Characterization of mitochondrial transcripts associated with cytoplasmic male sterility in Phaseolus vulgaris L. PDF
Preview Characterization of mitochondrial transcripts associated with cytoplasmic male sterility in Phaseolus vulgaris L.
CHARACTERIZATIONOFMITOCHONDRIAL TRANSCRIPTSASSOCIATEDWITHCYTOPLASMIC MALESTERILITYINPHASEOLUSVULGARISL. By BYOUNGOKKIM ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT OFTHEREQUIREMENTSFORTHEDEGREEOF DOCTOROFPHILOSOPHY LTNIVERSITYOFFLORIDA 1998 ACKNOWLEDGEMENTS Iwanttoexpressmydeepappreciationfirsttomyadvisor,Dr. ChristineD.Chase,forhersupportfinanciallyandscientificallyduringmy pursuitofthedoctoratedegree. Herencouragementandpatienceenabled metofinishtheseprojects. MybelovedChristianfellow,VictorOrtega,and alllabmembersweremyjoyandhopewhenwewereworkingtogether. Theirtolerancewasbeyondmyimagination,andtheirsmileswere unforgettable. Ihavetogivemysinceregratitudetothem. Mycommittee members.Dr.KennethCline,Dr.DarylPring,Dr.RobertFerl,andDr. Alfi-edLewin,wereveryspecialtomeduringthecourseofmyresearch. Theirinstructionswereindispensableandinvaluable. Withoutthem,I wouldnothavesurvived. Ithankyou,all. Youaregreat. Mywife,JaeYeounPark,andkids,JooHyunandJungKyu,were special,arespecialandwillbespecialforever. Byoung-sook,Byoung-chun, andByoung-hyunKim(brothersandsister)aremyhistory. Theyarethe oneswhodeservetohaveallthecredit. Godblessmyfamilyandweshall alwaysbewithJesusChrist,ourSavior. ii 5 TABLEOFCONTENTS ACKNOWLEDGMENTS ii LISTOFTABLES vi LISTOFFIGURES viii ABSTRACT ix CHAPTERS INTRODUCTION 1 CHAPTER RNAEDITINGOFMITOCHONDRIAL TRANSCR1:IPTSASSOCIATEDWITHCYTOPLASMICMALE STERILITYINPHASEOLUSVULGARISL. LiteratureReview MitochondrialGenomesinHigherPlants 6 TranscriptionofMitochondrialGenes 9 CytoplasmicMaleSterility(CMS) 12 NuclearFertilityRestoration 14 MitochondrialRNA(mtRNA)Processingin FertilityRestoration 1 CMSmP.vulgaris 17 RNAEditing 19 MaterialsandMethods PlantMaterial 24 IsolationofMitochondriaandExtractionofNucleic Acids 25 GelElectrophoresis,NucleicAcidTransfer,andBlot Hybridization 25 cDNASynthesisandAmplification 26 iii ListofPrimers 27 SequencingofDNAandcDNA 27 Results NorthernHybridizationDetectedCMS-Unique atpA/orf209RNAs 28 RNAEditingoftheatpA/orf209Transcript 31 RNAEditingoforf239andthecobSegment inconfig-pvs 33 Homologsoforf239WerePresentinOtherPlant Species 35 Discussion CMS-UniqueatpATranscripts 36 RNAEditingImprovesProteinConservation 38 SilentEditingIsPartial 39 RNAEditingIsSequence-Specific 40 TheOriginoforf239 43 CHAPTER2: IDENTIFICATIONOFALOW-ABUNDANTDNA MOLECULEASSOCIATEDWITH PHASEOLUS VULGARIS. LiteratureReview MitochondrialGenomesinHigherPlants 60 GenerationofSub-stoichiometricMoleculesthrough Recombination 62 PhysicalStructureoftheMitochondrialDNA 63 GeneContentsandNuclearInfluencesinPlant Mitochondria 65 CMS 67 MaterialsandMethods PlantMaterial 70 IsolationofMitochondriaandExtractionof NucleicAcids 71 GelElectrophoresis,NucleicAcidTransfer, andBlotHybridization 71 PCRAmplificationandCloning 73 iv 1 Sequencing 73 RNaseHDigestionAssay 74 Results TheUnidentified2.1kbSegmentWasLocatedatthe 5'Endofthe7.0kbpvsTranscript 75 5'RACEtoIdentifytheUnidentified2.1kbof thepvsTranscript. 78 TranscriptionMap 80 IdentificationoftheSublimonConfigurationContaining thepvsSequence 82 TheMtDNASublimonContainingthePvsSequence WasTranscribed 85 Discussion The2.1kbSequenceofUnknownOriginWas Locatedatthe5'endofthe7.0kbpvsTranscript 87 SearchfortheUnidentified2.1kbSegment by5'RACE 89 TranscriptionMappingoftheCMS-Sprite MitochondrialGenome 90 SublimonsContainingthepvsSequenceand18S/5S rRNAGenes 92 CONCLUSIONANDFUTUREPROSPECTIVES 11 REFERENCES 113 BIOGRAPHICALSKETCH 129 V LISTOFTABLES Table page 1-LMitochondrialgenomesineukaryotes 6 1-2.GeneticMaterials 24 1-3.PrimersusedforsynthesisofcDNA/DNAfragments 45 1-4.ComparisonoftheatpARNAeditingbetween commonbeanandsugarbeet 58 1-5.Comparisonoftheeditingsitesfoundinacobgenesegment amongdifferentplantspecies 58 1-6.Frequencyofsilenteditinginplantmitochondria 59 2-1.GeneticMaterials 70 2-2.Oligonucleotides 96 vi LISTOFFIGURES Fig1-1. MitochondrialDNAconfigurationsofCMS-unique pvsregionandflankingrepeats 46 Fig.1-2.RNAgelblotanalysisofthepvsandatpA/orf209 transcriptsinCMSlinesandmale-fertilerevertantlines—47 Fig.1-3.DiagrammaticrepresentationofRNAeditinginthe atpA/orf209region 48 Fig.1-4.NucleotidesequencesandRNAeditingsitesinthe atpA/orf209segmentofP.vulgaris 49 Fig.1-5. Autoradiographsofsequencinggelsforcomparisonof themitochondrialgenomicDNA(gDNA)andcDNAin theorf239and3'cobsegmentofP.vulgaris 52 Fig.1-6. DiagrammaticrepresentationofRNAeditingin theorf239lcobsegment 53 Fig.1-7.NucleotidesequencesandRNAeditingsitesinthe orf239/cobsegment 54 Fig.1-8. SouthernblothybridizationassayoftotalDNA withtheorf239probe 56 Fig.1-9.RNAgelblotanalysiswiththeorf239probein soybeanandbroadbean 57 Fig.2-1.MitochondrialDNAconfigurationcontaining thepvssequenceandflankingrepeats 97 vii Fig.2-2.Diagrammaticrepresentationoftlie oligonucleotide-directedRNaseHcleavageassay 98 Fig.2-3.Oligonucleotide-directedRNaseHdigestionassay 100 Fig.2-4.PrimerextensionanalysisofCMS-SpriteRNA witholigo2(Fig.2-1) 102 Fig.2-5.HybridizationofmitochondrialRNAswithWPR-3 cosmidlibraryclones 103 Fig.2-6.Identificationofsub-stoichiometricfragments — containingthepvssequenceincommonbeanmtDNA 105 Fig.2-7.PGRamplificationfrommtDNA containing18S/5Sgenesandthepvssequence 106 Fig.2-8.AlignmentofthenovelsublimonmtDNAcontainingthe 18S/5SrRNAgenes,3'orf209andthepvssequencein P.vulgariswithcorrespondingsoybeanpSB2-2 mitochondrialDNAsequences 108 Fig.2-9.ComparisonofthemtDNAorganizationscontaining atpA/orf209sequenceand18S/5S/3'orf209between commonbeanandsoybean 109 Fig.2-10.RT-PCRanalysisrevealedthepresenceofRNA transcribedfromthe18S/5SrRNA andthepvsconfiguration 110 viii AbstractofDissertationPresentedtotheGraduateSchool oftheUniversityofFloridainPartialFulfillmentofthe RequirementsfortheDegreeofDoctorofPhilosophy CHARACTERIZATIONOFMITOCHONDRIALTRANSCRIPTS ASSOCIATEDWITHCYTOPLASMICMALESTERILITYIN PHASEOLUSVULGARISh. By BYOUNGOKKIM May,1998 Chair:Dr.ChristineD.Chase MajorDepartment:PlantMolecularandCellularBiology MitochondrialgenesareassociatedwiththeCMStraitandare transmittedthroughthematernalparent. Inthecommonbean, mitochondrialDNA(mtDNA)sequencesof3.7kbdesignatedpvsare associatedwithCMS. A7.0kbRNAisthemostabundantamong transcriptscontainingorf239ofthepvs,andhasbeenknowntobe associatedwithpolyribosomes. A2.1kbsegmentofthe7.0kbtranscript wasnotaccountedforbyhybridizationwithpvsandflankingmtDNA clones. Inthepresentstudy,Ihaveinvestigatedtheoriginofthis2.1kb segmentandtheRNAeditingpatternsofthepvstranscripts. ix CMS-uniqueatpA/orf209transcripts(3.0,2.8,and2.5kb)werefound bynorthernanalysisinadditiontopreviouslyidentifiedtranscripts. Three C-to-UpartialRNAeditingeventswererevealedintheorf2S9codingregion ofthepvstranscripts,whereasthreesiteswerecompletelyeditedinthe3' cobsegmentco-transcribedwiththeorf239. Theconcomitantoccurrenceof partialandcompleteeditingpatternssuggestedthattranscriptabundance doesnotaffectthefrequencyofRNAediting. TheRNAeditingeventsand presenceofor/2iP-homologoussequencesinmitochondriaofotherplants suggestedthatorf239couldbepartofanas-yet-unidentifiedmitochondrial proteincodinggene. TheoriginoftheCMS-unique7.0kbtranscriptwasinvestigated. Oligonucleotide-directedRNaseHdigestionassaysindicatedthatthe physicalstructureofthe7.0kbpvsRNAwaslinearandthattheunidentified 2.1kbsegmentwaslocated5'ofthepvssequenceonthislinearmolecule. SouthernhybridizationandPCRanalysisrevealedaCMS-unique 'sublimon'containingthemitochondrial18S/5SrRNAsequence,a3' portionoforf209andthepvssequence. RT-PCRanalysisverifiedthatthis sublimonconfigurationwastranscribedinCMScytoplasms. Thepvs sublimonmoleculeistranscribedtogiverisetopossiblythe7.0kbpvs RNA. X
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