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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, lea
18、ky 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.,Promoters used to drive fusion protein expression in two-h
19、ybrid cloning vectors,cDNA的生成（RT-PCR及RACE）,一般提取的RNA中，都含有少量的植物DNA。在RT-PCR时，为避免基因组DNA的存在而造成扩增产物的污染问题，需在RNA溶液中加入少量无RNase的DNase，于37消化30min后，在进行反转录。建议应在无菌操作台上进行反转录体系的配制。在实验中，往往需要获得基因的全长，那么就需做5RACE（Rapid Amplification of cDNA End）与3RACE。我们就以Clontech公司的SMART（Switching Mechanism at 5 end of RNA Transcript)技
20、术来探讨RACE反应。何为SMART技术？顾名思义， SMART RNA转录5端转换机制，它实际上指RNA在MuMLV反转录酶的作用下，将RNA反转录为第一链cDNA，引物一般用Oligo(dT)；当MuMLV反转录酶遇到RNA的5端时，它就展示出其末端转移酶活性，一般是在cDNA的3端加上几个碱基，首先并且主要是胞嘧啶 （C），而BD SMART引物的3端具有Oligo(G),与胞嘧啶互补配对，则创造出一个延伸性的模板SMART引物的5端（如下图所示）；接着反转录酶转换模板，也就是以SMART引物的5端序列为模板继续合成第一条cDNA链，这样就使得cDNA具有mRNA的完整5末端并同时含有S
21、MART Oligo 的互补序列，然后通过长距离PCR扩增出具有完整5端的双链cDNA。,反转录酶可以说是一种特殊的聚合酶，前人研究表明，聚合酶一般在新合成链的3端加上同聚尾（几个相同的碱基），不同种类的聚合酶所加的碱基种类和数目不一样，如Taq酶一般在扩增片段的3端加上一个“A”，而反转录酶是加上3个 “C” 。对此，研究人员巧妙地应用了反转录酶的天然活性，并精心设计了SMART Oligo和CDS引物，开发出SMART技术。当前，SMART技术在新基因全长获得的实验中具有广泛地应用。,ds cDNA(0.44kb)电泳图,Destroy RNA secondary structure,se
22、condary structure not to be formed again,Screening by cotransformation,Screening By Yeast Mating,Y187 suspension,Pull-down,Troubleshooting Guide,A.Constructing DNA-BD fusions DNA-BD/bait activates reporter genes Bait protein is toxic to yeast cellsB. Generating cDNA libraries Low yield of dsDNA Size
23、 distribution of dsDNA is less than expected Presence of low molecular weight (0.1kb) in dsDNA fragmentsC. Constructing & Screening Y2H Low transformation efficiency Low mating efficiency Excessive background growth on library screening medium Failure to detect known protein-protein interaction,酵母单杂
24、交的实验过程（附）,报告载体的构建（pHIS2/target DNA),插入片段为60bp左右,报告载体酶切鉴定图,感病组织RNA的提取,通过SMART（Switching Mechanism at the 5 end of the RNA Transcript) 技术进行 cDNA的合成，通过LD-PCR扩增ds cDNA,取1g 总RNA,具有接头（SMART III & CDSIII)的ds cDNA,总RNA电泳图,ds cDNA(0.44kb)电泳图,酵母感受态的制备,在含有不同浓度3-AT的营养缺陷型培养基（SD/-His/-Trp)上筛选，以确定抑制背景的适当3-AT浓度,报告载体转化酵母Y187,将1：100与1：1000稀释液分别铺在营养缺陷型SD/-Trp 及SD/-Trp/-Leu的平板上以确定感受效率,以下三个培养皿从左到右3-AT的浓度分别为20mM、40mM、60mM；可以说背景用60mM的3-AT抑制不住。这说明插入报告载体的元件可能与酵母的内源蛋白互作，则这个元件不宜用酵母单杂交来分析。,1.SD/-Trp: 3.4106 cfu/5g pHIS2/target DNA2.SD/-Leu: 3.6106 cfu/3g pGADT7-Rec2 DNA3. SD/-Leu/-Trp: 8.0105 个/文库,
