Genome structure and gene content in protist mitochondrial ...

This review surveys the 23 complete protist mtDNA sequences that have been determined to date, commenting on such aspects as mitochondrial genome structure, ... 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1PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,DepartmentofBiochemistry,DalhousieUniversity,Halifax,NovaScotiaB3H4H7,Canada *Towhomcorrespondenceshouldbeaddressed.Tel:+19024942521;Fax:+19024941355;Email:[email protected] Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar B.FranzLang, B.FranzLang 2PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,DépartementdeBiochimie,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar RobertCedergren, RobertCedergren 2PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,DépartementdeBiochimie,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar G.BrianGolding, G.BrianGolding 5PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,DepartmentofBiology,McMasterUniversity,Hamilton,OntarioL8S4K1,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar ClaudeLemieux, ClaudeLemieux 6PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,DépartementdeBiochimie,UniversitéLaval,Québec,QuébecG1K7P4,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar DavidSankoff, DavidSankoff 3PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,CentredeRechercheMathématique,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar MoniqueTurmel, MoniqueTurmel 6PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,DépartementdeBiochimie,UniversitéLaval,Québec,QuébecG1K7P4,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar NicolasBrossard, NicolasBrossard 4PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,OGMPSequencingUnit,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar EricDelage, EricDelage 2PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,DépartementdeBiochimie,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar TimG.Littlejohn, TimG.Littlejohn 4PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,OGMPSequencingUnit,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar ...Showmore IsabellePlante, IsabellePlante 4PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,OGMPSequencingUnit,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada +Presentaddress:AustralianNationalGenomicInformationService(ANGIS),UniversityofSydney,Sydney,NewSouthWales2006,Australia Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar PierreRioux, PierreRioux 4PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,OGMPSequencingUnit,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar DianeSaint-Louis, DianeSaint-Louis 4PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,OGMPSequencingUnit,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar YunZhu, YunZhu 4PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,OGMPSequencingUnit,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar GertraudBurger GertraudBurger 2PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,DépartementdeBiochimie,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada4PrograminEvolutionaryBiology,CanadianInstituteforAdvancedResearch,OGMPSequencingUnit,UniversitédeMontréal,Montréal,QuébecH3C3J7,Canada Searchforotherworksbythisauthoron: OxfordAcademic PubMed GoogleScholar +Presentaddress:AustralianNationalGenomicInformationService(ANGIS),UniversityofSydney,Sydney,NewSouthWales2006,Australia AuthorNotes NucleicAcidsResearch,Volume26,Issue4,1February1998,Pages865–878,https://doi.org/10.1093/nar/26.4.865 Published: 01February1998 Articlehistory Received: 23October1997 Accepted: 21November1997 Published: 01February1998 PDF SplitView Views Articlecontents Figures&tables Video Audio SupplementaryData Cite Cite MichaelW.Gray,B.FranzLang,RobertCedergren,G.BrianGolding,ClaudeLemieux,DavidSankoff,MoniqueTurmel,NicolasBrossard,EricDelage,TimG.Littlejohn,IsabellePlante,PierreRioux,DianeSaint-Louis,YunZhu,GertraudBurger,GenomestructureandgenecontentinprotistmitochondrialDNAs,NucleicAcidsResearch,Volume26,Issue4,1February1998,Pages865–878,https://doi.org/10.1093/nar/26.4.865 SelectFormat Selectformat .ris(Mendeley,Papers,Zotero) .enw(EndNote) .bibtex(BibTex) .txt(Medlars,RefWorks) Downloadcitation Close PermissionsIcon Permissions Share Email Twitter Facebook More NavbarSearchFilter ThisissueAllNucleicAcidsResearch AllNARJournalsAllJournals MobileMicrositeSearchTerm Search SignIn Register Close searchfilter Thisissue AllNucleicAcidsResearch AllNARJournals AllJournals searchinput Search AdvancedSearch SearchMenu Abstract Althoughthecollectionofcompletelysequencedmitochondrialgenomesisexpandingrapidly,onlyrecentlyhasaphylogeneticallybroadrepresentationofmtDNAsequencesfromprotists(mostlyunicellulareukaryotes)becomeavailable.Thisreviewsurveysthe23completeprotistmtDNAsequencesthathavebeendeterminedtodate,commentingonsuchaspectsasmitochondrialgenomestructure,genecontent,ribosomalRNA,introns,transferRNAsandthegeneticcodeandphylogeneticimplications.WealsoillustratetheutilityofacomparativegenomicsapproachtogeneidentificationbyprovidingevidencethatorfBinplantandprotistmtDNAsisthehomologofatp8,thegeneinanimalandfungalmtDNAthatencodessubunit8oftheF0portionofmitochondrialATPsynthase.AlthoughseveralprotistmtDNAs,likethoseofanimalsandmostfungi,areseentobehighlyderived,othersappeartobehaveretainedanumberoffeaturesoftheancestral,proto-mitochondrialgenome.SomeoftheseancestralfeaturesarealsosharedwithplantmtDNA,althoughthelatterhaveevidentlyexpandedconsiderablyinsize,ifnotingenecontent,inthecourseofevolution.ComparativeanalysisofprotistmtDNAsisprovidinganewperspectiveonmtDNAevolution:howtheoriginalmitochondrialgenomewasorganized,whatgenesitcontained,andinwhatwaysitmusthavechangedindifferenteukaryoticphyla. Introduction MitochondrialDNA(mtDNA)isextraordinarilydiverseinsize,genecontentandgenomeorganization(1–5)anditisadauntingpathwaysbywhichthisevolutionarydiversificationhasoccurred.ThepreferredapproachtoansweringsuchevolutionaryquestionsisthroughcomparativeanalysisofcompletemtDNAsequences,whichprovidesagenome-levelperspectiveonsuchissuesaswhatgenesarepresent,howtheyarearranged,whetherthereareintrons(and,ifso,whattypes),howspacersequencesaredistributedandhowlargetheyare,whethersegmentsofthegenomearerepeatedandotherrelevantinformation.Currently,63completemtDNAsequencesareavailablethroughpublicdomaindatabases;however,thephylogeneticrangethatthesesequencesrepresentisbothnarrowandbiased:47(75%)arefromanimalspecies(31vertebrate,16invertebrate);five(8%)arefromfungi;two(3%)arefromplants;onlynine(14%)arefromprotists,inspiteofthefactthatthelattergroupoforganisms(mostlyunicellular)comprisesthebulkofthebiologicaldiversityoftheeukaryoticlineage(6).Thislimitedandhighlynon-representativedatasethasmadeitdifficulttodrawmeaningfulconclusionsabouttheancestralformofthemitochondrialgenome,anecessarystartingpointforinferencesaboutsubsequentmitochondrialgenomeevolution.Toredressthisimbalance,theOrganelleGenomeMegasequencingProgram(OGMP)wasestablishedin1992,havingasaspecificaimthesystematicandcomprehensivedeterminationofcompleteprotistmtDNAsequences.[BriefdescriptionsoftheOGMPandtwoallieddatabases,theProtistImageDatabase(PID)andtheOrganelleGenomeDatabaseProject(GOBASE),appearattheendofthisreview].Atthattimeonlythreecompleteprotistmitochondrialgenomesequenceshadbeenpublished:the6kbmtDNAsequencesoftheapicomplexansPlasmodiumyoelii(arodentparasite)(7)andPlasmodiumfalciparum(thehumanmalariaparasite)(8)andthe40kbmtDNAsequenceoftheciliateprotozoanParameciumaurelia(9).PartialbutextensivemtDNAsequenceinformationwasalsoavailableforanotherciliateprotozoan,Tetrahymenapyriformis,severaltrypanosomatidprotozoa(inthegeneraTrypanosoma,LeishmaniaandCrithidia)andthegreenalga(chlorophyte)Chlamydomonasreinhardtii.TheselimiteddatasuggestedthatprotistmtDNAsmightbeevenmorestructurallyvariablethantheircounterpartsinthemulticellulareukaryoticlineages(1).Intheensuing5years,alargerselectionofcompleteprotistmtDNAsequenceshasbecomeavailablethroughtheeffortsoftheOGMP,acomplementaryFungalMitochondrialGenomeProject(FMGP)(5)andotherresearchgroups.Thisreviewsummarizesandcommentsuponvariousaspectsofprotistmitochondrialgenomestructure,particularlygenecontent,thathaveemergedfromthesenewsequences.Inrecentyearscomprehensivereviewsofanimal(10),fungal(5,11)andplant(12,13)mtDNAshavebeenpublished,butreviewsofprotistmtDNAshavebeenlimitedtospecificgroups,e.g.ciliates(14),trypanosomatids(15)andapicomplexans(16).Becauseprotistsencompassmostofthephylogeneticbreadthoftheeukaryoticlineageand,bydefinition,containanumberofcladeswhoseevolutionarydepthexceedsthatofthetraditionalanimal,plantandfungalkingdoms,itisimportanttosamplewidelywithinthisdisparateassemblagetoobtainaclearperspectiveontherangeofmtDNAstructuraldiversityinprotists,incomparisonwiththemorewidelystudiedmitochondrialgenomesfromothereukaryotes.Thedataassembledhereemphasizethatmostnon-protistmtDNAs,particularlythoseofanimals,aresubstantiallyderivedrelativetomostoftheirprotistcounterparts,havinglostmanygenesthatarecommonlystillfoundinprotistmitochondrialgenomes.Thecompilationprovidedherebetterdefinesthepropertiesofatypicalancestral(i.e.minimallydiverged)protistmtDNAandallowsustosuggestwithgreaterconfidencewhatgeneswerelikelycontainedintheproto-mitochondrialgenome(i.e.thelastcommonancestorofcontemporarymitochondrialgenomes).ScopeofTheReview Table1identifiesthe23completeprotistmtDNAsequencesthattoourknowledgehavebeendeterminedtodate.Thesesequencesencompassareasonablybroadselectionofprotisttaxa,althoughtheystillrepresentonlyafractionofrecognizedprotistlineages(6).Nineofthesesequencesareinthepublicdomain;theremainderareunpublishedonesdeterminedbytheOGMP(eight),theFMGP(two)orotherresearchgroups(four).Aswell,weincludecompletemtDNAsequencesfromrepresentativenon-protistsforpurposesofcomparison.Figure1displaystherelativephylogeneticpositions(totheextentthatthesecanbeinferredorproposedatpresent)oftheprotistslistedinTable1,togetherwithotherprotistspecies,includingfuturecandidatesselectedbytheOGMPforcompletemtDNAsequencing.Methodology Datacollectionandanalysis InthecaseofcompletemtDNAsequencespublishedbyothergroupsanddepositedinthepublicdomainwehaveusedthestandardizedandcorrectedversionsavailableinGOBASE(17;seebelow).Importantly,annotationsaccompanyingthesesequenceshavebeenunifiedwithrespecttogeneandproductnomenclature.Theseparticularsequenceshavealsobeenre-analyzedbyususinginformaticstoolsdevelopedin-houseanddescribedbelow.WiththeexceptionofBLAST(usedforremotedatabasesearches)(18),FASTA(usedfordetailedsequencecomparison)(19)andNIP(theStadennucleotidesequenceanalysispackage)(20),alloftheinformaticstoolsemployedforthiscompilationhavebeendevelopedbytheOGMPSequencingUnit.ManyoftheprogramsmakeuseoftheOGMP‘masterfile’(mf)concept,anASCII-basedsequencefileformatthatintegratesnucleotidesequence,geneannotationsandtechnicalnotes.ThesequenceretrievalandanalysistoolsdevelopedbytheOGMPhaveforthemostpartbeenwritteninthePerlprograminglanguage.Thesetoolsinclude:BBLAST[batchmodeBLASTsearchoftheNationalCenterforBiotechnologyInformation(NCBI)GenBankdatabase];BOB(BLASToutputbrowser);FERRET,BADGERandCLEVER,retrievaltoolsusedinconjunctionwiththeNCBIEntrezdatabase;GOBASE2MF[aprogramforconvertingfromsequencerecordsstoredinSybasetablesofGOBASE(17)intomfformat];CLEANMF(usedtoverifysequencefilesinmfformatastoannotationsyntaxandlogic);PEPPER(fortranslationofproteincodingsequencesandextractionofnon-codingregions);ONIP(commandlineinterfacetotheStadenNIPprogram,usedinthecreationofcodonusagetablesofvariousgeneclasses);CN(sequencecounterandchecker).ForcompilingthebodyofdatapresentedinTable2,anumberofwrapperscriptswerewrittenintheBourneshellscriptlanguage;theseprogramscallupontheabovetoolsandproduceoutputfilesofappropriatelayout.Scriptsthatusegenomesequencefilesinmfformatasinputinclude:CODAT(calculationofA+Tcontentofcodingandnon-codingregions);COTAB[creationofcodonusagetablesofthreetypesofproteincodingregions:genes,intronicopenreadingframes(ORFs)anduniqueORFs];BFASTA(batchFASTAsearch,usedincomparingtheproteinsequencesoftwolibraryfiles);TRNLIST(whichcreatesalistoftRNAgenespresentinagenome).FurtherinformationabouttheseprogramsisavailableattheOGMPwebsite(seebelow).ResultsandDiscussion Mitochondrialgenomestructure CompletesequenceanalysishasprovidedevidenceofbothcircularmappingandlinearmappingprotistmtDNAs,withcircularmappinggenomespredominating(Table2).Amongtheprotistmitochondrialgenomescharacterizedaslinear,nocommonendstructureshavebeenidentified(seeTable2fordetails).TheprotistmtDNAslistedinTable2haveamediansizeof∼40kb,rangingfrom6kbinthethreeapicomplexanspecies(thesmallestknownmtDNAs)to77kbinthechoanoflagellateMonosigabrevicollis.ThemajorityofprotistmtDNAsarecompact,gene-richgenomes,withfewornolargenon-codingregions.Intergenicspacersaregenerallysmallandsparse,accountinginninecasesfor<10%ofthemtDNA,withcodingregionssometimesoverlapping.InAcanthamoebacastellanii,Dictyosteliumdiscoideum,M.brevicollis,ChlamydomonaseugametosandPedinomonasminorallgenesaretranscribedfromthesamestrandofthemtDNA;otherwise,morethanonepotentialtranscriptionunitispresentinprotistmitochondrialgenomes.TheoverallA+Tcontentishigh(>70%in15cases)inprotistmtDNAsandisusuallyelevatedinnon-codingintergenicregionscomparedwithcodingregions(upto1.2-foldhigherinM.brevicollismtDNA).ThenumbersinTable2suggestthat,ingeneral,protistmtDNAshaveevolvedinthedirectionofhigherA+Tcontent. Table1OpeninnewtabDownloadslideCompletelydeterminedmitochondrialgenomesequencesaDescriptionsofanddetailedinformationaboutmanyofthesespeciesmaybefoundattheProtistImageDatabase(PID;URLhttp://megasun.bch.umontreal.ca/protists/).bWherethecompletesequenceisreportedinoneortwopapers,thereferencesarelistedhere;otherwise,relevantcitationscanbeobtainedbyconsultingtheannotationprovidedintheNCBIentry.Datafromunpublishedsequenceswereprovidedby:OGMP,OrganelleGenomeMegasequencingProgram;FMGP,FungalMitochondrialGenomeProject(URLhttp://megasun.bch.umontreal.ca/People/lang/FMGP);RWL,R.W.Lee(DepartmentofBiology,DalhousieUniversity,Halifax,NovaScotia,Canada);YT,Y.Tanaka(InstituteofBiologicalSciences,UniversityofTsukuba,Japan);CL/MT(C.LemieuxandM.Turmel,DépartementdeBiochimie,UniversitéLaval,Québec,Canada);PJM,P.J.Myler(SeattleBiomedicalResearchInstitute,Seattle,WA);DRW,D.R.Wolstenholme(DepartmentofBiology,UniversityofUtah,SaltLakeCity,UT).cDatasummariesandgenemapsfortheindividualOGMPsequencingprojectsareavailableatURLhttp://megasun.bch.umontreal.ca/ogmp/.dP.J.Myler,personalcommunication.Adifferentsequence,assembledfromanumberofseparatesources,isavailableasNCBIaccessionno.M94286.ThesequenceofthetranscribedregionofLeishmaniatarentolaemaxicircleDNAisalsoavailable(accessionno.M101026).Table1OpeninnewtabDownloadslideCompletelydeterminedmitochondrialgenomesequencesaDescriptionsofanddetailedinformationaboutmanyofthesespeciesmaybefoundattheProtistImageDatabase(PID;URLhttp://megasun.bch.umontreal.ca/protists/).bWherethecompletesequenceisreportedinoneortwopapers,thereferencesarelistedhere;otherwise,relevantcitationscanbeobtainedbyconsultingtheannotationprovidedintheNCBIentry.Datafromunpublishedsequenceswereprovidedby:OGMP,OrganelleGenomeMegasequencingProgram;FMGP,FungalMitochondrialGenomeProject(URLhttp://megasun.bch.umontreal.ca/People/lang/FMGP);RWL,R.W.Lee(DepartmentofBiology,DalhousieUniversity,Halifax,NovaScotia,Canada);YT,Y.Tanaka(InstituteofBiologicalSciences,UniversityofTsukuba,Japan);CL/MT(C.LemieuxandM.Turmel,DépartementdeBiochimie,UniversitéLaval,Québec,Canada);PJM,P.J.Myler(SeattleBiomedicalResearchInstitute,Seattle,WA);DRW,D.R.Wolstenholme(DepartmentofBiology,UniversityofUtah,SaltLakeCity,UT).cDatasummariesandgenemapsfortheindividualOGMPsequencingprojectsareavailableatURLhttp://megasun.bch.umontreal.ca/ogmp/.dP.J.Myler,personalcommunication.Adifferentsequence,assembledfromanumberofseparatesources,isavailableasNCBIaccessionno.M94286.ThesequenceofthetranscribedregionofLeishmaniatarentolaemaxicircleDNAisalsoavailable(accessionno.M101026).Inanimals,asexemplifiedbyHomosapiensandMetridiumsenileinTable2,theevolutionarytrendhasclearlybeentowardafurthercompactionofthemitochondrialgenome,bothbylossofgenesandbyvirtualeliminationofintergenicspacers.Conversely,inplants(e.g.Marchantiapolymorpha)thetrendhasbeenintheoppositedirection,withthemtDNAtendingtoincreaseinsize,primarilybyacquisitionofalargeamountofapparentlynon-codingDNAofcurrentlyunknownoriginandfunction(Table2).Intherecentlysequenced366924bpmitochondrialgenomeoftheangiospermArabidopsisthaliana(21),fewergenesareencodedthanarefoundinM.polymorphamtDNA,whichishalfthesize(Table2);overall<10%oftheA.thalianamtDNAhasanassignedcodingfunction.Akeyquestionishowandwhyevolutionhasproducedsuchdivergentmitochondrialgenomepatternsindifferenteukaryoticlines.Genecontent Invertebrateanimals,e.g.H.sapiens(Hsa),themitochondrialgenomecontainsgenesfor13innermitochondrialmembraneproteinsinvolvedinelectrontransportandcoupledoxidativephosphorylation(nad1-6and4L,cob,cox1-3andatp6and8)(Table3),aswellasgenesforlargesubunit(LSU)andsmallsubunit(SSU)rRNAs(rnlandrnsrespectively;Table4).This‘standardset’ofmtDNA-encodedgenes(plusatp9)isalsofoundinfungal(e.g.Allomycesmacrogynus,Ama)mtDNAs,exceptthatcertainascomycetefungi(e.g.Schizosaccharomycespombe,Spo)lackallnadgenes.AnimalandfungalmtDNAsdonotencodea5SrRNA(Table4)nor,withtheexceptionofrps3inA.macrogynusmtDNA(22),dotheycarryanyribosomalproteingenes(Table5).InlandplantmtDNAsafewextrarespiratorychainproteingenesarefound(e.g.nad9andatp1inM.polymorpha;Table3);however,themostnotabledeparturefromanimalandfungalmtDNAsisthepresenceinplantmtDNAofasetofribosomalproteingenes(Table5)aswellasagenefor5SrRNA(rrn5;Table4).InthecaseofM.polymorphamtDNAseveralhomologsofknownmitochondrialgenes(e.g.sdh3,4andyejR,U,V;Tables3and6)wereinitiallyconsideredtobeuniqueORFs(23).Withrespecttogenecontent,protistmtDNAsgenerallyresembleplantratherthananimalorfungalmtDNAs.ThelargestgenerepertoiresofaridentifiedinanymtDNAisthatfoundinthemitochondrialgenomeoftheheterotrophicflagellateReclinomonasamericana(Ram,Tables3–7;24).GenesintheothersequencedmtDNAsareallsubsetsoftheR.americanaset,implyingthattheR.americanapatternisclosesttotheancestralpatternofgenescarriedbytheproto-mitochondrialgenome(24).TheR.americanaresultsalsoindicatethatgeneloss(presumablybytransfertothenucleus)hasoccurredtodifferentextentsindifferentlineages(25),withmanyrespiratorychaingenesandalmostallribosomalproteingeneshavingalreadybeeneliminatedinthecommonancestorofanimalandfungalmtDNAs.InsupportoftheviewthatR.americanamtDNAisancestral(i.e.minimallydiverged)isthehighlyeubacterialcharacterofcertainofitsgenes(e.g.rnpB,encodingtheRNAcomponentofRNaseP)aswellasthepresenceofputativeeubacterialtranslationinitiationsignals(Shine-Dalgarnomotifs;24).Inaddition,asinthecaseofchloroplastgenomes(3,26,27),R.americanamtDNAencodessubunitsofamulti-component,eubacteria-like(α2ββ4)coreRNApolymerase.Incontrast,inothereukaryotesthecoremitochondrialRNApolymeraseisasinglepolypeptide,nuclearDNA-encodedenzymehomologoustobacteriophageT3andT7RNApolymerases(28–32).AlthoughR.americanamtDNAhasalargernumberofgenesthanothersequencedprotistmtDNAs,itisnotablethattheseadditionalgenesareallinvolvedinmitochondrialbiogenesisand/orfunction. Figure1OpeninnewtabDownloadslidePhylogenetichypothesisoftheeukaryoticlineagebasedonultrastructuralandmoleculardata.Organismsaredividedintothreemaingroupsdistinguishedbymitochondrialcristalshape(eitherdiscoidal,flattenedortubular).Unbrokenlinesindicatephylogeneticrelationshipsthatarefirmlysupportedbyavailabledata;brokenlinesindicateuncertaintiesinphylogeneticplacement,resolutionofwhichwillrequireadditionaldata.Colorcodingoforganismalgenusnamesindicatesmitochondrialgenomesthathavebeencompletely(Table1),almostcompletely(Jakoba,NaegleriaandThraustochytrium)orpartially(*)sequencedbytheOGMP(red),theFMGP(black)orothergroups(green).NamesinblueindicatethosespecieswhosemtDNAsarecurrentlybeingsequencedbytheOGMPorarefuturecandidatesforcompletesequencing.Amitochondriateretortamonadsarepositionedatthebaseofthetree,withbrokenarrowsdenotingtheendosymbioticorigin(s)ofmitochondriafromaRickettsia-likeeubacterium.Macrophar.,Macropharyngomonas.Figure1OpeninnewtabDownloadslidePhylogenetichypothesisoftheeukaryoticlineagebasedonultrastructuralandmoleculardata.Organismsaredividedintothreemaingroupsdistinguishedbymitochondrialcristalshape(eitherdiscoidal,flattenedortubular).Unbrokenlinesindicatephylogeneticrelationshipsthatarefirmlysupportedbyavailabledata;brokenlinesindicateuncertaintiesinphylogeneticplacement,resolutionofwhichwillrequireadditionaldata.Colorcodingoforganismalgenusnamesindicatesmitochondrialgenomesthathavebeencompletely(Table1),almostcompletely(Jakoba,NaegleriaandThraustochytrium)orpartially(*)sequencedbytheOGMP(red),theFMGP(black)orothergroups(green).NamesinblueindicatethosespecieswhosemtDNAsarecurrentlybeingsequencedbytheOGMPorarefuturecandidatesforcompletesequencing.Amitochondriateretortamonadsarepositionedatthebaseofthetree,withbrokenarrowsdenotingtheendosymbioticorigin(s)ofmitochondriafromaRickettsia-likeeubacterium.Macrophar.,Macropharyngomonas.TheemergingdatasuggestthatlossofparticulargenesfrommtDNAhappenedanumberoftimes,independently,inthecourseofmitochondrialgenomeevolution.Forexample,sdhgeneshaveonlybeenfoundsofar(Table3)inthemtDNAofacryptophyte[Rhodomonassalina(33)],rhodophytes[theredalgaePorphyrapurpurea(33),Chondruscrispus(34)andCyanidiumcaldarium(35)]andlandplants[M.polymorpha(33,36)],aswellasinR.americanamtDNA(24,33).ThesegenesarenotpresentinA.thalianamtDNA(21)andsofarhavenotbeenidentifiedinother,partiallysequencedangiospermmitochondrialgenomes.Consideringtheproposedphylogeneticpositionsoftheselineages(Fig.1)andthecurrentlimiteddistributionofmtDNA-encodedsdhgenes,weinferthatthesegenesmusthavebeenlostfrommtDNAondifferentoccasions(33). Table2OpeninnewtabDownloadslideCharacteristicsofsequencedmitochondrialgenomesaC,circularmapping;L,linearmapping.bIncludesidentifiedgenes,unidentifiedORFs,intronsandintronORFs.cIncludes492bpsubterminalinvertedrepeatsandterminal40nt3′single-strandextensions(78).dIncludes2208bpterminalinvertedrepeats(OGMP,unpublishedresults).eSequencestartsattheDNAreplicationinitiationloop,whichcontainsatandemarrayof1134bpA+T-richrepeatunits.TerminationsequenceattheotherendofthelinearDNA(estimatedtobe∼200bp)remainsunsequenced(14).fHead-to-tailtandemrepeatsofa6kbunit(82).gLengthofrepeatunit.hExcludingtandemlyarrayedtelomericsequences(31bprepeatunit)ofvariablelength(OGMP,unpublishedresults).i7.1kbDNAelementcontainingincompletelycharacterizedterminalinvertedrepeats(79).jExcludesterminalinvertedrepeatsequences(residues1–59and5783–5895ofZ23263).kIdentificationoffragmentedandscrambledrRNAcodingmodules(seeTable4)isincompleteforthesegenomes;forthatreasontheproportionofcodingversusnon-codingDNAcannotbecalculatedatpresent.Table2OpeninnewtabDownloadslideCharacteristicsofsequencedmitochondrialgenomesaC,circularmapping;L,linearmapping.bIncludesidentifiedgenes,unidentifiedORFs,intronsandintronORFs.cIncludes492bpsubterminalinvertedrepeatsandterminal40nt3′single-strandextensions(78).dIncludes2208bpterminalinvertedrepeats(OGMP,unpublishedresults).eSequencestartsattheDNAreplicationinitiationloop,whichcontainsatandemarrayof1134bpA+T-richrepeatunits.TerminationsequenceattheotherendofthelinearDNA(estimatedtobe∼200bp)remainsunsequenced(14).fHead-to-tailtandemrepeatsofa6kbunit(82).gLengthofrepeatunit.hExcludingtandemlyarrayedtelomericsequences(31bprepeatunit)ofvariablelength(OGMP,unpublishedresults).i7.1kbDNAelementcontainingincompletelycharacterizedterminalinvertedrepeats(79).jExcludesterminalinvertedrepeatsequences(residues1–59and5783–5895ofZ23263).kIdentificationoffragmentedandscrambledrRNAcodingmodules(seeTable4)isincompleteforthesegenomes;forthatreasontheproportionofcodingversusnon-codingDNAcannotbecalculatedatpresent.AsthesortsofcomparativedatabeinggeneratedbycompleteprotistmtDNAsequencingcontinuetoaccumulate,weshouldbeabletodocumentmorepreciselythenumberandtimingofindividualinstancesofmitochondrialgeneloss,manyofwhichundoubtedlyinvolvemitochondriontonucleusgenetransfer.Evennow,theresultssuggestthatgenefluxfrommitochondrialtonucleargenomesisnotonlyawidespreadandon-goingphenomenon,butthatithasbeenbothmoregradualandmorefrequentthanpreviouslyappreciated.Thecox2gene,asoneexample,appearstohavebeenlostfrommtDNAatleastthreetimes(seeTable3):inthelineageleadingtotheApicomplexa,inthePedinomonas/Chlamydomonaslineageofgreenalgaeandincertainlegumes(dicotyledonousplants)(37,38).MostprotistmtDNAscontainanumberofconservedbutunidentifiedORFs(Table6).Especiallynotableinthisregardareymf16(whichhasbeenshowntocodeforamembraneproteinofunknownfunction;39)andymf39,whicharepresentinthemtDNAofmanyprotistsandplants(butnotinanimalorfungalmtDNA).However,mostoftheunidentifiedORFsencounteredduringmitochondrialgenomesequencingareunique:theydonotmatchanysequenceintheproteindatabases.Consideringthenatureanddistributionofidentifiedrespiratorychain(Table3)andribosomalproteingenes(Table5),wesuspectthatatleastsomeoftheseunidentifiedORFsmayrepresenthighlydivergedversionsofknownmtDNA-encodedgenes,nolongerrecognizablebysimilaritysearches.AdditionalcomparativedatashouldhelptoaddressthisquestionandmayultimatelypermitthefunctionalassignmentofconservedORFs,asinthecaseofymf19(orfB;seebelow).Assumingthatfurthergeneassignmentsofthistypecanbemadethroughthiscomparativeapproach,differencesinprotistmtDNAgenecontentcouldturnouttobelesspronouncedthantheyappeartobeatthemoment.RibosomalRNA Withonlyafewexceptions,protistmtDNAsencodeLSUandSSUrRNAswhosepotentialsecondarystructuresdeviateminimallyfromtheireubacterialcounterparts(OGMP,unpublishedresults).ThiscorrespondstowhathasbeenobservedwithplantmitochondrialrRNAs,butstandsinmarkedcontrasttomostfungalbutparticularlyanimalmitochondrialrRNAs(40,41).ClearlyrecognizableinmostprotistmitochondrialLSUrRNAsarethe5′-and3′-terminalregionscorrespondingtothe‘5.8S’and‘4.5S’domainsofaeubacterialcounterpartsuchasEscherichiacoli23SrRNA.TheseterminalregionshavelargelybeeneliminatedfromanimalmitochondrialLSUrRNAs(41).Theseobservationsreinforcetheemergingviewthatthemostancestral(minimallyderived)mitochondrialgenomeswillbefoundamongtheprotists. Table3OpeninnewtabDownloadslideMitochondrialDNA-encodedgenesinvolvedinelectrontransportandcoupledoxidativephosphorylationaaFullorganismnamesarelistedinTable1.▪,genepresent;□pseudogene;○geneabsent.bArabidopsisthalianamtDNA(accessionnosY08501andY08502)lackssdhgenesbutencodesafunctionalcopyofnad7(21).cPyoandTpamtDNAs,whichhavethesamegenecontentasPfamtDNA,arenotlistedinthistable.dThesamegenesarefoundinthemaxicircleDNAofLeishmaniatarentolae(accessionno.M10126).Transcriptsoftrypanosomatidmitochondrialgenesundergopost-transcriptionalUaddition/deletionRNAeditingtogeneratetranslatablemRNAs(83).eInbothT.pyriformisandP.aureliamitochondriathenad1geneissplitintotwopiecesandrearranged(OGMP,unpublishedresults).InT.pyriformis,correspondingtranscriptshavebeenidentified,one(nad1_a)encodingtheN-terminalportionandtheother(nad1_b)specifyingtheC-terminalportionofNADHdehydrogenasesubunit1(J.EdqvistandM.W.Gray,unpublishedresults).fIdentificationofnad3intrypanosomatidmtDNA(84)shouldberegardedastentative(P.J.Myler,personalcommunication).gGenecontainssixin-frameTGAcodons(23);transcriptdetectedbutnotfurtherprocessed(85).hAsingleopenreadingframe(cox1_cox2)encodesbothsubunits1and2ofcytochromecoxidaseinA.castellanii(86)andD.discoideum(87,88)mtDNAs.iorf172(ymf19;89)inM.polymorphamtDNAandorfBinangiospermmtDNA(seetext).Table3OpeninnewtabDownloadslideMitochondrialDNA-encodedgenesinvolvedinelectrontransportandcoupledoxidativephosphorylationaaFullorganismnamesarelistedinTable1.▪,genepresent;□pseudogene;○geneabsent.bArabidopsisthalianamtDNA(accessionnosY08501andY08502)lackssdhgenesbutencodesafunctionalcopyofnad7(21).cPyoandTpamtDNAs,whichhavethesamegenecontentasPfamtDNA,arenotlistedinthistable.dThesamegenesarefoundinthemaxicircleDNAofLeishmaniatarentolae(accessionno.M10126).Transcriptsoftrypanosomatidmitochondrialgenesundergopost-transcriptionalUaddition/deletionRNAeditingtogeneratetranslatablemRNAs(83).eInbothT.pyriformisandP.aureliamitochondriathenad1geneissplitintotwopiecesandrearranged(OGMP,unpublishedresults).InT.pyriformis,correspondingtranscriptshavebeenidentified,one(nad1_a)encodingtheN-terminalportionandtheother(nad1_b)specifyingtheC-terminalportionofNADHdehydrogenasesubunit1(J.EdqvistandM.W.Gray,unpublishedresults).fIdentificationofnad3intrypanosomatidmtDNA(84)shouldberegardedastentative(P.J.Myler,personalcommunication).gGenecontainssixin-frameTGAcodons(23);transcriptdetectedbutnotfurtherprocessed(85).hAsingleopenreadingframe(cox1_cox2)encodesbothsubunits1and2ofcytochromecoxidaseinA.castellanii(86)andD.discoideum(87,88)mtDNAs.iorf172(ymf19;89)inM.polymorphamtDNAandorfBinangiospermmtDNA(seetext).AminorityofprotistmtDNAsencoderRNAgeneswhosestructureand/orthestructureoftheirproductsisveryunusual.The9S(SSU)and12S(LSU)mitochondrialrRNAsoftrypanosomatidprotozoa(e.g.LeishmaniatarentolaeandTrypanosomabrucei)areamongthesmallestandstructurallymostdivergentofknownrRNAs,havingpotentialsecondarystructuresinwhichonlyafewoftheexpectedconservedstructuralelementsareidentifiable(40,41).AlsounusualarethemitochondrialrnlgenesofParameciumaurelia(42,43),Tetrahymenapyriformis(43)andPedinomonasminor(OGMP,unpublishedresults),whicharesplitintotwopiecesthatareseparatedinthegenomeandinterspersedwithothergenes(Table4).ThePedinomonassituationisparticularlyintriguingbecauseamoreextremecaseofrnlfragmentationandscramblingisseeninthemtDNAofaphylogeneticallylaterbranchinggreenalgalgenus,Chlamydomonas(44–46).FragmentedanddispersedrRNAgeneelements,encodedonbothstrandsofthemtDNA,havealsobeenfoundinthesmallapicomplexanmtDNAs(8,47).BecausemostprotistmtDNAsencodeconventional,16S-likeand23S-likerRNAs(theancestralstate),thesedeviantexamplesmustrepresentderivedpatternsofmitochondrialrRNAgenestructureandorganizationwithinthespecificlineagesinwhichtheyoccur.LikeanimalandfungalmtDNAs,mostprotistmtDNAslacka5SrRNAgene,thecurrentexceptions(Table4)beingthechlorophytealgaeProtothecawickerhamii(48)andNephroselmisolivacea(Nol)(M.Turmel,C.OtisandC.Lemieux,unpublishedresults),theredalgaC.crispus(seeTable4,footnoteg)andthejakobidflagellateR.americana(49).Asinthecaseofsdhgenesnotedabove,thesporadicphylogeneticdistributionofmitochondrialrrn5suggeststhatthisgenewaslostfrommtDNAanumberoftimes. Table4OpeninnewtabDownloadslideRNA-encodinggenesinmtDNAaaFullorganismnamesarelistedinTable1.▪,genepresent;○geneabsent.bThesamegenesarepresentinA.thalianamtDNA(21).cPyoandTpamtDNAshavethesamegenecontentasPfamtDNA.dMultiplysplitandrearrangedrnlandrnsgenes→multiplyfragmentedLSUandSSUrRNAs(44–47).eSplit(2piece)andrearrangedrnl(42,43;OGMP,unpublishedresults).fSplit(2piece)rns→split(2piece)SSUrRNA(90,91).gTheoriginalclaimthatC.crispusmtDNAencodesa5SrRNA(34)hassincebeendiscounted(49;seealso4)However,re-analysisoftheC.crispusmtDNAsequencehasnowrevealedageneforabonafide5SrRNA,differentfromthe5SrRNA-likestructureoriginallyproposedbyLeblancetal.(34).TheC.crispusrrn5(complementofresidues16043–16152inZ47547)islocatedbetweenandinthesametranscriptionalorientationasnad3andrps11(G.Burger,unpublishedresults).hB.F.Lang,unpublishedresults.iSmallRNAsthatfunctioninUaddition/deletionRNAediting(83).jThenumberofguideRNAsencodedbytheT.bruceiandL.tarentolaemaxicircleDNAsisthreeand15respectively.ForacompilationoftrypanosomatidguideRNAsseehttp://www.biochem.mpg.de/~goeringe/gRNA/gRNAseqs.html).kGeneencodinga129ntRNAofunknownfunctionislocatedimmediatelydownstreamofrnl(Y.Tanaka,personalcommunication).Table4OpeninnewtabDownloadslideRNA-encodinggenesinmtDNAaaFullorganismnamesarelistedinTable1.▪,genepresent;○geneabsent.bThesamegenesarepresentinA.thalianamtDNA(21).cPyoandTpamtDNAshavethesamegenecontentasPfamtDNA.dMultiplysplitandrearrangedrnlandrnsgenes→multiplyfragmentedLSUandSSUrRNAs(44–47).eSplit(2piece)andrearrangedrnl(42,43;OGMP,unpublishedresults).fSplit(2piece)rns→split(2piece)SSUrRNA(90,91).gTheoriginalclaimthatC.crispusmtDNAencodesa5SrRNA(34)hassincebeendiscounted(49;seealso4)However,re-analysisoftheC.crispusmtDNAsequencehasnowrevealedageneforabonafide5SrRNA,differentfromthe5SrRNA-likestructureoriginallyproposedbyLeblancetal.(34).TheC.crispusrrn5(complementofresidues16043–16152inZ47547)islocatedbetweenandinthesametranscriptionalorientationasnad3andrps11(G.Burger,unpublishedresults).hB.F.Lang,unpublishedresults.iSmallRNAsthatfunctioninUaddition/deletionRNAediting(83).jThenumberofguideRNAsencodedbytheT.bruceiandL.tarentolaemaxicircleDNAsisthreeand15respectively.ForacompilationoftrypanosomatidguideRNAsseehttp://www.biochem.mpg.de/~goeringe/gRNA/gRNAseqs.html).kGeneencodinga129ntRNAofunknownfunctionislocatedimmediatelydownstreamofrnl(Y.Tanaka,personalcommunication).TransferRNAsandthegeneticcode CompletesequencingofanorganellegenomeistheonlywaytodetermineunequivocallywhetherthatgenomeencodesallofthetRNAspeciesnecessarytosupportorganellarproteinsynthesis.SeveralprotistmtDNAs(thoseofM.brevicollis,P.wickerhamii,R.salinaandMalawimonasjakobiformisinTable1)doappeartoencodetheminimalrequiredtRNAset,ifoneallowsthatasingletRNAisabletodecodethefour-codonfamilyspecifyingagivenaminoacid(seeTable7).However,inmostcases,tRNAsrecognizingoneormorecodonsareevidentlyabsentfromthemitochondrialgenome,andtRNAimportfromthecytosolisusuallyinvokedasthemechanismformakingupthedeficit.ImportofnuclearDNA-encodedcytosolictRNAsintomitochondriaisclearlyrequiredinthecaseofA.castellanii,D.discoideum,P.aurelia,T.pyriformis,Chlamydomonasspp.andP.minor,whosemtDNAsencodesubstantiallyfewerthantheminimalrequiredset(Table7);infact,importoftRNAintoTetrahymenamitochondria,longinferredonthebasisoftRNApopulationstudies(50),hasrecentlybeendocumentedexperimentally(51).NotRNAgeneshavebeenfoundinthemitochondrialgenomesofapicomplexanortrypanosomatidprotists,whereimportofafullsetoftRNAsfromthecytoplasmisassumed(52,53).ThedatainTable7indicatethatmitochondrialtRNAimportisnotonlylikelytobewidespreadamongprotists[asitisalsoinplants(54)andseveralchytridiomycetefungi(5)],butthatitemergedearlyintheevolutionofthemitochondrialtranslationsystem,probablyanumberoftimesindependently.GenesforcertaintRNAs(e.g.MetandTrp)areencodedbythemitochondrialgenomesofvirtuallyallprotists,whereasgenesforothertRNAs(notablyThr)arefoundinfrequentlyamongprotistmtDNAs(Table7).Severalprotistmitochondrialgenomes,aswellasthatofM.polymorpha,lackonlyoneortwooftheminimalrequiredsetoftRNAgenes.Again,inthesecasesitisgenerallyheldthatimportofcytosolictRNAsmakesupthedeficit.Indeed,importintoM.polymorphamitochondriahasrecentlybeendocumentedinthecaseofnucleus-encodedtRNAIle(aau)(55)andtRNAThr(agu)(56),genesforwhichhavenotbeenidentifiedinM.polymorphamtDNA(23).However,analternativepossibilitythatshouldbeconsideredisthattheanticodonsequenceinasinglemtDNAencodedtRNAmightbesubjecttopartialediting,suchthattheuneditedandeditedversionsacceptdifferentaminoacidsandpairwithcodonscorrespondingtotheseaminoacids.PartialC→UeditingofatRNA‘Gly’(gcc)togenerateatRNAAsp(guc)inopossummitochondria(57)servesasaprecedentforthispossibility.InA.castellanii,sequencingofthemtDNAhasprovidedevidenceofanoveltypeoftRNAeditingthataffectsmostofthemtDNA-encodedtRNAs(58–62;D.H.PriceandM.W.Gray,unpublishedresults).Thiseditingisconfinedtooneormoreofthefirstthreepositionsatthe5′-endofthetRNA(62).Exceptforthemismatchingintheacceptorstemthatiscorrectedbythisediting,thesecondarystructuresofAcanthamoebamitochondrialtRNAsarequiteconventional(58–62).WhatappearstobethesametypeofmitochondrialtRNAeditinghasrecentlybeendocumentedinthechytridiomycetefungusSpizellomycespunctatus(63)andseveralotherprimitivefungi(B.F.Lang,unpublishedresults);moreover,inthecaseoftRNAsencodedbyD.discoideummtDNAsecondarystructuremodelingstronglysuggeststhatseveraloftheseundergoasimilartypeofediting.OrthodoxcloverleafsecondarystructuresaretheruleformitochondrialtRNAsthroughouttheprotists,onenotablevariantbeinganunusualtRNAMetinTetrahymenamitochondria(64).ThestructurallyaberranttRNAscharacteristicofanimalmitochondria(65,66)arethereforeexceptional,representingahighlyderivedformofmitochondrialtRNAwhich,nevertheless,isabletoassumetherequiredL-shapedtertiarystructure(67). Table5OpeninnewtabDownloadslideRibosomalproteingenesencodedbymtDNAaaFullorganismnamesarelistedinTable1.▪genepresent;□pseudogene;○geneabsent.Smallsubunit-associatedribosomalproteinsarealsoencodedbythemtDNAsofyeast(Saccharomycescerevisiae;var1)andNeurosporacrassa(S-5)(seetableIIIin2);however,theseproteinssharenoobvioussequencesimilaritywithanyknowneubacterialsmallsubunitribosomalprotein.bSeveralofthesegeneshavenotbeenidentifiedinthecompletelysequencedA.thalianamitochondrialgenome(accessionnosY08501andY08502);theseincluderps1,rps2,rps8,rps10,rps11,rps13andrpl6.Twoadditionalgenes(rps14andrps19)arepresentaspseudogenesinA.thalianamtDNA(21).cLikethePfamitochondrialgenome,PyoandTpamtDNAsdonotencodeanyribosomalproteingenes.dSameribosomalproteingenecontentinL.tarentolaemaxicircleDNA(accessionno.M10126).eorf227(previouslynamedurfa;92);G.BurgerandB.F.Lang,unpublishedresults.fNotranscriptdetected(22).gNotreportedintheoriginalpublicationdescribingthisgenome(34).Table5OpeninnewtabDownloadslideRibosomalproteingenesencodedbymtDNAaaFullorganismnamesarelistedinTable1.▪genepresent;□pseudogene;○geneabsent.Smallsubunit-associatedribosomalproteinsarealsoencodedbythemtDNAsofyeast(Saccharomycescerevisiae;var1)andNeurosporacrassa(S-5)(seetableIIIin2);however,theseproteinssharenoobvioussequencesimilaritywithanyknowneubacterialsmallsubunitribosomalprotein.bSeveralofthesegeneshavenotbeenidentifiedinthecompletelysequencedA.thalianamitochondrialgenome(accessionnosY08501andY08502);theseincluderps1,rps2,rps8,rps10,rps11,rps13andrpl6.Twoadditionalgenes(rps14andrps19)arepresentaspseudogenesinA.thalianamtDNA(21).cLikethePfamitochondrialgenome,PyoandTpamtDNAsdonotencodeanyribosomalproteingenes.dSameribosomalproteingenecontentinL.tarentolaemaxicircleDNA(accessionno.M10126).eorf227(previouslynamedurfa;92);G.BurgerandB.F.Lang,unpublishedresults.fNotranscriptdetected(22).gNotreportedintheoriginalpublicationdescribingthisgenome(34).InalmosthalfoftheprotistslistedinTable7weinfer,onthebasisofcodonusageandthepresenceofatRNATrphavingaCCAanticodon,thatthemitochondrialtranslationsystemusesthestandardgeneticcode,asisthecaseinlandplants.IntheremainingprotistsUGAappearstobedecodedastryptophanratherthanasstop(Table7),beingthepreferredTrpcodoninallbutP.aurelia;infact,UCAisusedalmostexlusivelytoencodeTrpinM.brevicollisandT.pyriformismitochondria.FromthephylogeneticdistributionofthiscodevariationitisevidentthatthechangeinUGAcodingmusthaveoccurredonmorethanoneoccasion.Introns ComparedwithplantmtDNA,protistmtDNAsseemtohaveremarkablyfewintrons(Table8).AtleasthalfofthesegenomesentirelylackgroupIandgroupIIintrons.Sofar,amongthe23completelysequencedprotistmtDNAslistedinTable1,groupIintronshaveonlybeenfound(andthenonlyinsmallnumbers)intheamoeboidprotozoaA.castellaniiandD.discoideum,thegreenalgaeP.wickerhamii,N.olivaceaandC.eugametosandthechoanoflagellateM.brevicollis.ProtothecawickerhamiiandM.polymorphamtDNAssharewithoneanother(andwithfungalmtDNA)positionallyequivalentandstructurallyhomologouscox1introns,suggestingthattheseintronshavebeeninheritedverticallyfromamitochondrialancestoroffungi,greenalgaeandplants(68).Ontheotherhand,horizontaltransferofothergroupIintronsissuggestedbythefactthatinthernlgeneofA.castellaniimtDNAandinthechloroplastDNAofcertainChlamydomonasspecies,severalmobilegroupIintronsarenotonlypositionallyidentical,buthavehomologousintroncorestructuresandintronORFs(69).VeryfewgroupIIintronshavebeenfoundinprotistmtDNAs(atotalofsevensuchintronsinfiveof23completelysequencedprotistmtDNAs).Again,wehavesomeevidencesuggestingacquisitionofcertainoftheseintronsbyhorizontaltransfer(OGMP,unpublishedresults),asappearsalsotobethecaseforcertaingroupIIintronsfoundinthernlgeneofthebrownalgaPylaiellalittoralis(70).InourviewthepaucityofgroupIIintronsinprotistmtDNAscoupledwiththeirsporadicdistributionandevidenceofhorizontaltransfermakesitquiteunlikelythattherewasawholesaleacquisitionofgroupIIintronsbytheeukaryoticcellviathea-proteobacteria-likeproto-mitochondrialendosymbiont. Table6OpeninnewtabDownloadslideAdditionalproteingenesencodedbymtDNAaaFullorganismnamesarelistedinTable1.▪genepresent;□pseudogene;○geneabsent.IntronORFsnotincluded(seeTable8).bSameinL.tarentolaemaxicircleDNA.cGeneissplitintothreeseparateORFsinbothM.polymorpha(orf509=ymf4;orf169=ymf3;orf322=ymf2)andA.thaliana(ccb382,ccb203andccb452).M.polymorphaorf509isequivalenttoA.thalianaccb382+ccb203,whereasA.thalianaccb452ishomologoustoM.polymorphaorf169+orf322(21).dorf228=ymf5(ccb256inA.thalianamtDNA;21).eorf277=ymf6(ccb206inA.thalianamtDNA;21).forf244inMpomtDNA.gorf183inMpomtDNA(orf25inangiosperms).hAputativemutShomolog,identifiedinacoralmtDNA(93),hasnotbeenfoundinanyofthesequencedprotistmtDNAslistedinTable1.iORFshowingsimilaritytomitochondrialplasmid-encodedDNApolymerase.jRemnantsofdpogene(94).kCodingsequencedistributedoverthreeseparateORFs(OGMP,unpublishedresults).lORFshowingsimilaritytoreversetranscriptase.mOdaetal.(23).nBoerandGray(95).oCodingsequencedistributedbetweentwoseparateORFs(OGMP,unpublishedresults).pORFshowingsimilaritytoDNAendonucleaseoftypeGIF-YIG(96).qThreeORFsofthistypehavebeenfoundinAmamtDNA(22).rComprising>60codonsandnotoverlappingoneanotherorotheridentifiedgenes.sOnly29ORFs>60codonswerepredictedaspossiblegenesusingadefinedindexofG+Ccontentinthefirst,secondandthirdpositionsofcodons(23).tInthecourseofre-analyzingtheCcrmtDNAsequenceoneofthetwopreviouslyannotated(34)uniqueORFs,orf94,hasbeenidentifiedasrpl20(G.Burger,unpublishedresults).uAnadditional13ORFsinTpy(equivalentto14PauORFs)aredefinedas‘ciliate-specific’(sharedbetweenTpyandPaubutnotothermtDNAs).Ofthe25ORFs(unique+ciliate-specific)inPaumtDNA12werepreviouslyannotated(9),whereasanadditional13havebeenfoundinthecourseofre-analyzingthePaumtDNAsequence(G.Burger,unpublishedresults).Table6OpeninnewtabDownloadslideAdditionalproteingenesencodedbymtDNAaaFullorganismnamesarelistedinTable1.▪genepresent;□pseudogene;○geneabsent.IntronORFsnotincluded(seeTable8).bSameinL.tarentolaemaxicircleDNA.cGeneissplitintothreeseparateORFsinbothM.polymorpha(orf509=ymf4;orf169=ymf3;orf322=ymf2)andA.thaliana(ccb382,ccb203andccb452).M.polymorphaorf509isequivalenttoA.thalianaccb382+ccb203,whereasA.thalianaccb452ishomologoustoM.polymorphaorf169+orf322(21).dorf228=ymf5(ccb256inA.thalianamtDNA;21).eorf277=ymf6(ccb206inA.thalianamtDNA;21).forf244inMpomtDNA.gorf183inMpomtDNA(orf25inangiosperms).hAputativemutShomolog,identifiedinacoralmtDNA(93),hasnotbeenfoundinanyofthesequencedprotistmtDNAslistedinTable1.iORFshowingsimilaritytomitochondrialplasmid-encodedDNApolymerase.jRemnantsofdpogene(94).kCodingsequencedistributedoverthreeseparateORFs(OGMP,unpublishedresults).lORFshowingsimilaritytoreversetranscriptase.mOdaetal.(23).nBoerandGray(95).oCodingsequencedistributedbetweentwoseparateORFs(OGMP,unpublishedresults).pORFshowingsimilaritytoDNAendonucleaseoftypeGIF-YIG(96).qThreeORFsofthistypehavebeenfoundinAmamtDNA(22).rComprising>60codonsandnotoverlappingoneanotherorotheridentifiedgenes.sOnly29ORFs>60codonswerepredictedaspossiblegenesusingadefinedindexofG+Ccontentinthefirst,secondandthirdpositionsofcodons(23).tInthecourseofre-analyzingtheCcrmtDNAsequenceoneofthetwopreviouslyannotated(34)uniqueORFs,orf94,hasbeenidentifiedasrpl20(G.Burger,unpublishedresults).uAnadditional13ORFsinTpy(equivalentto14PauORFs)aredefinedas‘ciliate-specific’(sharedbetweenTpyandPaubutnotothermtDNAs).Ofthe25ORFs(unique+ciliate-specific)inPaumtDNA12werepreviouslyannotated(9),whereasanadditional13havebeenfoundinthecourseofre-analyzingthePaumtDNAsequence(G.Burger,unpublishedresults).Acomparativegenomicsapproachtogeneidentification:thecaseoforfBandatp8 AccumulatingsequencedataareaidingintheidentificationofsomeoftheunassignedORFsthathavebeenuncoveredinthecourseofsequencingmitochondrialgenomes.AsanexampleweprovideevidenceherethatorfB,aconservedgeneofunknownfunctionoriginallyidentifiedinplantmtDNA(seeTable3,footnotei),isthehomologofatp8,whichencodessubunit8oftheF0portionoftheATPsynthase.ThelattergenehasbeenfoundinanumberofanimalandfungalmtDNAs,butuptonowhasnotbeenidentifiedinplantorprotistmitochondrialgenomes.Conversely,orfBisfoundinalmostallplantandprotistmtDNAs,butnotinthoseofanimalsorfungi.BothAtp8andOrfBproteinsarecharacterizedbythesameblockofthreeidenticalaminoacidsattheN-terminus,followedbyanotherwisequitevariablesequence(Fig.2).TheknownOrfBproteinsofplantsdifferfromAtp8essentiallyintheirincreasedlength.BecausethereisalsomuchlengthvariationamongOrfBhomologsinsomeprotistmtDNAs,wewerepromptedtoassessthepossibilitythatatp8andorfBarehomologousgenes. Table7OpeninnewtabDownloadslideTransferRNAgenesencodedbymtDNAaaSeeTable1forcompleteorganismnames.▪genepresent;○geneabsent.Aminoacylationspecificity(a.a.)isindicatedbythestandardonelettersymbolsforaminoacids(Me,elongatormethionine;Mf,initiatormethionine).ThepredictedanticodonofeachtRNAisshowninlowercaseletters,withthepredictedcodon(s)thatwouldberecognizedshowninuppercaseletters(N=anynucleotide;R=AorG;Y=CorU).Expandedwobblebasepairingisassumed,suchthatanticodonsbeginningwithuridineareconsideredtorecognizeallcodonsinafour-codonfamily.bDuplicateidenticalgenes.cDuplicatenon-identicalgenes.dTriplicategenes,twoofwhichareidentical,thethirddifferingbyasingleT→Ctransition.eGenomespecifiesasingletrnM(cau).fCinthefirstpositionoftheanticodonpresumedtobemodifiedtolysidine,whichconvertsthetRNAtoanAUA-decodingisoleucineacceptor(97).gAinfirstthepositionoftheanticodonpresumedtobemodifiedtoinosine,withtheresultingtRNAabletopairwithcodonsendinginC,UandA,andperhapsalsoG(see98).htrnK(cuu),thecorrespondingtRNAofwhichwouldbeexpectedtorecognizeAAGbutnotAAA(61).iOnlyUGGTrpcodonsappearinconservedproteincodinggenesinS.pombemtDNA,however,severalUGAcodonsoccurinrps3andintronORFs(92).jBothUGGandUGAaredecodedasTrpinA.castellaniimitochondria(61),whereasthetRNAspecifiedbytrnW(cca)wouldbeexpectedtorecognizeonlyUGG.kIncludesatrnL(aag)notlistedinthetable.lIncludesapresumptivetrnEpseudogene,unrelatedinsequencetoauthentictrnE.mIncludesatrnI(uau)notlistedinthetable.nTranscriptsofmostAcamitochondrialtRNAgenes(12of15)undergosubstitutionalRNAeditingatoneormoreofthefirstthreepositionsoftheacceptorstem(61,64;D.H.PriceandM.W.Gray,unpublishedresults).TranscriptsofatleasthalfoftheDdimitochondrialtRNAgenesarepredictedtoundergoasimilartypeofediting.oIncludesatrnX(uuua)pseudogene(D.H.PriceandM.W.Gray,unpublishedresults),thetranscriptofwhichispredictedtohavean8ntanticodonloop(61).pIncludesanunusualtRNA-likeelementwhoseanticodonsequencewouldpairwithUAAandUAG(99),whicharenormallyterminationcodons.qIncludesatrnI(aau)notlistedinthetable.rIncludesatrnX(cua),thecorrespondingtRNAofwhichwouldbeexpectedtorecognizeUAG(normallyaterminationcodon).sIncludesatrnL(gag)notlisted.Table7OpeninnewtabDownloadslideTransferRNAgenesencodedbymtDNAaaSeeTable1forcompleteorganismnames.▪genepresent;○geneabsent.Aminoacylationspecificity(a.a.)isindicatedbythestandardonelettersymbolsforaminoacids(Me,elongatormethionine;Mf,initiatormethionine).ThepredictedanticodonofeachtRNAisshowninlowercaseletters,withthepredictedcodon(s)thatwouldberecognizedshowninuppercaseletters(N=anynucleotide;R=AorG;Y=CorU).Expandedwobblebasepairingisassumed,suchthatanticodonsbeginningwithuridineareconsideredtorecognizeallcodonsinafour-codonfamily.bDuplicateidenticalgenes.cDuplicatenon-identicalgenes.dTriplicategenes,twoofwhichareidentical,thethirddifferingbyasingleT→Ctransition.eGenomespecifiesasingletrnM(cau).fCinthefirstpositionoftheanticodonpresumedtobemodifiedtolysidine,whichconvertsthetRNAtoanAUA-decodingisoleucineacceptor(97).gAinfirstthepositionoftheanticodonpresumedtobemodifiedtoinosine,withtheresultingtRNAabletopairwithcodonsendinginC,UandA,andperhapsalsoG(see98).htrnK(cuu),thecorrespondingtRNAofwhichwouldbeexpectedtorecognizeAAGbutnotAAA(61).iOnlyUGGTrpcodonsappearinconservedproteincodinggenesinS.pombemtDNA,however,severalUGAcodonsoccurinrps3andintronORFs(92).jBothUGGandUGAaredecodedasTrpinA.castellaniimitochondria(61),whereasthetRNAspecifiedbytrnW(cca)wouldbeexpectedtorecognizeonlyUGG.kIncludesatrnL(aag)notlistedinthetable.lIncludesapresumptivetrnEpseudogene,unrelatedinsequencetoauthentictrnE.mIncludesatrnI(uau)notlistedinthetable.nTranscriptsofmostAcamitochondrialtRNAgenes(12of15)undergosubstitutionalRNAeditingatoneormoreofthefirstthreepositionsoftheacceptorstem(61,64;D.H.PriceandM.W.Gray,unpublishedresults).TranscriptsofatleasthalfoftheDdimitochondrialtRNAgenesarepredictedtoundergoasimilartypeofediting.oIncludesatrnX(uuua)pseudogene(D.H.PriceandM.W.Gray,unpublishedresults),thetranscriptofwhichispredictedtohavean8ntanticodonloop(61).pIncludesanunusualtRNA-likeelementwhoseanticodonsequencewouldpairwithUAAandUAG(99),whicharenormallyterminationcodons.qIncludesatrnI(aau)notlistedinthetable.rIncludesatrnX(cua),thecorrespondingtRNAofwhichwouldbeexpectedtorecognizeUAG(normallyaterminationcodon).sIncludesatrnL(gag)notlisted. Table8OpeninnewtabDownloadslideIntronsandintronORFsinmtDNAaaFullorganismnamesarelistedinTable1.bAgroupIintroninnad5containsnad1andnad3genes(100).Table8OpeninnewtabDownloadslideIntronsandintronORFsinmtDNAaaFullorganismnamesarelistedinTable1.bAgroupIintroninnad5containsnad1andnad3genes(100).TheN-terminalfunctionaldomain(71)ofATPsynthasesubunit8iswellconservedindifferentfungicomparedwiththecentralhydrophobicdomain(72)andtheC-terminaldomain(73).Thelatterdomaincontainsaregionenrichedinpositivelychargedaminoacidresidues(73),whicharethoughttoplayanimportantroleinassemblyoftheF0complex(seebelow).IfOrfBisindeedhomologoustoAtp8,weshouldfindsimilaraminoacidsignaturesinamultiplealignmentofaphylogeneticallydiversecollectionofbothtypesofsequences.SuchacollectionhasrecentlybecomeavailablethroughthesequencingeffortsoftheOGMPandFMGP.AsshowninFigure2,thehighlyconservedN-terminaldomainprovidesthebestevidenceforhomologybetweenorfBandatp8.Furtherevidencesupportingthisinferenceisthepresenceofperfectlyalignedcentralhydrophobicandpositivelychargeddomains.Basedonthealignmentofthefirst57aminoacidsshowninFigure2,wesuggestthatthereislittlebasisforadistinctionbetweenthe‘Atp8’and‘OrfB’classesofprotein.Withtwonotableexceptions,thissequencecompilationfurtherdemonstratesthatalongC-terminalextension(position78andbeyondinFig.2)isonlyfoundamongplantsandprotists.InthestramenopilesCafeteriaroenbergensisandOchromonasdanicathemtDNAcodesforashorterprotein,aboutaslongasthelongestfungalsequences.Thisfeatureisnotcladespecificbecauseinanotherstramenopile,Phytophthorainfestans,themitochondrialgenomespecifiesanAtp8proteinthatisrathertypicalinsizeforprotists.TheC-terminalextensionisnotonlyquitevariableinsize,butindeedissodivergentinsequencethatitcanonlybereasonablywellalignedamongverycloselyrelatedspecies(e.g.landplants).ThusthepresenceorabsenceofaC-terminalextensionalsodoesnotdistinguishbetween‘Atp8’and‘OrfB’classes.ConservedsequencemotifswithinthehydrophobicandC-terminaldomainsoftheAtp8/OrfBproteinarerestrictedtotheboundariesbetweenthesedomains,the‘LPmotif’(71),whichisimmediatelyfollowedbyaregionwithoneorseveralpositivelychargedaminoacids.Previousstudiesinfungihaveshownthatthesepositivelychargedaminoacidsplayanimportantroleinassemblyofsubunits6,8and9(73).Insummary,plantandprotistmitochondrialOrfBproteinscontainalloftheconservedsequenceelementscharacteristicofanimalandfungalAtp8proteins.ThustheorfBgenerepresentsthebestcandidateforthepreviously‘missing’atp8homologinplantandprotistmtDNAs.Phylogeneticimplications ThemitochondrialgenecontentandgenomeorganizationdatabeinggeneratedbytheOGMPandothergroupsareservingtofurtherclarifyourviewsabouttheoriginandevolutionofthemitochondrialgenome.OneexampleinvolvestherelationshipbetweenlandplantandChlamydomonasmtDNAs,whicharesodifferentinstructure,organizationandmodeofexpressionthattheyshowlittleevidenceofhavingacommonevolutionaryorigin(1,2,74).Intheabsenceofaphylogeneticallybroaddatabaseofcomparativeinformationweatonetimeentertainedthepossibilitythattheplantmitochondrialgenomemighthavehadadifferent,morerecentevolutionaryancestrythanChlamydomonasandothermitochondrialgenomes(75).However,sequencingofP.wickerhamii(48)andother(24,34,61)protistmtDNAshasclearlydemonstratedthatplantmtDNAhasretainedanancestralpatternthathasevidentlybeenlostinthemorerapidlyevolvingandhighlyderivedChlamydomonasmtDNA(74).ItisworthemphasizingthatthemajorityoftheprotistmtDNAssequencedtodatebytheOGMP,particularlythosefrommoreobscureprotistsselectedfromthewildonthebasisofultrastructuralorotherphylogeneticconsiderations,retainamoreorlessancestralpatternofgenecontentandorganization.Incontrast,mostofthemtDNAsthathadbeensequencedpriortotheinceptionoftheOGMP(thosefromanimals,mostfungi,chlamydomonadaleangreenalgae,ciliatesandtrypanosomatidprotozoa)arehighlyderived.Itiscuriousthatthemajorityoftheprotiststhathavebeenselectedasmodelsforbiochemical,geneticandmolecularbiologicalresearchhappentohavemtDNAsthataretheleastrepresentativeoftheancestralform.Descriptions OrganelleGenomeMegasequencingProgram(OGMP)(http://megasun.bch.umontreal.ca/ogmp/).TheOGMPwasinitiatedasamulti-disciplinaryandinter-universityconsortiumofCanadianinvestigatorsinterestedinorganellegenomeevolutionandeukaryoticphylogeny.AscurrentlyconstituteditconsistsofaTeam(B.F.Lang,administrativecoordinator;M.W.Gray,scientificcoordinator;G.Burger,C.LemieuxandM.Turmel)andanAdvisoryBoard(R.Cedergren,G.B.Golding,D.Sankoff,T.G.LittlejohnandC.J.O'Kelly),withexternalcollaboratorsonsomeindividualprojects.TheexperimentalarmoftheOGMP,theSequencingUnit(directedbyG.Burger),islocatedintheDépartementdeBiochimie,UniversitédeMontréal.TheSequencingUnitcomprisestwodivisions:MolecularBiology(I.Plante,D.Saint-LouisandY.Zhu),whichconstructsclonelibraries,performstheactualsequencingandworksoutimprovedcloningandsequencingmethods;Informatics(N.BrossardandP.Rioux),whichdevelopsandimplementstoolsrequiredforprojectmanagement,datahandling,sequenceanalysisandannotation.AsthedataproductionarmoftheOGMP,theSequencingUnitdeliversanalyzedandfullyannotatedmitochondrialgenomesequencesforsubmissiontopublicdomaindatabases.TheOGMPwebsite(URLgivenabove)containsadditionalinformationabouttheprogram,aswellasdatasummariesandgenemapsfortheindividualOGMPsequencingprojectscompletedtodate(Table1). Figure2OpeninnewtabDownloadslideAlignmentofAtp8andOrfBaminoacidsequences.Sequencesfrombacteria(B),protists(P),landplants(L),fungi(F)andanimals(A)arecompared.ThreeletterabbreviationsoforganismnamesarelistedinTable1.Additionalabbreviations:Rru,Rhodospirillumrubrum;Bvu,Betavulgaris;Rst,Rhizopusstolonifer;Rss,Rhizophydiumssp.;Hss,Harpochytriumssp.;Sco,Schizophyllumcommune;Spu,Spizellomycespunctatus;Ani,Aspergillusnidulans;Sce,Saccharomycescerevisiae;Pli,Paracentrotuslividus.SequenceswereobtainedfromtheNCBIdatabasesexceptforPin,Mbr,Rst,Rru,Hss,ScoandSpu,whichareunpublishedFMGPsequences,andMja,Rsa,Cro,OdaandPpu,whichareunpublishedOGMPsequences.Colorhighlightingisasfollows:blue,invariantaminoacids;magenta,identicalresiduescomprisingatleast10(40%ormore)ofthetotalnumberofresiduesinagivencolumn(alsocoloredinmagentaarethoseresiduesthataccordingtothePAMmatrixarepositiveorneutralexchangeswithreferencetothemostabundantresidueinthecolumn);yellow,positivelychargedaminoacids.Dashes(−)denoteamissingresidueatthispositionincomparisonwithothersequence(s).Asterisks(*)marktranslationterminationcodons;numbersprecedinganasteriskindicatetheremaininglengthofsequencethatisnotshown.Figure2OpeninnewtabDownloadslideAlignmentofAtp8andOrfBaminoacidsequences.Sequencesfrombacteria(B),protists(P),landplants(L),fungi(F)andanimals(A)arecompared.ThreeletterabbreviationsoforganismnamesarelistedinTable1.Additionalabbreviations:Rru,Rhodospirillumrubrum;Bvu,Betavulgaris;Rst,Rhizopusstolonifer;Rss,Rhizophydiumssp.;Hss,Harpochytriumssp.;Sco,Schizophyllumcommune;Spu,Spizellomycespunctatus;Ani,Aspergillusnidulans;Sce,Saccharomycescerevisiae;Pli,Paracentrotuslividus.SequenceswereobtainedfromtheNCBIdatabasesexceptforPin,Mbr,Rst,Rru,Hss,ScoandSpu,whichareunpublishedFMGPsequences,andMja,Rsa,Cro,OdaandPpu,whichareunpublishedOGMPsequences.Colorhighlightingisasfollows:blue,invariantaminoacids;magenta,identicalresiduescomprisingatleast10(40%ormore)ofthetotalnumberofresiduesinagivencolumn(alsocoloredinmagentaarethoseresiduesthataccordingtothePAMmatrixarepositiveorneutralexchangeswithreferencetothemostabundantresidueinthecolumn);yellow,positivelychargedaminoacids.Dashes(−)denoteamissingresidueatthispositionincomparisonwithothersequence(s).Asterisks(*)marktranslationterminationcodons;numbersprecedinganasteriskindicatetheremaininglengthofsequencethatisnotshown.ProtistImageDatabase(PID)(http://megasun.bch.umontreal.ca/protists/).ThePID(T.G.LittlejohnandC.J.O'Kelly)isacompilationofimagesandshortdescriptionsofselectedprotistgenera,especiallythosewhosespeciesarefrequentlyusedasexperimentalorganismsorareimportantinstudiesoforganismalevolution.TheintentofthePIDistoprovideintegratedon-lineinformationaboutthemorphology,taxonomyandphylogeneticrelationshipsoftheseorganisms.ThePID,whichwasinitiatedwithintheOGMP,containsdescriptionsofmostofthespecieswhosemtDNAshavebeensequencedbytheOGMP.ThePIDisbeingcontinuedindependentlyfrombutinclosecollaborationwiththeOGMP,withitswebpagesmaintainedontheOGMPwebserver.OrganelleGenomeDatabaseProject(GOBASE)(http://megasun.bch.umontreal.ca/gobase/).ShortlyaftertheOGMPwasestablisheditbecameapparentthattherewereseriouslimitationsinaccessingalloftherelevantinformationassociatedwithorganelles.Dataaredispersedamonganumberofsources(WorldWideWeb,publicdatarepositories,scientificjournalsandbooks)andinmanycasesaredifficulteventolocate.Usuallyonlylimitedlinksexistamongdatasources(e.g.thereisnoeasywaytoconnectfromaGenBankrecordcontaininganrRNAsequencetothecorrespondingsecondarystructurecontainedinanotherdatabase).Itisevenmoredifficulttoperformthesortofcross-genomecomparisonsthatwereessentialforthepresentreview.Further,thedatasetsareoftenincompleteand/orcontainerrors,whicharesometimeshardtoidentifyandtorectifyintheunderlyingdatasource.Insuchadisorganizedstateorganellegenomicdataconstituteamajorunderexploitedinformationresource.TheGOBASEproject(17)wasinitiatedbyasubsetofOGMPmembers(B.F.Lang,M.W.Gray,G.BurgerandT.G.Littlejohn)torectifythissituation.GOBASE,whichisataxonomicallybroaddatabasethatorganizesandintegratesdiversedatarelatedtoorganelles,hasbeenconstructedasarelationaldatabasewithaweb-baseduserinterface.Thecurrentversionfocusesonthemitochondrialsubsetofdata.References 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