酵母雙雜交原理與實(shí)驗(yàn)具體流程ppt課件

1、酵母雙雜交系統(tǒng)原理及具體操作流程,酵母雙雜交系統(tǒng)可進(jìn)行兩個(gè)蛋白互作分析，可用一個(gè)已知的蛋白因子（在雙雜交系統(tǒng)中稱為誘餌蛋白）去釣取與其結(jié)合的蛋白；也可用進(jìn)一步驗(yàn)證兩個(gè)蛋白之間的互作。應(yīng)用Clontech第三代酵母雙雜交系統(tǒng)，并在按實(shí)驗(yàn)手冊(cè)要求的嚴(yán)格操作下進(jìn)行蛋白互作分析，我們的篩選結(jié)果將具有較好的重復(fù)性與可靠性,單、雙雜交的方法是基于許多真核生物轉(zhuǎn)錄因子都是以模塊形式存在的，它們的轉(zhuǎn)錄激活域和DNA結(jié)合域在結(jié)構(gòu)和功能上都有區(qū)別。這就允許研究者去構(gòu)建不同的融合基因，當(dāng)在酵母中表達(dá)融合蛋白，能立即結(jié)合DNA靶序列激活下游啟動(dòng)子的轉(zhuǎn)錄（圖1所示），BD Matchmaker系統(tǒng)應(yīng)用酵母中已經(jīng)研究透徹

2、的轉(zhuǎn)錄因子GAL4的轉(zhuǎn)錄激活域和DNA結(jié)合域來進(jìn)行研究,單雜與雙雜的異同點(diǎn),酵母單雙雜，都基于許多真核生物轉(zhuǎn)錄因子的轉(zhuǎn)錄激活域和DNA結(jié)合域在結(jié)構(gòu)和功能上都有區(qū)別。這就允許研究者去構(gòu)建不同的融合基因，當(dāng)在酵母中表達(dá)融合蛋白，就能結(jié)合DNA靶序列激活下游啟動(dòng)子的轉(zhuǎn)錄。單雜交是文庫中的轉(zhuǎn)錄因子直接與靶序列結(jié)合，使與轉(zhuǎn)錄因子融合的GAL4AD激活報(bào)告基因HIS3的轉(zhuǎn)錄，而雙雜是借助與誘餌蛋白與文庫中調(diào)控因子的互作，使得GAL4BD和AD通過這個(gè)“橋梁”共同起作用，激活報(bào)告基因（ADE2、HIS3 、 lacZ和 MEL1）的轉(zhuǎn)錄,推薦使用Clontech公司的第三代載體，pGADT7-Rec 和pG

3、BKT7進(jìn)行雙雜交篩選，因?yàn)樗鼈儺a(chǎn)生更少的假陽性。對(duì)于cDNA合成，構(gòu)建一個(gè)與GAL4激活域的融合文庫，在雙雜交中推薦使用pGADT7-Rec，這一克隆是通過體內(nèi)同源重組來實(shí)現(xiàn)的（圖2），這一步驟是利用酵母中的高效重組系統(tǒng)使ds DNA與GAL4 AD質(zhì)粒融合。借助于同源重組克隆，文庫的構(gòu)建和篩選能快速接連地進(jìn)行（步驟3和4），不需任何細(xì)菌轉(zhuǎn)化步驟。用cDNA文庫和pHIS2載體進(jìn)行簡單的酵母轉(zhuǎn)化，接著在選擇性培養(yǎng)基上進(jìn)行酵母雙雜交的篩選,圖2. BD MatchmakerTM雙雜交文庫構(gòu)建和篩選。上圖所示，借助于重組克隆使文庫構(gòu)建和篩選快速有效,Yeast promoters and oth

4、er 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 d

5、omain 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 a

6、bility 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

7、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

8、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 gene re

9、gulation 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 te

10、xt, 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

11、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 prom

12、oters 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 be r

13、ecognized 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 reporter al

14、one 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 -galactosid

15、ase 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 of th

16、e 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 repo

17、rter 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 ex

18、pression 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-hybrid

19、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時(shí)，為避免基因組DNA的存在而造成擴(kuò)增產(chǎn)物的污染問題，需在RNA溶液中加入少量無RNase的DNase，于37消化30min后，在進(jìn)行反轉(zhuǎn)錄。建議應(yīng)在無菌操作臺(tái)上進(jìn)行反轉(zhuǎn)錄體系的配制。 在實(shí)驗(yàn)中，往往需要獲得基因的全長，那么就需做5RACE（Rapid Amplification of cDNA End）與3RACE。我們就以Clontech公司的SMART（Switching Mechanism at 5 end of RNA Transcript)技術(shù)來探討RA

20、CE反應(yīng)。 何為SMART技術(shù)？顧名思義， SMART RNA轉(zhuǎn)錄5端轉(zhuǎn)換機(jī)制，它實(shí)際上指RNA在MuMLV反轉(zhuǎn)錄酶的作用下，將RNA反轉(zhuǎn)錄為第一鏈cDNA，引物一般用Oligo(dT)；當(dāng)MuMLV反轉(zhuǎn)錄酶遇到RNA的5端時(shí)，它就展示出其末端轉(zhuǎn)移酶活性，一般是在cDNA的3端加上幾個(gè)堿基，首先并且主要是胞嘧啶 （C），而BD SMART引物的3端具有Oligo(G),與胞嘧啶互補(bǔ)配對(duì)，則創(chuàng)造出一個(gè)延伸性的模板SMART引物的5端（如下圖所示）；接著反轉(zhuǎn)錄酶轉(zhuǎn)換模板，也就是以SMART引物的5端序列為模板繼續(xù)合成第一條cDNA鏈，這樣就使得cDNA具有mRNA的完整5末端并同時(shí)含有SMART

21、Oligo 的互補(bǔ)序列，然后通過長距離PCR擴(kuò)增出具有完整5端的雙鏈cDNA,反轉(zhuǎn)錄酶可以說是一種特殊的聚合酶，前人研究表明，聚合酶一般在新合成鏈的3端加上同聚尾（幾個(gè)相同的堿基），不同種類的聚合酶所加的堿基種類和數(shù)目不一樣，如Taq酶一般在擴(kuò)增片段的3端加上一個(gè)“A”，而反轉(zhuǎn)錄酶是加上3個(gè) “C” 。對(duì)此，研究人員巧妙地應(yīng)用了反轉(zhuǎn)錄酶的天然活性，并精心設(shè)計(jì)了SMART Oligo和CDS引物，開發(fā)出SMART技術(shù)。當(dāng)前，SMART技術(shù)在新基因全長獲得的實(shí)驗(yàn)中具有廣泛地應(yīng)用,ds cDNA(0.44kb)電泳圖,Destroy RNA secondary structure,secondary

22、 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 cells B. Generating cDNA libraries Low yield of dsDNA Size distribution of dsDNA is less than expected Presence of low molecular weight (0.1kb) in dsDNA fragments C. Constructing & Screeni