Validating TbRAP1-Interacting Candidates


Validating TbRAP1-Interacting Candidates



Telomeres are specialized protein-DNA complexes located at the ends of eukaryotic chromosomes. They act as caps on the ends of chromosomes to preserve DNA from degradation and rearrangements and are therefore essential for genome stability. Trypanosoma brucei, a protozoan parasite that causes sleeping sickness in humans and nagana in cattle, evades the host immune responses by regularly switching its surface antigen, Variant Surface Glycoproteins (VSG). VSGs are expressed exclusively from regions adjacent to telomeres. Hence, understanding the VSG regulation by the telomere complex would help in developing means to eliminate this parasite. Our recent studies indicate that TbRAP1 is a protein associated with the telomere complex and is important for VSG silencing and VSG switching. In order to better understand the underlying mechanisms of TbRAP1-mediated VSG regulation, we attempted to identify proteins that interact with TbRAP1. By using TbRAP1 as bait, we identified a number of TbRAP1-interacting candidates in a yeast-2-hybrid screen. We have now subcloned these candidates into yeast expression vectors and tested their interactions with TbRAP1 directly by the liquid yeast 2-hybrid assay using Ortho-Nitrophenyl-β-galactoside (ONPG) as the substrate of the reporter gene product -glactosidase.

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Validating TbRAP1-Interacting Candidates