Project description:Bloodstream-form trypanosomes (Lister 427) constitutively expressing ZC3H5-TAP (Tb927.3.740) were used. The protein was pulled down with an IgG column , then the protein and bound RNA was eluted using TEV protease. RNA was then sequenced from unbound (flow-through) and bound (eluate) fractions.
Project description:Trypanosoma brucei developed a sophisticated life cycle to adapt to different host environments. Although developmental differentiation of T. brucei has been the topic of intensive research for decades, the mechanisms responsible for adaptation to different host environments are not well understood. We developed stable isotope labeling by amino acids in cell culture in trypanosomes to compare the proteomes of two different life cycle stages. Quantitative comparison of 4364 protein groups identified many proteins previously not known to be stage-specifically expressed. The identification of stage-specific proteins helps to understand how parasites adapt to different hosts and provides new insights into differences in metabolism, gene regulation, and cell architecture. A DEAD-box RNA helicase, which is highly up-regulated in the bloodstream form of this parasite and which is essential for viability and proper cell cycle progression in this stage is described as an example.
Project description:The protozoan parasite Trypanosoma evansi is responsible for causing Surra in a variety of mammalian hosts over a wide geographical area. In order to identify which genes and processes are required to establish disease in mice, parasites were irradiated over a range using a Cobalt60 gamma source. A custom Trypanosome spp. array that covers the genomes of three trypanosome species, T. brucei, T. evansi and T. congolense was designed by Affymetrix with an average of 9300 whole gene transcripts from all three species were targeted. Irradiation differentially affected the abundance of gene transcripts in a dose-dependent trend. We present these genes as necessary for repair from irradiation damage, and essential for disease establishment in mice post irradiation.
Project description:To analyse the impact of URT1-mediated uridylation on miRNA and siRNA tailing, we deep-sequenced small RNA libraries for WT and urt1 duplicate samples at the same developmental stage that was analyzed by TAIL-seq, i.e., two-week-old seedlings. Examination of miRNA and siRNA tailing in WT and urt1 samples.
Project description:We have analysed the genome-wide distribution of RNA polymerase in mechanosenstive deletion strains of E.coli upon hyperosmotic stress. We have used ChIP-chip of the ß subunit of RNA polymerase and we have assessed changes in RNA polymerase distribution in Frag1 MJF612 and MJF641 strains that are deleted for incresing numbers of mechanosensitive channels in response to hyperosmotic stress .
Project description:The first step of glycosylphosphatidylinositol (GPI) anchor biosynthesis in all eukaryotes is the addition of N-acetylglucosamine (GlcNAc) to phosphatidylinositol (PI) which is catalysed by a UDP-GlcNAc : PI α1-6 GlcNAc-transferase. This enzyme has been shown to be a complex of at least seven subunits in mammalian cells and a similar complex of homologous subunits has been postulated in yeast. Homologs of most of these mammalian and yeast subunits were identified in the Trypanosoma brucei predicted protein database. The putative catalytic subunit of the T. brucei complex,TbGPI3, was epitope tagged with three consecutive c-Myc sequences at its C-terminus. Immunoprecipitation of TbGPI3-3Myc followed by native polyacrylamide gel electrophoresis and anti-Myc Western blot showed that it is present in a ~240 kDa complex. Label-free quantitative proteomics were performed to compare anti-Myc pull-downs from lysates of TbGPI-3Myc expressing and wild type cell lines. TbGPI3-3Myc was the most highly enriched protein in the TbGPI3-3Myc lysate pull-down and partner proteins TbGPI15, TbGPI9, TbGPI2, TbGPI1 and TbERI1 were also identified with significant enrichment. Our proteomics data also suggest that an Arv-1 like protein (TbArv1) is a subunit of the T. brucei complex. Yeast and mammalian Arv1 have been previously implicated in GPI biosynthesis, but here we present the first experimental evidence for physical association of Arv1 with GPI biosynthetic machinery. A putative E2-ligase has also been tentatively identified as part of the T. brucei UDP-GlcNAc : PI α1-6 GlcNAc-transferase complex.
Project description:A disproportionate number of predicted proteins from the genome sequence of the protozoan parasite Trypanosoma brucei, an important human and animal pathogen, are hypothetical proteins of unknown function. This work describes a protein correlation profiling mass spectrometry approach, using two size exclusion and one ion exchange chromatography systems, to derive sets of predicted protein complexes in this organism by hierarchical clustering and machine learning methods. We provide examples of both potential new subunits of known protein complexes and of novel trypanosome complexes of suggested function, contributing to improving the functional annotation of the trypanosome proteome. These hypothesis-generating proteomic data are provided in an open access online data visualisation environment (http://134.36.66.166:8083/complex_explorer)
Project description:Previous BioID experiments targeting the nucleoporin (NUP) NUP158 and the nuclear transport factor MEX67 indicated (PMID: 36410438; PXD031245) that proximity labelling delivers highly specific interactome data in the confined localisation of the nuclear pore, with a labelling radius well below the size of the nuclear pore. Here we extended this approach to target NUPs NUP110, NUP76 and NUP96 with a TurboID-HA tandem tag fused to both, the N and C-terminus of each respective target protein. While the central NUP96 biotinylated most nuclear pore proteins with the marked exception of the nuclear basket, NUP158, NUP110 and NUP76 caused biotinylation of specific sub-complexes of the pore and Mex67 labelled NUPs lining the pore channel.