary protein, mucin, or glucose. Our controls were glucose and mucin, and our experimental protein sources were soy protein, egg-white protein, and Torula yeast protein. We grew four B. theta cultures per carbon source statically at 37°C in a Coy anaerobic chamber (2.5 % H2 /10 % CO2 /88.5 % N2) in minimal medium (100 mM KH2PO4, 8.5 mM [NH2]4SO4, 15 mM NaCl, 5.8 μM vitamin K3, 1.44 μM FeSO4⋅7H2O, 1 mM MgCl2, 1.9 μM hematin, 0.2 mM L-histidine, 3.69 nM vitamin B12, 208 μM L-cysteine, and 7.2 μM CaCl2⋅2H2O) with one of the above mentioned nutrients added at 0.5% (wt/v) concentration. In order to aid the solubilization of the dietary proteins, we pre-prepared the proteins in 200 mM NaOH at 37°C for four days, the glucose control was also dissolved in 200 mM NaOH. After 8 hours, we enumerated CFUs to confirm growth, and we pelleted cells by centrifuging at 4,000 g for 10 minutes. We then removed the supernatant and froze the pellets at -80°C within 30 minutes.
2024-12-18 | PXD050296 | Pride
Project description:Purpose: Investigation of genome-wide changes in R-loop levels after knockdown of DDX41 U2OS cells in comparison to a control knockdown. Method: Concanavalin A-coated beads were activated in Binding Buffer (20 mM Hepes-KOH pH 7.9, 10 mM KCl, 1mM CaCl2, 1 mM MnCl2). 5*105 U2OS cells were washed twice with Wash Buffer (Hepes-NaOH pH7.5, 150 mM NaCl, 0.5 mM Spermidine, 1 mM protease inhibitor) at room temperature and afterwards immobilized on the activated beads in 50 µl Wash Buffer containing 0.05% Digitonin. Either pA-MNase or RHΔ-MNase were added to the cells overnight at 4°C on a rotating wheel. After three washes with Wash Buffer containing 0.05% Digitonin, samples resuspended in 100 µl Dig-Wash-Buffer were equilibrated on ice. Activity of the MNase was triggered by adding 2mM CaCl2 to the samples for 30 minutes. 2x Stop Buffer (68 µl 5M NaCl, 40 µl 0.5 M EDTA, 20 µl 0.2 M EGTA, 10 µl 5% digitonin, 5 µl 10mg/ml RnaseA, 20 pg/ml spike-in Drosophila DNA (Active Motif)) was mixed with the samples to stop the reaction. Chromatin fragments were released by incubating the samples for 20 minutes at 37°C and centrifugation at 16.000×g for 10 minutes at 4°C. Supernatants were incubated at 70°C in the presence of 0.1% SDS and 5 µg proteinase K. Before library preparation, the DNA was recovered by phenol-chloroform extraction Results: We were able to map R-loop dynamics genome-wide in U2OS cells in biological triplicates. MapR signal was significantly increased around the transcription start site in the absence of DDX41 and decreased after treatment with ActinomycinD.
2021-05-04 | GSE168170 | GEO