ABSTRACT: We determine gene expression profile for the knockout of Rv3263 (DNA Methyltransferase) and compared to wildtype M. tuberculosis. Two RNA-Seq datasets are included: wildtype (H37Rv_SS1) and knockout (delta_Rv3263)
DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M. tuberculosis, which we term MamA. MamA creates N⁶-methyladenine in a six base pair recognition sequence present in approximately 2,000 copies on each strand of the genome. Loss of MamA reduces th ...[more]
Project description:To elucidate responses of myeloid cells to SAMHD1 deficiency in the absence of exogenous viral infection, we performed global gene expression analysis of SAMHD1 deficient macrophages. Peritoneal macrophages from nine mutants and nine controls were FACS sorted. Cells from three animals were pooled to yield three poolls per group. RNA from these pools was subjected to next generation mRNA sequencing
Project description:To investigate the contribution of type-1 IFN signalling to the upregulation of IFN- stimulated genes in SAMHD1-deficient cells, we performed global gene expression analysis of SAMHD1-deficient IFNAR-/- macrophages. Peritoneal macrophages from ten SAMHD1-deficient IFNAR-/- and six SAMHD1-deficient controls were FACS sorted. RNA was subjected to next generation mRNA sequencing.
Project description:To elucidate responses of myeloid cells to SAMHD1 deficiency in the absence of exogenous viral infection, we performed global gene expression analysis of SAMHD1 deficient macrophages. Peritoneal macrophages from 10 mutants and 10 controls were FACS sorted. Isolated RNA was subjected to next generation mRNA sequencing.
Project description:Gene expression in adult male and female mouse liver assayed by RNA-seq, as part of a study on chromatin states in male and female mouse and their role in sex-biased liver gene expression (A Sugathan and DJ Waxman (2013) Molec Cell Biol, in press). Total liver RNA was prepared from 12 individual male and 12 individual female mice. Four RNA pools, comprised of RNA isolated from 6 individual male or female livers (2 pooled biological replicates for each sex) were then prepared and used for RNA-seq.
Project description:The molecular mechanisms of ethanol toxicity and tolerance in bacteria, while important for biotechnology and bioenergy applications, remain incompletely understood. Genetic studies have identified potential cellular targets for ethanol and revealed multiple mechanisms of tolerance, but it remains difficult to separate direct and indirect effects of ethanol. We used adaptive evolution to generate spontaneous ethanol-tolerant strains of Escherichia coli, then characterized the mechanisms of toxicity and resistance associated with select mutations. Evolved alleles of metJ, rho, and rpsQ were sufficient to recapitulate much of the observed ethanol tolerance, implicating translation and transcription as key processes affected by ethanol. We found that ethanol induces mistranslation errors during protein synthesis, and that the evolved rpsQ allele protects cells by rendering the ribosome hyper-accurate. Ribosome profiling and RNAseq analyses of the ethanol-tolerant strain versus the wild type established that ethanol negatively affects transcriptional and translational processivity. Ethanol-stressed cells exhibited ribosomal stalling at internal AUG codons, which may be ameliorated by the adaptive inactivation of the MetJ repressor of methionine biosynthesis genes. Ethanol also caused aberrant intragenic transcription termination for mRNAs with low ribosome density, which was reduced in a strain with the adaptive rho mutation. Furthermore, ethanol inhibited transcript elongation by RNA polymerase in vitro. We propose that ethanol-induced inhibition and uncoupling of mRNA and protein synthesis are major contributors to ethanol toxicity in E. coli, and that adaptive mutations in metJ, rho, and rpsQ protect central dogma processes in the presence of ethanol. RNA-seq comparison of wild-type and mutant strains to assess readthrough of Rho-dependent transcriptional terminators
Project description:Retinoic-acid receptor-related orphan receptor-γt-positive (RORγt+) innate lymphoid cells (ILCs) produce interleukin (IL)-22 and IL-17, which are critical for protective immunity against enteric pathogens. The molecular mechanism underlying the development and survival of RORγt+ ILCs is not thoroughly understood. Here we show that Dedicator of cytokinesis 8 (DOCK8), a scaffolding protein involved in cytoskeletal rearrangement and cell migration, is essential for the protective immunity against Citrobacter rodentium. A comparative RNA sequencing-based analysis reveals an impaired induction of antimicrobial peptides in the colon of DOCK8-deficient mice, which correlates with high susceptibility to infection and a very low number of IL-22-producing RORγt+ ILCs in their GI tract. Furthermore, DOCK8-deficient RORγt+ ILCs are less responsive to IL-7 mediated signaling, more prone to apoptosis and produce less IL-22 due to a defect in IL-23-mediated STAT3 phosphorylation. Our studies reveal an unsuspected role of DOCK8 for the function, generation and survival of RORγt+ ILCs. Control and DOCK8 KO mice were infected with 2X109 CFU of Citrobacter rodentium and day 8 post infection mice were sacrificed and their colons were harvested (n=5) . Total RNA was purified from the infected colons with RNeasy mini kit (Qiagen). RNA sequencing was performed (pooled RNA sample from five mice in each group) at Genomic Core Facility Southwestern Medical Center, University of Texas.
Project description:Purpose: The purpose of this experiment is to expand the repertoire of C. elegans edited transcripts and identify the roles of ADR-1 as indirect regulator of editing and ADR-2 as the only active deaminase in vivo. Methods: Strand-specific RNA sequencing of wild-type and adr mutant worms, followed by a novel RNA variant calling and comparative analysis pipeline. Results: Despite lacking deaminase function, ADR-1 affects editing of over 60 adenosines within the 3’ UTRs of 16 different mRNAs. Furthermore, ADR-1 interacts directly with ADR-2 substrates, even in the absence of ADR-2; and mutations within its dsRNA binding domains abolished both binding and editing regulation. Conclusions: ADR-1 acts as a major regulator of editing by binding ADR-2 substrates in vivo and raises the possibility that other dsRNA binding proteins, including the inactive human ADARs, regulate RNA editing by deaminase-independent mechanisms. Strand-specific RNA sequencing of wild-type and adr mutant worms, followed by a novel RNA variant calling and comparative analysis pipeline.
Project description:To compensate for the increased dosage of X chromosomes in females one of the two X chromosomes is inactivated in eutherian mammals in an Xist-dependent manner. Xist is, however, not found in metatherians. Here we describe long non-coding RNA Rsx (RNA-on-the-silent X) that exhibits properties consistent with a role in X-inactivation in opossum. Rsx is abundantly expressed in females but not males and is transcribed from, and coats, the inactive X chromosome copy. Furthermore, when both X chromosomes are active, Rsx is silenced. We used RNA-Seq to determine exon-intron structure and expression level of Rsx in males and females. RNA sequencing was used to detect and characterize transcripts with strongly biased female expression.
Project description:We sequenced mRNA from Wangiella dermatitidis at two pH conditions (2.5 and 6), including three biological replicates of each sample. Examination of transcript level changes in reponse to low pH stress.