资源描述：
《Slovakia的链霉菌基因破坏实验》由会员上传分享，免费在线阅读，更多相关内容在行业资料-天天文库。
1、JournalofGeneralMicrobiology(1993),139,2525-2529.PrintedinGreatBritain2525OptimizationofStreptomycesaureofacienstransformationanddisruptionofthekrdAgeneencodingahomologueoftheprincipaltrfactorJANKORMANEC,*BRONISLAVA~~UCHOVAandMARIANFARKA~OVSK+Instituteo
2、fMolecularBiology,SlovakAcademyofSciences,Dubravska'cesta21,84251Bratislava,Slovakia(Received11February1993;revised19April1993;accepted10May1993)~~~~ConditionsforthepreparationofprotoplastsfromStreptomycesaureofaciensandtransformationwithseveralreplicat
3、iveandintegrativeplasmidswereoptimized.Ofallthereplicativeplasmidsused,carryingvariousStreptomycesreplicationorigins,onlyplasmidpGM9producedtransformants,withatransformationefficiencyof2x103(pgDNA)-'.WhenplasmidDNAisolatedfromS.aureofacienswasused,thetr
4、ansformationefficiencyincreasedtolo5transformants(pgDNA)-'.TransformationofprotoplastswithintegrativeplasmidscontainingpartofthehrdAgenegaverisetotransformantswithanefficiencyofapproximately30transformants(pgDNA)-'.Integrationfrequencywasstronglyreduced
5、whenplasmidDNAwasmodifiedbydamordcmmethylase.Byintegrativetransformation,viaadoublecrossover,astablenullmutantofthehrdAgenewasprepared.Thismutationappearedtohavenoobviouseffectongrowth,morphology,differentiationandproductionofchlortetracycline.Introduct
6、ionMatsushima,1981;Ogawaetal.,1983;Hopwoodetal.,1985).However,wecouldnotprepareprotoplastsofS.InStreptomycesaureofaciensCCM3239(ATCC10762),aureofaciensCCM3239byanyofthetechniquesmen-fourgenes(hrdA,hrdB,hrdDandhrdE)encodingtioned.Also,therecentlypublishe
7、dmethodsforprep-homologuesoftheprincipalfactorofRNApolymerasearationofprotoplastsfromindustrialS.aureofacienswereidentified(Kormanecetal.,1992).strains(Isaeva&Voeikova,1990;Muchovaetal.,1991)Severalmethodshavebeendevelopedfordisruptingwereunsuccessful.T
8、herefore,wehadtooptimizechromosomalcopiesofclonedStreptomycesgenes.conditionsforpreparationandtransformationofproto-Phage(9C31withadeletedattPsitewasusedforplastsofS.aureofaciensCCM3239.disruptionviahomologousrecombinationinS.coe
