啟動(dòng)子活性分析

啟動(dòng)子活性分析基因表達(dá)調(diào)控的研究策略和常用方法2015-9-22基因表達(dá)調(diào)控是多層次的復(fù)雜過(guò)程啟動(dòng)子（Promoters）是位于結(jié)構(gòu)基因5‘端上游的DNA序列，其長(zhǎng)度從100bp到200bp不等，與轉(zhuǎn)錄起始時(shí)RNA聚合酶識(shí)別、結(jié)合和啟動(dòng)轉(zhuǎn)錄有關(guān)。啟動(dòng)子控制基因轉(zhuǎn)錄的起始時(shí)間和表達(dá)的程度。啟動(dòng)子的組成promoter普遍轉(zhuǎn)錄因子啟動(dòng)子活性分析ReporterAssayEMSA

ChIPReporterAssaypromoterstructuralgenepromoterreportergene報(bào)告基因(reporter)是一種編碼可被檢測(cè)的蛋白質(zhì)或酶的基因。（1）已被克隆和全序列已測(cè)定；（2）表達(dá)產(chǎn)物在受體細(xì)胞中本不存在，即無(wú)背景，在被轉(zhuǎn)染的細(xì)胞中無(wú)相似的內(nèi)源性表達(dá)產(chǎn)物；（3）其表達(dá)產(chǎn)物能進(jìn)行定量測(cè)定。（一）氯霉素乙酰轉(zhuǎn)移酶基因（CAT）可催化乙酰CoA的乙?；D(zhuǎn)移到氯霉素3羥基，而使氯霉素解毒。線性范圍較窄，靈敏性較低

（二）β半乳糖苷酶由大腸桿菌lacZ基因編碼，可催化半乳糖苷水解。（三）熒光素酶（Luciferase）是自然界中能夠產(chǎn)生生物熒光的酶的統(tǒng)稱。熒光的產(chǎn)生是來(lái)自于熒光素（底物）的氧化，哺乳細(xì)胞無(wú)內(nèi)源性熒光素酶。最常用的熒光素酶有細(xì)菌熒光素酶、螢火蟲熒光素酶和Renilla熒光素酶。螢火蟲熒光素酶靈敏度高，檢測(cè)線性范圍寬，是最常用于哺乳細(xì)胞的報(bào)告基因。（四）熒光蛋白家族：GFP,BFP,RFPpromoterluc熒光素酶報(bào)告基因檢測(cè)缺失（deletion）突變（mutation）熒光素酶報(bào)告基因檢測(cè)GelShiftAssayElectrophoreticalMobilityShiftAssayEMSABandShiftAssay1.Introduction2.Principle3.Application4.Materials5.Procedure6.ProofofSpecificityContentsRegulatoryRegionCodingSequenceGeneRegulationbyTranscriptionFactorsIntroductionDNA-proteincomplexesmigrateslowerthannon-boundDNAinanativepolyacrylamideoragarosegel,resultingina“shift”inmigrationofthelabeledDNAband.PrincipleR.Voll09/01Adouble-strandedoligonucleotidecontainigaNF-B-bindingsiteislabeled

witharadioactiveisotopeandincubatedwithdifferentnuclearextract.Duringgel-electrophoresis,NF-Bboundtotheoligonucleotidecausesashiftcomparedtothefreeprobe.NF-

BFreeProbeRadioactivelylabeledoligonucleotidewithNF-B-bindingsite(probe)andboundNF-BcomplexesRadioactivelylabeledoligonucleotidewithNF-B-bindingsite(probe)Nuclearextract

ofnon-activated

cellsNuclearextractofactivatedcellsBiotin-labeledDNABiotin-labeledDNA+ProteinextractBiotin-labeledDNA+Proteinextract+200-foldmolarexcessofunlabeledDNAApplicationDetectionofDNA-bindingfactors/proteins1.AnalysisofDNAsequences(e.g.promoteror

enhancerregions)fortheirpotentialtobind

specificallytoproteins/nuclearextracts2.Analysisof(sub-)cellularextractsforthepresence

ofcertainDNA-bindingproteins(e.g.atranscription

factorwithaknownrecognitionsequence)Materials3'or5'end-labeledDNAtarget.ProteinextractunlabeledDNAtargetPolyacrylamidegelin0.5XTBEElectrophoresisapparatusPositivelychargednylonmembraneElectroblottertransferapparatusUVlampX-rayfilmPreparetheProbeR.Voll09/01AP-1biotin-5’-GCTTGATGACTCAGCCGGAAC-3’3’-CGAACTACTGAGTCGGCCTTG-5’-biotinNF-kBbiotin-5’-AGTTGAGGGGACTTTCCCAGGC-3’3’-TCAACTCCCCTCAAAGGGTCCG-5’-biotinBindingmotif1.Synthesizecomplementarysinglestrandedbiotin-labeledandunlabeledoligonucleotidescontainingatranscriptionfactorbindingsite.2.AnnealingtheOligos