This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Variant surface glycoproteins (VSG's) comprise the majority of the proteins on the surface of Trypanosoma brucei. Although VSG expression levels are very high, at any given time there is only one active VSG expression site (out of twenty) and only a single VSG gene is expressed (from a collection of over a thousand genes). The mechanism and components of this highly selective process are still unknown. Using affinity, DNA and ion exchange chromatographies, as well as ultracentrifugation, we have succeeded in purifying a protein complex that binds to one of the VSG expression sites. The proteins present were subsequently resolved by SDS-PAGE and analyzed by liquid chromatography-electrospray mass spectrometry. Due to the incompleteness of the T. brucei's genome and the low degree of homology to proteins from other organisms, there was no immediate evidence that a transcription factor was present in the purified protein complex. We are currently investigating the components of this complex by various techniques such as pullouts by tagged proteins, fluorescence in situ hybridization, Western blotting and RNA interference.
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