Unknown,Transcriptomics,Genomics,Proteomics

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The Transcriptionally Active Regions in the Genome of Bacillus subtilis

ABSTRACT: The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3,217 strand-specific Transcriptionally Active Regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome-wide expression during mid-exponential growth on rich (LB) and minimal (M9) medium. The identified TARs account for 81.5% of the genes as they are currently annotated and additionally we find 44 novel protein-coding genes, 85 putative non-coding RNAs (ncRNAs) and 184 antisense transcripts. Hybridization of labeled genomic DNA (gDNA) has revealed a few thousand regions giving rise to very low signals. These are mostly caused by sequence errors, as is observed in the highly degenerate trp operon and the folding of stable structures such as the regions containing Rho-independent terminators. Through this work we have discovered a group of membrane-associated genes, among these having a long conserved 3â UnTranslated Region (UTR) predicted to fold into a large and highly stable structure. Among these are YwbN, a target of the twin-arginine translocase (Tat) protein translocation system. The transcriptionally active regions were determined at two different conditions, rich medium (Luria Broth) and minimal medium (M9). Cells were harvested at mid-exponential phase (OD600 = 0.5) and each condition were replicated three times. From each replicate RNA was extracted using FastRNA pro blue kit (Qbiogene) and labelled with Cy3 using a strand-specific protocol. The cDNA was hybridized to 385K NimbleGen tiling arrays 'BaSysBio Bsub T1' covering the entire genome with 45-65 nt isothermal probes, spaced by 22 nt on each strand. Additionally genomic DNA was extracted from 4 replicates at OD600 = 0.5, labelled and hybridized to the tiling arrays. These serve as a reference for the mRNA hybridizations. The normalized RNA hybridization signals were used to create segments using a structural change model (Huber et al, 2006) and transcriptionally active regions were identified by a segment joining-scheme.

ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168

SUBMITTER: Simon Rasmussen

PROVIDER: E-GEOD-16086 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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The transcriptionally active regions in the genome of Bacillus subtilis.

Rasmussen Simon S Nielsen Henrik Bjørn HB Jarmer Hanne H

Molecular microbiology 20090804 6

The majority of all genes have so far been identified and annotated systematically through in silico gene finding. Here we report the finding of 3662 strand-specific transcriptionally active regions (TARs) in the genome of Bacillus subtilis by the use of tiling arrays. We have measured the genome-wide expression during mid-exponential growth on rich (LB) and minimal (M9) medium. The identified TARs account for 77.3% of the genes as they are currently annotated and additionally we find 84 putativ ...[more]

PMID: 19682248

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Project description:Experiments conducted on this tiling array are used to (1) validate the frozen gene sets of the current genome annotation, (2) improve the predicted gene structures by empirically determining UTRs and intron-exon boundaries, identifying missing upstream, internal, and downstream exons and alternative transcripts, (3) propose gene structure models in transcribed regions containing no predicted genes and (4) delineate transcriptionally active regions of the genome from intergenic, intronic and genic regions. Signal to background ratios were determined by first calling probes that fluoresced at intensities greater than 99% of the random probes’ signal intensities; therefore only 1% of fluorescing experimental probes should be false positives. We conducted two-color competitive hybridizations that measure differential expression from three replicates, each using RNA from independent biological extractions. Transcriptional active regions (TARs) were defined by stringing together overlapping probes showing fluorescence above a 1% false positive rate (FPR). Positive probes were joined into a TAR if they were adjacent (maxgap=0, no intermittent non-positive probe) and a TAR’s length had to be at least 45 bp (minrun=45, mid-point first positive probe to mid-point last positive probe, resulting in at least 3 adjacent positive probes for a TAR). Transcriptional active regions (TARs) were defined by stringing together overlapping probes showing fluorescence above a 1% false positive rate (FPR). The data analysis to measure differential expression of genes and of unannotated TARs was performed using the statistical software package R and Bioconductor with additions and modifications. The signal distributions across chips, samples and replicates were adjusted to be equal according to the mean fluorescence of the random probes on each array. All probes including random probes were quantile-normalized across replicates. Expression-level scores were assigned for each predicted gene based on the median log2 fluorescence over background intensity of probes falling within the exon boundaries.

Dataset Information

The Transcriptionally Active Regions in the Genome of Bacillus subtilis

Publications

The transcriptionally active regions in the genome of Bacillus subtilis.

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