Shigella flexneri ntrBC and nac mutant expression analysis
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ABSTRACT: Investigate whole genome gene expression level changes in an S. flexneri ntrBC and nac mutants compared to wild type strain. NtrBC is a two component regulatory system, nac is a transcriptional activator Mutations in ntrBC and nac result in small plaque formation in Henle cell monolayers compared to wild type bacteria. Gene expression studies were pursued to identify the genes regulated by these transcriptional regulators. A 9 array study using total RNA recovered from the wild type, ntrBC and nac mutant strains in three independent experiments. Bacterial RNA was isolated from infected Henle cells at four hours post-infection. Gene expression from the mutant strains was compared to that of wild type.
Project description:During early neurogenesis, multiple whole animal gene expression profiling studies revealed widespread changes in developmental mRNA and miRNA abundance in ethanol-exposed embryos. Consistent with a role for miRNAs in neurobehavioral development, miRNA target prediction analyses identified multiple miRNAs misexpressed in the ethanol exposed cohorts that were also predicted to target inversely expressed transcripts known to influence brain morphogenesis. [mRNA] A twelve chip study using total RNA recovered from pools of 75 tropical 5D zebrafish embryos at 24 hours post fertilization (hpf). Embryos were exposed to control embryo medium, 100 mM ethanol, or 300 mM ethanol from 4-24 hpf, with four-fold biological redundancy per condition. A single 12x135K Nimblegen array was used to measure the expression level of 38,489 genes from Danio rerio using 60-mer probes, with three-fold technical redundancy. [miRNA] A twelve chip study using miRNA recovered from pools of 75 tropical 5D zebrafish embryos at 12, 24, 36, and 48 hours post fertilization (hpf). Embryos were exposed to control embryo medium or 300 mM ethanol from 4-24 hpf, with two-fold biological redundancy per condition. Control samples were pooled and hybridized to a single array. 12 miRZebrafish arrays (based on MirBase release 12.0) were used to measure the expression level of 218 mature miRNA from Danio rerio, with 12-fold technical redundancy.
Project description:In the past century, recently emerged infectious diseases have become major drivers of species decline and extinction. Amphibian declines have occurred due to the fungal disease chytridiomycosis, which has exacerbated the conservation crisis of this taxonomic group. Biologists are beginning to understand what traits are important for susceptibility to this disease, but more work is needed to determine why some species succumb to disease while others do not. We conducted a laboratory experiment to examine how two toad species respond to infection in controlled environment. We selected two related species thought to differ in susceptibility â?? Bufo marinus (an invasive and putatively resistant species) and B. boreas (an endangered and putatively susceptible species). We measured infection intensity, body weight, histological changes at the site of infection, and genome-wide gene expression changes using a custom assay developed from transcriptome sequencing. Our results confirmed that the two species differ in susceptibility. The more susceptible species, B. boreas, experienced higher infection intensities, loss in body weight, more dramatic histological changes, and larger perturbations in gene expression. We found key differences in skin expression responses in multiple pathways including up-regulation of skin integrity-related genes in the resistant B. marinus. Together our results show intrinsic differences in host response between related species, which are likely to be an important factor in explaining variation in response to a deadly emerging pathogen in wild populations. We processed 72 tissue samples in total: six biological replicates, three tissue types (ventral skin, liver, spleen), two treatment groups (pathogen exposed, control), and two host species (Bufo marinus, Bufo boreas). The custom Nimblegen microarray design included 135,200 60-bp probes (excluding control probes) targeting 31,367 transcript contigs from Bufo marinus, Bufo boreas, and model species Xenopus tropicalis, with 4 probes per probe-set. We used the 12-plex microarray platform (12 arrays per glass slide). Differential expression analyses were performed separately for each tissue type and host species.
Project description:Reversible protein phosphorylation is one of the most important posttranslational modifications (PTMs) due to its vital role in cellular functions and signaling pathways. Protein phosphorylation is also known to be involved in the regulation of apoptosis. Previously, we have performed a SILAC-based analysis of phosphotyrosinylation of cisplatin-induced apoptotic Jurkat T cells. To further expand this study, we used the same experimental setup and analyzed the global phosphorylation profile. The different steps of the phosphopeptide enrichment protocol using TiO2 beads were tested and the best approach was applied in combination with LC-MS with different normalized collision energy (NCE) values for HCD fragmentation. In total, more than 2,000 phosphoproteins of more than 4,000 phosphopeptides were identified in the four biological replicates of both states. HCD with NCE 25 accounted for 64% and NCE 35 for 36 % of identified phosphopeptides. 425 phosphopeptides were found to be significantly regulated in cisplatin-treated against control samples. KEGG pathway analysis revealed splicosomal proteins and the MAPK signaling pathway to be upregulated and cell cycle proteins to be downregulated. The transcription factors Atf2 and Jun, which are both involved in the MAPK signaling pathway, were further investigated. For Jun, more than 90 spectral counts were found only in apoptotic cells and covered the well-known stress induced phosphorylation sites at S-63/S-73, as well as S-243 which reduces the ability to bind DNA and is required for GSK3 mediated ubiquitinylation at S-239. For Atf2, more than 60 spectral counts were found in apoptotic cells vs. 8 in control cells. Most significantly upregulated sites for Atf2 have been identified at S-90 and S-112, which are less well studied phosphorylation sites of Atf2 as several other sites. Western blot analyses using non-phosphorylated and phosphorylated antibodies were performed to validate the phosphorylation events of Jun and Atf2. Our findings contribute to the current knowledge of the phosphorylation profiles of apoptotic cells.
Project description:Investigation of whole genome gene expression level changes during a 44 hour time period in Aedes aegypti observed under light/dark and constant dark conditions. This is a 12-plex high defination NimbleGen array design. The Higgs White Eye (Wh) strain of Aedes aegypti was investigated for rhythmic expression during the light/dark phase and constant darkness. Samples were collected every 4 hours for 44 hours in each treatment, and RNA collected from whole head tissue.
Project description:Transcript abundance was measured in whole-body virgin male Drosophila serrata from 41 inbred lines that had diverged through 27 generations of mutation accumulation. Pleiotropic mutations are the ultimate source of genetic variation in complex traits, including many human diseases. However, the nature and extent of mutational pleiotropy remain largely unknown. Here, we investigate the variation in 11,604 gene expression traits among 41 mutation accumulation lines of Drosophila serrata, which had diverged for 27 generations. We detected significant mutational variance in 4.6% of ESTs, but 70% of ESTs were invariant among lines, allowing us to reject a null hypothesis of phenome-wide universal pleiotropy. Mutational covariance among ESTs was detected at a frequency of only 1 in 193 random pairs of variable EST, bu t was detected among random combinations of five ESTs in 1 in 5 cases, revealing that mutational covariance among multiple ESTs was common. The observed frequency of significant multivariate covariance among random ESTs implied that a substantial number of ESTs (>70) must be pleiotropically affected by at least some mutations. We measured gene expression of male Drosophila serrata from 41 mutation accumulation lines (whole-body). Data from two replicates for each line are presented.
Project description:Transcript abundance was measured in whole-body virgin male Drosophila serrata from 41 inbred lines that had diverged through 27 generations of mutation accumulation that were sexually selected Sexual selection is predicted to have widespread effects on the genetic variation generated by new mutations as a consequence of the genic capture of condition by male sexual traits. We manipulated the opportunity for sexual selection on males during 27 generations of mutation accumulation in inbred lines of Drosophila serrata, and used a microarray platform to investigate the effect of sexual selection on the expression of 2685 genes, representing a broad coverage of biological function. Sexual selection had little effect on mean gene expression levels, with only 4 genes diverging significantly at a false discovery rate of 5% . In contrast, sexual selection impacted on both the magnitude and nature of mutational variance accumulating in these genes. The magnitude of mutational variance increased under sexual selection by an average of 29%. Mutational variance was less commonly generated by extreme phenotypes less commonly under sexual selection. Furthermore, analysis of random sets of five genes revealed that the mutational variance that accumulated under sexual selection was less pleiotropic in nature than that found in the absence of sexual selection. The generation of greater mutational variance without a general concomitant change in mean expression under sexual selection suggested that gene expression traits were be under apparent rather than direct sexual selection. We discuss two main explanations for the broad-based increase in mutational variance under sexual selection that both require extensive pleiotropy between traits affecting male mating success, standard metric traits represented here by gene expression traits, and general fitness. We measured gene expression of male Drosophila serrata from 41 mutation accumulation lines (whole-body) that were sexually selected. Data from two replicates for each line are presented.
Project description:To identify BVES-interacting proteins from mouse skeletal muscle, we performed IP-mass from AAV9-BVES-HA injected mouse skeletal muscle using the anti-HA magnetic beads.
Project description:Analysis of CPEB translational regulator target mRNAs Microarray analysis of mRNAs associated with polysomes in wild type (WT) and Cpeb1 KO MEFs
Project description:Investigation of whole genome gene expression to identify overlooked sRNAs and sORFs. Background The completion of numerous genome sequences has introduced an era of whole-genome study. However, many real genes, including small RNAs (sRNAs) and small ORFs (sORFs), are missed in genome annotation. In order to improve genome annotation, we sought to identify novel sRNAs and sORFs in Shigella, the principal etiologic agents of bacillary dysentery or shigellosis. Results Firstly, we identified 64 sRNAs in Shigella which is experimentally validated in other bacteria based on sequence conservation. Secondly, among possible approaches to search for sRNAs, we employed computer-based and tiling array based methods, followed by RT-PCR and northern blots. This allowed us to identify 12 sRNAs in Shigella flexneri strain 301. We also find 29 candidate sORFs. Conclusions This investigation provides an updated and comprehensive annotation of the Shigella genome, increases the expected numbers of sORFs and sRNAs with the corresponding impact on future functional genomics and proteomics studies. Our method can be used for the large scale reannotation of sRNAs and sORFs in any microbe whose genome sequence is available. Study using total RNA recovered from five conditions.