Abstract
BackgroundThe protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis. Its cell cycle is complex and not fully understood at the molecular level. The T. brucei genome contains over 6000 protein coding genes with >50% having no predicted function. A small scale RNA interference (RNAi) screen was carried out in Trypanosoma brucei to evaluate the prospects for identifying novel cycle regulators.ResultsProcyclic form T. brucei were transfected with a genomic RNAi library and 204 clones isolated. However, only 76 RNAi clones were found to target a protein coding gene of potential interest. These clones were screened for defects in proliferation and cell cycle progression following RNAi induction. Sixteen clones exhibited proliferation defects upon RNAi induction, with eight clones displaying potential cell cycle defects. To confirm the phenotypes, new RNAi cell lines were generated and characterised for five genes targeted in these clones. While we confirmed that the targeted genes are essential for proliferation, we were unable to unambiguously classify them as cell cycle regulators.ConclusionOur study identified genes essential for proliferation, but did not, as hoped, identify novel cell cycle regulators. Screening of the RNAi library for essential genes was extremely labour-intensive, which was compounded by the suboptimal quality of the library. For such a screening method to be viable for a large scale or genome wide screen, a new, significantly improved RNAi library will be required, and automated phenotyping approaches will need to be incorporated.
Highlights
The protozoan parasite, Trypanosoma brucei, is spread by the tsetse fly and causes Human African Trypanosomiasis
Where potential cell cycle defects were identified, new RNA interference (RNAi) cell lines were generated and the analysis repeated in an attempt to confirm the original phenotype in the Procyclic form (PF) and to determine whether these genes were involved in cell cycle regulation in bloodstream stage (BS) trypanosomes
Of the 155 sequenced inserts, 52 contained sequences of no interest for this screen and a further 25 inserts could not be identified by BLAST, which, since the library was made from total genomic DNA, could have come from intermediate or mini-chromosomes that were not sequenced in the T. brucei genome project [15]
Summary
RNAi library vector inserts (integrated into the rDNA spacer region of the genome) were PCR-amplified from genomic DNA of clones, sequenced and analysed by BLAST analysis at GeneDB http://www.genedb.org/. Initial screening Sixteen of the 76 clones targeting non-VSG/ESAG proteincoding genes gave proliferation defects following RNAi induction [see Additional file 2] and [Additional file 3]. Clone 33 (targeting RSP3 [16]) acted as a positive control and upon induction, displayed proliferation and cell cycle defects, consistent with previously published data [see Additional file 4]. The hypothetical ORF, Tb927.5.3260, and PP1 (Tb11.01.0450) are essential for proliferation but may not be required for cell cycle control In PF parasites, RNAi of the hypothetical ORF (Tb927.5.3260) caused changes to the cell cycle profile (Fig. 1D), but these defects only accumulated in significant numbers at late time points, suggesting that they could be downstream effects of another defect. Depletion of all seven PP1 genes simultaneously in PF trypanosomes, reduced proliferation but did not effect the cell cycle [25], okadaic acid (a PP1 and PP2A inhibitor) treatment disrupts kinetoplast segregation [26]
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