Project description:Strigomonas culicis is a kinetoplastid parasite of insects that maintains a mutualistic association with an intracellular symbiotic bacterium, that is highly integrated into the protozoa metabolism: it furnishes essential compounds and divides in synchrony with the nuclear host. The protozoa, conversely, can be rid of the endosymbiont, producing a cured cell line, which presents a diminished ability to colonize the insect host. This obligatory association can represent an intermediate step of the evolution towards the formation of a organelle, therefore representing an interesting model to understand the symbiogenesis theory. Here, we used shotgun proteomics to compare the S. culicis endosymbiont-containing and aposymbiotic strains, revealing a total of 11,305 peptides, and up to 2,213 proteins (2,029 and 1,452 for wild and aposymbiotic, respectively). Gene ontology associated to comparative analysis between both strains revealed that the biological processes most affected by the elimination of the symbiont were the amino acid metabolism, as well as protein synthesis and folding. This large-scale comparison of the protein expression in S. culicis marks a step forward in the comprehension of the role of endosymbiotic bacterium in monoxenic trypanosomatid biology, particularly because these organisms have a polycistronic open reading frame organization and post-transcriptional gene regulation.
Project description:Abstract: The Kinetoplastida (Euglenozoa) are unicellular flagellates that include the trypanosomatid parasites, most notably Trypanosoma brucei, T.cruzi and Leishmania spp. These organisms cause substantial mortality and morbidity in humans and their livestock worldwide as the causative agents of African sleeping sickness, Chagas disease and leishmaniasis respectively. Draft genome sequences are available for several species of both Trypanosoma and Leishmania. Bodo saltans is a free-living heterotroph found worldwide in freshwater and marine habitats, and it is among the closest bodonid relatives of the trypanosomatids. The purpose of a B. saltans genome sequence is to provide an 'out-group' for comparative genomic analysis of the trypanosomatid parasites. It will provide a model of the ancestral trypanosomatid to distinguish those derived parts of the parasite genomes (i.e., unique trypanosomatid adaptations) from those which are a legacy of the free-living ancestor. To aid annotation of the B.saltans genome sequence, total genomic RNA was extracted on four occasions from the total cellular mass of 160ml of B.saltans cell culture, for the purposes of transcription profiling by high throughput sequencing. Cells were unmodified. B.saltans cells were grown in water at 4oC. Total genomic RNA was extracted from a cell pellet using TRIZOL reagent and ethanol precipitated. Poly A+ mRNA was purified from total RNA using oligo dT dyna bead selection and libraries were created using the Illumina RNA-seq protocol. The samples were sequenced on an Illumina HiSeq 2000. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
Project description:We report the application of CAGE (Cap Analysis of Gene Expression) on collections of Daphnia pulex individuals representing three major developmental states. This submission comes from a project of Michael Lynch and was funded by a grant from the National Institutes of Health entitled 'Population Genomics of Daphnia pulex' (Project Number: 1R01GM101672-01A1).
Project description:RNA polymerase III (RNAP III) synthetizes small essential non-coding RNA molecules such as tRNAs and 5S rRNA. In yeast and vertebrates, RNAP III needs general transcription factors TFIIIA, TFIIIB and TFIIIC to initiate transcription. TFIIIC, composed of six subunits, binds to internal promoter elements in RNAP III-dependent genes. Limited information is available about RNAP III transcription in the trypanosomatid protozoa Trypanosoma brucei and Leishmania major, which diverged early from the eukaryotic lineage. Analyses of the first draft of the trypanosomatid genome sequences failed to recognize orthologs of any of the TFIIIC subunits, suggesting that this transcription factor is absent in these parasites. However, a single putative TFIIIC subunit was recently annotated in the databases. Here we characterize this subunit in T. brucei and L. major and demonstrate that it corresponds to Tau95. In silico analyses showed that both proteins possess the typical Tau95 sequences: the DNA binding region and the dimerization domain. As anticipated for a transcription factor, Tau95 localized to the nucleus in insect forms of both parasites. Chromatin immunoprecipitation (ChIP) assays demonstrated that Tau95 binds to tRNA and U2 snRNA genes in T. brucei. Remarkably, by performing tandem affinity purifications we identified orthologs of TFIIIC subunits Tau55, Tau131 and Tau138 in T. brucei and L. major. Thus, contrary to what was assumed, trypanosomatid parasites do possess a TFIIIC complex. Other putative interacting partners of Tau95 were identified in T. brucei and L. major.
Project description:Comparative genomics studies in primates are extremely restricted due to our limited access to samples from non-human apes. In order to gain better insight into the genetic processes that underlie variation in complex phenotypes in primates, we must have access to faithful model systems for a wide range of cell types. To facilitate this, we have generated a panel of 7 fully characterized chimpanzee induced pluripotent stem cell (iPSC) lines derived from healthy donors. To begin demonstrating the utility of comparative iPSC panels, we collected RNA-sequencing and DNA methylation data from the chimpanzee iPSCs and the corresponding fibroblast lines, as well as from 7 human iPSCs and their source lines, which encompass multiple populations and cell types. We observe much less within-species variation in iPSCs than in somatic cells, indicating that the reprogramming process erases many inter-individual differences. The low within-species regulatory variation in iPSCs allowed us to identify many novel inter-species regulatory differences of small magnitude. We used ChIP-seq to characterize the genome-wide distribution of two types of histone modifications (H3K27me3 and H3K27ac) in three of our chimpanzee iPSCs and compared them to histone modification data from three human iPSC lines from the Roadmap Epigenomics project: