Replication of mitochondrial DNA in the early development of Misgurnus fossilis

摘要

MitochondriaisolatedfromMisgurnusfossilisembryosatvariousdevelopmentalstageswereincubatedwith3H-dTTPinvitroandtheincorporationintomtDNAwasdetermined.IthasbeenfoundthattherateofmtDNAlabelingincreasesexponentiallywithadoublingtimeof7hrfrom0.01pmoleof3H-dTMP/mgprotein/hrinmitochondriafromunfertilizedeggsto0.4pmolesof3H-dTMP/mgprotein/hrinmitochondriaof35hrembryos.ThepoolofintramitochondrialdTTPdecreases2.5timesduringthefirst10hrafterfertilization,thenremainspracticallyconstantupto35hrofdevelopment.Therateofexogenous3H-dTTPincorporationintotheacidsolublepoolofisolatedmitochondriaattwostagesisapproximatelyproportionaltothepoolsize.Thusidenticalspecificactivitiesof3H-dTTPinsidemitochondriawouldbeobtainedevenwithpoolsofdifferentsizes.Weconcludethattheincreaseof3H-dTMPincorporationintomtDNAindevelopmentreflectsgenuineactivationofmtDNAsynthesis.Asearlyas6hrafterfertilizationthebulkofthelabelincorporatedintomtDNAisfoundinthefractionassociatedwithcovalentlyclosedmolecules.ThispatternoflabelingcharacteristicforreplicatingmtDNAismaintainedthroughoutearlydevelopment.Incontrastsuchpreferentiallabelincorporationintotheclosedcircularfractionwasnotfoundwithmitochondriaofunfertilizedeggs.ClosedmtDNAfromunfertilizedeggscontainsnotmorethan1%ofmoleculeswithD-loops.In35hrembryosthecorrespondingvalueisequaltoabout4%.ActivationofmtDNAreplicationinembryogenesisisprobablyduetotheactivationofmechanismsresponsibleforthegenerationofprimersforreplication.DNApolymeraseactivitysolubilizedfrommitochondriaremainsunchangedinthecourseofembryogenesis.