Transcription profiling of E. coli cells overexpressing GraL for short and long periods of time
Ontology highlight
ABSTRACT:
ORGANISM(S): Escherichia coli
SUBMITTER: Katarzyna Potrykus
PROVIDER: E-GEOD-15406 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:While studying greA expression, we noted presence of an intrinsic terminator in the leader region of greA mRNA transcript. We found this terminator to be quite efficient both in vivo and in vitro. This region seems to be conserved among many enteric bacteria. Judging from fitness experiments, the resulting short RNAs (50-59nt long) exert biological effects. This lead us to propose that greA leader region encodes a novel small non-coding RNA that we collectively call GraL. We used microarrays to detail the effects of GraL after short and longterm overproduction of GraL. Experiment Overall Design: E. coli cells harbouring plasmids overexpressing GraL or vector control, were monitored in an effort to elucidate a longterm (constitutive overexpression) or short term (15 min induction with IPTG) effects. This was done in two backgrounds- either in wt E. coli cells, or ppGpp0 cells (delta relA, delta spoT).
2009-12-19 | E-GEOD-15405 | biostudies-arrayexpress
Project description:While studying greA expression, we noted presence of an intrinsic terminator in the leader region of greA mRNA transcript. We found this terminator to be quite efficient both in vivo and in vitro. This region seems to be conserved among many enteric bacteria. Judging from fitness experiments, the resulting short RNAs (50-59nt long) exert biological effects. This lead us to propose that greA leader region encodes a novel small non-coding RNA that we collectively call GraL. We used microarrays to detail the effects of GraL after short and longterm overproduction of GraL. Experiment Overall Design: E. coli cells harbouring plasmids overexpressing GraL or vector control, were monitored in an effort to elucidate a longterm (constitutive overexpression) or short term (15 min induction with IPTG) effects. This was done in two backgrounds- either in wt E. coli cells, or ppGpp0 cells (delta relA, delta spoT).
2009-12-19 | E-GEOD-15404 | biostudies-arrayexpress
Project description:MgrR is a newly characterized Hfq dependent small RNA RNA. The expression of MgrR is regulated by Two component system, PhoPQ regulon, which senses low Mg2+ in environment. It has been reported that Hfq-binding sRNAs base pair with target RNAs, frequently leading to rapid degradation of target messages or, less frequently, to stabilization, both of which can be assayed by using microarrays. In order to search for the target genes of MgrR, we therefore examined the consequences of MgrR expression on mRNA abundance under two conditions. In condition 1, the chromosomal copy of mgrR was deleted and MgrR was expressed for 15â from an induced plac-mgrR plasmid and compared to cells carrying a vector induced for the same period. In condition 2, the expression of mRNAs was compared in wild-type cells (mgrR+) and the mgrR deletion strain, both grown in LB; because MgrR levels are fairly high under our normal growth conditions, this allowed analysis of both the direct and indirect (long-term) effects of MgrR. Experiment Overall Design: The strains used are either wild type which is derivatives of MG1655 or mgrR::kn knockout to restrict MgrR expression to the inducible pBR Plac-mgrR vector. The mgrR knockout mutants carried either pBR Plac-mgrR or the control vector pBR Plac. A Fresh colony of bacteria was inoculated in LB media with ampicilline at final concentration of 100ug/ml at 37°C with agitation. To induce MgrR expression, cultures carrying the pBR Plac-mgrR construct were grown to an O.D.600 of 0.5 and IPTG was added to the culture at a final concentration of 100uM. Both cultures for wild type and mgrR deletion mutant were grown to O.D. 600 of 0.5 and cells were collected. Total RNA was extracted from cells at the indicated time using the hot phenol procedure.
2009-11-14 | E-GEOD-18935 | biostudies-arrayexpress
Project description:Bacteria selectively consume some carbon sources over others through a regulatory mechanism termed catabolite repression. Here, we show that the base pairing RNA Spot 42 plays a broad role in catabolite repression in Escherichia coli by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse non-preferred carbon sources. Many of the genes repressed by Spot 42 are transcriptionally activated by the global regulator CRP. Since CRP represses Spot 42, these regulators participate in a specific regulatory circuit called a multi-output feedforward loop. We found that this loop can reduce leaky expression of target genes in the presence of glucose and can maintain repression of target genes under changing nutrient conditions. Our results suggest that base pairing RNAs in feedforward loops can help shape the steady-state levels and dynamics of gene expression. MG1655 lacIq cells harboring the control vector pBRplac or the Spot 42-inducible vector pSpot42 were grown in LB to an ABS600 of ~0.4 and treated with 1 mM IPTG. After 7 min, total RNA was isolated using the hot phenol extraction procedure.
2011-01-01 | E-GEOD-24875 | biostudies-arrayexpress
Project description:Strains devoid of ppGpp (ΔrelA ΔspoT; called ppGpp0), and ppGpp0 dksA- exhibit several amino acid requirements for growth on minimal media. We found that overexpression of DksA can complement some of those requirements. Since DksA is a factor that binds to the RNA polymerase secondary channel, we wondered if other secondary channel proteins might also exert a similar role with respect to growth on minimal media. In our study we found that GreA and partially GreB can in fact complement these requirements under certain conditions. Here, we wished to investigate a broader effect of GreA and GreB on ppGpp0 and ppGpp0 dksA- strains. Since the parent strains are unable to grow in minimal media, we had to supplement the M9 glucose medium with a set of amino acids (DFHILQSTV). We found that both, GreA and GreB can affect a much larger set of genes in the absence of dksA, than in its presence. Also, GreA seems to affect more genes than GreB, under both conditions. We used microarrays to detail the effects of overproducing either GreA or GreB in cells devoid of ppGpp, in the dksA+ or dksA- background E. coli cells harbouring plasmids carrying either greA (pA = pHM1873) or greB (pB = pHM1874) genes, or vector control (pGB2), were monitored in an effort to elucidate the effects of their protein products. This was done in two backgrounds- either in E. coli ppGpp0 cells (ΔrelA, ΔspoT) carrying wt dksA gene or a dksA deletion.
2011-10-26 | E-GEOD-28795 | biostudies-arrayexpress
Project description:ABSTRACT: BACKGROUND: While changes in chromosome number that result in aneuploidy are associated with phenotypic consequences such as Down syndrome and cancer, the molecular causes of specific phenotypes and genome-wide expression changes that occur in aneuploids are still being elucidated. RESULTS: We employed a segmental aneuploid condition in maize to study phenotypic and gene expression changes associated with aneuploidy. Maize plants that are trisomic for 90% of the short arm of chromosome 5 and monosomic for a small distal portion of the short arm of chromosome 6 exhibited a phenotypic syndrome that includes reduced stature, tassel morphology changes and the presence of knots on the leaves. The knotted-like homeobox gene knox10, which is located on the short arm of chromosome 5, was shown to be ectopically expressed in developing leaves of the aneuploid plants. Expression profiling revealed that ~40% of the expressed genes in the trisomic region exhibited the expected 1.5 fold increased transcript levels while the remaining 60% of genes did not show altered expression even with increased gene dosage. CONCLUSIONS: We found that the majority of genes with altered expression levels were located within the chromosomal regions affected by the segmental aneuploidy and exhibits dosage-dependent expression changes. A small number of genes exhibit higher levels of expression change not predicted by the dosage, or display altered expression even though they are not located in the aneuploid regions. Experiment Overall Design: There are four biological replicates of pooled wild-type siblings and four biological replicates of pooled DpDf plants. The DpDf plants are segmental aneuploids that contain three copies of the short arm of chromosome 5. These plants are all in the B73 inbred genetic background.
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Project description:We wanted to test the effect on global gene expression of depleting the essential cell cycle regulator CtrA in order to determine the genes both indirectly and directly transcriptionally regulated by CtrA Gene expression changes in S. meliloti 1,2,4 and 6 hours post CtrA depletion Log-phase S. meliloti cultures carrying an IPTG-inducible allele of CtrA were split and transferred to growth media lacking IPTG (depletion) and with 1mM IPTG (control). Samples for RNA extraction were taken 1,2,4, and 6 hours after the start of the depletion experiment to monitor gene expression changes in the population after different periods of CtrA depletion.
2015-04-24 | E-GEOD-68218 | biostudies-arrayexpress
Project description:The replicative helicase, DnaC, was immunoprecipitated in cell extract prior to, or following conditional depletion of PcrA The differential DnaC association at each genomic location was determined before/after PcrA depletion
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