African trypanosomes (<i>Trypanosoma brucei spp.</i>) are the causative agents of Human African Trypanosomiasis and Nagana in cattle and livestock. The complex biphasic lifecycle of <i>T. brucei</i> involves differentiation from the mammalian host to the tsetse fly vector, a process that involves highly regulated changes in gene expression, morphology and surface antigen expression. Three members of the <i>T. brucei </i>Zinc Finger Protein (TbZFP1, 2 and 3) family have been characterised, which contain the unusual C<sub>x8</sub>C<sub>x5</sub>C<sub>x3</sub>H RNA binding zinc finger motif, as well as a PY or WW protein-protein interacting motif. The TbZFP family are important regulators of differentiation and all three molecules have been shown <i>in vivo</i> to be essential for effective differentiation, although no proteins or RNA transcripts involved in these mechanisms have as yet been identified. Here, data obtained from screens for proteins and RNA transcripts that interact with the TbZFP family is presented. Using the yeast two-hybrid system, TbZFP1 was shown to interact directly with TbZFP2 and 3, this interaction being dependent on the WW domain. Furthermore, a yeast two-hybrid screen to identify novel protein interactions using TbZFP2 and 3 was undertaken, though no genuine interactions were identified. In addition, a systematic evolution of ligands by an exponential enrichment (SELEX) approach was used to identify the sequence TCAGT/C as a putative RNA binding motif for TbZFP3 and the ability of TbZFP3 to bind RNA was verified using an <i>in vitro</i> EMSA. Finally, a screen was undertaken for RNA transcripts whose abundance was affected by the ablation of TbZFP2.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:645081 |
| Date | January 2007 |
| Creators | Craddy, Paul |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14731 |
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