酵母双杂交原理与实验具体流程.pptxVIP

酵母双杂交系统原理及具体操作流程; 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。 ;;; Yeast promoters and other cis-acting regulatory elements play a crucial role in yeast-based expression systems and transcriptional assays such as the MATCHMAKER One- and Two-Hybrid Systems. Differences in the promoter region of reporter gene constructs can significantly affect their ability to respond to the DNA-binding domain of specific transcriptional activators; promoter constructs also affect the level of background (or leakiness) of gene expression and the level of induced expression. Furthermore, differences in cloning vector promoters determine the level of protein expression and, in some cases, confer the ability to be regulated by a nutrient (such as galactose in the case of the GAL1promoter). UAS and TATA regions are basic building blocks of yeast promoters The initiation of gene transcription in yeast, as in other organisms, is achieved by several molecular mechanisms working in concert. All yeast structural genes (i.e., those transcribed by RNA polymerase II) are preceded by a region containing a loosely conserved sequence (TATA box) that determines the transcription start site and is also a primary determinant of the basal transcription level. Many genes are also associated with cis-acting elements—DNA sequences to which transcription factors and other trans-acting regulatory proteins that bind and affect transcription levels. ;The term “promoter” usually refers to both the TATA box and the associated cis-regulatory elements. This usage is especially common wh