Project description:Caenorhabditis elegans is a nematode widely used in biology and genomics as a model organism. We provide an integrated, quantitative reference map for the transcriptome of whole, wild type Bristol N2 strain C. elegans worms. The map has been obtained by meta-analysis of 110 gene expression profiles available in Gene Expression Omnibus (GEO) repository and integrated using the computational biology tool Transcriptome Mapper (TRAM). Following probe assignment to the relative locus and intra- and inter-sample normalization (in particular using the scaled quantile method), a mean, consensus reference value is provided for 45,932 transcripts, along with standard deviation. Expression values are all mapped in the context of genomic coordinates. The map provides easy access to relationships among expression values of different genes in this standard condition, highlights genomic segments with relatively high over-/under-expression and may serve as a reference to test for gene expression variation for both individual genes and the whole transcriptome in specific biological conditions (e.g. mutated strains or differently grown worms).
Project description:The molecular regulatory mechanisms of host responses to Mycobacterium avium subsp. paratuberculosis (MAP) infection during the early subclinical stage are still not clear. In this study, surgically isolated ileal segments in newborn calves (n?=?5) were used to establish in vivo MAP infection adjacent to an uninfected control intestinal compartment. RNA-Seq was used to profile the whole transcriptome (mRNAs) and the microRNAome (miRNAs) of ileal tissues collected at one-month post-infection. The most related function of the differentially expressed mRNAs between infected and uninfected tissues was "proliferation of endothelial cells", indicating that MAP infection may lead to the over-proliferation of endothelial cells. In addition, 46.2% of detected mRNAs displayed alternative splicing events. The pre-mRNA of two genes related to macrophage maturation (monocyte to macrophage differentiation-associated) and lysosome function (adenosine deaminase) showed differential alternative splicing events, suggesting that specific changes in the pre-mRNA splicing sites may be a mechanism by which MAP escapes host immune responses. Moreover, 9?miRNAs were differentially expressed after MAP infection. The integrated analysis of microRNAome and transcriptome revealed that these miRNAs might regulate host responses to MAP infection, such as "proliferation of endothelial cells" (bta-miR-196?b), "bacteria recognition" (bta-miR-146?b), and "regulation of the inflammatory response" (bta-miR-146?b).
Project description:Mitochondria are essential organelles that harbor a reduced genome, and expression of that genome requires regulated metabolism of its transcriptome by nuclear-encoded proteins. Despite extensive investigation, a comprehensive map of the yeast mitochondrial transcriptome has not been developed and all of the RNA-metabolizing proteins have not been identified, both of which are prerequisites to elucidating the basic RNA biology of mitochondria. Here, we present a mitochondrial transcriptome map of the yeast S288C reference strain. Using RNAseq and bioinformatics, we show the expression level of all transcripts, revise all promoter, origin of replication, and tRNA annotations, and demonstrate for the first time the existence of alternative splicing, mirror RNAs, and a novel RNA processing site in yeast mitochondria. The transcriptome map has revealed new aspects of mitochondrial RNA biology and we expect it will serve as a valuable resource. As a complement to the map, we present our compilation of all known yeast nuclear-encoded ribonucleases (RNases), and a screen of this dataset for those that are imported into mitochondria. We sought to identify RNases that are refractory to recovery in traditional mitochondrial screens due to an essential function or eclipsed accumulation in another cellular compartment. Using this in silico approach, the essential RNase of the nuclear and cytoplasmic exosome, Dis3p, emerges as a strong candidate. Bioinformatics and in vivo analyses show that Dis3p has a conserved and functional mitochondrial-targeting signal (MTS). A clean and marker-less chromosomal deletion of the Dis3p MTS results in a defect in the decay of intron and mirror RNAs, thus revealing a role for Dis3p in mitochondrial RNA decay.
Project description:A high-resolution genetic linkage map is an essential tool for decoding genetics and genomics in non-model organisms. In this study, a linkage map was constructed for the swimming crab (Portunus trituberculatus) with 10,963 markers; as far as we know, this number of markers has never been achieved in any other crustacean. The linkage map covered 98.85% of the whole genome with a mean marker interval of 0.51?cM. The de novo assembly based on genome and transcriptome sequencing data enabled 2,378 explicit annotated markers to be anchored to the map. Quantitative trait locus (QTL) mapping revealed 10 growth-related QTLs with a phenotypic variance explained (PVE) range of 12.0-35.9. Eight genes identified from the growth-related QTL regions, in particular, RE1-silencing transcription factor and RNA-directed DNA polymerase genes with nonsynonymous substitutions, were considered important growth-related candidate genes. We have demonstrated that linkage mapping aided by de novo assembly of genome and transcriptome sequencing could serve as an important platform for QTL mapping and the identification of trait-related genes.
Project description:Rapid growth of brain tumors such as glioblastoma often results in oxygen deprivation and the emergence of hypoxic zones. In consequence, the enrichment of reactive oxygen species occurs, harming nonmalignant cells and leading them toward apoptotic cell death. However, cancer cells survive such exposure and thrive in a hypoxic environment. As the mechanisms responsible for such starkly different outcomes are not sufficiently explained, we aimed to explore what transcriptome rearrangements are used by glioblastoma cells in hypoxic areas. Using metadata analysis of transcriptome in different subregions of the glioblastoma retrieved from the Ivy Glioblastoma Atlas Project, we created the reactive oxygen species-dependent map of the transcriptome. This map was then used for the analysis of differential gene expression in the histologically determined cellular tumors and hypoxic zones. The gene ontology analysis cross-referenced with the clinical data from The Cancer Genome Atlas revealed that the metabolic shift is one of the major prosurvival strategies applied by cancer cells to overcome hypoxia-related cytotoxicity.
Project description:BACKGROUND: While many genome sequences are complete, transcriptomes are less well characterized. We used both genome-scale tiling arrays and massively parallel sequencing to map the Caenorhabditis elegans transcriptome across development. We utilized this framework to identify transcriptome changes in animals lacking the nonsense-mediated decay (NMD) pathway. RESULTS: We find that while the majority of detectable transcripts map to known gene structures, >5% of transcribed regions fall outside current gene annotations. We show that >40% of these are novel exons. Using both technologies to assess isoform complexity, we estimate that >17% of genes change isoform across development. Next we examined how the transcriptome is perturbed in animals lacking NMD. NMD prevents expression of truncated proteins by degrading transcripts containing premature termination codons. We find that approximately 20% of genes produce transcripts that appear to be NMD targets. While most of these arise from splicing errors, NMD targets are enriched for transcripts containing open reading frames upstream of the predicted translational start (uORFs). We identify a relationship between the Kozak consensus surrounding the true start codon and the degree to which uORF-containing transcripts are targeted by NMD and speculate that translational efficiency may be coupled to transcript turnover via the NMD pathway for some transcripts. CONCLUSIONS: We generated a high-resolution transcriptome map for C. elegans and used it to identify endogenous targets of NMD. We find that these transcripts arise principally through splicing errors, strengthening the prevailing view that splicing and NMD are highly interlinked processes.
Project description:BACKGROUND: Recent studies have identified in Mycobacterium avium subsp. paratuberculosis (MAP), already known as a pathogen in ruminants, a potential zoonotic agent of some autoimmune diseases in humans. Therefore, considering the possible risk for public health, it is necessary a thorough understanding of MAP's gene expression during infection of human host as well as the identification of its immunogenic and/or virulence factors for the development of appropriate diagnostic and therapeutic tools. RESULTS: In order to characterize MAP's transcriptome during macrophage infection, we analyzed for the first time the whole gene expression of a human derived strain of MAP in simulated intraphagosomal conditions and after intracellular infection of the human macrophage cell line THP-1 by using the DNA-microarray technology. Results showed that MAP shifts its transcriptome to an adaptive metabolism for an anoxic environment and nutrient starvation. It up-regulates several response factors to oxidative stress or intracellular conditions and allows, in terms of transcription, a passive surface peptidoglycan spoliation within the macrophage along with an intensification of the anabolic activity for lipidic membrane structures. CONCLUSIONS: These results indicate a possible interactive system between MAP and its host cell based on the internal mimicry unlike other intracellular pathogens, bringing new hypothesis in the virulence and pathogenicity of MAP and its importance in human health.
Project description:Johne's disease, caused by infection with Mycobacterium avium subsp. paratuberculosis, (MAP), is a chronic intestinal disease of ruminants with serious economic consequences for cattle production in the United States and elsewhere. During infection, MAP bacilli are phagocytosed and subvert host macrophage processes, resulting in subclinical infections that can lead to immunopathology and dissemination of disease. Analysis of the host macrophage transcriptome during infection can therefore shed light on the molecular mechanisms and host-pathogen interplay associated with Johne's disease. Here, we describe results of an in vitro study of the bovine monocyte-derived macrophage (MDM) transcriptome response during MAP infection using RNA-seq. MDM were obtained from seven age- and sex-matched Holstein-Friesian cattle and were infected with MAP across a 6-h infection time course with non-infected controls. We observed 245 and 574 differentially expressed (DE) genes in MAP-infected versus non-infected control samples (adjusted P value ≤0.05) at 2 and 6 h post-infection, respectively. Functional analyses of these DE genes, including biological pathway enrichment, highlighted potential functional roles for genes that have not been previously described in the host response to infection with MAP bacilli. In addition, differential expression of pro- and anti-inflammatory cytokine genes, such as those associated with the IL-10 signaling pathway, and other immune-related genes that encode proteins involved in the bovine macrophage response to MAP infection emphasize the balance between protective host immunity and bacilli survival and proliferation. Systematic comparisons of RNA-seq gene expression results with Affymetrix(®) microarray data generated from the same experimental samples also demonstrated that RNA-seq represents a superior technology for studying host transcriptional responses to intracellular infection.
Project description:Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of severe gastroenteritis in cattle. To gain a better understanding of MAP virulence, we investigated the role of leuD gene in MAP metabolism and stress response. For this, we have constructed an auxotrophic strain of MAP by deleting the leuD gene using allelic exchange. The wildtype and mutant strains were then compared for metabolic phenotypic changes using Biolog phenotype microarrays. The responses of both strains to physiologically relevant stress conditions were assessed using DNA microarrays. Transcriptomic data was then analyzed in the context of cellular metabolic pathways and gene networks. Our results showed that deletion of leuD gene has a global effect on both MAP phenotypic and transcriptome response. At the metabolic level, the mutant strain lost the ability to utilize most of the carbon, nitrogen, sulphur, phosphorus and nutrient supplements as energy source. At the transcriptome level, more than 100 genes were differentially expressed in each of the stress condition tested. Systems level network analysis revealed that the differentially expressed genes were distributed throughout the gene network, thus explaining the global impact of leuD deletion in metabolic phenotype. Further, we find that leuD deletion impacted metabolic pathways associated with fatty acids. We verified this by experimentally estimating the total fatty acid content of both mutant and wildtype. The mutant strain had 30% less fatty acid content when compared to wildtype, thus supporting the results from transcriptional and computational analyses. Our results therefore reveal the intricate connection between the metabolism and virulence in MAP.
Project description:Genome structural annotation, i.e., identification and demarcation of the boundaries for all the functional elements in a genome (e.g., genes, non-coding RNAs, proteins and regulatory elements), is a prerequisite for systems level analysis. Current genome annotation programs do not identify all of the functional elements of the genome, especially small non-coding RNAs (sRNAs). Whole genome transcriptome analysis is a complementary method to identify "novel" genes, small RNAs, regulatory regions, and operon structures, thus improving the structural annotation in bacteria. In particular, the identification of non-coding RNAs has revealed their widespread occurrence and functional importance in gene regulation, stress and virulence. However, very little is known about non-coding transcripts in Histophilus somni, one of the causative agents of Bovine Respiratory Disease (BRD) as well as bovine infertility, abortion, septicemia, arthritis, myocarditis, and thrombotic meningoencephalitis. In this study, we report a single nucleotide resolution transcriptome map of H. somni strain 2336 using RNA-Seq method.The RNA-Seq based transcriptome map identified 94 sRNAs in the H. somni genome of which 82 sRNAs were never predicted or reported in earlier studies. We also identified 38 novel potential protein coding open reading frames that were absent in the current genome annotation. The transcriptome map allowed the identification of 278 operon (total 730 genes) structures in the genome. When compared with the genome sequence of a non-virulent strain 129Pt, a disproportionate number of sRNAs (?30%) were located in genomic region unique to strain 2336 (?18% of the total genome). This observation suggests that a number of the newly identified sRNAs in strain 2336 may be involved in strain-specific adaptations.