DNA BINDING PROTEINS & REGULATION OF SURFACE GLYCOPROTEINS IN TRYPANOSOMA BRUCE

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。变异型表面糖蛋白(VSG‘s)是布氏锥虫表面蛋白的主要成分。虽然VSG的表达水平非常高，但在任何给定的时间，只有一个活性的VSG表达位点(20个)，并且只有一个VSG基因表达(从超过1000个基因的集合)。这一高度选择性的过程的机制和成分仍不清楚。利用亲和层析、DNA和离子交换层析以及超速离心法，我们成功地纯化了与VSG表达位点之一结合的蛋白质复合体。存在的蛋白质随后经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分离，并用液相色谱-电喷雾质谱仪进行分析。由于布鲁氏毛滴虫基因组的不完整，以及与其他生物蛋白质的同源性很低，目前还没有直接证据表明在纯化的蛋白质复合体中存在转录因子。我们目前正在通过各种技术来研究这个复合体的组成，如标记蛋白拔出、荧光原位杂交、Western blotting和RNA干扰。

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.