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African trypanosomes are major pathogens of humans and livestock and represent a model for studies of unusual protozoal biology. We describe a high-throughput phenotyping approach termed RNA interference (RNAi) target sequencing, or RIT-seq that, using Illumina sequencing, maps fitness-costs associated with RNAi. We scored the abundance of >90,000(More)
Antigenic variation in African trypanosomes requires monoallelic transcription and switching of variant surface glycoprotein (VSG) genes. The transcribed VSG, always flanked by '70 bp'-repeats and telomeric-repeats, is either replaced through DNA double-strand break (DSB) repair or transcriptionally inactivated. However, little is known about the(More)
Silent information regulator 2 (Sir2)-related proteins or sirtuins function as NAD(+)-dependent deacetylases or ADP ribosylases that target a range of substrates, thereby influencing chromatin structure and a diverse range of other biological functions. Genes encoding three Sir2-related proteins (SIR2rp1-3) have been identified in the parasitic(More)
Eukaryotic chromosomes are capped with telomeres which allow complete chromosome replication and prevent the ends from being recognized by the repair machinery. The African trypanosome, Trypanosoma brucei, is a protozoan parasite where antigenic variation requires reversible silencing of a repository of telomere-adjacent variant surface glycoprotein (VSG)(More)
Antigenic variation in African trypanosomes involves monoallelic expression and reversible silencing of variant surface glycoprotein (VSG) genes found adjacent to telomeres in polycistronic expression sites (ESs). We assessed the impact on ES silencing of five candidate essential chromatin-associated factors that emerged from a genome-wide RNA interference(More)
The concept of disease-specific chemotherapy was developed a century ago. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work, and the drugs that emerged remain in use for treating human African trypanosomiasis (HAT). The importance of understanding the mechanisms underlying selective drug action and(More)
In Trypanosoma brucei, RNA interference (RNAi) and recombinant protein expression are established as powerful approaches for functional genomics, particularly when combined with inducible expression. The favoured methods involve exploiting homologous recombination to target expression cassettes to a chromosome sub-set to establish stable cell lines.(More)
To be effective, therapeutic compounds must typically enter target cells and, in some cases, must be concentrated or modified. Thus, uptake and activation mechanisms often form the basis of selectivity against infectious agents. Loss-of-function screens can be used to identify proteins involved in drug uptake and metabolism and may also identify clinically(More)
Chromatin modification is important for virtually all aspects of DNA metabolism but little is known about the consequences of such modification in trypanosomatids, early branching protozoa of significant medical and veterinary importance. MYST-family histone acetyltransferases in other species function in transcription regulation, DNA replication,(More)
Allelic exclusion underpins antigenic variation and immune evasion in African trypanosomes. These bloodstream parasites use RNA polymerase-I (pol-I) to transcribe just one telomeric variant surface glycoprotein (VSG) gene at a time, producing superabundant and switchable VSG coats. We identified trypanosome VSG exclusion-1 (VEX1) using a genetic screen for(More)