ChAP-chip by array of Bacillus subtilis to obtain genome-wide profiles of AbrB and Abh
Ontology highlight
ABSTRACT:
ORGANISM(S): Bacillus subtilis
SUBMITTER: Shu Ishikawa
PROVIDER: E-MEXP-2774 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Transcriptome analysis of abrB, abh, and abrB abh deletion mutant strains
2010-12-24 | E-MEXP-2776 | biostudies-arrayexpress
Project description:We constructed the B. subtilis strains in which the expression of the intact and the C-terminal truncated RNAP alpha subunit were induced by different stimulus, IPTG and xylose, under the control of Pspac and Pxyl promoters. Then, we performed ChAP-chip analysis to analyze the impact of alpha-CTD defect on genome-wide transcriptome profile.
2015-06-25 | E-MTAB-3385 | biostudies-arrayexpress
Project description:We constructed the B. subtilis strains in which the expression of the intact and the C-terminal truncated RNAP alpha subunit were induced by different stimulus, IPTG and xylose, under the control of Pspac and Pxyl promoters. Then, we performed ChAP-chip analysis to analyze the impact of M-^UM-^AM-A-CTD defect on genome-wide transcriptome profile.
2015-06-25 | E-MTAB-3384 | biostudies-arrayexpress
Project description:Coordination of chromosome segregation and cytokinesis is crucial for efficient cell proliferation. In Bacillus subtilis the nucleoid occlusion protein Noc protects chromosomes by associating with the chromosome and preventing cell division in its vicinity. Using protein localization, ChAP-on-Chip and bioinformatics, we have identified a consensus Noc-binding DNA sequence (NBS), and show that Noc is targeted to about 70 discrete regions scattered around the chromosome, though absent from a large region around the replication terminus. Purified Noc bound specifically to an NBS in vitro. NBSs inserted near the replication terminus bound Noc-YFP and caused a delay in cell division. An autonomous plasmid carrying an NBS recruited Noc-YFP and conferred a severe Noc-dependent inhibition of cell division. This shows that Noc is a potent inhibitor of division but that its activity is strictly localized by interaction with NBS sites in vivo. We propose that Noc not only serves as a spatial regulator of cell division to protect the nucleoid, but also a timing device with an important role in the co-ordination of chromosome segregation and cell division.
2009-07-01 | E-MEXP-2133 | biostudies-arrayexpress
Project description:The direct roles of the NO-sensitive NsrR repressor and the ResD response regulator in transcriptional control in Bacillus subtilis
2013-11-22 | E-MEXP-3882 | biostudies-arrayexpress
Project description:Bacterial Gre factors associate with RNA polymerase (RNAP) and stimulate intrinsic cleavage of the nascent transcript at the active site of RNAP. Biochemical and genetic studies to date have shown that E. coli Gre factors prevent transcriptional arrest during elongation and enhance transcription fidelity. Furthermore, Gre factors participate in stimulation of promoter escape and suppression of promoter-proximal pausing during beginning of RNA synthesis in E. coli. Although Gre factors are conserved in general bacteria, limited functional studies have been performed in bacteria other than E. coli. In this investigation, ChAP-chip analysis was conducted to visualize the distribution of B. subtilis GreA on the chromosome and determine the effects of GreA inactivation on core RNAP trafficking. Our data show that GreA is uniformly distributed in the transcribed region from the promoter to coding region with core RNAP, and its inactivation induces RNAP accumulation at many promoter or promoter-proximal regions. Based on these findings, we propose that GreA would constantly associate with core RNAP during transcriptional initiation and elongation, and resolves its stalling at promoter or promoter-proximal regions, thus contributing to the even distribution of RNAP along the promoter and coding regions in B. subtilis cells.
2011-04-10 | E-MEXP-3056 | biostudies-arrayexpress
Project description:To obtain an insight into the in vivo dynamics of RNA polymerase (RNAP) on the B. subtilis genome, we analyzed the distribution of ?A and ? subunits of RNAP and the NusA elongation factor on the genome in exponentially growing cells, using the ChAP (Chromatin Affinity Precipitation)-chip method. In contrast to E. coli RNAP, which often accumulates at the promoter-proximal region, B. subtilis RNAP is evenly distributed from the promoter to the coding sequences in the majority of genes. This finding suggests that B. subtilis RNAP recruited to the promoter promptly translocates away from the promoter to form the elongation complex. We detected RNAP accumulation in the promoter-proximal regions of some genes, most of which are attributed to transcription attenuation systems in the leader region. Our findings suggest that the differences in RNAP behavior during initiation and early elongation steps between E. coli and B. subtilis result in distinct strategies for post-initiation control of transcription. The E. coli mechanism involves trapping at the promoter and promoter-proximal pausing of RNAP in addition to transcription attenuation, whereas transcription attenuation in leader sequences is mainly employed in B. subtilis. Wild-type strain, Bacillus subtilis 168, was also used for RNA and genomic DNA extraction and analysis - RNA data was divided by genome DNA data to normalize (1) PCR bias and (2) copy number of RNA molecule per genome for multi-copy genome of exponentially growing bacteria.
2010-06-29 | E-MEXP-2649 | biostudies-arrayexpress
Project description:Preparation of nuclear extracts<br><br>D. melanogaster Schneider cells were prepared as previously described by Andrews and Faller (1991). Briefly, in a typical preparation, 8x106 cells (grown in Schneider medium plus 10% fetal calf serum; GIBCO-BRL) were pelleted and resuspended in 1,5 mL cold PBS1X; the cell suspension is then tranferred to a microfuge tube. Cells are pelleted for 10 seconds and resuspended in 400 M-5L cold Buffer A (10mM HEPES-KOH pH 7.9 at 4M-0C, 1.5mM MgCl2, 10mM KCl, 0.5mM duthiothreitol, 0.2mM PMSF, 0.2U/microL RNasin). The cells are allowed to swell on ice for 10 minutes, and then vortexed for 10 seconds. Samples are centrifuged for 10 seconds, and the supernatant fraction is discarded. The pellet is resuspended in 100 M-5L of cold Buffer C (20mM HEPES-KOH pH 7.9, 25% glycerol, 420mM NaCl, 1.5mM MgCl2, 0.2mM EDTA, 0.5mM dithiothreitol, 0.2mM PMSF) and incubated on ice for 20 min for high-salt extraction. Cellular debris is removed by centrifugation for 2 min at 4M-0C and the supernatant fraction is stored at M-^V70M-0C. All buffers were prepared from double-distilled autoclaved water that had been treated with 0.1% DEPC (Sigma).<br><br>RNA immunoprecipitation and Reverse Transcription<br><br>For RNA immunopreciptation, the nuclear extract was incubated whit 50M-5g of monoclonal C1A9 anti HP1 antibody and the mixture was kept at 4M-0C overnight under continuous gentle movement. One-hundred microliters ofprotein G-Sepharose (Sigma) suspension (50% packed Sepharose in Buffer C) was added and the incubation was continued overnight as described.The beads were pelleted by 2 min centrifugation at 240 g at 4M-0C; the pellet was washed briefly three times whit each 1mL of IP Wash Solution (150mM NaCl, 50mM Tris pH 7.5, 0.5% NP40) and the Sepharose was transferred for eluition into a fresh plastic tube , pelleted again and then the supernatant was removed completely. To elute the immunocomplexes for protein analysis, an aliquot of beads were suspended in 50M-5L SDS-PAGE sample buffer and incubated for 10 min at 90M-0C; following centrifugation the supernatant was removed and used for Western blot for testing the presence of HP1 protein.<br>To elute the immunoprecipitated RNAs, the pelleted beads were boiled in 200M-5L of DEPC wather for 5 min, spun, and the supernatant recovered; 1mL of Trizol (Invitrogen) was added to 200 M-5L of supernatant and mixed well, followed by the addition of 200M-5L of chloroform. This mixture was incubated at 4M-0C for 5 min and then centrifuged at 12000 g for 15 min; the RNAs in the aqueous phase was precipitated whit half volume of isopropanol; after precipitation, the RNAs was resuspend in 10M-5L of DEPC wather. Contaminating DNA was digested whit RNase-free DNase I (Sigma). The RNA purified from the previous step is used as a template to synthesize cDNA using oligo dT , random hexamers and SuperScript reverse transcriptase III (Invitrogen) according to the manufacturer's protocol. <br><br> cDNA amplification and labeling<br><br>The cDNA was used as template for a two-step random PCR amplification; in Round A, Sequenase is used to extend randomly annealed primers (Primer A) to generate templates for subsequent PCR; during Round B, the specific primer B is used to aplify the templates previously generated and finally round C consist of additional PCR cycles to inorporate the amino allyl dUTP nucleotide.<br> About 25 ng of each cDNA sample was used for two 8 min extension with 2,7 mM Round A primer (5M-^R-GTT TCC CAG TCA CGA TCN NNN NNN NN-3M-^R, N being a mixture of all four nucleotides with 60% A+T and 40% G+C) at 37M-0C with 267U/ml Sequenase version 2.0 (usb). DNA was denatured at 94M-0C for 2 min and cooled to 10M-0C , and Sequenase 2.0 was added between extensions. The resulting products were used as template for 25 cycles of PCR using 1U/100M-5l Taq polymerase (Platinum Taq Invitrogen) and 10mM Round B primer (5M-^R-GTT TCC CAG TCA CGA TC-3M-^R). Finally this DNA was used as template for 25 cycles PCR to inorporate the amino allyl dUTP nucleotides to which the fluorescent dye may then be attached. To remove Tris buffer which interferes with the indirect coupling, the aminoallyl-cDNA samples were desalted by filtering through a Microcon M-^V30 and then mixed with the succinimidyl esters of the Cy3 or Cy5 dyes (Amersham Biosciences) in 0,1M sodium bicarbonate buffer (pH 9); the coupling reaction was icubated overnight in the dark at room temperature.<br>Each dye labeled sample was purified by AutoSeq MicroSpin G-50 columns (Amersham Biosciences) following the manufacterers directions.<br><br>Microarrays Hybridization and washing <br><br>The dried labeled cDNAs pellet were resuspended in 80M-5l of DIG Easy Hyb (Roche) hybridization buffer containg 0,5 mg/ml yeast tRNa and 0,5 mg/ml salmon sperm DNA. Finally the DNA probe was denaturated by incubation at 65M-0C for 10 min and, after a brief centrifugation to spin down the drops, the mixture was pipetted onto a microarray; a coverslip was applied and the slide was placed in a microarray hybridation chamber (BioRad) and incubated overnight at 37M-0C.<br>After hybridization, the slide was submerged in 0,01% SDS, 1%SSC until the coverslip slipped off the surface; The slide was transferred to a solution of 1%SSC and shaken at 50M-0C for 10 min, then washed once more by shaking in 0,1%SSC.<br>Slide was dried by centrifugation for 3 min at 550 rpm and scanned with an ScanArray Lite Microarray Scanner (Packard Bioscience) with laser intensities chosen to maximize signals while avoiding pixel saturation.<br>ScanArray express softwere was used to quantify hybridation signals; bad spots were flagged automatically by softwere and subsequently each slide was inspected manually.<br>
2009-10-12 | E-MEXP-1556 | biostudies-arrayexpress
Project description:Expression data from Kc167 cells under normal conditions. Used to assess expression levels of genes with ORC bound at promoter. Control experiments in support of our previous modencode submssion GSE17282. Kc167 cells were harvested from log phase gorwth. RNA extraction and hybridization on Affymetrix microarrays. Chromatin immunoprecipitation with IgG against TAP-ORC2 or control cells.
2010-06-20 | E-GEOD-18942 | biostudies-arrayexpress
Project description:P388D1 murine macrophages were cultured in 85 mm tissue culture plates to about semi confluency. L. monocytogenes serotypes (1/2a EGD-e, 4a L99, 4b CLIP80459 and 4b F2365) were infected to the P388D1 cell monolayer at a MOI of 100 per eukaryotic cell. Infection was carried for 45 min and followed by addition of fresh medium containing 20 M-5g/ml gentamicin. The medium of the plates (containing 20 M-5g/ml gentamicin) infected with L. monocytogenes serotypes were replaced after 2 h post infection with fresh medium containing 50 M-5g/ml gentamicin. At each step the plates were washed extensively with 1x PBS. Incubation of the bacterial tissue culture plates was carried out in a humidified incubator for up to 4 h post infection.
2010-11-25 | E-MEXP-1947 | biostudies-arrayexpress