酵母双杂交原理与实验具体流程.

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
பைடு நூலகம்
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
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.
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。
two-hybrid assay because it is under the control of 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 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

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 gene's promoter will 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 reporters—ADE2, HIS3, MEL1, and lacZ—under the control of three distinct GAL4 upstream activating sequences (UASs) and TATA boxes . The ADE2 reporter 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 α-
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.
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。
galactosidase. MEL1 is endogenous to both Y187 and AH109. Because αgalactosidase is a secreted enzyme, its activity can be detected by adding X-α-Gal to the selection plate: If MEL1 is active and X-α-Gal is present, the colony will turn blue. lacZ in Y187 exhibits a high level of induced β-galactosidase activity in a positive
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 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
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 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 g al actose in the case of the G AL 1p romoter) .