DNA BINDING PROTEINS & REGULATION OF SURFACE GLYCOPROTEINS IN TRYPANOSOMA BRUCE

DNA结合蛋白

• 批准号: 7355044
• 负责人: GEORGE ALAN MARTIN CROSS
• 金额: $ 0.12万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7355044
• 关键词: DNA; BINDING; PROTEINS; REGULATION; SURFACE

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.

该子项目是利用 NIH/NCRR 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中得到体现。列出的机构是中心的机构，不一定是研究者的机构。布氏锥虫表面的大部分蛋白质由变异表面糖蛋白 (VSG) 组成。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 基因被表达（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。使用亲和层析、DNA 层析和离子交换层析以及超速离心，我们成功纯化了一种与 VSG 表达位点之一结合的蛋白质复合物。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。由于布氏锥虫基因组的不完整性以及与其他生物体蛋白质的同源性较低，没有直接证据表明纯化的蛋白质复合物中存在转录因子。我们目前正在通过各种技术研究该复合物的成分，例如标记蛋白提取、荧光原位杂交、蛋白质印迹和 RNA 干扰。