酵母单杂交的原理与应用实例

2001,Vol.21,No.4(100084),,,DREB,DNA(),(),,,,(YeastOne2hybridmethod)DNA()DNA(cDNA)[1](invivo)1,(minimalpromoter,Pmin),PmincDNA,cDNA,(),Pmin,,WangReed,OLF21cDNA[2],,REST[3],Dc3ABAbZIP[4],1,Pmin,3,Yamaguchi2Shinozaki9,RD(re2sponsivetodehydration)rd29A,,,rd29A,DRE(dehydration2respon2siveelement)[5],TACCGACATLiuDRE,DREDREB[6]DREB(cDNA),cDNA,1cDNAAD(cDNAADfusionli2brary)75cDNA,cDNAADAD(activationdomain),GAL4cDNAADpGAD426(Clontech)pAD2GAL4phagemidVector(HybridZAPTM,STRATA2GENE),,,mRNAmRNA,,,mRNAcDNA,(adaptor),GAL4AD(2A),(packaging),XL21(invivoexcision),cDNA2(Yeastreporter),,(Pmin),,22Pmin222cDNAAD2.1(),Pmin332B4DRE:DNA,Hind,PCR,rd29ADRE75bpDNA(50bp,)Hind75bpDNA,HindDNA(DRE):52AGCT2TACATCAGTTTGAAAGAAAAGGGAAAAAAAGAAAAAAT2AAATAAAAGATATACTACCGACATGAGTTCCAAAAA2GCA23T4DNA,DRE,2DRE2DRE2,DRE2DRE2DRE2DRE2DRE2DRE2DRE2DRE2DRE2(2%)DREpBluescriptSK+Hind,4DRE53,EcoRHinc4DREpBluescriptSK+,DNA:52GAATTC2DRE2DRE2DRE2DRE2GTCGAC23(5EcoR,3Hinc)2.2,pHISi2185pLacZi(MATCHMAKEROne2HybridSystem,Clontech)pHISi21PminHIS3HIS3,PminHIS3,,PminHIS3,HIS,SD(SDPHis-),32AT(32aminotri2azole,)pLacZiPCYC1LacZ,PCYC1,,PCYC1,LacZ,ZPX2gal,,pLacZiURA3,,(SDPUra-)4DRE52GAATTC2DRE2DRE2DRE2DRE2GTCGAC23pHISi21PminHIS3pLacZiPCYC1,XhoNcopHISi21pLacZipHISi21(YM4271,ClontechOne2HybridSystem),SDPHis-(Yeasttrans2formant),4DREpLacZiSDPHis-,Ura-,2BpHISi21pLac2Zi,One2HybridSystem(Clontech)3cDNApHISi21pLacZicDNAAD,cDNAAD,cDNA(sence)(antisence),mRNA,5,cDNAAD,cDNAcDNA,cDNAAD10,1,601,mRNA(),mRNA,,cDNAmRNA1020,60,1020HIS3URA3(2B),cDNAADpAD2GAL4LEU2(2A),,SD(SDPHis-,Ura-,Leu-)(2C)PminHIS3PCYC1,10mmolPL3AT,cDNAADOne2HybridSystem(Clontech),cDNAAD,,(2BDRE),PminHIS3PCYC1,HIS3LacZ,10mmolPL3ATSDPHis-,Ura-,Leu-(3),562mm3SDPHis-,Ura-,Leu-43AT,200lTE,6095mmolPL3ATSDPHis-,Ura-,Leu-,60mmolPL3ATSDPHis-,Ura-,Leu-,10,X2galZ,3030,,5cDNA,,GAL4(activationdomain,AD)cDNAPmin,ADcDNAGAL4AD,cDNA,cDNA,(invivoanalysis)44ADYEP2GAPDRE(MDRE)cDNA2ApAD2GAL4AD,ADYEP2GAP,cDNA(4A)AD,cDNADRE,HIS3LacZ,cDNA,cDNA,PCRDRETAC2CGACATTATTTTCAT(MDRE),2.1,3MDRE(4B),cDNADREDREB,cDNAAD7(No.1,No.18,No.63,No.75,No.104,No.125,No.127)YEP2GAP,10mmolPL3ATSDPHis-,Ura-,Leu-,118,ZPX2gal(5A)GAL4AD,118cDNADRE,HIS3LacZ5B11810mmolPL3ATSDPHis-,Ura-,Leu-,118(W1W18),(M1M18),,(5B)118cDNADRE,DRE,cDNA065([6])6cDNASTRATAGENEcD2NA(YeastcDNAisolationSystem)cDNA,PCR(cDNA),cDNA,(pBlue2scriptSK+),OLF21DREB,DNA,ETSSpETS4[7],MRE(metalresponseelement)[8],BOBF21[9],HMG2like[10],BBob1[11]IgHBZLF[12],RNA[13];,P2DNA2[14];RNA2[15]90,(G1999011703)(39970166)[1]LiJ.J.andHerskowitzI.,Science,1993,5141:1870-1874.[2]WangM.N.andReedR.R.,Nature,1993,6433:121-126.[3]ChongJ.A.,Tapia-RamirezJ.,KimS.,etal.,Cell,1995,3:947-957.[4]KimS.Y.,ChungH-J.,andThomasT.L.,PlantJournal,1997,6:1237-1251.[5]Yamaguchi-ShinozakiK.andShinozakiK.,PlantCell,1994,6:251-264.[6]LiuQ.,KasugaM.,SakumaY.,etal.,PlantCell,1998,10:1391-1406.[7]WeiZ.,AngererR.C.,andAngerer,L.M.,MolecularandCellularBiology,1999,2:1271-1278.[8]InouyeC.,RemondelliP.,KarinM.,etal.,DNAandCellBiology,1994,7:731-742.[9]StrubinM.,NewellJ.W.,andMatthiasP.,Cell,1995,3:497-506.[10]LehmingN.,ThanosD.,Brickman.J.M.,etal.,Nature,1994,6493:175-178.[11]GstaigerM.,KnoepfelL.,GeorgievO.,etal.,Nature,1995,6512:360-362[12]GstaigerM.,HovensC.,GeorgievO.,etal.,Biol.Chem.,1996,10:669-673.[13]KlagesN.,StrubinM.,Nature,1995,6525:822-823.[14]VidalM.,BrachmannR.K.,FattaeyA.,etal.,ProcNatAcadSci16USA,1996,19:10315-10320[15]WilhelmJ.E.andValeR.D.,GenestoCells,1996,3:317-323TheMechanismandApplicationofYeastOne2hybridSystemChenFengLiJieZhangGui2youLiuQiang(DepartmentofBiologicalSciencesandBiotechnology,TsinghuaUniversity,Beijing100084)AbstractTheinducedexpressionsofmanygenesarecontrolledbyspecifictranscriptionfactorsandcis2actingele2ments.Oneofthekeystounderstandthemolecularmechanismofgeneexpressionandthepathwayofsignaltransductionistocloneandidentifythetranscriptionfactors.Yeastone2hybridsystemisanewmethodforisolationandidentificationofcDNAclonesencodingspecificDNA2bindingproteins.HeretheprocedureofscreeningDREBtranscriptionfactorbyyeastone2hybridsystemwasdescribedtointroducethisapproach.KeywordsYeastone2hybridsystem,Geneexpression,Cis2actingelement,Transcriptionfactor(65)ConstructionofGeneticallyEngineeredAntibodyFusionProteinsZhangXue2hongZhangWei2jie(CollegeofLifeScienceandBiotechnology,ShanghaiJiaotongUniversity,Shanghai,200030)AbstractAntibodyfusionproteinscanretainbiologicalfunctionofantibodyandfunctionalcharacterofproteinsfusedtotheantibody,canbeusedwidelyinimmuno2therapy,immuno2diagnosis,purificationofantibodyandanalysisofantibodyorantigen.Antibodyfusionproteincanbeparticularlyusedtopreparetheimmuno2directeddrug.Geneticallyen2gineeredantibodyfusionproteinshavemoreadvantagesovertraditionalchemicalcrosslinkingantibodyfusionproteins.Heretheconstructionandcharacterizationofgeneticallyengineeredantibodyfusionproteinswerereviewed.KeywordsAntibodyfusionproteinAntibodyGeneticengineeringConstructionISBN7253522268127P114,,,20016,180100:8(100080):010-62534585,62562548():[e-mail],[e-mail]26