Transcritptome of a revR deletion mutant during Clostridium perfringens exponential phase growth.
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ABSTRACT: RevR is a putative orphan response regulator with a high degree of similarity to YycF from Bacilus subtilis and PhoB from Clostridium kluyveri. A revR deletion mutant of C. perfringens strain 13 was generated and the transcriptome analysed using microarrays. Total RNA was isolated from exponentially growing cells from the revR mutant and the wild-type control. Gene expression levels were compared between the revR mutant and wild-type strain 13
Project description:The MalNO is a putative two-component signal transduction system, previously known as the VirJI sytem. MalO is the putative cognate response regulator of the MalN sensor histidine kinase. Based on previous evidence that suggested the plc gene, encoding ?-toxin, was upregulated during stationary phase in the malO mutant, microarrays were used to analyse the transcriptome of a malO mutant during stationary phase growth. Total RNA was isolated from stationary phase cells of the malO mutant and the wild-type control. Gene expression levels were compared between the malO mutant and wild-type strain 13
Project description:ReeS, previously named as CPE1512, was originally annotated as the only hybrid sensor histidine kinase/response regulator in Clostridium perfringens. Further evidence suggests that ReeS is more likely to function as an orphan sensor histidine kinase. A reeS deletion mutant was constructed and the transcriptome analysed using microarrays. Total RNA was isolated from the reeS mutant and the wild-type control cells during exponential phase growth. Gene expression levels were compared between the reeS mutant and wild-type strain 13
Project description:Transcriptome changes were measured in S. pyogenes strain HSC5 in response to glucose availability, taking samples from the wild type strain (HSC5) grown in carbohydrate poor medium (C medium) versus this same medium with 1% (w/v) Glucose added. Furthermore, the effects of deletion of glucose responsive regulators CcpA and LacD.1 were measured by using isogenic mutants in these genes (Delta CcpA and Delta LacD.1 respectively) compared to the wild type parent strain (HSC5), both grown in C medium with 1% glucose. Experiments were carried out using two biological replicates for each variable tested (ie two independent RNA samples gathered on separate days), and dye exchange experiments for each RNA sample tested were carried out.
Project description:Mutation of the ASHH2 gene has pleiotropic developmental effects. The aim of the study was to identify changes in gene expression in seedlings of the ashh2-1/sdg8-1 mutant (SALK_065480, insertion in the gene At1g77300, SDG8/EFS/ASHH2) and look for correlation wth changes in methylation marks on histone tails. Wild type Arabidopsis thaliana (ecotype Colombia) seedlings were used as reference. For low-expresssed tissue-specific genes there was a correlation between reduced H3K36me3 levels and reduction in expression. Although some highly expressed genes with low tissue-specificity show reduced H3K36me3 levels in the mutant, expression levels were, however, not affeccted. Six biological replicates with 12 plants each both of Arabidopsis thaliana wild type (ecotype Colombia-0) and ashh2-1/sgd8-1 mutant (SALK_065480, insertion in the gene At1g77300, SDG8/EFS/ASHH2) were sown out on soil and grown under the following conditions; 22oC day and 18oC night, 16 hr day length with 30 min adjustment of light to on and off, and 85 μmol/m2/sek in light intensity. The inflorescences of each biological replikate were harvested in bulk from 30-36 days old plants at same time of the day (between 12:45 and 13:45) and at the same developmental stage in bulk.
Project description:Gene expression profiling of biopsied human lymph node (LN) tissue comparing each patient sample against mobilised peripheral blood stem cells (PBSC), the reference channel Evaluate whether gene expression microarray can diagnose lymph node biopsies as reactive or as one of three main types of lymphoma: classical Hodgkin’s lymphoma (cHL), diffuse large B cell lymphoma (DLBCL) or follicular lymphoma (FL). Two condition experiment, LN vs mobilised PBSC, 116 cases assayed, 1 replicate per array
Project description:Candida albicans is a ubiquitous opportunistic pathogen that is the most prevalent cause of hospital-acquired fungal infections. In mammalian hosts, C. albicans is engulfed by phagocytes that attack the pathogen with DNA-damaging reactive oxygen species (ROS). Acetylation of histone H3 lysine 56 (H3K56) by the fungal-specific histone acetyltransferase Rtt109 is important for yeast model organisms to survive DNA damage and maintain genome integrity. To assess the importance of Rtt109 for C. albicans pathogenicity, we deleted the predicted homologue of Rtt109 in the clinical C. albicans isolate, SC5314. C. albicans rtt109 -/- mutant cells lack acetylated H3K56 (H3K56ac) and are hypersensitive to genotoxic agents. Additionally, rtt109 -/- mutant cells constitutively display increased H2A S129 phosphorylation and elevated DNA repair gene expression, consistent with endogenous DNA damage. Four independent pairs of biological replicate samples were analyzed to compare RNA levels in wild-type and rtt109 -/- cells. Two experiments were performed: first, in unperturbed cells grown in YPD and second, in cells exposed to H2O2. To account for dye effects, two of the four samples for each experiment were analyzed using Cy3 labeling of the wild-type sample and Cy5 labeling of the mutant sample; the dyes were swapped for the other two biological replicates.
Project description:Little is known about transcriptional control of neurite branching or presynaptic differentiation, events that occur relatively late in neuronal development. Using the C. elegans mechanosensory circuit as an in vivo model, we show that SAM-10, an ortholog of mammalian Single-Stranded DNA binding Protein (SSDP), functions cell-autonomously in the nucleus to regulate synaptic differentiation as well as positioning of a single neurite branch. PLM mechanosensory neurons in sam-10 mutants exhibit abnormal placement of the neurite branch point, and defective synaptogenesis, characterized by an overextended synaptic varicosity, underdeveloped synaptic morphology and disrupted co-localization of active zone and synaptic vesicles. SAM-10 functions coordinately with LDB-1 (Lim Domain Binding protein-1), demonstrated by our observations that 1) mutations in either gene show similar defects in PLM neurons; and 2) LDB-1 is required for SAM-10 nuclear localization. SAM-10 regulates PLM synaptic differentiation by suppressing transcription of prk-2, which encodes an ortholog of the mammalian Pim kinase family. PRK-2-mediated activities of SAM-10 are specifically involved in PLM synaptic differentiation, but not other sam-10 phenotypes such as neurite branching. Thus, these data reveal a novel transcriptional signaling pathway that regulates neuronal specification of neurite branching and presynaptic differentiation. Animals were grown on fresh 8P plates (Wormbook.org) until gravid. Eggs were collected by bleaching and hatched in M9 buffer overnight. L1 animals were fed on fresh food for 2 hours at room temperature, harvested and separated from worm debris using a 25μm mesh. RNA was isolated using a standard Trizol protocol. RNA preps were stored at -80C. Quality of RNA preps was first estimated using Agilent 2100 Bioanalyzer. RNA was then reverse-transcribed using the Genisphere Array 350 kit, which generate tagged cDNA. Resulted products are hybridized to the microarrays. Four independent high quality RNA preps (RIN>=7) were chosen for microarray assay using long oligomer-based spotted microarray (Washington University, St. Louis, MO). For dye-swap controls, we ran a technical replicate hybridization for each pair of samples in which the dyes are reversed. Gene spots with “found/good” signals (defined by ScanArray, channel flag 3) in all scans were chosen for gene expression analysis.
Project description:Erwinia carotovora subsp. atroseptica (Eca) is an enterobacterial phytopathogen causing economically-significant soft rot disease. Pathogenesis is mediated by multiple secreted virulence factors, many of which are secreted by the Type II (Out) secretion system. DsbA catalyses the introduction of disulphide bonds into periplasmic and secreted proteins. In this study, the extracellular proteome (secretome) of wild type Eca SCRI1043 and dsbA and out mutants was analysed by spectral counting mass spectrometry. This revealed that dsbA inactivation had a huge impact on the secretome and identified diverse DsbA- and Out-dependent secreted proteins, representing known, predicted and novel candidate virulence factors. Further characterisation of the dsbA mutant showed that secreted enzyme activities, motility, production of the quorum sensing signal and virulence were absent or greatly reduced. The impact of DsbA on secreted virulence factor production was mediated at multiple levels, including impacting on the Out secretion system and the virulence gene regulatory network. Transcriptome analyses revealed that the abundance of a broad, but defined, set of transcripts, including many virulence factors, was altered in the dsbA mutant, identifying a new virulence regulon responsive to extracytoplasmic conditions. In conclusion, DsbA plays a crucial, multi-faceted role in the pathogenesis of Eca. Keywords: Mutant comparison RNA samples used for genome-wide transcriptional profiling using microarrays: RNA was hybridised to four Eca genomic arrays, with each array hybridising one wild type and one dsbA mutant sample (from four independent cultures of each strain) and incorporating a dye-swap (i.e. wild type labelled with Cy3 in 2/4 and with Cy5 in 2/4).
Project description:Chickens were kept under a regimen of 12 hours of light and 12 hours of dark for 7 days. At LD2, 2 hours into the light cycle on the 8th day, 3 chickens were sacrificed by decapitation and their retinas dissected and pooled. Similarly, at LD18, 6 hours into the dark cycle on the 8th day, retinas were harvested from 3 chickens. Total RNA was extracted from each set of retinas using Trizol Reagent (Invitrogen). The total RNA samples were amplified to produce aRNA using the Message Amp Kit (Ambion). Randomly primed fluorescent probes were produced from the aRNA samples using a Genisphere 3DNA Array 900MPX expression array detection kit. The fluorescent dye on the probe derived from the experimental aRNA (LD2) was Cy5 while the dye on the control probe (LD18) was Cy3. Hybridizations and washes were as suggested by Genisphere. Labeled arrays were scanned in an Affymetrix 428 array scanner. The images obtained were subsequently analyzed by GenePixPro software (Axon Instruments). The GenePix results (.gpr) file was further analyzed by GeneSpring 5 (Silicon Genetics) which applied intensity dependent LOWESS normalization to the results and calculated normalized Cy5 to Cy3 ratios. This GEO Series was created by the GEO staff as part of a cleanup effort to ensure that all GEO Samples are included within a Series entry.
Project description:Leptospirosis is a globally significant zoonotic disease caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since availability of iron is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. Virulence genes in some bacteria have been shown to be iron-regulated. In many bacteria, expression of iron-uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutant in one of these, la1857. We conducted microarray analysis to identify differentially expressed genes in L. interrogans serovar Manilae grown under low iron compared with normal iron conditions found in EMJH medium. We also compared the transcriptional profile of the la1857 mutant versus wild-type Manilae under normal and low iron conditions. Analysis used RNA derived from L. interrogans serovar Manilae grown under low iron (EMJH medium + 40 µM 2,2-dipyridyl) and normal iron as experimental and control samples, respectively. We have a serovar Manilae mutant in la1857, one of four predicted fur homologs. The mutant was also grown under low iron and normal iron conditions and compared with wild-type Manilae grown under low and normal iron conditions respectively. Three independent RNA samples (biological replicates) from parent and mutant strains grown with or without 2,2-dipyridyl were compared in a loop design resulting in 12 arrays. The orientation of Cy3 to Cy5 labeling was the same for replicates 1 and 3, while replicate 2 was a dye swap. Direct comparisons were made between arrays of experimental and control samples using raw data pulled from two different channels for data analysis. Data from wild-type Manilae grown under low iron versus normal iron and mutant versus wild-type under normal iron conditions is reported in the manuscript.