酵母雙雜交原理與實(shí)驗(yàn)具體流程課件

1、酵母雙雜交原理與實(shí)驗(yàn)具體流程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)行兩個蛋白互作分析，酵母雙雜交系統(tǒng)可進(jìn)行兩個蛋白互作分析，可用一個已知的蛋白因子（在雙雜交系統(tǒng)中稱為誘餌可用一個已知的蛋白因子（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)證兩個蛋白之間的互作。應(yīng)用個蛋白之間的互作。應(yīng)用clontechclontech第三代酵母雙雜交第三代酵母雙雜交系統(tǒng)，并在系統(tǒng)，并在按實(shí)驗(yàn)手冊要求按實(shí)驗(yàn)手冊要求的嚴(yán)格操作下進(jìn)行蛋白互的嚴(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)性與可靠作分析，我們的篩選結(jié)果將具有

2、較好的重復(fù)性與可靠性。性。酵母雙雜交原理與實(shí)驗(yàn)具體流程2 單、雙雜交的方法是基于許多真核生物轉(zhuǎn)錄因子都是以單、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)錄激活域和模塊形式存在的，它們的轉(zhuǎn)錄激活域和dnadna結(jié)合域在結(jié)構(gòu)和功能上結(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)合達(dá)融合蛋白，能立即結(jié)合dnadna靶序列激活下游啟動子的轉(zhuǎn)錄（圖靶序列激活下游啟動子的轉(zhuǎn)錄（圖1 1所所示），示），bd matchmakerbd matchmaker系統(tǒng)應(yīng)用酵母中已經(jīng)研

3、究透徹的轉(zhuǎn)錄因子系統(tǒng)應(yīng)用酵母中已經(jīng)研究透徹的轉(zhuǎn)錄因子gal4gal4的轉(zhuǎn)錄激活域和的轉(zhuǎn)錄激活域和dnadna結(jié)合域來進(jìn)行研究。結(jié)合域來進(jìn)行研究。酵母雙雜交原理與實(shí)驗(yàn)具體流程3單雜與雙雜的異同點(diǎn) 酵母單雙雜，酵母單雙雜，都基于許多都基于許多真核生物轉(zhuǎn)錄因子的轉(zhuǎn)錄激活真核生物轉(zhuǎn)錄因子的轉(zhuǎn)錄激活域和域和dna結(jié)合域在結(jié)構(gòu)和功能結(jié)合域在結(jié)構(gòu)和功能上都有區(qū)別上都有區(qū)別。這就允許研究者。這就允許研究者去構(gòu)建不同的融合基因，當(dāng)在去構(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)酵母中表達(dá)融合蛋白，就能結(jié)合合dna靶序列激活下游啟動子靶序列激活下游啟動子的轉(zhuǎn)錄。單雜交是文庫中的的轉(zhuǎn)錄。單雜交是文庫中的轉(zhuǎn)轉(zhuǎn)錄

4、因子直接與靶序列結(jié)合錄因子直接與靶序列結(jié)合，使使與轉(zhuǎn)錄因子融合的與轉(zhuǎn)錄因子融合的gal4ad激激活報告基因活報告基因his3的轉(zhuǎn)錄，而的轉(zhuǎn)錄，而雙雙雜是借助與誘餌蛋白與文庫中雜是借助與誘餌蛋白與文庫中調(diào)控因子的互作，使得調(diào)控因子的互作，使得gal4bd和和ad通過這個通過這個“橋橋梁梁”共同起作用共同起作用，激活報告基，激活報告基因（因（ade2、his3 、 lacz和和 mel1）的轉(zhuǎn)錄。）的轉(zhuǎn)錄。酵母雙雜交原理與實(shí)驗(yàn)具體流程4酵母雙雜交原理與實(shí)驗(yàn)具體流程5酵母雙雜交原理與實(shí)驗(yàn)具體流程6 推薦使用推薦使用clontechclontech公司的公司的第三代載體，第三代載體，pgadt7-re

5、c pgadt7-rec 和和pgbkt7pgbkt7進(jìn)行雙雜交篩選，因?yàn)樗M(jìn)行雙雜交篩選，因?yàn)樗鼈儺a(chǎn)生更少的假陽性。對于們產(chǎn)生更少的假陽性。對于cdnacdna合成，構(gòu)建一個與合成，構(gòu)建一個與gal4gal4激活域的激活域的融合文庫，在雙雜交中推薦使用融合文庫，在雙雜交中推薦使用pgadt7-recpgadt7-rec，這一克隆是通過體，這一克隆是通過體內(nèi)同源重組來實(shí)現(xiàn)的（圖內(nèi)同源重組來實(shí)現(xiàn)的（圖2 2），），這一步驟是利用酵母中的高效重這一步驟是利用酵母中的高效重組系統(tǒng)使組系統(tǒng)使ds dnads dna與與gal4 adgal4 ad質(zhì)粒質(zhì)粒融合。借助于同源重組克隆，文融合。借助于同源重組

6、克隆，文庫的構(gòu)建和篩選能快速接連地進(jìn)庫的構(gòu)建和篩選能快速接連地進(jìn)行（步驟行（步驟3 3和和4 4），不需任何細(xì)菌），不需任何細(xì)菌轉(zhuǎn)化步驟。用轉(zhuǎn)化步驟。用cdnacdna文庫和文庫和phis2phis2載體進(jìn)行簡單的酵母轉(zhuǎn)化，接著載體進(jìn)行簡單的酵母轉(zhuǎn)化，接著在選擇性培養(yǎng)基上進(jìn)行酵母雙雜在選擇性培養(yǎng)基上進(jìn)行酵母雙雜交的篩選。交的篩選。圖圖2. bd matchmakertm雙雜交文庫構(gòu)建和篩選。上圖所示，借雙雜交文庫構(gòu)建和篩選。上圖所示，借助于重組克隆使文庫構(gòu)建和篩選快速有效助于重組克隆使文庫構(gòu)建和篩選快速有效酵母雙雜交原理與實(shí)驗(yàn)具體流程7酵母雙雜交原理與實(shí)驗(yàn)具體流程8酵母雙雜交原理與實(shí)驗(yàn)具體流程

7、9 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 res

8、pond 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

9、 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 worki

10、ng 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 as

11、sociated with cis-acting elementsdna sequences to which transcription factors and other trans-acting regulatory proteins that bind and affect transcription levels. 酵母雙雜交原理與實(shí)驗(yàn)具體流程10 the term “promoter” usually refers to both the tata box and the the term “promoter” usually refers to both the tata box

12、 and the associated cis-regulatory elements. this usage is especially associated cis-regulatory elements. this usage is especially common when speaking of yeast gene regulation because the cis common when speaking of yeast gene regulation because the cis regulatory elements are relatively closely as

13、sociated with the regulatory elements are relatively closely associated with the tata box (yoccum, 1987). this is in contrast to multicellular tata box (yoccum, 1987). this is in contrast to multicellular eukaryotes, where cisregulatory elements (such as enhancers) can eukaryotes, where cisregulator

14、y elements (such as enhancers) can be found very far upstream or downstream from the promoters they be found very far upstream or downstream from the promoters they regulate. in this text, minimal promoter will refer regulate. in this text, minimal promoter will refer specifically to the tata region

15、, exclusive of other cis-acting specifically to the tata region, exclusive of other cis-acting elements.elements. the minimal promoter (or tata box) in yeast is typically the minimal promoter (or tata box) in yeast is typically approximately 25 bp upstream of the transcription start site. approximat

16、ely 25 bp upstream of the transcription start site. yeast tata boxes are functionally similar to prokaryotic pribnow yeast tata boxes are functionally similar to prokaryotic pribnow boxes, but are not as tightly conserved. furthermore, some yeast boxes, but are not as tightly conserved. furthermore,

17、 some yeast transcription units are preceded by more than one tata box. the transcription units are preceded by more than one tata box. the yeast his3 gene, for example, is preceded by two different tata yeast his3 gene, for example, is preceded by two different tata boxes: tr, which is regulated, a

18、nd tc, which is constitutive.boxes: tr, which is regulated, and tc, which is constitutive.酵母雙雜交原理與實(shí)驗(yàn)具體流程11 uas and tata regions can be switched to create novel promoters uas and tata regions can be switched to create novel promoters for gal4-based systems, either a native gal uas or a synthetic uasg

19、 17- 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 mer consensus sequence (heslot & gaillardin, 1992) provides the binding site for the gal4 dna-bd. if you are putting together your own

20、one- or two-hybrid system, 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 recognized by the you must make sure that the reporter genes promoter will be recognized by the dna-bd moiety encoded in your dna-bd

21、 fusion vector.dna-bd moiety encoded in your dna-bd fusion vector.reporter genes under the control of gal4-responsive elementsreporter genes under the control of gal4-responsive elements ah109 contains four reportersade2, his3, mel1, and laczunder the ah109 contains four reportersade2, his3, mel1, a

22、nd laczunder the control of three distinct gal4 upstream activating sequences (uass) and tata control of three distinct gal4 upstream activating sequences (uass) and tata boxes . the ade2 reporter alone provides strong nutritional selection. for higher boxes . the ade2 reporter alone provides strong

23、 nutritional selection. for higher stringency, and to reduce the incidence of false positives, select for ade2 and 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 his3 (james et al., 1996)

24、. you also have the option of assaying for mel1, which encodes -galactosidase. mel1 is endogenous to both y187 and ah109. because -encodes -galactosidase. mel1 is endogenous to both y187 and ah109. because -galactosidase is a secreted enzyme, its activity can be detected by adding x-galactosidase is

25、 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 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 will

26、 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 the activity in a positive two-hybrid assay because it is under the control of the intact gal1 uas.intact gal1 uas.酵母雙雜交原理與實(shí)驗(yàn)具體流程12酵母雙雜交原理與實(shí)驗(yàn)具體流程13repor

27、ter genes under the control of a reporter genes under the control of a minimal his3 promoterminimal his3 promoter the his3 reporter gene in yeast strain y190 is unusual the his3 reporter gene in yeast strain y190 is unusual among the gal4 two-hybrid reporter gene constructs in that it is among the g

28、al4 two-hybrid reporter gene constructs in that it is under the control of the gal1 uas and a minimal promoter containing under the control of the gal1 uas and a minimal promoter containing both his3 tata boxes the his3 reporter plasmids phisi and phisi-1 both his3 tata boxes the his3 reporter plasm

29、ids phisi and phisi-1 used in the matchmaker one hybrid system also have both of the his3 used in the matchmaker one hybrid system also have both of the his3 tata boxes present in the minimal promoter. by inserting a cis-tata boxes present in the minimal promoter. by inserting a cis-acting element i

30、n the mcs, the regulated tata box (tr) can be acting element in the mcs, the regulated tata box (tr) can be affected, but there is still a significant amount of constitutive, affected, but there is still a significant amount of constitutive, leaky expression due to the his3 tc. the leaky his3 expres

31、sion of leaky expression due to the his3 tc. the leaky his3 expression of these one-hybrid plasmids is first used to help construct his3 these one-hybrid plasmids is first used to help construct his3 reporter strains, and later is controlled by including 3-reporter strains, and later is controlled b

32、y including 3-aminotriazole in the medium to suppress background growth.aminotriazole in the medium to suppress background growth.酵母雙雜交原理與實(shí)驗(yàn)具體流程14promoters used to drive fusion protein expression in two-hybrid cloning vectors酵母雙雜交原理與實(shí)驗(yàn)具體流程15cdna的生成（rt-pcr及race） 一般提取的一般提取的rnarna中，都含有少量的植物中，都含有少量的植物dn

33、adna。在。在rt-pcrrt-pcr時，為避免基因組時，為避免基因組dnadna的存在而造成擴(kuò)增產(chǎn)的存在而造成擴(kuò)增產(chǎn)物的污染問題，需在物的污染問題，需在rnarna溶液中加入少量無溶液中加入少量無rnasernase的的dnasednase，于，于3737消化消化30min30min后，在進(jìn)行反轉(zhuǎn)錄。后，在進(jìn)行反轉(zhuǎn)錄。建議應(yīng)在無菌操作臺上進(jìn)行反轉(zhuǎn)錄體系的配制。建議應(yīng)在無菌操作臺上進(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，那么就需做在實(shí)驗(yàn)中，往往需要獲得基因的全長，那么就需做5race5race（rapid amplification of cdna endrapid amp

34、lification of cdna end）與與3race3race。我們就以。我們就以clontechclontech公司的公司的smartsmart（switching mechanism at 5 end of rna switching mechanism at 5 end of rna transcript)transcript)技術(shù)來探討技術(shù)來探討racerace反應(yīng)。反應(yīng)。 何為何為smartsmart技術(shù)？顧名思義，技術(shù)？顧名思義， smart rnasmart rna轉(zhuǎn)錄轉(zhuǎn)錄55端轉(zhuǎn)換機(jī)制，它實(shí)際上指端轉(zhuǎn)換機(jī)制，它實(shí)際上指rnarna在在mumlvmumlv反轉(zhuǎn)錄反轉(zhuǎn)錄酶的

35、作用下，將酶的作用下，將rnarna反轉(zhuǎn)錄為第一鏈反轉(zhuǎn)錄為第一鏈cdnacdna，引物一般用，引物一般用oligo(dt)oligo(dt)；當(dāng)；當(dāng)mumlvmumlv反轉(zhuǎn)錄酶遇到反轉(zhuǎn)錄酶遇到rnarna的的55端時，它就展示出其末端轉(zhuǎn)移酶活性，一般是在端時，它就展示出其末端轉(zhuǎn)移酶活性，一般是在cdnacdna的的33端加上幾個堿基，首先并且主要是胞嘧端加上幾個堿基，首先并且主要是胞嘧啶啶 （c c），而），而bd smartbd smart引物的引物的33端具有端具有oligo(g),oligo(g),與胞嘧啶互補(bǔ)配對，則創(chuàng)造出一個延伸性的模與胞嘧啶互補(bǔ)配對，則創(chuàng)造出一個延伸性的模板板sma

36、rtsmart引物的引物的55端（如端（如下圖下圖所示）；接著反轉(zhuǎn)錄酶轉(zhuǎn)換模板，也就是以所示）；接著反轉(zhuǎn)錄酶轉(zhuǎn)換模板，也就是以smartsmart引物的引物的55端端序列為模板繼續(xù)合成第一條序列為模板繼續(xù)合成第一條cdnacdna鏈，這樣就使得鏈，這樣就使得cdnacdna具有具有mrnamrna的完整的完整55末端并同時含有末端并同時含有smart smart oligo oligo 的互補(bǔ)序列，然后通過長距離的互補(bǔ)序列，然后通過長距離pcrpcr擴(kuò)增出具有完整擴(kuò)增出具有完整55端的雙鏈端的雙鏈cdnacdna。酵母雙雜交原理與實(shí)驗(yàn)具體流程16反轉(zhuǎn)錄酶可以說是一種特殊的聚合酶，前人研究表明，

37、聚合酶一般在新合成鏈的3端加上同聚尾（幾個相同的堿基），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“a”，而反轉(zhuǎn)錄酶是加上3個 “c” 。對此，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。酵母雙雜交原理與實(shí)驗(yàn)具體流程17ds cdna(0.44kb)電泳圖destroy rna secondary structuresecondary structure not to be formed again酵母雙雜交原理與實(shí)驗(yàn)具體流程18

38、酵母雙雜交原理與實(shí)驗(yàn)具體流程19酵母雙雜交原理與實(shí)驗(yàn)具體流程20screening by cotransformation酵母雙雜交原理與實(shí)驗(yàn)具體流程21酵母雙雜交原理與實(shí)驗(yàn)具體流程22酵母雙雜交原理與實(shí)驗(yàn)具體流程23screening by yeast mating酵母雙雜交原理與實(shí)驗(yàn)具體流程24酵母雙雜交原理與實(shí)驗(yàn)具體流程25y187 suspension 酵母雙雜交原理與實(shí)驗(yàn)具體流程26酵母雙雜交原理與實(shí)驗(yàn)具體流程27酵母雙雜交原理與實(shí)驗(yàn)具體流程28酵母雙雜交原理與實(shí)驗(yàn)具體流程29酵母雙雜交原理與實(shí)驗(yàn)具體流程30 pull-down酵母雙雜交原理與實(shí)驗(yàn)具體流程31troubleshoot

39、ing guide酵母雙雜交原理與實(shí)驗(yàn)具體流程32a.constructing dna-bd fusionsa.constructing dna-bd fusions dna-bd/bait activates reporter genes dna-bd/bait activates reporter genes bait protein is toxic to yeast cells bait protein is toxic to yeast cellsb. generating cdna librariesb. generating cdna libraries low yield of dsdna low yield of dsdna size distribution of dsdna is less than expected size distribution of dsdna is less than expected presence of low molecular weight (0.1kb) in dsdna fragments presence of low molecular weight (0.1kb) in dsdna fragmentsc. constructing &a