Progression of the eukaryotic cell cycle requires the regulation of hundreds of genes to ensure that they are expressed at the required times. Integral to cell cycle progression in yeast and animal cells are temporally controlled, progressive waves of transcription mediated by cell cycle-regulated transcription factors. However, in the kinetoplastids, a group of early-branching eukaryotes including many important pathogens, transcriptional regulation is almost completely absent, raising question ...[more]
Project description:Cy3 and Cy5 direct labelled RNA from Bloodstream MiTat1.1 trypanosomes and Procyclic 427 Lister were hybridized onto JCVI Trypanosoma brucei oligoarrays (version2). Procyclic RNA were used as control for data analysis.
Project description:We analysed differentiation of the EATRO1125 strain of Trypanosoma brucei brucei, which was first isolated in 1966 from a bushbuck (Tragelaphus scriptus) in Uganda (origin stated (Bouteillea, 1995) without an original reference.<br>To analyse gene expression, we isolated at least 3 x 10e8 trypanosomes at different differentiation states, using two independent biological replicates. Bloodstream forms were harvested at a density of 2 x 10e5/ml (low density, logarithmic growth), and 2 x 10e6/ml (high density, logarithmic growth). Cells were also taken immediately upon attaining the density of 2 x 106/ml, treated with 3 mM cis-aconitate and moved to a room at 27°C. Samples were taken 30 min, 60 min, 12h and 24h after this. At 24 h the cells were centrifuged, resuspended (at 27°C) in MEM-Pros medium, which contains proline as the major energy source. Samples were taken again at 48h and 72h. A culture that had been maintained for several weeks after transformation was used as a source of established procyclic trypanosomes.
Project description:The first 4 samples belong to the RNA-IP using in situ TAP tagged ZC3H30 in procyclic (insect) form of the parasite T. brucei Lister 427, 2 samples are Elu or eluate, and 2 are FL or flowthrough (unbound) sample. The other 8 samples are also from procyclic cells. 4 samples belong to DKO(ZC3H30 gene double knockout), 2 are non-stressed and 2 are heat shocked samples; the rest 4 samples are DKO-ectopic (ZC3H30 double knockouts, expressing, ectopic copy of ZC3H30) 2 are non-stressed and 2 are heat shocked samples. Heat Shock experiment was done at 39 degree Celsius.
Project description:The life cycle of Trypanosoma brucei involves several cell differentiation transitions that allow transmission, survival and proliferation of these parasites. One of these transitions, the differentiation of growth-arrested stumpy forms in the mammalian blood into proliferating insect-stage procyclic forms, can be induced synchronously in vitro by addition of cis-aconitate (CA). Using single-cell analysis by flow-cytometry to follow differentiation, we show that this transition is an irreversible bistable switch where cells commit to differentiation after 1-3 hours of exposure to CA. This irreversibility implies the existence of positive feedback mechanisms that allow commitment to differentiation: i.e. the establishment of “memory” of exposure to the differentiation signal. Such mechanisms probably depend on post-translational modifications (e.g. phosphorylation) and/or synthesis of regulatory proteins. Using the reversible protein synthesis inhibitor cycloheximide, we find that protein synthesis is required for establishment of signal memory and normal commitment to differentiation. To characterize the ‘commitment proteome’, we performed SILAC phosphoproteomics to provide a detailed map of the protein expression and phosphorylation events during the early stages of differentiation in a synchronised parasite population. Using a rigorous candidate gene approach we have also demonstrated that the stumpy form enriched serine-throenine protein kinases TbNRKA/B stringently control the earliest events in differentiation identifying these kinases as major regulators of trypanosome development.
Project description:Organization of the genome into compacted chromatin is a eukaryotic innovation facilitating increased sophistication in transcriptional regulation. In metazoa coiled-coil lamin proteins are major components of the chromatin organizer at the nuclear periphery and maintain nuclear integrity. While identifiable lamin homologues are restricted to metazoans, morphologically analogous structures maintaining nuclear organization in other eukaryotic lineages are known, but the molecular constituents remain undefined. Trypanosoma brucei NUP-1 is a large coiled-coil protein associated with fibrils at the inner face of the nuclear envelope. Using transcriptome analysis in combination with RNA interference and various imaging techniques, we demonstrate that NUP-1 forms a stable immobile cage around the nucleus, is required for viability and nuclear structural integrity, directs the positional organization of nuclear pore complexes, and serves to organize chromatin and specifically repress genes located at the nuclear periphery involved in immune evasion. Based on architectural similarity and functionality, we propose that NUP-1 is a novel, highly divergent lamin The effect of Nup-1 depletion on the transcriptome was examined in three independent experiments (A, B, & C). T. brucei cultures were either treated with RNAi (plus) or left untreated (minus) and RNA was extracted from each sample at the indicated time point (0h, 6h, 12h, 24h, or 48h). Two color microarrays were performed comparing treated and untreated samples at each time point. Dye swaps were performed and are indicated. Replicates of t=12h and t=24h for sample B were also included.
Project description:T. brucei PF cells were treated with several chemical reagents and anti-trypanosomatid drugs. The effect of each chemical perturbation on the transcriptome of T. brucei was examined by transcript profiling of treated vs. control cells. The results indicated widespread changes, suggesting that the transcriptome of T. brucei is highly responsive to environmental factors that perturb its metabolic and biological pathways. 11 chemical perturbations, each co-hybridized with a common reference RNA from control non-treated cells. One array per treatment.
Project description:Rats (four per isolate) were infected with Trypansooma rhodesiense taken from the CNS of two different patients from Lwala. Blood was taken at parasitaemias ranging from 3-20 E7. The results can be compared with those from human patient samples.
Project description:Protein kinase A (PKA), the main effector of second messenger cAMP, is highly conserved among eukaryotes and a paradigm for the mechanisms of regulation of protein kinases by ligands. The unique PKA holoenzymes in the phylogenetically distant protozoan parasite Trypanosoma are unresponsive to cAMP in vitro and in vivo. By small molecule screening and optimization, we designed direct, membrane-permeable activators binding with a kD of 9 nM to the CNB pockets of the T. brucei regulatory PKA subunit. 7-Cyano-7-deazainosine has low toxicity and thus is a perfect tool to explore cAMP-independent PKA signaling in these important pathogens. This project describes PKA-induced phosphoproteome changes of the bloodstream stage of T. brucei.