Project description:Two lines of Trypanosoma brucei were isolated from cattle in Uganda. The DNA is from parasites that had undergone two rodent passages.
Project description:Eukaryotes have an array of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, histones from Kinetoplastids are highly divergent. The structural and functional consequences of this variation are unknown. Here, we have biochemically characterised nucleosome core particles (NCPs) from the Kinetoplastid parasite Trypanosoma brucei. A structure of the T. brucei NCP reveals that global histone architecture is conserved, but specific sequence alterations lead to distinct DNA and protein interaction interfaces. The T. brucei NCP is unstable and has weakened DNA binding overall. However, dramatic changes at the H2A-H2B interface introduce local reinforcement of DNA contacts. The T. brucei acidic patch has altered topology and is refractory to known binders, indicating that the nature of chromatin interactions in T. brucei may be unique. Overall, our results provide a detailed molecular basis for understanding evolutionary divergence in chromatin structure.
Project description:5-Hydroxymethyluracil (5hmU) is a thymine modification existing in the genomes of a number of living organisms. The post-replicative formation of 5hmU occurs via hydroxylation of thymine, which can be mediated by the ten-eleven translocation (TET) dioxygenases in mammalian and J-binding proteins (JBPs) in protozoan genomes, respectively. In addition, 5hmU also can be generated through oxidation of thymine by reactive oxygen species or from deamination of 5hmC by activation-induced cytidine deaminase (AID) or APOBEC family enzymes. While the biological roles of 5hmU have not been fully explored, identifying its genomic location will assist in elucidating its functions. Herein, we report a method of enzyme-mediated bioorthogonal labeling to selectively enrich genomic regions containing 5hmU. 5hmU DNA kinase (5hmUDK) was utilized to selectively install an azide group or alkynyl group into the hydroxyl group of 5hmU followed by incorporation of the biotin linker through click chemistry and capture of 5hmU-containing DNA fragments via streptavidin pull-down. The enriched fragments were applied to deep sequencing to map the location of 5hmU. With this established enzyme-mediated bioorthogonal labeling strategy, we achieved the genome-wide mapping of 5hmU in Trypanosoma brucei (T. brucei) genomes. The method described here will allow for a better understanding of the functional roles and dynamics of 5hmU in genomes
Project description:The host range of African trypanosomes is influenced by innate protective molecules in the blood of primates. A subfraction of human high-density lipoprotein (HDL) containing apolipoprotein A-I, apolipoprotein L-I, and haptoglobin-related protein is toxic to Trypanosoma brucei brucei but not the human sleeping sickness parasite Trypanosoma brucei rhodesiense. It is thought that T. b. rhodesiense evolved from a T. b. brucei-like ancestor and expresses a defense protein that ablates the antitrypanosomal activity of human HDL. To directly investigate this possibility, we developed an in vitro selection to generate human HDL-resistant T. b. brucei. Here we show that conversion of T. b. brucei from human HDL sensitive to resistant correlates with changes in the expression of the variant surface glycoprotein (VSG) and abolished uptake of the cytotoxic human HDLs. Complete transcriptome analysis of the HDL-susceptible and -resistant trypanosomes confirmed that VSG switching had occurred but failed to reveal the expression of other genes specifically associated with human HDL resistance, including the serum resistance-associated gene (SRA) of T. b. rhodesiense. In addition, we found that while the original active expression site was still utilized, expression of three expression site-associated genes (ESAG) was altered in the HDL-resistant trypanosomes. These findings demonstrate that resistance to human HDLs can be acquired by T. b. brucei. Keywords: Trypanosoma, VSG, antigenic switching, HDL-resistance