TurkJBiol 31(2007)81-86 ©TÜB‹TAK IdentificationandCharacterizationoftheRatDVL2GeneUsing BioinformaticTools LokmanVARIfiLI,OsmanÇEN DepartmentofBiology,FacultyofArtsandScience,HarranUniversity,fianl›urfa-TURKEY Received:02.10.2006 Abstract: WeidentifiedandcharacterizedtheratDVL2geneusingbioinformatics.Inadditiontothestructureandchromosomal localizationoftheratDVL2gene,thetranscribedandtranslatedproteinproductofthegenewasanalyzedinsilico.Resultss howed thattheratDVL2geneconsistsof15exonsandislocatedontheratgenomiccontigWGA1854.3onchromosome10.Database searchesusingtheratDVL2aminoacidsequenceasaqueryshowedanumberofhomologousproteinsequencesindifferentspecies, includingM.musculus,P.troglodytes,C.familiaris,H.sapiens,B.taurus,D.rerio,X.laevis,and T.nigroviridis.DAX,PDZsignaling, andDEP-conserveddomainstructureswereidentifiedwithintheratDVL2protein. KeyWords: Bioinformatics,DVL2,ratgenome,Wntsignaling BiyoinformatikMetodlarKullanarakS›çanDVL2GenininTan›mlanmas›veKarakterizasyonu Özet: Buçal›flmadabiyoinformatikyaklafl›mlarkullanaraks›çanDVL2geninitan›mlad›kvekarakterizeettik.S›çanDVL2genininyap› vekromozomallokalizasyonunaekolarakgeninkodlad›¤›proteindeinsilicoolarakanalizedildi.Sonuçlar›m›zagöres›çanDVL2geni 15eksondanoluflmuflve10.kromozomdakigenomikkontigWGA1854.3üzerindebulunmaktad›r.S›çanDVL2proteininin M. musculus,P.troglodytes,C.familiaris,H.sapiens,B.taurus,D.rerio,X.laevis ve T.nigroviridis türlerindekihomologlar›vebunlar aras›ndakihomolojioranlar›aminoasitduzeyindebelirlendi.RatDVL2proteinindeDAX,PDZ-SinyalizasyonveDEPkorunmufl domeynyap›lar›oldu¤ubelirlendi. AnahtarSözcükler: Biyoinformatik,DVL2,ratgenomu,Wntsinyalizasyonu Introduction noinformationaboutthegene,suchasstructure, Wntsignalingisconservedinvariousspeciesandplays sequencelength,thepredictedproteinsequence,or importantrolesindevelopmentanddifferentiation(1-5); functionaldomains,ispresent.Therefore,weaimedto however,abnormalWntsignalingmaycause identifyandfurthercharacterizeit. abnormalities,suchascancer(6).Wntsignalsare Herein,weanalyzedtheratDVL2gene,itsstructure, transducedthroughfrizzledreceptors.Theytransduce thechromosomallocalization,predictedpeptide signalstotheintracellularspacebyactivatingdisheveled sequence,anddomainstructures.Wealsoanalyzedits (DVL)proteins(7).TheDVLfamilyproteinsaremulti- homologuesinotherspeciesandtheirevolutionary domain,intracellulartransducerswithimportant relationship. signalingfunctions(8).AllDVLproteinfamilymembers contain3highlyconserveddomains(9):NterminalDAX (alsocalledDIX)(10),centralPDZ(11),andCterminal MaterialsandMethods DEP(12)domains.Thereare3DVLgenes(DVL1,DVL2, IdentificationDVL2geneinratgenome andDVL3)inthehuman(13)andmousegenomes(14- Newgenefragmentswereidentifiedusinginsilico 16),and2DVLgenes(DVL1andDVL3)intherat toolsanddatabasesearches,aspreviouslydescribed(17- genome.Recently,theratDVL2genehasbeengrossly 20).Toidentifytherelatedgenomicclones,aTBLASTN predictedinsilicoandaddedtotheNCBIGenebank searchthroughtheNCBIserver(http://www.ncbi.nlm. database(GeneID:303251);however,inthisannotation, nih.gov/blast)againstnon-redundantdatabaseswas 81 IdentificationandCharacterizationoftheRatDVL2GeneUsingBioinformaticTools performedusingtheaminoacidsequencesofhuman exon-intronboundariesweredeterminedbyexamining DVL2asthequerysequence.Withinthedetermined theconsensussequenceofexon-intronboundariesand genomicregion,putativeCDSwasdeterminedbyusing thecodonusagewithinthecodingregion(Figure1b). theWise2program(http://www.ebi.ac.uk/wise2).To Analysesofdeducedamino-acidsequence identifythecompletemRNA,BLASTnsearchesagainst ESTdbwereperformedusingthenucleotidesequenceof RatDVL2mRNAwasfoundtoconsistofatleast34 putativeCDSofratDVL2asaquerysequence.This bpof5’UTR,2208bpofcodingregion,andatleast575 processwasrepeatedseveraltimesfortheprecise bpofa3’UTRregion(Figure2).TheratDVL2mRNA determinationofthemRNAsequence. waspredictedtoencodeapeptideof736aminoacids. Inordertoestablishthegenomicorganizationofthe TheBLASTPprogramrevealedthatratDVL2has DVL2gene,themRNAsequencewasBLASTedagainstthe 98%,96%,96%,92%,94%,78%,77%,and74% genomeof R.norvegicus .UsingGenomatixsoftware totalaminoacididentitywiththecorrespondingproteins (http://www.genomatix.de)andinspectingthegt-agrule ofM.musculus (CAI35165),H.sapiens (AAH14844),C. ofintronicsequence,usageoftheexonswasdefined. Deducingandanalyzingtheamino-acidsequence 10q.24 ofDVL2gene A Predictionofthecodingregionwasperformedusing theORFFinderprogram(http://www.ncbi.nlm.nih.gov/ 43.660K 43.720K projects/gorf/).TheBLASTPprogram(http://www. Gabarap Acadvl MGC94192 ncbi.nlm.nih.gov/blast/)atNCBIwasusedtoidentifythe Dv12 homologousproteinsinotherspecies. LOC497670 Amino-acidsequencealignmentswereperformed usingtheClustalWprogram(http://www.ebi.ac.uk/ clustalw).RPS-BLAST(http://www.ncbi.nlm.nih.gov/ blast)wasusedforproteindomainapplicationanalysis. Phylogeneticanalysis Thephylogenetictreeswereconstructedusingthe 43.670K 43.680K neighbor-joiningmethod(NJ)withJones-Taylor- DVL2Gene Thornton(JTT)distances.NJsearcheswereconducted usingtheMEGA3program(21).Thereliabilityofinternal brancheswasassessedusing500bootstrapreplicates. B ExpressionAnalysis TheESTscorrespondingtotheratDVL2genewere searchedforwiththeBLASTnprogram(http// www.ncbi.nlm.nih.gov/blast).Then,thetissuesourcesof ESTscorrespondingtothenewgenewerelisted. Results IdentificationofratDVL2gene TBLASTNsearchesusinghumanDVL2mRNArevealed thattheratDVL2genefragmentswerelocatedwithinthe ratgenomecontigWGA1854.3onchromosome10at thenucleotideposition43.670kb-43.680kb,andthat Figure1. (A)StructureandchromosomallocalizationoftheratDVL2 theywereabout10kbinsize(Figure1a).Theprecise gene.(B)Exon–intronboundariesoftheratDVL2gene. 82 L.VARIfiLI,O.ÇEN familiaris (XP_546582),P.troglodytes (XP_511978),B. ExpressionofratDVL2 taurus (XP_870854), X.laevis (AAH90218,XDSH), D. Weinvestigateddifferentialtissueandorgan rerio (NP_997813),and T.nigroviridis (CAG00725), expressionofratDVL2byanalyzingthesourcesofrat respectively.ClustalWalignmentoftheseproteins DVL2ESTs,asdefinedbytheBLASTnprogram(Table). displayedveryhighsimilarityintheNterminalhalfand morediversityattheCterminus(Figure3). RPS-BLASTresultsrevealedthatratDVL2proteins Discussion includeDAX,PDZsignaling,andDEP-conserveddomains TheDVLgenesofvertebratesarehomologouswiththe spanningfromaminoacid13to93,265to352,and DrosophilaDSHgeneandtheyarehighlyconserved 433to505,respectively(Figure2). throughoutevolution.TheproductsofDVLgenesare Phylogeneticsanalysis requiredforthetransductionofWntsignalsfromthecell surfacetothecytoplasm.AlthoughthemechanismofWnt WeconstructedthephylogenetictreeoftheDVL2 signalingfromfrizzledreceptorstocytoplasmisnot proteinsfromthespeciesgivenabovewithMEGA3 understoodveryclearly,DVLproteinsareconsideredthe (Figure4).TheresultsindicatedthatDVL2isconserved intersectionofWntsignaling(24).Therefore,identification amongallorganismsinvestigated,withveryhigh ofWnt-signalingcomponentsinexperimentalanimals,such conservationamongthemammals. astherat,isveryimportantforunderstandingofthe Figure2. RatDVL2mRNAandproteins.RatDVL2proteinsincludeDAX,PDZsignaling,andDEP- conserveddomains.Thesedomainsareboxed. 83 IdentificationandCharacterizationoftheRatDVL2GeneUsingBioinformaticTools Mus musculus MAGSSAGGGGVGETKVIYHLDEEETPYLVKIPVPAERITLGDFKSVLQRPAGAKYFFKSMDQDFGVVKEEISDDNARLPCFNGRVVSWLVSSDTP Rattus norvegicus MAGSSAGGGGVGETKVIYHLDEEETPYLVKIPVPAERITLGDFKSVLQRPAGAKYFFKSMDQDFGVVKEEISDDSARLPCFNGRVVSWLVSSESP Pan troglodytes MAGSSTGGGGVGETKVIYHLDEEETPYLVKIPVPAERITLGDFKSVLQRPAGAKYFFKSMDQDFGVVKEEISDDNARLPCFNGRVVSWLVSSDNP Homo sapiens MAGSSTGGGGVGETKVIYHLDEEETPYLVKIPVPAERITLGDFKSVLQRPAGAKYFFKSMDQDFGVVKEEISDDNARLPCFNGRVVSWLVSSDNP Canis familiaris MAGSGAGGGGVGETKVIYHLDEEETPYLVKIPVPAERITLGDFKSVLQRPAGAKYFFKSMDQDFGVVKEEISDDNARLPCFNGRVVSWLVSSENP Bos taurus MAGSGAGGGGVGETKVIYHLDEEETPYLVKIPVPAERITLGDFKSVLQRPAGAKYFFKSMDQDFGVVKEEISDDNARLPCFNGRVVSWLVSSDNP Xenopus laevis ----------MAETKVIYHLDEEETPYLVKVPVPATDIRLRDFKAALGR-GHAKYFFKAMDQDFGVVKEEISDDNAKLPCFNGRVVSWLVSSEGS Danio rerio ----------MAETKIIYHIDEEETPYLVKIPIAAEKITLLDFKQVLNK-PNYKFFFKSMDQDFGVVKEEISDDSAKLPCFNGRVVSWLVSSDTP Tetraodon nigroviridis ----------MAETKIIYHIDEEETPYLVKIPIAAENITLLDFKQVLNK-PNYKFFFKSMDQDFGVVKEEISDDGAKLPCFNGRVVSWLVSSDTL *** *** ********** * * * * *** * * *** *************** * *************** Mus musculus QPEV---APPAHESRTELVPPP---PPLPPLPPERTSGIGDSRPPSFHPNVSSSHENLEPETETESVVSLRRDRPRRRDSSEHGAGGHRPGGPSR Rattus norvegicus QPET---APPAHESRTELVPPP---PPLPPLPPERTSGIGDSRPPSFHPNVSSSHENLEPETETESVVSLRRDRPRRRDSSEHGAGGHRPGVPSR Pan troglodytes QPEM---APPVHEPRAELAPPA---PPLPPLPPERTSGIGDSRPPSFHPNVSSSHENLEPETETESVVSLRRERPRRRDSSEHGAGGHRTGGPSR Homo sapiens QPEM---APPVHEPRAELAPPA---PPLPPLPPERTSGIGDSRPPSFHPNVSSSHENLEPETETESVVSLRRERPRRRDSSEHGAGGHRTGGPSR Canis familiaris QPEM---APPAHEPRAELAPPP---PPLPPLPPERTSGIGDSRPPSFHPNVSSSRENLEPETETESVVSLRRERPRRRDSSEHGAGGHRPGGPSR Bos taurus QPEM---APPAHEPRTDLAPPP---PPLPPLPPERTSGIGDSRPPSFHPNVSSSRENLEPETETESVVSLRRERPRRRDSSEHGAGGHRPSGPSR Xenopus laevis QPDS---APPAPATEVRPEPPPPVPPPIPPPPAERTSGIGDSRPPSFHPNVSGSTEQLD--QDNESVISMRRDRVRRRESSEQAGVGRGVNG--R Danio rerio AAEP---VAPPVEVPSQPSPPP---PPLPPPPAERMAGIGDSRPPSFHPNTTGSLESLDDQTETESVVSFRRERPRPRESLEQ--HGPRMNGQSH Tetraodon nigroviridis AAEPPVAVVPPVETTAQPSPPP---PPLPPPPAERTGGIGDSRPPSFHPNATGSVETLDEQTETESVVSFRRERPRHRENMEQ--HGPRMNGQSR * ** ** ** * ** ************* * * * *** * ** * * * * * Mus musculus LERHLAGYESSSTLMTSELESTSLGDSDEDDTMSRFSSSTEQSSASRLLKRHRRRRKQRPPRMERTSSFSSVTDSTMSLNIITVTLNMEKYNFLG Rattus norvegicus LERHLAGYESSSTLMTSELESTSLGDSDEDDSMSRFSSSTEQSSASRLLKRHRRRRKQRPPRMERTSSFSSVTDSTMSLNIITVTLNMEKYNFLG Pan troglodytes LERHLAGYESSSTLMTSELESTSLGDSDEEDTMSRFSSSTEQSSASRLLKRHRRRRKQRPPRLERTSSFSSVTDSTMSLNIITVTLNMEKYNFLG