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1、TheEMBOJournalvol.9no.12pp.4173-4179,1990Aninvitrosystemfortheleader-primedtranscriptionofcoronavirusmRNAsSusanC.BakerandMichaelM.C.Laiofcoronaviruses(Laietal.,1984).ThismechanismisproposedtoinvolvethesynthesisofaleaderRNAwhichHowardHughesMedicalInstitute,DepartmentofMicrobiology,isusedasaprimerfort
2、hesynthesisofsubgenomicmRNAs.UniversityofSouthernCaliforniaSchoolofMedicine,LosAngeles,Themurinecoronavirus;mousehepatitisvirus(MHV),hasCA90033,USAbeenstudiedmostextensivelyasamodelsystemofleader-CommunicatedbyD.Kolakofskyprimedtranscription.MHVcontainsasingle-strandedinfectiousRNAgenomeof32kb(Pachu
3、ketal.,1989).InWehavedevelopedaninvitrotranscriptionsystemwhichinfectedcellsthevirionRNAisfirsttranscribedintocanutilizeexogenousleaderRNAformousehepatitisnegative-strandedRNA,whichthenservesasthetemplatevirusIHV)'leader-primed'mRNAtranscription.Cyto-foragenomicsizemRNAandsixorsevensubgenomicplasmic
4、extractscontainingviralproteinsandtemplatemRNAs(FigureIA).ThesemRNAshavea3'-coterminal,RNAwerepreparedbylysolecithinpermeabilizationofnested-setstructure(Laietal.,1981;Leibowitzetal.,1981)MHV-infectedcells.SyntheticleaderRNAwhichdifferedandeachmRNAcontainsanidentical5'-endleadersequenceinsequencefro
5、mtheendogenousleaderRNAwasaddedof72-77nucleotides(Laietal.,1983,1984;Spaanetal.,totheextractsanddemonstratedtobeincorporatedinto1983).Onlythe5'-endopenreadingframe(ORF)ofeachMHVmRNAs.IrrespectiveofthesizeofleaderRNAsmRNAistranslated,althoughthesemRNAscontainmultipleadded,theexogenousleaderRNAwasjoin
6、edtotheORFs.IntheintergenicregionsbetweenORFsexistsaendogenousmRNAatthesamesite,whichcorrespondsstretchofsequencevaryingfrom7to18nucleotideswhichtoaUCUAApentanucleotiderepeatregion.Onlyleaderarehomologoustothe3'-endoftheleaderRNA.ThisregionRNAscontaingthepentanucleotidesequencescouldbepresumablyisin
7、volvedintheregulationoftranscriptionofutilizedfortranscription.Mismatchesbetweentheinter-mRNAs.Thereisasignificantcorrelationbetweentheextentgenicsiteandtheexogenousleadersequencewithintheofsequenceho
