Transcriptomic response of Dickeya dadantii to DNA relaxation by novobiocin
ABSTRACT: We analyze the transcriptomic response of the phytopathogenic enterobacterium Dickeya dadantii to a DNA supercoiling relaxation shock using the gyrase inhibitor novobiocin. The shock was applied to Dickeya cells grown in minimal medium supplemented with sucrose, in exponential or transition to stationary phase, and in minimal medium with sucrose + PGA (a pectin derivative) in transition to stationary phase.
Project description:Small non-coding RNAs function in concert with Argonaute (Ago) proteins to regulate gene expression at the level of transcription, mRNA stability or translation. Ago proteins bind small RNAs and form the core of silencing complexes. Here we report the analysis of small RNAs associated with human Ago1 and Ago2 revealed by immunoprecipitation and deep sequencing. Among the reads we find small RNAs originating from the small nucleolar RNA (snoRNA) ACA45. Moreover, processing of ACA45 requires Dicer activity but is independent of Drosha/DGCR8. Using bio-informatic prediction algorithms and luciferase reporter assays, we uncover the mediator subunit CDC2L6 as one potential mRNA target of ACA45 small RNAs suggesting a role for ACA45 processing products in post-transcriptional gene silencing. We further identify a number of human snoRNAs with microRNA (miRNA)- like processing signatures. We have therefore identified a novel class of small RNAs in human cells that originate from snoRNAs and can function like miRNAs. Two samples examined. Small RNAs associated to human Argonaute 1 and human Argonaute 2
Project description:We have analysed the global gene expression of the plant pathogen Dickeya dadantii 3937 when grown in vitro under different growth and stress conditions. A multi-factorial design with 32 different experimental conditions was constructed, with two biological replicates for each condition. Cells were grown to exponential phase or stationary phases in four different growth media: M63 supplemented with 0.2% sucrose as carbon source, with or without 0.2% (w/v) polygalacturonate (PGA) and with or without 0.1% Saintpaulia leaf extracts (1 g leaves in 1 L M63). Cells grown in these four media were subjected to different stresses: (i) acid stress, by an incubation of 15 min in the presence of 30 mM malic acid; (ii) oxidative stress, by an incubation of 15 min in the presence of 100 _M H2O2 or (iii) osmotic stress, by an incubation of 15 min in the presence of 300 mM NaCl. The micro-arrays used in this study were custom designed and produced by Roche NimbleGen, Inc. (Madison, WI) based on the annotated sequence of D. dadantii, available at Genbank accession number n¡ CP002038, which comprises 4597 CDS. The 4plex expression micro-arrays consist of 60-mer oligonucleotides, triplicated in three blocks on the array (5 oligonucleotides per CDS).
Project description:In this study transcriptional start sites (TSS) of E. coli were determined for several growth conditions To detect the complement of transcripts expressed from E. coli, we collected two independent biological replicates (B1 and B2 samples) from MG1655 wild type strain grown to exponential (OD 600 ~0.4) or stationary phase (OD 600 ~2.0) in LB medium (samples LB 0.4 and LB 2.0, respectively) as well as grown to exponential phase (OD 600 ~0.4) in M63 minimal glucose medium (sample M63 0.4). For all six samples, total RNA was extracted and subjected to differential RNA-seq (dRNA-seq) library preparation for primary transcriptome analysis as described previously (Sharma et al., 2010). Specifically, prior to cDNA library construction half of each RNA sample was treated with 5’ terminator exonuclease (+TEX samples), which degrades RNAs containing a 5’-mono-phosphate (5’-P) and, thus, enriching enriches for primary transcripts containing 5’-tri-phosphates (5’-PPP). The other half of each sample was left untreated (-TEX samples) and thus contains both primary transcripts (5’-PPP) and processed RNAs (5’-P).
Project description:We have analyzed the transcriptomes of Dickeya dadantii wild type, fis and hns strains in condition of DNA relaxation induced by novobiocin, a DNA gyrase inhibitor. For DNA relaxation novobiocin was added for 15 min to 100µg/ml final concentration to exponentially growing wild type, hns and fis strains (OD600= 0.2) grown in M63 medium supplemented by sucrose 0.2% (w/v). At this concentration, novobiocin has no impact on D. dadantii growth The micro-arrays used in this study were custom designed and produced by Roche NimbleGen, Inc. (Madison, WI) based on the annotated sequence of D. dadantii, available at Genbank accession number n° CP002038, which comprises 4597 CDS. The 4plex expression micro-arrays consist of 60-mer oligonucleotides, triplicated in three blocks on the array (5 oligonucleotides per CDS). For micro-array analyses, cDNA was synthesized, labeled and hybridized by Roche NimbleGen Inc.
Project description:RNA-Seq data encompass transcriptomes from two sequential isolates of B. contaminans ST872. Each isolate was cultivated in three conditions (serum, sputum and BSM medium) in three biological replicates.
Project description:We report the transcriptome profiles (RNA seq) of three different co-isolates of S.aureus that have been identified and isolated in both laboratory and infective scenarios. The transcriptome profiles were generated by deep sequencing and transcript levels assessed. For this the raw reads underwent quality-trimming (using the FastX suite), polyA-clipping, size filtering, mapping to the reference genome, coverage calculation, gene wise expression quantification followed by differential gene expression analysis (all done by the tool "READemption" (Förstner et al., unpublished) using "segemehl" (Hoffmann et al., 2009) and "DESeq" (Anders et al., 2010). RT-PCR as well as phenotypical assays were further used to validate the data. Differential gene expression between the isolates was observed in 4% (116 of 2774 genes). This had a significant impact on the physiology of each strain. RNA profiles of 3 different subpopulations of S.aureus generated by deep sequencing
Project description:Attempt to identify small non-coding RNAs that change in levels as a result of viral infection of C.elegans Small non-coding RNA (18-30nt) was extracted from animals either infected with Orsay virus or uninfected as indicated.
Project description:An attempt to identify small non-coding RNAs that change with increasing generations after becoming homozygous for the loss of PRG-1 Small non-coding RNA (18-30nt) was extracted from animals the indicated number of generations after homozygosity was established
Project description:piRNAs are required to maintain germline integrity and fertility but their mechanism of action is poorly understood. Here we demonstrate that C. elegans piRNAs silence transcripts in trans through imperfectly complementary sites. We find that target silencing is independent of Piwi endonuclease activity or “slicing”. Instead, we show that piRNAs initiate a localized secondary endogenous small interfering RNA (endo-siRNA) response. Endogenous protein-coding gene, pseudogene and transposon transcripts exhibit Piwi-dependent endo-siRNAs at sites complementary to piRNAs and are derepressed in Piwi mutants. Genomic loci of piRNA biogenesis are depleted of protein-coding genes but not pseudogenes or transposons. Our data suggest that nematode piRNA clusters are evolving to generate piRNAs against active mobile elements. Thus, piRNAs provide heritable, sequence-specific triggers for RNAi in C. elegans. 7 small RNA libraries were sequenced as part of 25 flow cell lanes on the Illumina GA II platform. Samples were treated with tobacco acid pyrophosphatase to allow cloning of small RNAs with a 5'-triphosphate. Samples were labelled for multiplexing using 4-bp 5'-barcodes or barcodes included in Illumina TruSeq adapters. In most cases a single flow cell lane included several multiplexed libraries.