酵母双杂交原理与实验具体流程.

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-actingelements—DNAsequencestowhichtranscriptionfactorsandothertrans-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,youmustmakesurethatthereportergene'spromoterwillberecognizedbytheDNA-BDmoietyencodedinyourDNA-BDfusionvector.ReportergenesunderthecontrolofGAL4-responsiveelementsAH109containsfourreporters—ADE2,HIS3,MEL1,andlacZ—underthecontrolofthreedistinctGAL4upstreamactivatingsequences(UASs)andTATAboxes.TheADE2reporteraloneprovidesstrongnutritionalselection.Forhigherstringency,andtoreducetheincidenceoffalsepositives,selectforADE2andHIS3(Jamesetal.,1996).YoualsohavetheoptionofassayingforMEL1,whichencodesα-galactosidase.MEL1isendogenoustobothY187andAH109.Becauseα-galactosidaseisasecretedenzyme,itsactivitycanbedetectedbyaddingX-α-Galtotheselectionplate:IfMEL1isactiveandX-α-Galispresent,thecolonywillturnblue.lacZinY187exhibitsahighlevelofinducedβ-galactosidaseactivityinapositivetwo-hybridassaybecauseitisunderthecontroloftheintactGAL1UAS.ReportergenesunderthecontrolofaminimalHIS3promoterTheHIS3reportergeneinyeaststrainY190isunusualamongtheGAL4two-hybridreportergeneconstructsinthatitisunderthecontroloftheGAL1UASandaminimalpromotercontainingbothHIS3TATAboxesTheHIS3reporterplasmidspHISiandpHISi-1usedintheMATCHMAKEROneHybridSystemalsohavebothoftheHIS3TATAboxespresentintheminimalpromoter.Byinsertingacis-actingelementintheMCS,theregulatedTATAbox(TR)canbeaffected,butthereisstillasignificantamountofconstitutive,leakyexpressionduetotheHIS3TC.TheleakyHIS3expressionoftheseone-hybridplasmidsisfirstusedtohelpconstructHIS3reporterstrains,andlateriscontrolledbyincluding3-aminotriazoleinthemediumtosuppressbackgroundgrowth.Promotersusedtodrivefusionproteinexpressionintwo-hybridcloningvectorscDNA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DNaseⅠ，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5‘RACE（RapidAmplificationofcDNAEnd）与3’RACE。我们就以Clontech公司的SMART（SwitchingMechanismat5’endofRNATranscript)技术来探讨RACE反应。何