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1、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
2、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,酵母单双杂，都基于许多真核生物转录因子的转录激活域和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，就能结合DNA靶序列激活下游启动子的转录。单杂交是文库中的转录因子直接与靶序列结合，使与转录因子融合的GAL4AD激活报告基因HIS3的转录，而双杂是借助与诱饵蛋白与文库中调控因子的互作，使得GAL4BD和AD通过这个“桥梁”共同起作用，激活报告基因（ADE2、HIS3 、 lacZ和 MEL1）的转录。,推荐使用Clontech公司的第三代载体，pGADT7-Rec
3、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。,图2. BD MatchmakerTM双杂交文库构建和筛选。上图所示，借助于重组克隆使文库构建和筛选快速有效,Yeast promoters and
4、 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-bindi
5、ng 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 t
6、he 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 ye
7、ast 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-act
8、ing elementsDNA 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 when speaking of yeast ge
9、ne 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 th
10、is 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,
11、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
12、 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 promoter will
13、 be recognized by the DNA-BD moiety encoded in your DNA-BD fusion vector.Reporter genes under the control of GAL4-responsive elements AH109 contains four reportersADE2, HIS3, MEL1, and lacZunder the control of three distinct GAL4 upstream activating sequences (UASs) and TATA boxes . The ADE2 reporte
14、r alone provides strong nutritional selection. For higher stringency, and to reduce the incidence of false positives, select for ADE2 and HIS3 (James et al., 1996). You also have the option of assaying for MEL1, which encodes -galactosidase. MEL1 is endogenous to both Y187 and AH109. Because -galact
15、osidase is a secreted enzyme, its activity can be detected by adding X-Gal to the selection plate: If MEL1 is active and X-Gal is present, the colony will turn blue. lacZ in Y187 exhibits a high level of induced -galactosidase activity in a positive two-hybrid assay because it is under the control o
16、f the intact GAL1 UAS.,Reporter genes under the control of a minimal HIS3 promoter The HIS3 reporter gene in yeast strain Y190 is unusual among the GAL4 two-hybrid reporter gene constructs in that it is under the control of the GAL1 UAS and a minimal promoter containing both HIS3 TATA boxes The HIS3
17、 reporter plasmids pHISi and pHISi-1 used in the MATCHMAKER One Hybrid System also have both of the HIS3 TATA boxes present in the minimal promoter. By inserting a cis-acting element in the MCS, the regulated TATA box (TR) can be affected, but there is still a significant amount of constitutive, leaky expression due to the HIS3 TC. The leaky HIS3 expression of these one-hybrid plasmids is first used to help construct HIS3 reporter strains, and later is controlled by including 3-aminotriazole in the medium to suppress background growth.,
