启动子活性分析

启动子活性分析基因表达调控的研究策略和常用方法2015-9-22基因表达调控是多层次的复杂过程启动子（Promoters）是位于结构基因5‘端上游的DNA序列，其长度从100bp到200bp不等，与转录起始时RNA聚合酶识别、结合和启动转录有关。启动子控制基因转录的起始时间和表达的程度。启动子的组成promoter普遍转录因子启动子活性分析ReporterAssayEMSAChIPReporterAssaypromoterstructuralgenepromoterreportergene报告基因(reporter)是一种编码可被检测的蛋白质或酶的基因。（1）已被克隆和全序列已测定；（2）表达产物在受体细胞中本不存在，即无背景，在被转染的细胞中无相似的内源性表达产物；（3）其表达产物能进行定量测定。（一）氯霉素乙酰转移酶基因（CAT）可催化乙酰CoA的乙酰基转移到氯霉素3羟基，而使氯霉素解毒。线性范围较窄，灵敏性较低（二）β半乳糖苷酶由大肠杆菌lacZ基因编码，可催化半乳糖苷水解。（三）荧光素酶（Luciferase）是自然界中能够产生生物荧光的酶的统称。荧光的产生是来自于荧光素（底物）的氧化，哺乳细胞无内源性荧光素酶。最常用的荧光素酶有细菌荧光素酶、萤火虫荧光素酶和Renilla荧光素酶。萤火虫荧光素酶灵敏度高，检测线性范围宽，是最常用于哺乳细胞的报告基因。（四）荧光蛋白家族：GFP,BFP,RFPpromoterluc荧光素酶报告基因检测缺失（deletion）突变（mutation）荧光素酶报告基因检测GelShiftAssayElectrophoreticalMobilityShiftAssayEMSABandShiftAssay1.Introduction2.Principle3.Application4.Materials5.Procedure6.ProofofSpecificityContentsRegulatoryRegionCodingSequenceGeneRegulationbyTranscriptionFactorsIntroductionDNA-proteincomplexesmigrateslowerthannon-boundDNAinanativepolyacrylamideoragarosegel,resultingina“shift”inmigrationofthelabeledDNAband.PrincipleR.Voll09/01Adouble-strandedoligonucleotidecontainigaNF-B-bindingsiteislabeledwitharadioactiveisotopeandincubatedwithdifferentnuclearextract.Duringgel-electrophoresis,NF-Bboundtotheoligonucleotidecausesashiftcomparedtothefreeprobe.NF-BFreeProbeRadioactivelylabeledoligonucleotidewithNF-B-bindingsite(probe)andboundNF-BcomplexesRadioactivelylabeledoligonucleotidewithNF-B-bindingsite(probe)Nuclearextractofnon-activatedcellsNuclearextractofactivatedcellsBiotin-labeledDNABiotin-labeledDNA+ProteinextractBiotin-labeledDNA+Proteinextract+200-foldmolarexcessofunlabeledDNAApplicationDetectionofDNA-bindingfactors/proteins1.AnalysisofDNAsequences(e.g.promoterorenhancerregions)fortheirpotentialtobindspecificallytoproteins/nuclearextracts2.Analysisof(sub-)cellularextractsforthepresenceofcertainDNA-bindingproteins(e.g.atranscriptionfactorwithaknownrecognitionsequence)Materials3'or5'end-labeledDNAtarget.ProteinextractunlabeledDNAtargetPolyacrylamidegelin0.5XTBEElectrophoresisapparatusPositivelychargednylonmembraneElectroblottertransferapparatusUVlampX-rayfilmPreparetheProbeR.Voll09/01AP-1biotin-5’-GCTTGATGACTCAGCCGGAAC-3’3’-CGAACTACTGAGTCGGCCTTG-5’-biotinNF-Bbiotin-5’-AGTTGAGGGGACTTTCCCAGGC-3’3’-TCAACTCCCCTCAAAGGGTCCG-5’-biotinBindingmotif1.Synthesizecomplementarysinglestrandedbiotin-labeledandunlabeledoligonucleotidescontainingatranscriptionfactorbindingsite.2.AnnealingtheOligos：Heatupanequimolarmixtureofthe2oligosto95°Candletthemslowlycooldownbyturningofftheheatblock.PlanBindingReactionsR.Voll09/01Procedure1.Preparenuclearproteinextracts2.PrepareBiotin-labeledandunlabeledtargetDNA3.PlanBindingReactions4.PrepareandPre-RunGel5.PrepareandPerformBindingReactions6.ElectrophoreseBindingReactions7.ElectrophoreticTransferofBindingReactionstoNylonMembrane8.Cross-linkTransferredDNAtoMembrane9.DetectBiotin-labeledDNAbyChemiluminescenceProofofSpecificityR.Voll09/011.SupershiftusingantibodiesagainsttheDNA-bindingprotein(proteinSpecificity)2.Competitionforbindingtothelabeledprobeusingunlabeledwildtypeandmutatedoligos(DNASpecificity)R.Voll09/01SupershiftAdouble-strandedoligonucleotidecontainigaNF-B-bindingsiteislabelledradioactiveandincubatedwithanuclearextract.Duringgel-electrophoresis,NF-Bboundtotheoligonucleotidecausesashiftcomparedtothefreeprobe.RadioaktivlabelledoligonucleotidewithNF-B-bindingsite(probe)andboundNF-BRadioactivlabelledoligonucleotidewithNF-B-bindingsite(probe)NuclearextractofactivatedcellsNuclearextractofactivatedcellswithanti-p50antibodyp50/p65Freeprobep50/p65+anti-p50R.Voll09/01CompetitionwithUnlabeledOligosIncreasingamountsofunlabeledoligoscontainingtheNF-Bbindingsiteorunlabeledoligoswithamutatedbindingsitewereaddedtothereactionmixpriortogelelectrophoresis.Specificbindingisextinguishedonlybythenon-mutatedoligo.Freeprobep50/p65WildtypeoligoMutatedoligoGGGGACTTTCCCGGAGACTTTCCCChIPChromatinImmunoprecipitationChromatinimmunoprecipitationUsedtodeterminewhetheragivenproteinbindstoagivenDNAsequenceinvivoLikeallproteinanalysisinvolvingantibodies(includingwesterns)aspecificantibodyisrequiredIfthereisnospecificantibody,thenepitopetaggingcanbeemployed(FLAG,MYC,HIS)Procedure启动子活性分析ReporterAssayEMSAChIP