酵母双杂交原理和实验具体流程.pptVIP

酵母双杂交系统原理及具体操作流程 酵母双杂交系统可进行两个蛋白互作分析，可用一个已知的蛋白因子（在双杂交系统中称为诱饵蛋白）去钓取与其结合的蛋白；也可用进一步验证两个蛋白之间的互作。应用Clontech第三代酵母双杂交系统，并在按实验手册要求的严格操作下进行蛋白互作分析，我们的筛选结果将具有较好的重复性与可靠性。 单、双杂交的方法是基于许多真核生物转录因子都是以模块形式存在的，它们的转录激活域和DNA结合域在结构和功能上都有区别。这就允许研究者去构建不同的融合基因，当在酵母中表达融合蛋白，能立即结合DNA靶序列激活下游启动子的转录（图1所示），BD Matchmaker系统应用酵母中已经研究透彻的转录因子GAL4的转录激活域和DNA结合域来进行研究。 单杂与双杂的异同点 推荐使用Clontech公司的第三代载体，pGADT7-Rec 和pGBKT7进行双杂交筛选，因为它们产生更少的假阳性。对于cDNA合成，构建一个与GAL4激活域的融合文库，在双杂交中推荐使用pGADT7-Rec，这一克隆是通过体内同源重组来实现的（图2），这一步骤是利用酵母中的高效重组系统使ds DNA与GAL4 AD质粒融合。借助于同源重组克隆，文库的构建和筛选能快速接连地进行（步骤3和4），不需任何细菌转化步骤。用cDNA文库和pHIS2载体进行简单的酵母转化，接着在选择性培养基上进行酵母双杂交的筛选。 The term “promoter” usually refers to both the TATA box and the associated cis-regulatory elements. This usage is especially common when speaking of yeast gene regulation because the cis regulatory elements are relatively closely associated with the TATA box (Yoccum, 1987). This is in contrast to multicellular eukaryotes, where cisregulatory elements (such as enhancers) can be found very far upstream or downstream from the promoters they regulate. In this text, minimal promoter will refer specifically to the TATA region, exclusive of other cis-acting elements. The minimal promoter (or TATA box) in yeast is typically approximately 25 bp upstream of the transcription start site. Yeast TATA boxes are functionally similar to prokaryotic Pribnow boxes, but are not as tightly conserved. Furthermore, some yeast transcription units are preceded by more than one TATA box. The yeast HIS3 gene, for example, is preceded by two different TATA boxes: TR, which is regulated, and TC, which is constitutive. UAS and TATA regions can be switched to create novel promoters For GAL4-based systems, either a native GAL UAS or a synthetic UASG 17-mer consensus sequence (Heslot Gaillardin, 1992) provides the binding site for the GAL4 DNA-BD. If you are putting together your own one- or two-hybrid system, you must make sure that the reporter genes p