Project description:To investigate the relationship between RNA polymerase binding and transcription ChIP-seq on the common house-keeping SigmaD and RNA polymerase beta subunit were coupled with RNA-seq at various growth phases of Escherichia coli in rich media.
Project description:We have analyzed the genome-wide redistribution of RNA polymerase in E.coli upon methylglyoxal stress. Herefore, we have used ChIP-chip against the beta subunit of RNA polymerase and we have assessed changes in RNA polymerase distribution upon sub-lethal and lethal concentrations of methylglyoxal.
Project description:Chromatin immunoprecipitation experiment to identify H-NS and RNA polymerase (beta-subunit) binding sites on the Salmonella Typhimurium chromosome
Project description:Sensorimotor dysfunction following incomplete spinal cord injury (SCI) is often characterized by paralysis, spasticity and pain. Previously, we showed that intrathecal (i.t.) administration of the albumin-oleic acid (A-OA) complex in rats with SCI produced partial improvement of these symptoms and that oral 2-hydroxyoleic acid (HOA), a non-hydrolyzable OA analogue), was efficacious in the modulation and treatment of nociception and pain-related anxiety, respectively. Here we observed that intrathecal treatment with the complex albumin-HOA (A-HOA) every 3 days following T9 spinal contusion injury promoted significant recovery in locomotor function and marked an inhibition of TA noxious reflex activity (i.e., nociception) in Wistar rats. To investigate the mechanism of action of A-HOA, microarray analysis was carried out in the spinal cord lesion area. Representative genes involved in pain and neuroregeneration were selected to validate the changes observed in the microarray analysis by quantitative real-time RT-PCR. Comparison of the expression between healthy rats, SCI rats, and SCI treated with A-HOA rats revealed relevant changes in the expression of genes associated with neuronal morphogenesis and growth, neuronal survival, pain and inflammation. Thus, treatment with A-HOA not only induced a significant overexpression of growth and differentiation factor 10 (GDF10), tenascin C (TNC), aspirin (ASPN) and sushi-repeat-containing X-linked 2 (SRPX2), but also a significant reduction in the expression of prostaglandin E synthase (PTGES) and phospholipases A1 and A2 (PLA1/2). Currently, SCI has very important unmet clinical needs. A-HOA proved to downregulate genes involved in inflammation and upregulate genes involved in neuron growth, which balanced the important body response to medular lesion and allowed recovery from paralysis and pain.
Project description:Background: The intracellular pathogen Mycobacterium tuberculosis is known to encounter several stress conditions during infection, including low pH, hypoxia, starvation and oxidative and nitrosative stress. Mycobacterial adaptation to stress conditions is, in part, mediated by several nucleic acid-associated proteins. In this study, we sought to establish an affinity purification-mass spectrometry (AP-MS) approach that would enable the collective identification of nucleic acid-associated proteins in mycobacteria. We hypothesized that targeting the RNA polymerase complex through affinity purification would allow for the identification of RNA- and DNA-associated proteins that not only maintain the bacterial chromosome but also enable transcription and translation. Results: AP-MS analysis of the RNA polymerase β-subunit cross-linked to nucleic acids identified 275 putative nucleic acid-associated proteins in the model organism Mycobacterium smegmatis under standard culturing conditions. The AP-MS approach successfully identified proteins that are known to make up the RNA polymerase complex, as well as several other known RNA polymerase complex-associated proteins such as a DNA polymerase, sigma factors, transcriptional regulators, and helicases. Gene ontology enrichment analysis of the identified proteins revealed that this approach selected for proteins with GO terms associated with nucleic acids and cellular metabolism. Importantly, we identified several proteins of unknown function not previously known to be associated with nucleic acids. Validation of several candidate nucleic acid-associated proteins demonstrated for the first time DNA association of ectopically expressed MSMEG_1060, MSMEG_2695 and MSMEG_4306 through affinity purification. Conclusions: Effective identification of nucleic acid-associated proteins, which make up the RNA polymerase complex as well as other DNA- and RNA-associated proteins, was facilitated by affinity purification of the RNA polymerase β-subunit in M. smegmatis. The successful identification of several transcriptional regulators suggest that our approach could be sensitive enough to investigate the nucleic acid-associated proteins that maintain cellular functions and mediate transcriptional and translational change in response to environmental stress.
Project description:These data were used to infer the genome-wide localization of sigma70 and core RNAP beta subunit for the study "The transition between transcriptional initiation and elongation in E. coli is highly variable and often rate-limiting" (Reppas et al. 2006). This study analyzes transfrags with respect to the ChIP profile of the sigma70 and the beta subunit of RNA polymerase. Keywords: ChIP-chip; tiled analysis of mRNA expression
Project description:In this study, we performed a ChIP-chip experiment to determine the regulon of FnrL in Rhodobacter sphaeroides. We grew R. sphaeroides under anaerobic photosnthetic conditions, in which FnrL is expected to bind DNA and contol gene expression, and immuno-precipitated FnrL, but also sigma70 and the Beta' subunits of RNA polymerase to determine transcription activity. DNA immunoprecipitated with polyclonal antibodies against FnrL, sigma70, or the Beta' subunit of RNA polymerase was labelled with Cy5 and hybridized on two-color tilling arrays (triplicates for each) with genomic DNA as an input control labelled with Cy3.