recognizedwww.haole008.com

NewlydiscoveredyoungCORE-SINEsinmarsupialgenomesMaruoMunemasaa,MasatoNikaidoa,HidenoriNishiharaa,StephenDonnellanb,ChristopherC.
Austinc,NorihiroOkadaa,aGraduateSchoolofBioscienceandBiotechnology,TokyoInstituteofTechnology,Yokohama,JapanbEvolutionaryBiologyUnit,SouthAustralianMuseum,NorthTerrace,SA5000,AustraliacMuseumofNaturalScience,LouisianaStateUniversity,LA70803,USAReceived3July2007;receivedinrevisedform2October2007;accepted4October2007ReceivedbyTakashiGojoboriAvailableonline12October2007AbstractAlthoughrecentmammaliangenomeprojectshaveuncoveredalargepartofgenomiccomponentofvariousgroups,severalrepetitivesequencesstillremaintobecharacterizedandclassifiedforparticulargroups.
Theshortinterspersedrepetitiveelements(SINEs)distributedamongmarsupialgenomesareoneexample.
WehaveidentifiedandcharacterizedtwonewSINEsfrommarsupialgenomesthatbelongtotheCORE-SINEfamily,characterizedbyahighlyconserved"CORE"domain.
PCRandgenomicdotblotanalysesrevealedthatthedistributionofeachSINEshowsdistinctpatternsamongthemarsupialgenomes,implyingdifferenttimingoftheirretropositionduringtheevolutionofmarsupials.
ThemembersofMar3(Marsupialia3)SINEaredistributedthroughoutthegenomesofallmarsupials,whereastheMac1(Macropodoidea1)SINEisdistributedspecificallyinthegenomesofkangaroos.
SequencealignmentoftheMar3SINEsrevealedthattheycanbefurtherdividedintofoursubgroups,eachofwhichhasdiagnosticnucleotides.
TheinsertionpatternsofeachSINEatparticulargenomicloci,togetherwiththedistributionpatternsofeachSINE,suggestthattheMar3SINEshaveintensivelyamplifiedaftertheradiationofdiprotodontians,whereastheMac1SINEhasamplifiedonlyslightlyafterthedivergenceofhypsiprimnodonsfromothermacropods.
BycompilingtheinformationofCORE-SINEscharacterizedtodate,weproposeacomprehensivepictureofhowSINEevolutionoccurredinthegenomesofmarsupials.
2007ElsevierB.
V.
Allrightsreserved.
Keywords:CORE-SINE;Marsupials;Phylogeny;Evolution1.
IntroductionMammaliangenomesharboralargeamountofretroposonsthatpropagatetheircopiesinthehostgenomeviaanRNAintermediategeneratedfroma"copyandpaste"mechanismcalledretroposition(Rogers,1985;Weineretal.
,1986;Brosius,1991;Okada,1991a,b).
Shortinterspersedrepetitiveelements(SINEs)belongtoaclassofretroposonsthataccountformorethantenpercentofnuclearDNA.
TheroleofSINEsinthehostgenomestillremainstobeclarified;however,recentstudies,includingthosefromourlaboratory,havefoundthatsomeSINE-derivednon-codingsequencesarehighlyconserved(Nishiharaetal.
,2006a).
ThisimpliesthattheseSINEsmighthaveacquiredsomefunctionalityduringtheevolution(Nishi-haraetal.
,2006a;Bejeranoetal.
,2006;Mikkelsenetal.
,2007).
ItmaythereforebeusefultocharacterizeandcategorizethegenomiccomponentsofvariousmammalswithrespecttoSINEs.
Recentcomprehensivegenomesequencingprojectshaveallowedustoinvestigateparticularanimalsonthewhole-genomelevel(e.
g.
Marguliesetal.
,2005),providingaverypowerfultoolforrevealingacompletepictureofSINEevolution.
Indeed,owingtothecompletionofthehumangenomeproject,thecontributionofSINEstothehumangenomehasbeenclarifiedindetail—theAlufractioncoversAvailableonlineatwww.
sciencedirect.
comGene407(2008)176–185www.
elsevier.
com/locate/geneAbbreviations:SINE,shortinterspersedrepetitiveelement;LINE,longinterspersedrepetitiveelement;PCR,polymerasechainreaction;mya,millionyearsago.
ThenucleotidesequencesreportedinthispaperhavebeensubmittedtoGenBankandhavebeenassignedaccessionnumbersAB326393toAB326416.
Correspondingauthor.
DepartmentofBiologicalSciences,GraduateSchoolofBioscienceandBiotechnology,TokyoInstituteofTechnology,4259-B21,Nagatsuta-cho,Midori-ku,Yokohama226-8501,Japan.
Tel.
:+81459245742;fax:+81459245835.
E-mailaddress:nokada@bio.
titech.
ac.
jp(N.
Okada).
0378-1119/$-seefrontmatter2007ElsevierB.
V.
Allrightsreserved.
doi:10.
1016/j.
gene.
2007.
10.
008morethan13%andlonginterspersedrepetitiveelements(LINEs)comprisemorethan20%ofthewholegenome(InternationalHumanGenomeSequencingConsortium,2001).
Furthermore,recentgenomeprojectonshort-tailedopossum(Monodelphisdomestica)haverevealedthatSINEscovermorethan10%andLINEscomprisemorethan29%ofitsgenome(Gentlesetal.
,2007).
Morethan30SINEfamilieshavebeencharacterizedbasedontheirstructure.
UsuallySINEsarecomposedofa5′terminaltRNA-or7SLRNA-relatedregioncontainingapolIIIpromoterandapartnerLINE-related3′tail.
Furthermore,severalSINEfamiliesaregroupedintoasuperfamilybasedonthepresenceofacentralconserveddomain.
Todate,threesuperfamilieshavebeencharacterizedasV-SINEs(Ogiwaraetal.
,2002),Due-SINEs(Nishiharaetal.
,2006a)andCORE-SINEs(GilbertandLabuda,1999),whichexistinvertebrateandinvertebrategenomes.
Amongthesethreesuperfamilies,CORE-SINEsareconsideredtobearatheryounggroupandsomeintactCORE-SINEsarethoughttopossessretropositionalactivityinmammalian(especiallynon-eutherian)genomes(GilbertandLabuda,2000).
TheCOREelement,whichisthecentralconserveddomainofCORE-SINEs,wasinitiallyreportedasmammalianinterspersedrepeats(MIRs)andiswidelydistrib-utedamongmammaliangenomes(Jurkaetal.
,1995;SmitandRiggs,1995).
Later,thisMIRwasdividedintotwofamilies,Ther1(MIRinRepBaseReports)andTher2(MIR3),whicharedistributedamongthegenomesofTheria(extant"Theria"consistsofallmammalsexceptforplatypusandechidnas)(GilbertandLabuda,2000).
MIRsarethemostprevalentrepeatinthehumangenomenexttoAlu,inthatTher1shares2.
2%andTher2shares0.
3%ofthedrafthumangenomesequence(InternationalHumanGenomeSequencingConsortium,2001).
TheTher1andTher2arehighlydivergent,andseemtohavelosttheirretropositionalactivitybeforethesplitofmonotremes,marsupialsandeutherians,whichoccurredmorethan110mya(millionyearsago).
AlthoughtheCORE-SINEslackretro-positionalactivityinthegenomesofeutherians,theyarestillactiveinnon-eutheriangenomes.
GilbertandLabuda(2000)reportedthepresenceofthreeadditionalCORE-SINEfamilies(Mon1,Mar1andOpo1).
ThemembersofthesefamiliesareFig.
1.
SequencealignmentsofthenewlyidentifiedSINEsubfamilies.
(A)TheMar3subfamilyissubdividedintoMar3a,b,c,andd.
(B)TheMac1subfamily.
Thedotsindicatenucleotidesidenticaltotheconsensussequenceatthetop.
TheAboxandBbox,whicharetypicalforthetRNAregionofeachSINEareshownbythickbar.
Thediagnosticnucleotidesforeachsubfamilyareshadedinblack.
TheinsertionsimmediatelyupstreamofallMar3COREdomainscausedbytheduplicationofthe3′endofthetRNA-relatedregionareindicatedbyarrows.
UnderlinednucleotidesindicatethetargetsiteduplicationsofeachSINEloci.
177M.
Munemasaetal.
/Gene407(2008)176–185morerecentthanthoseofTher1andTher2.
Mon1isspecificallydispersedinthegenomesofmonotremesandfurthersubdividedintothreesubfamilies(Mon1a,Mon1bandMon1c).
Mar1isdistributedinthegenomesofalllivingmarsupials.
Incontrast,Opo1hasbeenshowntobedistributedinthegenomesofAmericanmarsupial(Virginiaopossum:Didelphisvirginiana,andGrayShort-tailedOpossum:M.
domestica),andabsentinAustralianmarsupialgenomes.
TheOpo-1ofM.
domesticaisdepositedinRepBaseasSINE_1MD.
Inthepresentstudy,basedongenomiclibraryscreening,wefoundtwoCORE-SINEsthatarecloselyrelatedtoMar1.
ExplorationofthegenomedatabasefortheseSINEs,togetherwithPCRandgenomicdotblotanalysesrevealedthattheyarenewlyrecognizedCORE-SINEs,Mar3andMac1,whicharedistinct,butcloselyrelatedtothemembersofMar1.
Furthermore,detailedsequencealignmentandnucleotidediversityestimationoftheCORE-SINEscharacterizedtodatehaveenabledustoprovideanearlycompletepictureoftheevolutionofthesetwonewSINEs.
DescriptionsofrepetitivesequencessuchasMar3andMac1maybeveryusefulinunderstandingthegeneticcomponentsofnon-eutherianmammalsandforadvancingcomparativegenomeanalysisbetweenbroadmammaliantaxa.
2.
Materialsandmethods2.
1.
DNAsamplesTheDNAsamplesusedinthestudywerefromthefollowinggenomes:Red-neckedwallaby(Macropusrufogriseus),swampwallaby(Wallabiabicolor),westernharewallaby(Lagorchesteshirsutus),northernNailtailwallaby(Onychogaleaunguifera),quokka(Setonixbrachyurus),duskypademelon(Thylogalebrunii),littlerockwallaby(Peradorcusconcinna),yellow-footedrockwallaby(Petrogalexanthopus),Goodfellow'streekangaroo(Dendrolagusgoodfellowi),greaterforestwallaby(Dorcopsishageni),brush-tailedratkangaroo(Bettongiapencillata),long-nosedpotoroo(Potoroustridactylus),muskyratkangaroo(Hypsiprimnodonmoschatus),spottedcuscus(Phalangermacla-tus),HerbertRiverringtailpossum(Pseudochirulusherbertensis),sugarglider(Petaurusbreviceps),mountainpygmypossum(Burramysparvus),feathertailglider(Acrobatespygmaeus),koala(Phascolarctoscinereus),fat-tailedantechinus(Antechinusagilis),tigerquoll(Dasyurusmaclatus),brindledbandicoot(Isoodonmacrourus),Virginiaopossum(D.
virginiana),domesticpig(Susdomesticus),horse(Equuscaballus),housemouse(Musmusculus),andchicken(Gallusgallus).
TotalgenomicDNAswereisolatedbyphenolandchloroformextractionandethanolprecipitation(BlinandStafford,1976)andstoredat4°C.
2.
2.
ScreeningofgenomiclibrariestoidentifynewSINEsWeusedtheP.
breviceps,P.
maclatus,andM.
rufogriseusgenomesasrepresentativesofmarsupialsforscreeningCORE-SINEs.
TotalgenomicDNAswerefirstdigestedwithHindIIIrestrictionendonuclease.
DigestedDNAfragmentswerefractionatedbyultracentrifugationthroughsucrosedensitygradients(10–40%w/v),andtheoptimalsizefractions(about2kbp)wereascertainedbyagarose-gelelectrophoresis.
Fig.
1(continued).
178M.
Munemasaetal.
/Gene407(2008)176–185GenomiclibrarieswereconstructedbyligationoftheDNAfragmentsintotheplasmidvectorpUC18.
DNAprobesusedforscreeningthegenomiclibrarieswerepreparedbyPCRwiththeprimersetMar1For:5′-AGCTAGGTGGCGCAGTGGA-3′andMar1Rev:5′-AGTCGGACACGACTGAAACG-3′usingthegenomeoftheM.
rufogriseusasatemplate.
ThePCRproductswerelabeledinternallywith[α-32P]dCTPbytheprimerextensionmethod.
ThelabeledDNAprobeswerepurifiedusingQIAquickPCRPurificationKit(QIAGEN)andusedforsubsequentscreening.
Southernhybridizationwasperformedat50°Covernightinasolutionof6*SSC,1%SDS,2*Denhardt'ssolution,and100mg/mlherringspermDNAandwashedat50°Cfor50mininasolutionof2*SSCand1%SDS(Sambrook,Fritsch,andManiatis1989).
ThepositivelyhybridizedclonesthatappearedtocontainCORE-SINEsequencewereisolatedandpurified.
PurifiedplasmidsweresequencedusingtheBigDyeterminatorcyclesequencingkit(AppliedBiosystems)intheforwarddirectionusingtheprimerM4:5′-GTTTTCCCAGTCACGAC-3′andinthereversedirectionusingtheprimerRV:5′-CAGGAAACAGCTAT-GAC-3′usinganautomatedsequencer(ABI3100,AppliedBiosystems).
2.
3.
GenBanksearchesTheCORE-SINEsequencesidentifiedbythegenomiclibraryscreenabovewereappliedtotheFASTAprogramwithdefaultparametersinordertoobtainadditionalnewCORE-SINEsequences.
TheseCORE-SINEsequenceswerecompliedandalignedusingClustalX(Thompsonetal.
,1997)andaconsensussequencewasdeducedfromthealignment.
ThisresultedintheestablishmentoftheMar1,Mar3andMac1consensusSINEsequences.
Wealsocollected50sequencesofOpo1SINE,whose3′tailwasnotclarifiedinthepreviousstudybyGilbertandLabuda(2000).
Thealignmentofthese50Opo1SINEsequencesenabledustoclarifythe3′tail.
2.
4.
DotblotanalysisGenomicDNAofG.
gallus,M.
musculus,E.
caballus,S.
domesticus,D.
virginiana,D.
maclatus,I.
macrourus,A.
agilis,P.
cinereus,P.
breviceps,B.
parvus,A.
pygmaeus,P.
maclatus,H.
moschatus,P.
tridactylusandM.
rufogriseuswerespottedontoahybridizationtransfermembrane(GeneScreenPlus,PerkinElmer)usingadot-blotapparatus(modelDP-96,Fig.
2.
ComparisonofmammalianCORE-SINEs.
(A)AlignmentoftheconsensussequencesofmammalianCORE-SINEs.
Thethickunderlinesindicatetheannealingpositionoftheoligonucleotideprobesusedinthegenomicdotblothybridizationanalysis.
TheshorterCOREdomain,characteristicofthenewlyidentifiedSINEsandOpo1,isindicatedbythegraybar.
(B)SchematicrepresentationcomparingthestructureoftheCORE-SINEscharacterizedtodate.
ThepatternswithintheboxesindicatedistinctdiagnosticelementsforeachSINE.
179M.
Munemasaetal.
/Gene407(2008)176–185Advantech).
ThefollowingoligonucleotideprobesweredesignedtoannealspecificallytoconsensusCORE-SINEsequen-ces:Mar3b:5′-GGTCTCAGACACTTAACACTTCCTAGCTGTGT-3′,Mar3c:5′-AGCCTCAGACACTTGACA-CACTTACTAGCTGTGT-3′,Mar3d:5′-AAATCCCGCCT-CAGACACTTAACACAAAAACTAGCT-3′,Mac1:5′-CCCCAATTGCCTCATCCTGGGTCATCTCCAGTCATCC-3′(Fig.
2A).
Theoligonucleotideprobeswereend-labeledwith[γ-32P]ATP.
LabeledprobeswerepurifiedusingtheQIAquickNucleotideRemovalKit(QIAGEN).
TheplasmidDNAscontain-ingconsensusMar3andMac1sequencewereusedaspositivecontrols.
Southernhybridizationwasperformedat55°Covernight,andwashingwasperformedtwotimesat60°Cfor50minutes.
3.
Results3.
1.
NewlyisolatedCORE-SINEsinAustralianmarsupialsTheCORE-SINEsisolatedthroughscreeningofP.
brevi-ceps,P.
maclatus,andM.
rufogriseusgenomiclibrariesconsistedofMar1,Ther1anduncharacterizedSINEs.
TheseuncharacterizedSINEswerefurtherdividedintotwosub-groups.
ThefirstsubgroupconsistsofSINEswitha5′tRNA-derivedregion,aCORE-domain,anda3′polyAtail(Fig.
1A).
Thesecondsubgroupisdistinguishedbyaunique3′tailregioncontainingalong(CA)nterminalrepeat(Fig.
1B),whichisspecifictothissubgroup(Fig.
1B).
AlthoughtypicalCORE-SINEspossessahighlyconserved65bpCOREdomain,theCOREsofthenewlyisolatedSINEsare20bpshorterinlength.
ThisshorterCOREisalsodetectedinOpo1,implyingthesameancestryofthesenewSINEsandOpo1(Fig.
2A,B).
ThenewSINEswereclassifiedaseitherMar3family(Marsupialia3)orMac1family(Macropodoidea1)SINEStoindicatethedistributionoftheSINEfamiliesinthegenomesofallmarsupialsorofmacropods(themembersofthesuperfamilyMacropodoidea),respectively.
Recently,"MAR2_MD"SINEhasbeendepositedinRepBase,butthissequenceisjustashortfragmentofBov-BLINE(RTE-1)andhasnorelationshipwithCORE-SINEs.
Toavoidconfusion,wedonotusethename"Mar2"butuse"Mar3"forthenewlycharacterizedCORE-SINEs.
3.
2.
CharacterizationoftheMar3SINEFig.
1showsthesequencealignmentofMar3andMac1.
ThelengthofMar3variesfrom116to144bpwithoutthepolyAtail.
VariableregionsarelocatedatbothendsoftheCOREdomain.
MostofthemembersofMar3haveadiagnostic7bpinsertionimmediatelyupstreamoftheCOREdomain(Fig.
2B).
Thisdiagnosticinsertionisderivedfromduplicationofthe3′endofthetRNA-relatedregion(Fig.
1A,indicatedbyarrows).
ThemembersofMar3arefurthersubdividedintofoursubfamilies.
WenamedtheseSINEsubfamiliesMar3a,Mar3b,Mar3candMar3d(Figs.
1and2).
Mar3aistheonlysubfamilylackingthediagnosticinsertionupstreamoftheCOREdomain,implyingthatthisgroupisthemostprimitiveMar3SINE.
TheMar3csubfamilyalsohasa5′-GACACACTT-Fig.
2(continued).
180M.
Munemasaetal.
/Gene407(2008)176–1853′insertionimmediatelydownstreamoftheCOREdomain.
SeveralintermediatesbetweenMar3aandMar3cwereobtainedthroughoutthescreening(Fig.
1A),whichsuggeststhatMar3cwasderivedfromMar3a.
BecausewealsoobtainedsuchintermediateswhencomparingMar3awithMar3bandMar3d,thelattertwomembersalsomightbederivedfromMar3a.
Allmembersofthesesubfamilieshave3′polyAtailsthatare4to30bpinlength.
ThepresenceofthepolyAtailinthemembersoftheMar3familyimpliesthat,likeAluinthehumangenome,theyuseL1proteinsforretroposition.
Aftercompletionofourwork,Gentlesetal.
(2007)reportedthepresenceofaSINEfamilynamedSINE2_MDinthegenomeofshort-tailedopossum.
ThisSINE2_MDshareobvioussequencehomologywithMar3SINE,implyingthecommonancestryamongthem.
3.
3.
CharacterizationoftheMac1SINETheconsensussequenceofMac1is310bpinlength.
Mac1iscomposedofa5′tRNA-relatedregion,ashorterCOREdomainwithadiagnosticupstreaminsertionanda3′tailregionFig.
3.
ThedistributionofMar3andMac1amongthegenomesofvariousvertebratetaxa.
(A)DotblotanalysisonmarsupialgenomicDNA(leftpanel)andcontrolplasmidDNA(rightpanel).
Theradiolabeledprobeusedforeachsampleisindicatedatleft.
TheamountofinputDNAisindicatedtotherightofeachpanel.
(B)PCRanalysisofmarsupialgenomicDNAtoconfirmdotblotdata.
PCRproductsfromreactionscontaining10nggenomicDNAand5pmolofthePCRprimersindicatedatleft(seeSupp.
Table1)weresubjectedto3%agarosegelelectrophoresisandstainedwithethidiumbromide.
DNAsizemarkers(bp)areindicatedatright.
181M.
Munemasaetal.
/Gene407(2008)176–185thatisuniquetoMac1.
ThisSINEfamily,thesequencesofwhichcanbefoundinGenBank,wasalsofoundinthegenomesofTammarwallaby(Macropuseugenii)andswampwallaby(W.
bicolor).
The3′tailofMac1isabout250bpandendswith(CA)nrepeats.
ThesequencedivergenceamongthemembersofMac1isverysmall,showingthatthisSINEfamilyseemstobeveryyoung.
3.
4.
ThedistributionofnewSINEsWeinvestigatedthedistributionpatternofMar3andMac1amongvertebratesusingoligonucleotideprobesspecificforMar3b,Mar3c,Mar3dandMac1.
Fig.
3AshowsthedotblotpatterngeneratedbySouthernhybridizationusingeachprobeagainstthegenomicDNAofvariousvertebrates.
ItwasverydifficulttodesignprobestocompletelydistinguishbetweeneachMar3subfamilybecausethenucleotidedifferencesbetweenthesesubfamiliesareverysmall.
Indeed,althoughwedetectedcrosshybridizationofparticularprobestoseveralnegativecontrolplasmids,highlydensesignalswereclearlydetectedinthecaseofthecorrectcombinationofprobesandplasmids.
ThedotblotpatternsshowedtheexistenceoftheMar3b,Mar3candMar3dsubfamiliesinallmarsupialgenomes.
However,becauseofthepossibilityoffalsepositivesduetodotblotcross-hybridization,PCRanalysiswasusedtoconfirmthisconclusion(Fig.
3B).
WesequencedthePCRproductsforeachmarsupialandconfirmedthepresenceoftheMar3b,Mar3candMar3dsubfamilySINEsamongtheirgenomes.
Although,averyweaksignalwasdetectedinthemousegenomewiththeMar3cprobe(Fig.
3A),itwaslikelyanartifactbecausenoMar3csequencewasfoundinthemousegenomedatabaseandnoMar3c-specificPCRproductwasgeneratedfromthemousegenome(Fig.
3B).
AlthoughdotblotsignalsfortheMar3subfamilies'probeswereweakintheD.
virginiana,PCRproductsweresuccessfullyobtained.
ThisdatasuggeststhatMar3ispresentinthegenomeoftheD.
virginiana,butthecopynumberislowand/orthesequencedivergenceislarge,whichresultsinlesseffectivenessoftheprobe.
Mac1showedonlyalimiteddistributionwithinthegenomesofthesuperfamilyMacropodoidea,whichwasconfirmedbythePCRanalysis(Figs.
3A,B).
AlthoughweaksignalwasobtainedinB.
parvus,noobviousPCRbandwasobtained.
Onthecontrary,faintPCRproductsweredetectedinthegenomesofI.
macrourusandA.
agilis,whereasdotblotsignalswerenotobserved.
WeclonedandsequencedthePCRproductsofthesetwospecies,andfoundtheywerenon-specificbands,whichmightbeduetothemissannealingofthePCRprimers.
Therefore,wecanconcludethatMac1isdistributedspecificallyinthegenomesofthesuperfamilyMacropodoidea.
3.
5.
CharacterizationofparticularSINElociSupplementaryFigures1Aand1BshowtheinsertionpatternsoftwoMar3loci(PA031andPA023)andoneMac1locus(MS010).
InsertionofMar3atthePA023locuswasdetectedinbothP.
breviceps(Petauridae)andP.
herbertensis(Pseudocheiridae),implyingacloserelationshipbetweenthem.
AtthelocusPA031,theMar3insertionwasdetectedinallmembersofMacropodoidea,confirmingthemonophylyofthissuperfamily.
AtmanyofthelocirepresentedbyMS010(MS010,MS022,MS032,MS045andMS055),theinsertionofMac1wasdetectedinP.
tridactylus,B.
pencillata,D.
hageni,D.
goodfellowi,P.
xanthopus,P.
concinna,T.
brunii,S.
brachyurus,O.
unguifera,L.
hirsutus,W.
bicolor,andM.
rufogriseus,butnotinH.
moschatus.
ThesedataindicatethatHypsiprimnodonisthemostbasalgenusamongthekangaroosusedinthepresentanalysis.
ThisideaisconsistentwiththeresultofBurketal.
(1998).
4.
Discussion4.
1.
TherelationshipsofCORE-SINEfamiliesWediscoveredtwonovelCORE-SINEfamiliesandclarifiedthedistributionpatternsofthesefamiliesamongmarsupials.
DotblotandPCRanalysesindicatedthatmembersoftheMar3familyarepresentinallmarsupialgenomes.
CORE-SINEfamiliestypicallypossessa65bpCOREdomain,whichishighlyconservedinTher1,Ther2,Mon1andMar1.
However,theCOREdomainofthesenewlyidentifiedSINEfamiliesis20bpshorterthanthecanonicalCOREdomain(Fig.
1).
TheshorterCOREdomainiscommonlyobservedinseveralCORE-SINEs,includingtheOpo1family.
ThepresenceofCORE-SINEspossessingthisshorterCOREregionimpliesthat20bpofthe3′terminalregionoftheCOREdomainlackedimportanceforretropositionduringtheevolutionofmarsupials.
TheMar3familyconsistsofatleastfoursubfamilies.
Basedonthesequencedivergence,theoldestandmostprimitivesubfamilyisMar3a.
ThissubfamilyhasnodiagnosticinsertionimmediatelyupstreamoftheCOREdomain,whereasMar3b,Mar3c,andMar3dsharetheinsertionelement.
Therefore,wespeculatedthatthesethreesubfamiliesarederivedfromMar3a.
ThepresenceofseveralintermediatesequencesbetweenMar3aandMar3cenabledustodescribethetransitionfromMar3atoMar3c(Fig.
1A).
Mar3bandMar3dmightalsobederivedfromMar3a;namely,oneoftheintermediatesequencesbetweenMar3aandMar3ccouldbetheoriginofthesetwosubfamilies(Fig.
4).
TheinsertionintheupstreamregionoftheCOREdomain,whichisdiagnosticofMar3b,candd,isalsoobservedinMac1(Fig.
2A).
Furthermore,Mac1hastheshortCOREdomain,likeallmembersoftheMar3family.
ThesetwolinesofevidencesuggestthatMac1originatedfromoneofthemembersofMar3andproliferatedinthegenomeofthecommonancestorofthesuperfamilyMacropodoidea(Fig.
4).
4.
2.
ThedistributionofCORE-SINEfamiliesFig.
5illustratesthedistributionofeachCORE-SINEinMarsupials.
Mar1isamarsupial-specificSINE(GilbertandLabuda,2000)presentinbothAmericanandAustralianmarsupials.
Byusingthedatabasesearch,wefoundseveralorthologousloci,whereMar1SINEiscommonlypresentinD.
virginianaandM.
rufogriseus(datanotshown),whichindicatesthatthisSINEwasamplifiedbeforetheancestralsplitof182M.
Munemasaetal.
/Gene407(2008)176–185AmericanandAustralianmarsupialsandsuggestsarelativelyoldageforthisSINEgroup.
AccordingtothepreviousstudybyGilbertandLabuda(2000),Opo1showsadistributionspecifictoAmericanmarsupials.
OurresultsindicatethatallmembersofMar3aredistributedamongmarsupials.
However,SouthernblotanalysissuggeststhatMar3occursatalowerfrequencyintheAmericanmarsupial(representedbyD.
virginiana)genomethaninAustralianmarsupials.
Furthermore,severalinsertionsofMar3carespecifictoparticularlineagesofAustralianmarsupials.
Inaddition,averagenucleotidediversitiesofMar3c,andMar3daresmallerthanthatofMar1(Suppl.
Table2).
ThesethreelinesofevidencesuggestthattheproliferationofMar3ismorerecentthanMar1.
Mac1ispresentinthegenomesofallmembersofMacro-podoidea(Figs.
3A,B).
HoweverourSINEinsertionanalysesofeachparticularlocusrevealedthatMac1wasinsertedinallmembersofMacropodoidea(macropods)exceptH.
moschatus(Suppl.
Figs.
1Band2).
Therefore,majorityofthemembersofMac1werelikelyamplifiedsoonafterthedivergenceofH.
moschatusfromothermacropods,whichisestimatedtohavetakenplaceapproximately25myabasedonthemitochondrialDNAsequencesanalysis(BurkandSpringer,2000).
4.
3.
PhylogeneticimpactofSINEinsertionsThephylogenicrelationshipsofmarsupialsarestillenig-maticattheorderandfamilylevel,particularlyamongAustralianmarsupials(Osborneetal.
,2002;Nilssonetal.
,2004).
Toresolvetheseproblems,diagnosticSINEinsertionsprovideusefulinformation(Nikaidoetal.
,1999;ShedlockandOkada,2000;Nishiharaetal.
,2005;Nishiharaetal.
,2006b).
Inthepresentstudy,twophylogeneticallyinformativeinsertionsofMar3weredetected.
AtthelocusPA023,Mar3iscommontoPetauridaeandPseudocheiridae,butisnotfoundinotherdiprotodontianmarsupials,implyingthatthisSINEwasinsertedFig.
4.
IllustrationoftheevolutionaryrelationshipsamongCORE-SINEs.
Fig.
5.
ThesummaryofCORE-SINEevolution.
ThepresenceofactiveSINEsisindicatedbyshadedregions.
TheintensityofshadingisrelativetothespeculatedretropositionalactivityofeachSINEinferredfromthedistributionandthesequencedivergencecalculationsoftheseSINEs.
Thetimescaleisshownbelow.
183M.
Munemasaetal.
/Gene407(2008)176–185inacommonancestorofthesetwofamilies(Suppl.
Figs.
1,2).
Thatis,PA023suggeststhemonophylyofPetauridaeandPseudocheiridae,whicharegroupedinthesuperfamilyPe-tauroideabasedonmorphologicalclassification(AplinandArcher,1987).
ThePA031locussupportsthemonophylyofPotoroidaeandMacropodidae,whicharegroupedinthesuperfamilyMacropodoideabasedonmorphologicalstudy(Gray,1821)(Supp.
Figs.
1,2).
OurmoleculardataalsosupportthemonophyleticrelationshipsofthesuperfamiliesPetauroi-deaandMacropodoideaestablishedbynuclearandmitochon-drialsequencedata(Amrine-Madsenetal.
,2003;Burketal.
,1998;BurkandSpringer,2000;Osborneetal.
,2002).
TheconsistencyofthephylogenetictreesconstructedbySINEinsertions,morphologicalclassificationsorseveralDNAsequencecomparisonanalysesclearlyshowthereliabilityofSINEinsertionsasphylogeneticmarkers.
InthecaseofallMac1lociisolatedinthepresentstudy,SINEinsertionsweredetectedforallmembersofMacropodoideaexceptforH.
moschatus.
ThisSINEdatasuggeststhatPotoroidaearenon-monophyletic,whichhasalsobeensuggestedbyothermoleculardata(Burketal.
,1998).
ThecombinationofourdatawiththatofBurketal.
(1998)suggestsaneedfortaxonomicrevisionforH.
moschatus.
Thus,thesenewlycharacterizedMar3andMac1SINEsmightbeveryimportantforfurtherunderstandingofmarsupialphylogeny,particularlyregardingdiprotodontians.
Especially,Mar3SINEswillprovidereliableinformationfortheinter-familialphylogeny,andMac1SINEforintra-Macropodoideaphylogeny.
Inthisstudy,weidentifiedandcharacterizedtwonewCORE-SINEsinmarsupialgenomesandshowedthattheyarerelativelyyoungSINEscomparedwithTher1,Ther2andMar1,whichhavepreviouslybeencharacterized(GilbertandLabuda,1999,2000).
Thedistribution,sequencedivergenceandstructureofeachCORE-SINErevealedanearlycompletepictureoftheevolutionoftheseSINEs,suggestingtheirpromiseasaphylogenetictool.
OurSINEdatawillprovidethefoundationforfurtherunderstandingofthegenomesandevolutionofmarsupials,whosecompletegenomesequencesarenowrapidlyaccumulatinginthedatabase.
AcknowledgmentsWethankDrs.
KenAplin,YokoSattaandtheKanazawazooforvariousmarsupialsamples.
ThisworkwassupportedbyaGrant-in-AidtoN.
O.
fromtheMinistryofEducation,Science,SportsandCultureofJapan.
AppendixA.
SupplementarydataSupplementarydataassociatedwiththisarticlecanbefound,intheonlineversion,atdoi:10.
1016/j.
gene.
2007.
10.
008.
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