酵母双杂交原理与实验具体流程.ppt

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），BDMatchmaker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。,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dsDNA与GAL4AD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.BDMatchmakerTM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,Yeastpromotersandothercis-actingregulatoryelementsplayacrucialroleinyeast-basedexpressionsystemsandtranscriptionalassayssuchastheMATCHMAKEROne-andTwo-HybridSystems.DifferencesinthepromoterregionofreportergeneconstructscansignificantlyaffecttheirabilitytorespondtotheDNA-bindingdomainofspecifictranscriptionalactivators;promoterconstructsalsoaffectthelevelofbackground(orleakiness)ofgeneexpressionandthelevelofinducedexpression.Furthermore,differencesincloningvectorpromotersdeterminethelevelofproteinexpressionand,insomecases,confertheabilitytoberegulatedbyanutrient(suchasgalactoseinthecaseoftheGAL1promoter).UASandTATAregionsarebasicbuildingblocksofyeastpromotersTheinitiationofgenetranscriptioninyeast,asinotherorganisms,isachievedbyseveralmolecularmechanismsworkinginconcert.Allyeaststructuralgenes(i.e.,thosetranscribedbyRNApolymeraseII)areprecededbyaregioncontainingalooselyconservedsequence(TATAbox)thatdeterminesthetranscriptionstartsiteandisalsoaprimarydeterminantofthebasaltranscriptionlevel.Manygenesarealsoassociatedwithcis-actingelementsDNAsequencestowhichtranscriptionfactorsandothertrans-actingregulatoryproteinsthatbindandaffecttranscriptionlevels.,Theterm“promoter”usuallyreferstoboththeTATAboxandtheassociatedcis-regulatoryelements.ThisusageisespeciallycommonwhenspeakingofyeastgeneregulationbecausethecisregulatoryelementsarerelativelycloselyassociatedwiththeTATAbox(Yoccum,1987).Thisisincontrasttomulticellulareukaryotes,wherecisregulatoryelements(suchasenhancers)canbefoundveryfarupstreamordownstreamfromthepromoterstheyregulate.Inthistext,minimalpromoterwillreferspecificallytotheTATAregion,exclusiveofothercis-actingelements.Theminimalpromoter(orTATAbox)inyeastistypicallyapproximately25bpupstreamofthetranscriptionstartsite.YeastTATAboxesarefunctionallysimilartoprokaryoticPribnowboxes,butarenotastightlyconserved.Furthermore,someyeasttranscriptionunitsareprecededbymorethanoneTATAbox.TheyeastHIS3gene,forexample,isprecededbytwodifferentTATAboxes:TR,whichisregulated,andTC,whichisconstitutive.,UASandTATAregionscanbeswitchedtocreatenovelpromotersForGAL4-basedsystems,eitheranativeGALUASorasyntheticUASG17-merconsensussequence(Heslot&Gaillardin,1992)providesthebindingsitefortheGAL4DNA-BD.Ifyouareputtingtogetheryourownone-ortwo-hybridsystem,youmustmakesurethatthereportergenespromoterwillberecognizedbytheDNA-BDmoietyencodedinyourDNA-BDfusionvector.ReportergenesunderthecontrolofGAL4-responsiveelementsAH109containsfourreportersADE2,HIS3,MEL1,andlacZunderthecontrolofthreedistinctGAL4upstreamactivatingsequences(UASs)andTATAboxes.TheADE2reporteraloneprovidesstrongnutritionalselection.Forhigherstringency,andtoreducetheincidenceoffalsepositives,selectforADE2andHIS3(Jamesetal.,1996).YoualsohavetheoptionofassayingforMEL1,whichencodes-galactosidase.MEL1isendogenoustobothY187andAH109.Because-galactosidaseisasecretedenzyme,itsactivitycanbedetectedbyaddingX-Galtotheselectionplate:IfMEL1isactiveandX-Galispresent,thecolonywillturnblue.lacZinY187exhibitsahighlevelofinduced-galactosidaseactivityinapositivetwo-hybridassaybecauseitisunderthecontroloftheintactGAL1UAS.,ReportergenesunderthecontrolofaminimalHIS3promoterTheHIS3reportergeneinyeaststrainY190isunusualamongtheGAL4two-hybridreportergeneconstructsinthatitisunderthecontroloftheGAL1UASandaminimalpromotercontainingbothHIS3TATAboxesTheHIS3reporterplasmidspHISiandpHISi-1usedintheMATCHMAKEROneHybridSystemalsohavebothoftheHIS3TATAboxespresentintheminimalpromoter.Byinsertingacis-actingelementintheMCS,theregulatedTATAbox(TR)canbeaffected,butthereisstillasignificantamountofconstitutive,leakyexpressionduetotheHIS3TC.TheleakyHIS3expressionoftheseone-hybridplasmidsisfirstusedtohelpconstructHIS3reporterstrains,andlateriscontrolledbyincluding3-aminotriazoleinthemediumtosuppressbackgroundgrowth.,Promotersusedtodrivefusionproteinexpressionintwo-hybridcloningvectors,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（RapidAmplificationofcDNAEnd）与3RACE。我们就以Clontech公司的SMART（SwitchingMechanismat5endofRNATranscript)技术来探讨RACE反应。何为SMART技术？顾名思义，SMARTRNA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BDSMART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SMARTOligo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SMARTOligo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。,dscDNA(0.44kb)电泳图,DestroyRNAsecondarystructure,secondarystructurenottobeformedagain,Screeningbycotransformation,ScreeningByYeastMating,Y187suspension,Pull-down,TroubleshootingGuide,A.ConstructingDNA-BDfusionsDNA-BD/baitactivatesreportergenesBaitproteinistoxictoyeastcellsB.GeneratingcDNAlibrariesLowyieldofdsDNASizedistributionofdsDNAislessthanexpectedPresenceoflowmolecularweight(0.1kb)indsDNAfragmentsC.Constructing&ScreeningY2HLowtransformationefficiencyL