DNA BINDING PROTEINS & REGULATION OF SURFACE GLYCOPROTEINS IN TRYPANOSOMA BRUCE

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。变异表面糖蛋白（VSG）包括布氏锥虫表面上的大部分蛋白质。尽管VSG表达水平非常高，但在任何给定时间，只有一个活跃的VSG表达位点（二十个中），并且只有一个VSG基因表达（来自一千多个基因的集合）。这种高度选择性过程的机制和组成部分仍然未知。使用亲和，DNA和离子交换色谱，以及超离心，我们已经成功地纯化的蛋白质复合物，结合到VSG表达位点之一。随后通过SDS-PAGE解析存在的蛋白质，并通过液相色谱-电喷雾质谱法进行分析。由于T.由于这种蛋白质与布氏杆菌基因组的同源性很低，而且与其他生物体的蛋白质同源性很低，因此没有直接证据表明在纯化的蛋白质复合物中存在转录因子。目前，我们正在研究的各种技术，如拉脱标记蛋白，荧光原位杂交，蛋白质印迹和RNA干扰的这种复合物的组成部分。

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.