FERNANDAANTUNESALVESDACOSTA Isolamento e caracterização de DETs ( Differentially Expressed Transcripts ) em espécies de peixes: Leporinus macrocephalus () e Danio rerio (Cypriniformes)

Tese apresentada ao Instituto de Biociências da Universidade Estadual Paulista “Júlio de Mesquita Filho”, CampusdeBotucatu,paraobtençãodo títulodeDoutoremCiênciasBiológicas Genética

Orientadora:AdrianePintoWasko

BotucatuSP 2008

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FICHACATALOGRÁFICAELABORADAPELASEÇÃODEAQUIS.ETRAT.DAINFORMAÇÃO DIVISÃOTÉCNICADEBIBLIOTECAEDOCUMENTAÇÃOCAMPUSDEBOTUCATUUNESP BIBLIOTECÁRIARESPONSÁVEL: ROSEMEIREAPARECIDAVICENTE Costa,FernandaAntunesAlvesda. IsolamentoecaracterizaçãodeDETs(DifferentiallyExpressed Transcripts)emespéciesdepeixes: Leporinusmacrocephalus (Characiformes)e Daniorerio (Cypriniformes) / FernandaAntunes AlvesdaCosta.–Botucatu:[s.n.],2008 Tese(doutorado)–InstitutodeBiociênciasdeBotucatu,Universidade EstadualPaulista,2008. Orientador:Profª.DrªAdrianePintoWasko AssuntoCAPES:20200005 1.Genética.2.Peixes.3.Actina. CDD597.55 Palavraschave:Actina; Daniorerio ;DETs;Fabp6; Leporinus macrocephalus.

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...Quandoparamosdeaprenderede progredir,começamosamorrer realmente... C.TorresPastorino Aosmeuspais,JoséMariaeCecíliaeao meu esposo, Rogério, que me amam e me compreendem, sempre me apoiando emtodososgrandesepequenospassos donossocaminho. À minha querida orientadora, Adriane, exemplodepessoaeprofissional.

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AGRADECIMENTOS Agradeço a todos que contribuíram direta ou indiretamente à realização deste trabalho,emespecial: Àminhaorientadora,Profa.Dra.AdrianePintoWasko,pelaconfiança,orientação ededicaçãooferecidasparaelaboraçãoeconclusãodestetrabalhoeparaminha formaçãoprofissional; À FAPESP – Fundação de Amparo à Pesquisa do Estado de São Paulo, pelo apoiofinanceiroconcedido; AosProfs.Drs.CesarMartinseMaeliDalPaiSilvapeloapoiodiretonarealização destetrabalho; AosProfs.Drs.FaustoForesti,ClaudioOliveira,IvandeGodoyMaia,LígiaSousa Lima Silveira daMota,João Pessoa, Paulo EduardoRibolla, AlexandreSecorun Borges,peloapoioesugestões; À Dra. Debora Colombi, pela preciosas sugestões quanto às análises de PCR quantitativo; Aos Laboratórios de Biologia Molecular (Departamento de Genética), Biotecnologia e Genética Molecular – BIOGEM (Departamento de Genética) e Biologia e Genética de Peixes (Departamento de Morfologia). À Pósgraduação em Ciências Biológicas (Genética), ao Instituto de Biociências e à Universidade EstadualPaulista,pelaoportunidadecedidaparaarealizaçãodestetrabalho; Aos companheiros do Laboratório de Biologia Molecular Animal: Magali, Cassiane, Ana Teresa, Thaís, Juliana e alunos do Prof. Dr. Alexandre Secorun Borges–Fernanda,Peres,PauloeZéFilho; AoscompanheirosdoLaboratóriodeBiologiaeGenéticadePeixes:Alex,Andréia Alves, Andréia Poletto, Celso, Claudinha, Cristiane, Daniela, Danillo, Emanuel, Guilherme (Kabelo), Irani, Jefferson, Juliana, Karina, Kelly, Lessandra, Lígia, Luciana,LuizHenrique,Márcio,Marina,Marisa,PatríciaElda,Ricardo,Tatianee Welcy AosalunosdaProfa.MaeliDalPaiSilva,emespecial:Eduardo,FernandaLosie FernandaCarani,pelaenormeajuda; AosalunosdoLaboratóriodeBiotecnologiaeGenéticaMolecular–BIOGEM,em especialaoFlávio(Bonsai),Fábio,TâniaeVirgínia. À colega Magali, pela amizade, carinho e compreensão e por estar sempre presenteedispostaameajudarousimplesmenteparadizer“euentendo”,coisas quesomenteosamigossabemoverdadeirosignificado;

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Ao Prof. Dr. Luis Fernando Marins e ao Sr. Odval Stradioti, Piscicultura Água Milagrosa,pelogentilfornecimentodosanimais; AoProf.Dr.LuisFernandoMarinseseusalunos,peloapoioediscussõesepela acolhida; AosProfs.Drs.JonathanM.WrighteEileenDenovanWrighteStephenMockford pela grande oportunidade oferecida, pelos muitos ensinamentos e, em especial, pelocarinhoeatençãocomquemereceberam; ÀProfa.Dra.SilviaReginaRogattoeseusalunos,emespecialàElianeeGrace, peloauxílionodesenvolvimentodashibridaçõesfluorescentes insitu; Aos funcionários dos Departamentos de Genética e Morfologia pelo auxílio atribuídodurantearealizaçãodestetrabalho; Aos amigos e familiares pela presença e apoio durante os vários momentos de minhavida; Aosmeuspaispelocarinhoeapoioconstantes,pormeensinaremsobreavidae avivereportodadedicaçãoincondicionalàminhafelicidade; Ao meu amado esposo, Rogério, por completar minha felicidade, por me apoiar em todos os momentos e em todas minha decisões, por sempre buscar o meu bemepormeamarcomoeusou. Muitoobrigada!!

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SUMÁRIO

RESUMO ...... 01

ABSTRACT ...... 03

CONSIDERAÇÕES INICIAIS E OBJETIVOS ...... 05

INTRODUÇÃO GERAL ...... 08 Transcritosdiferencialmenteexpressos...... 08 Aespécie Leporinusmacrocephalus ...... 12 Aespécie Daniorerio ...... 15 Referências ...... 17

RESULTADOS E DISCUSSÃO ...... 30 CAPÍTULO 1 ...... 31 Molecularorganizationandexpressionanalysisofskeletalactingenesfrom white and red fish muscle tissues: insights into the musculature energetic demands ...... 32 Abstract ...... 32 Introduction...... 33 MaterialandMethods...... 36 ResultsandDiscussion ...... 42 Acknowledgements ...... 53 References ...... 53 Table,FiguresandLegends...... 60 CAPÍTULO 2 ...... 68 Identificationofsexuallydimorphicgeneexpressioninbraintissueofthefish Leporinus macrocephalus through mRNA differential display and real time PCRanalyses ...... 69 Abstract ...... 69

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Introduction...... 70 MaterialandMethods...... 73 ResultsandDiscussion ...... 77 Acknowledgements ...... 86 References ...... 87 Table,FiguresandLegends...... 97 CAPÍTULO 3 ...... 105 Spatiotemporal distribution of fatty acid binding protein 6 ( fabp6 ) gene transcriptsinthedevelopingandadultzebrafish(Danio rerio )……….106 Abstract ...... 106 Introduction...... 107 MaterialandMethods ...... 110 ResultsandDiscussion...... 112 Acknowledgements ...... 120 References ...... 120 Table,FiguresandLegends...... 124 CAPÍTULO 4 ...... 132 A comparative expression analysis of gene transcripts in brain tissue of wildtype and GHtransgenic zebrafish ( Danio rerio ) using DDRTPCR approach………………………………………………………………………133 Abstract ...... 133 Introduction...... 134 MaterialandMethods...... 136 ResultsandDiscussion ...... 139 Acknowledgements ...... 146 References ...... 147 Table,FiguresandLegends...... 155 CONSIDERAÇÕES FINAIS E CONCLUSÕES ...... 161 SÚMULA CURRICULAR ...... 163

RESUMO O presente estudo teve o intuito de isolar e caracterizar transcritos diferencialmenteexpressosemespéciesdepeixes,visandocontribuircomnovos dados genéticos acerca deste grupo basal de vertebrados. Desta forma, diferentes análises foram realizadas em Leporinus macrocephalus (piauçu) (Characiformes, ), espécie de peixe que representa um importante recursoalimentarquetemsidointensamenteexploradonaaqüiculturasubtropical equeapresentaumsistemadedeterminaçãosexualZZ/ZW,etambémem Danio rerio ( zebrafish ) (Cypriniformes, Cyprinidae), um organismo modelo já bem estabelecido em diferentes áreas da pesquisa científica. Os resultados indicaram que:(1)Asαactinasesqueléticasdetecidomuscularbrancoevermelhoparecem ser genética e funcionalmente distintasnão somente em L. macrocephalus como tambémemoutrasespéciesdepeixes,característicaque,atéomomento,nãofoi encontrada em outro grupo de vertebrados. Além disso, as características de cadatipodeactinapodemserrelacionadasàsdemandasenergéticasdostecidos musculares branco e vermelho; (2) Um expressão dimórfica entre machos e fêmeasocorreemtecidocerebralde L.macrocephalus ,relacionadaaogeneque codificaaproteínaAPBA2( amyloidbeta(A4)precursorproteinbinding,familyA, member2 ),queestáassociadaàproduçãodaproteínaβamilóideaqual,porsua vez, é relacionada à doença de Alzheimer; (3) A estrutura do gene fabp6 ( fatty acidbinding protein 6 ) de D. rerio é similar à maioria dos outros membros da família multigênica fabp de outros vertebrados, e sua distribuição tecidual em embriões,larvaseadultosdaespécietambémésimilaràencontradaemoutros organismos, especialmente mamíferos; (4) Os genes que codificam a proteína p300 ( E1A binding protein p300 ) e a proteína ADCY2 (adenilato ciclase 2) representam dois genes candidatos diferencialmente expressos entre amostras nãotransgêncas e homozigotas e hemizigotas transgênicas para o gene do hormônio do crescimento (GH), reforçando dados prévios que evidenciam que estas proteínas estão envolvidas na formação/crescimento muscular e liberação de GH. Desta forma, os dados obtidos contribuem para a compreensão da evolução, função e organizaçãomolecular de diversos genese podem servirde base a futurosestudos.Além disso, osresultadosobtidosconfirmamautilidade de Danio rerio como espécie modelo em estudos genéticos e indicam que 2

Leporinus macrocephalus representa um potencial modelo experimental em análises direcionadas a genes envolvidos no desenvolvimento de doenças humanas,comoalteraçõesneurodegenerativas,distrofiasmuscularesecâncer.

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ABSTRACT Thepresentstudyintenttoisolateandcharacterizedifferentiallyexpressed transcriptsinfishspeciesinordertocontributetonewgeneticdataonthisbasal vertebrate group. Therefore, different analyses were performed in Leporinus macrocephalus (piauçu) (Characiformes, Anostomidae), a fish species that represents an important food resource that has been intensively exploited in the subtropicalaquacultureandthatpresentsaZZ/ZWsexdeterminationsystem,and alsoin Daniorerio (zebrafish)(Cypriniformes,Cyprinidae),awellestablishedmodel organism in different areas of scientific research. The results indicated that: (1) Theαskeletalactinsfromwhiteandredmuscletissuesappeartobesomewhat geneticallyandfunctionally distinguishablenotonlyin L.macrocephalus butalso inotherfishspecies,afeaturethatwasnotfoundinothervertebrategroupssofar. Moreover,thecharacteristicsofeachactintypecouldberelatedtotheenergetic demands of white and red muscle tissues; (2) A sexually dimorphic gene expression occurs in brain tissue of males and females of L. macrocephalus relatedtothegenethatcodesfortheproteinAPBA2(amyloidbeta(A4)precursor proteinbinding,familyA,member2),thatisassociatedtotheproductionoftheβ amyloidproteinwhich,inturns,isrelatedtotheAlzheimerdisease;(3)The fabp6 (fatty acidbinding protein 6) gene structure of D. rerio is similar to most of the other members of the multigenic fabp family of other vertebrates, and its tissue distributioninembryos,larvae,andadultsofthespeciesisalsosimilartotheone found in other organisms, specially mammals; (4) The genes that code for the protein p300 (E1A binding protein p300) and for the ADCY2 protein (adenylate cyclase 2) represent two candidate differentially expressed genes among non transgenic and growth hormone (GH)transgenic homozygote and hemizygote samples of Danio rerio , reinforcing previous data that evidenced that these proteins are involved in muscle formation/growth and in GH release. Therefore, theobtaineddatacontributestothecomprehensionoftheevolution,function,and molecularorganizationofseveralgenesandcanbeusedasbasalinformationon futurestudies.Moreover, theresultsconfirmtheutilityof Daniorerio asamodel speciesingeneticanalysesandindicatethat Leporinusmacrocephalus represents apotentialexperimentalmodelinanalysesofgenesinvolvedinthedevelopment

4 of human diseases, as neurodegenerative alterations, muscle dystrophies and cancer.

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Considerações Iniciais e Objetivos

Nos últimos anos, vários estudos genéticos têm dedicado especial importânciaaanálisesdeexpressãogênicadiferencial.Estesestudospermitema caracterização de genes e identificação e avaliação do nível de expressão de determinadostranscritosemrelaçãoadiversasvariáveis.Destaforma,épossível analisarecomparar,porexemplo,amostrasrelacionadasadiferentesperíodosde desenvolvimento ou a tecidos distintos, submetidas a diferentes tratamentos, presentesemdistintosambientes,oumesmoamostrasdediferentessexosede organismostransgênicosenãotransgênicos.

Entre as técnicas mais utilizadas para isolamento e identificação de transcritos diferencialmente expressos (DETs Differentially Expressed

Transcripts ), destacamse: RTPCR ( Reverse Transcription Polymerase Chain

Reaction ), DD ( Differential Display), SH ( Suppression Hybridization ), SSH

(SuppressionSubtractiveHybridization ),RAPPCR( RNAArbitrarilyPrimedPCR ),

RDA ( Representational Difference Analysis ), SAGE ( Serial Analysis of Gene

Expression ), microarrays de DNA, PCR em tempo real, hibridação in situ e

Northernblotting .Dasmetodologiascitadasacima,RTPCR,DD,PCRemtempo realehibridação insitu foramutilizadasnopresentetrabalho.OsmétodosdeRT

PCR e DD, além de exibirem considerável eficiência e ampla aplicação em estudos de expressão diferencial, caracterizamse pela simplicidade, custo relativamentereduzidoe,emespecialparaDD,nãorequeremumconhecimento préviodogenomaquesedesejaestudar.

Os trabalhos relacionados à identificação e caracterização de DETs abrangem grande diversidade de organismos e incluem análises em vários modelos experimentais como bactérias, fungos, plantas, invertebrados e

6 vertebrados. Entretanto, esta área de pesquisa é ainda recente e, entre os vertebrados, embora os peixes representem um dos grupos mais diversificados, atéomomento,poucasespéciesapresentamseusgenomascaracterizados.

Entre os peixes mais amplamente utilizados em estudos genômicos, destacase a espécie Danio rerio , popularmente conhecida como zebrafish ou paulistinhaedegrandeimportânciaeconômicaparaaáreadeaquariofilia.Apesar de nativa do continente asiático, apresenta ampla distribuição geográfica, podendo ser encontrada em quase todos os países do mundo e representa um dosmaisimportantesmodelosanimaisutilizadosemestudosdedesenvolvimento embrionário, comportamentais, fisiológicos e genéticos. A facilidade de manipulação genética nesta espécie permitiu o desenvolvimento de várias linhagenstransgênicas,oquetemcontribuídoparaodesenvolvimentodenovos estudos relacionadosà expressão gênica.Atualmente,ogenomade Daniorerio apresentase quase totalmente caracterizado (~ 75%) devido ao projeto “ Danio rerio Sequencing ”, iniciado em 2001 pelo Wellcome Trust Sanger Institute para pesquisasgenômicas.Atualmente,inúmeraspesquisas,emdiferentesáreas,vêm sendodesenvolvidasem D.rerio ,especialmentedirecionadasàcriaçãodenovas linhagens transgênicas e à compreensão do surgimento e desenvolvimento de doençashumanas,comocâncereprocessosdegenerativos.

Por outro lado, trabalhos genéticos direcionados para diversos outros peixessãoextremamenteescassose,paraumgrandenúmerodeespécies,são aindainexistentes.Nestaperspectiva,oterritóriobrasileirorepresentaumafonte imensa de recursos ainda inexplorados, especialmente levandose em consideração que várias espécies apresentam estimável valor comercial e ecológico, sendo muitas delas endêmicas. Entre estas espécies, podese citar

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Leporinus macrocephalus , popularmente conhecida como piauçu ou piavuçu,

nativa do Pantanal e que representa uma importante fonte alimentar que, nos

últimos anos, tem sido intensivamente explorada como recurso natural e de

pisciculturanoBrasil.Apesardesuaimportânciaeconômica,osdadosgenéticos

acercadestaespéciesãoaindaextremamentereduzidos.

Dadaaimportânciade Daniorerio e Leporinusmacrocephalus ,opresente

trabalho teve o intuito de isolar e caracterizar transcritos diferencialmente

expressosnestasduasespécies,visandoampliarosdadosgenéticosempeixes,

grupoqueocupaumaposiçãobasalnahistóriaevolutivadosvertebrados.Desta

forma, a aquisição de um maior número de informações genéticas em espécies

de peixes pode contribuir à compreensão da evolução, função e organização

moleculardediversosgenes.Alémdesteobjetivogeral,foramtraçadosobjetivos

específicos, relacionados a diferentes subpropostas de pesquisa, detalhados

abaixo.

• Identificar e caracterizar genes de actina de tecido muscular esquelético

brancoevermelhode Leporinusmacrocephalus .

• Identificarecaracterizar transcritos diferencialmente expressosemtecido

cerebraldemachosefêmeasde Leporinusmacrocephalus .

• Determinaraestrutura,aorganizaçãoeadistribuiçãoespaçotemporaldo

gene fabp6 (fattyacidbindingproteintype6 )em Danio rerio .

• Identificarecaracterizar transcritos diferencialmente expressosemtecido

cerebraldelinhagensde Daniorerio transgênicasenãotransgênicaspara

ogenedohormôniodocrescimento(GH) .

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Introdução Geral

Transcritos diferencialmente expressos

Nasúltimasdécadas,pôdesepresenciarumconsiderávelcrescimentoda genômica,áreaqueserefereàaquisiçãodedadosdogenoma,especialmenteà obtençãodaseqüênciacompletadomaterialgenéticodosorganismos.Emboraa obtençãodestainformaçãonãosejaumatarefasimples,aampliaçãodestaárea depesquisaestáintimamenterelacionadaaosconhecimentosgenéticosgerados comoadventodosdenominadosProjetosGenomas.Atualmente,váriosprojetos quevisamdesvendarocódigogenéticovêmsendodesenvolvidosemorganismos diversos como bactérias, fungos, plantas, invertebrados e vertebrados e os resultadosgeradospodemrepresentardadosbásicosparaodesenvolvimentode trabalhos posteriores que visem compreender os mecanismos de expressão de diversos genes (Carneiro et al. 2006). Além dos dados de projetos de seqüenciamentototaldogenomadeváriasespécies,resultadosdeprojetosque objetivamaconstruçãodebibliotecasdeESTs( ExpressedSequenceTags )são diretamente utilizados em pesquisas voltadas à elucidação dos mecanismos biológicos envolvidos na expressão gênica e em sua regulação (Carneiro et al.

2006).

Emboraabordagensmaisamplasdeanálisedeexpressãogênicaemnível genômico ( GenomeWide Expression Profiling ) constituam o maior desafio atual para identificação e análise simultânea de um grande número de genes envolvidos nos mais diversos processos biológicos desde o desenvolvimento dosorganismosatésuasinteraçõescomfatoresambientais(Donson etal. 2002) projetos pontuais, direcionados à identificação e caracterização de genes

9 específicos, também vêm sendo realizados em diversas espécies. Através da aplicação de diferentes tecnologias, análises deste tipo podem gerar dados primordiais para futuros projetos genômicos de larga escala (Matz & Lukyanov

1998,Ponsuksili etal. 2001).

Nas últimas décadas, inúmeras técnicas em biologia molecular foram desenvolvidas visando facilitar o isolamento e a caracterização de transcritos diferencialmenteexpressos(DETsDifferentiallyExpressedTranscripts ),ouseja, genes expressos como RNAs mensageiros (RNAm) para síntese de proteínas que diferem em abundância entre amostras de tipos celulares ou tecidos específicos e cuja regulação pode estar relacionada a mecanismos ambientais, fisiológicose/ouquímicos.Entreestasdiversasmetodologias,podemsercitadas

RTPCR ( Reverse TranscriptionPolymerase Chain Reaction ) (Kawasaki 1990), hibridação subtrativa (Duguid et al. 1988), DD ( Differential Display ) (Liang &

Pardee1992),RAPPCR( RNAArbitrarilyPrimedPCR )(Welsh etal. 1992),RDA

(Representational Difference Analysis ) (Lisitsyn et al. 1993), SAGE ( Serial

Analysis of Gene Expression ) (Velculescu et al. 1995), microarrays de DNA

(Shena et al. 1995), SSH ( SuppressionSubtractiveHybridization )(Diatchenko et al.1996),PCRemtemporeal(Freeman etal. 1999),hibridação insitu (Wilkinson

1998)e Northernblotting (Alwine etal. 1979).

Emboraestavastagamadetécnicasparaisolamentoecaracterizaçãode transcritos diferencialmente expressos permita sua aplicação em estudos de diversas áreas, a escolha de uma destas metodologias deve considerar as características dos experimentos, os objetivos propostos e o organismo em estudo(Matz&Lukyanov1998).Alémdisso,apesardesuasinúmerasvantagens, normalmente, a aplicação de metodologias experimentais para prospecção de

10 transcritos diferencialmente expressos nem sempre representa uma tarefa fácil, pois,namaioriadasvezes,estesapresentamníveisdeexpressãoextremamente baixos. Entre os milhares de genes que compõem o genoma dos organismos vivos, somente cerca de 1015% são expressos em um determinado tipo de tecidoduranteumdadoperíodo,oquetornarelativamentedifícilsuaidentificação e caracterização (Liao & Zhang 2006). Além disso, as tecnologias disponíveis paraoisolamentodeDETssãogeralmentelaboriosasedealtocusto.

O crescente interesse por estudos de transcritos diferencialmente expressospodesercomprovadopelapublicaçãodemaisde30.000trabalhosnas mais diversas áreas de aplicação e pelas inúmeras revisões sobre o tema

(Medline, Julho de 2008 PubMed, NCBI Web site http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed ). Grande parte das pesquisas baseadas em genes que apresentam expressão diferencial entre amostras distintas vem sendo direcionada à biologia do desenvolvimento

(Kanamori2000,Xie etal .2001,Ando&Sakai2002,Anway etal .2003,Dann et al . 2003, Kim et al . 2004, Sunagawa & Magae 2005, Jianzhen et al . 2007,

Williams etal .2007,Chu etal .2008,Elis etal .2008,Sugimoto&Endoh2008), biologia celular e fisiologia de tecidos (Eriksson et al . 1999, Ahmed et al . 2000,

Boag etal .2000,Kim etal. 2001,Blásquez&Piferrer2004,Saunders&Magor

2004,Azumi etal. 2005,Venkatesh etal. 2005,Jin etal. 2006,Koury etal. 2007,

Bevan etal. 2008,Cervigni etal. 2008,Kim etal. 2008,Smartt&Erickson2008,

Wong et al. 2008, Zhang et al. 2008) e a processos tumorais e diagnóstico de diversas doenças (An et al . 2000, Miller et al. 2001, Lee & Cicila 2002, Hoei

Hansen etal. 2004,Li&Chen2004,Clinton etal. 2005,Kim etal. 2005,Poulin&

Labelle2005,Sharma etal. 2005,Zhang etal. 2005,Blaes etal. 2007,Collins et

11 al. 2007,Nagai etal. 2007,Kamalian etal. 2008,Shin etal. 2008),visandogerar informaçõessobreafunçãogênicaesuasrelaçõescomprocessosmetabólicose fisiológicos.

Embora análises nas áreas de biologia do desenvolvimento, fisiologia e carcinogênese representem a maior gama de dados relativos a transcritos diferencialmente expressos, outras áreas de pesquisa também têm merecido destaque. Uma destas áreas referese à caracterização de DETs em relação a alterações ambientais como contaminação, variação drástica de temperatura e/oudisponibilidadedeágua(Llado etal. 1998,Casagrande etal. 2001,D’Cotta etal. 2001,Carginale etal. 2002,Chang etal. 2004,Basile etal. 2005,Meyer et al. 2005,Markovskaya etal. 2007,Mishra etal. 2007,Li etal. 2008,SmithKeune

&Dove2008,Xu etal. 2008)eatratamentosquímicos(Denslow etal. 2001a,

Denslow et al. 2001b, Miller et al. 2001, Wong et al. 2002, Akbar et al. 2004,

HosoiTanabe et al. 2005, Badonel et al. 2007, CaamalVelázquez et al. 2007,

Zhang etal. 2007,Anderson etal. 2008,Karakurt&Huber2008,Kavar etal 2008,

Torelli etal. 2008).Estudoscomesteenfoquevêmtambémsendo,aolongodos anos, ampliados, especialmente devido ao crescente aumento dos problemas geradosporalteraçõesambientaisantrópicas.

Apesardograndenúmerodetrabalhosdirecionadosaanálisesdeexpressão gênicadiferencial,queabrangemumagrandediversidadedeorganismos,estaárea depesquisaéaindarecente.Particularmenteempeixes,atéomomento,existem poucos dados, especialmente se considerarmos que estes representam um dos grupos de vertebrados mais diversificados (Nelson 1994). Grande parte dos trabalhos de expressão gênica diferencial restringese a espécies nativas da

AméricadoNorteeEuropa,especialmentedepeixesdeinteressecomercial,como

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Salmoniformes, visandogerarbenefíciosparaapiscicultura(Parrington&Coward

2002). Estes estudos incluem o isolamento e a caracterização de genes relacionados à determinação sexual (D’Cotta et al. 2001, Denslow et al. 2001a,

Denslow etal. 2001b),aocrescimentoedesenvolvimento(Kanamori2000,Xie etal.

2001, Dann et al. 2003), à resistência a patógenos (Collins et al. 2007) e a condiçõesambientaisadversas(Carginaleetal. 2002,Larkin etal. 2003,Meyer et al. 2005).Alémdisso,espéciesdepeixesconsideradasmodelosanimaistambém têm sido alvo de estudos em expressão gênica diferencial. Devido ao genoma compacto(390Mb)eaoconteúdodegenessimilaraodehumanos, Fugurubripes

(baiacu ou pufferfish ) tem sido explorado como um modelo do genoma de vertebrados para identificação de genes e outros elementos funcionais nos genomasdeoutrosanimais(Brenner etal. 1993).

Desta forma, a ampliação de análises genéticas em peixes, que compreendem um grupo vertebrado basal, poderá levar à identificação e caracterizaçãodediversosgenes,assimcomofornecermaioresinformaçõessobre suaorganização,funçãoeevolução.

A espécie Leporinus macrocephalus

A família Anostomidae, grupo de peixes da ordem Characiformes relativamentericoemespéciesdistribuídasportodaaAméricadoSuleAmérica

Central, compreende 12 gêneros: Abramites, Anostomus, Gnathodolus,

Laemolyta, Leporellus, Leporinus, Rhytiodus, Schizodon, Synaptolaemus,

Anostomoides, Pseudanos e Sator . Entre estes, o gênero Leporinus é o mais diverso,possuindo87espéciesválidas(Garavello&Britski2003).

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Popularmente conhecida como piauçu ou piavuçu e nativa da região do

Pantanal, a espécie Leporinus macrocephalus (Figura 1) representa um dos principaispeixesdepescadesubsistênciaedepescaesportivanoBrasil(Garavello

&Britski1988,Martins&Yoshitoshi2003).Alémdisso,seucultivoemestaçõesde piscicultura tem se mostrado um empreendimento promissor, devido a seuhábito alimentar omnívoro, facilidade de adaptação às condições de cativeiro, bom desempenho reprodutivo e excelente taxa de crescimento (Corrêa Filho 2000,

Takahashi et al. 2004). A espécie é gonocorística, ou seja, os indivíduos se desenvolvem somente como machos ou somente como fêmeas e permanecem, assim,durantetodoseuciclodevida.Emboramachosefêmeasdaespécieatinjam, na maturidade, cerca de 60 cm de comprimento, diferenças de peso e tamanho podemserobservadasentreossexosemindivíduosadultosmachosdaespécie apresentam um maior tamanho corporal (J. Senhorini CEPTA/IBAMA, comunicaçãopessoal).

Dada a importânciade L. macrocephalus como recursoalimentar,diversos estudos voltados à criação da espécie em cativeiro vêm sendo realizados, especialmente análises de crescimento, alimentação e reprodução (Soares et al.

2000,Pinto etal. 2001,Nagae etal. 2002,Ribeiro&Godinho2003,Minucci etal.

2005, Normandes et al. 2006, Godinho 2007, Albinati etal. 2007). Além disso, L. macrocephalus tem sido, nos últimos anos, uma das espécies de destaque para pescaesportiva,especialmentenosestadosdosulesudestedoBrasil(Queiroz et al. 2002).

14

3,09cm

Figura 1 :Exemplaradultode Leporinusmacrocephalus (piauçuoupiavuçu).

Poroutrolado,osestudosgenéticosreferentesa Leporinusmacrocephalus são escassos e a maioria das análisesrefereseadadoscitogenéticos.Embora grande parte das espécies que compõem o gênero Leporinus não apresente cromossomos sexuais morfologicamente diferenciados entre machos e fêmeas

(Galetti et al. 1995, Venere et al. 2004), L. macrocephalus é caracterizado por apresentar um cariótipo composto por 54 cromossomos e, em especial, por apresentarumsistemadedeterminaçãosexualdotipoZZ/ZW,oquecaracteriza uma heterogametiafeminina. Assim, asfêmeaspossuemumparcromossômico heteromórficoconsistindodeumcromossomosubmetacêntrico(cromossomoZ), equivalenteaocromossomon o 2dosmachos,eumcromossomosubtelocêntrico

(cromossomo W), o maior do complemento cariotípico e ausente nos machos

(Galetti & Foresti 1987). Tal característica torna a espécie extremamente interessantepara estudosrelacionadosàdeterminação ediferenciaçãosexuale paraestudossobreaevoluçãodoscaracteresenvolvidosnestesprocessos.

As demais análises genéticas em L. macrocephalus referemse à caracterização do DNA ribossomal5S paraanálises filogenéticas(Ferreira etal.

2007) e isolamento e caracterização de locos microssatélites para utilização em

15 estudos de diversidade genética populacional (Morelli et al. 2007). Embora os dadosgenéticosem L.macrocephalus sejamaindaextremamenterestritos,este cenáriotendeamudarnospróximosanos.Ocrescentedesenvolvimentodosetor depisciculturanoBrasildevidoaograndepotencialhídrico,àenormeriquezade espéciesnativaseàsáreasdeclimapropícioàatividadeencontradasnopaís, associado à disponibilidade de diversas técnicas de biologia molecular, deverão levar à implementação de novas análises genéticas em espécies de peixes de interessecomercial,incluindo Leporinusmacrocephalus .

A espécie Danio rerio

A espécie Danio rerio (Figura 2) (Ordem Cypriniformes, Família

Cyprinidae), popularmente conhecida como paulistinha ou zebrafish , embora nativadoSuleSudestedaÁsia,tornouseamplamentedistribuída,podendoser encontrada em todos continentes. Características como a fácil adaptação ao ambiente de cativeiro, o espaço relativamente pequeno necessário para sua manutenção,aprolenumerosa,aproduçãocontínuadeovoseoperíodocurtode desenvolvimentoembrionárioelarvalrepresentamaspectosinteressantesparaos produtores,especialmentedaáreadeaquariofilia(Westerfield2000).Alémdestas características, Danio rerio é considerada uma espécie modelo em estudos de desenvolvimento embrionário, comportamentais, fisiológicos e genéticos de vertebrados, especialmente devido à transparência de estruturas como ovos e larvas,quepermitemanálisesdiretasdosestágiosembrionáriosedaexpressão espaçotemporal de determinados genes de interesse, através da aplicação de métodosdehibridação insitu (Detrich etal. 1998,Dahm&Geisler2006,Dahm et al. 2007,Bass&Gerlai2008,Fadool&Dowling2008).

16

0,53cm

Figura 2 :Exemplaradultode Daniorerio (paulistinhaou zebrafish ).

Aocontráriodediversasoutrasespéciesdepeixes,entreestas Leporinus macrocephalus , D.rerio apresentainúmerosdadosreferentesàcaracterizaçãode seugenoma.Aespécieapresentaumcariótipocompostopor50cromossomose nãoapresentacromossomossexuaismorfologicamentediferenciados,sendoseu mecanismo de determinação sexual desconhecido e, até o momento, nenhum generelacionadoàdeterminaçãosexualfoiidentificado(Amores&Postlethwaite

1999, Sola & Gornung 2001). Acreditase que fatores ambientais e ecológicos, comotemperaturaedensidadepopulacional,podeminfluenciarnadeterminação dosexode zebrafish (Lawrence etal. 2008).

Atualmente, aproximadamente 75% do genoma de Danio rerio já se encontra caracterizado, devido ao projeto de seqüenciamento iniciado em 2001 pelo Wellcome Trust Sanger Institute (Cambridge, Inglaterra) para pesquisas genômicas, e uma rede de dados ( Zebrafish Information Network ZFIN: http://www.zfin.org )foicriadacomointuitodeintegrardiversosdadosreferentes aogenomadestaespéciedepeixe(Spragueetal.2003).Alémdisso,nosúltimos cincoanos,comoadventodenovastécnicasbiotecnológicaseexperimentaisde análisesgenômicas,onúmerodeestudosgenéticosnestemodeloanimalcresceu

17 significativamente mais de 640 mil ESTs ( Expressed Sequence Tags ) que representamtranscritosde zebrafish foramseqüenciadosemaisde6.800RNAs mensageirosforamidentificadosecaracterizados(Zon&Peterson2005,Dahm&

Geisler2006).Afacilidadedemanipulaçãogenéticanestaespécieaindalevouao desenvolvimentodeváriaslinhagenstransgênicas,oquetemcontribuídoparao desenvolvimentodenovosestudosrelacionadosàexpressãogênicaejápermitiu umgrandeavançonaelucidaçãodasrelaçõesexistentesentrediversosgenesde zebrafish edeoutrosvertebrados(Sprague etal. 2003).

Atualmente, diversas pesquisas, em diferentes áreas, vêm sendo desenvolvidas em D. rerio , especialmente direcionadas à criação de novas linhagens transgênicas (Linney & Udvadia 2004, Ashworth & Brennan 2005,

Huang et al. 2005, Burket et al. 2008, Wu et al. 2008) e à compreensão do surgimento e desenvolvimento de doenças humanas, como câncer e processos degenerativos (Patton & Zon 2005, Lam & Gong 2006, Panulo et al. 2006,

Goessling etal .2007,Merlino&Khanna2007,Beckman2007,Feitsma&Cuppen

2008).Comoperspectiva,podesepreveracontinuaçãoeampliaçãodasanálises genéticasem Daniorerio ,tantonaáreabiológicacomonaáreademedicina.

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Resultados e Discussão Osresultadoseadiscussãodosdadosobtidosencontramseapresentados na forma de capítulos, referentes a trabalhos científicos. Visando facilitar a apresentação e compreensão de cada um destes capítulos, as figuras e tabelas foram numeradas em relação a cada trabalho específico. Da mesma forma, as referências bibliográficas citadas em cada capítulo não se encontram ao final do item“IntroduçãoGeral”dateseesimdiscriminadasnofinaldecadatrabalho.

Capítulo 1 : Molecular organization and expression analyses of skeletal actin genes from white and red fish muscle tissues: insights into the musculature energeticdemands; Capítulo 2: Identificationofsexuallydimorphicgeneexpressioninbraintissueof thefish Leporinusmacrocephalus throughmRNAdifferentialdisplayandrealtime PCRanalyses; Capítulo 3: Spatiotemporal distribution of fatty acidbinding protein 6 (fabp6) gene transcripts in the developing and adult zebrafish (Danio rerio ); Capítulo 4: Acomparativeexpressionanalysisofgenetranscriptsinbraintissue of wildtype and GHtransgenic zebrafish ( Danio rerio ) using a DDRTPCR approach.

31

CAPÍTULO 1

Organização molecular e análises de expressão de genes de actina

esquelética de tecidos musculares branco e vermelho de peixes:

compreensão sobre a demanda energética muscular

32

Molecular organization and expression analyses of skeletal actin genes from white and red fish muscle tissues: insights into the musculature energetic demands

Abstract

Two αskeletal actin genes were isolated from the neotropical fish Leporinus macrocephalus , referring to white and red muscle tissues. Similar actin isoforms were also identified in other fish species, mainly differing by a Ser/Ala 155 substitution, which can have a functional significance related to actinATP interaction.ASer 155 ,identifiedintheαskeletalwhitemuscleactin,seemstolead toahigheraffinitytoATPmoleculeswhich,inturns,couldberelatedtothefast contractionfibersofthistissue.However,anAla 155 ,asobservedintheαskeletal actinfromredmusclemightresultinadecreaseinactin’saffinityforATP,which couldalsobeassociatedtoitstissueslowcontractileperformance.FISHmapped the skeletal muscle actin genes to a pair of submetacentric chromosomes.

Comparisons on the predicted secondary protein structure evidenced no differences between the two analyzed actin isoforms, although a Phe/Ile 262 substitution at the red muscle actin leads to a hydrophobicity variation at the D plug of the protein which could alter its stability. Data on qRTPCR evidenced a significant gene expression variation between white and red muscle actin genes

(T=105MannWhitney;p<0.001).Moreover,althoughnosignificantvariationwas observed between adult males and females (white muscle: females

0.01175±0.00247 vs. males 0.01082±0.004447; red muscle: females

0.000847±0.000613 vs. males 0.000754±0.000374), there was a great variation ongeneexpressionwithinindividualsofeachsex.Availabledataonmuscleactins

33 lead to the proposal that white and red αskeletal actins are genetically and functionally distinguishable in fish species, a feature that is not found in other vertebrategroups.

Introduction

Muscles are a major source of energy consumption and heat generation and there has been a longstanding interest in the mechanisms that underlie muscleenergeticproperties.Skeletalmuscleisfoundthroughoutthebodyand,in highervertebrates,formanintegratednetworkwithaprominentskeletalsystemvia tendons, resisting gravity and facilitating mobility. Lower vertebrates, particularly thosethatareaquatic,haveaproportionallygreatermusclemass(e.g.Sambasivan

&Tajbakhsh2007).Infish,bodymassismainlycomposedbyskeletalmusculature thatconstitutes4075%ofthetotalweightoftheanimal.Inmostfishspecies,this tissueiscomposedbydifferentmusclefibersthatoccupydistinctaxialregionsa large portion of a deeper layer of white muscle (with fast contraction fibers and glycolytic metabolism, for energy supply), a superficial thin layer of red muscle

(withslowcontractionfibersandoxidativemetabolism),andanintermediatelayer between the red and white musculatures (with fibers of fast contraction and oxidative/glycolytic metabolism). While white muscle fibers are associated to fast swimming in fish, as predation and escape behaviors, the red muscle fibers are associated to slow movements, as migration and foraging habits (Driedzic &

Hochachka1976,Luther etal. 1995,Sänger&Stoiber2001).

One of the major components of muscle tissues is actinthat,together with myosin,tropomyosin,andtroponin,representsaproteinthatcomposestheparallel

34 fibrils(miofibrils)ofthestriatemusculature.Actinplaysacrucialroleinmaintaining cytoskeletal structure, contractile processes, cell motility and division, and intracellularmovements,andisessentialtoeukaryoticcells(Clarke&Spudich1977,

Lazarides&Revel1979).Morerecently,itwasevidencedthatactinisalsoinvolved indiversenuclearfunctions,includingtranscription,ribonucleoproteinpackagingand transport, chromatin remodeling, and the formation of karyoskeletal elements

(Pederson&Aebi2002,Bettinger etal. 2004,Kiseleva etal. 2004).Reflectingthe importanceofitsbiologicalfunctions,theactinstructureishighlyconservedamong differentorganisms.

Although the genes that code for actin proteins can occur in single copies, as observed in Sacharomyces cerevisae , some protozoans, and several greenalgae,actinproteinsareencodedbyamultigenefamilyin,plants, and in many protozoans examined to date (Vandeckerckhove & Weber 1984,

Hightower & Meagher 1986, Reece et al . 1992, Bhattacharya et al. 2000).

Mammalshavefourmuscleactinisoformstwoinstriatedmuscles( αskeletaland

αcardiac) and two in smooth muscles ( γenteric and αvascular) and two non muscleorcytoplasmictypes,the βand γisoforms(e.g.Vandekerckhove&Weber

1984, Kusakabe et al. 1997, Mounier & Sparrow 1997). However, teleost fish seems to contain a higher number of distinct actin isoforms, as revealed by the most extent research on actin’s diversity and tissue expression profile in fish genome performedto date on Takifugurubripes, revealingnineisoforms(two α skeletal,three αcardiac,an αanomaloustestistypeactin,two βcytoplasmic,and one βcytoplasmic vascular typeactin) (Venkatesh et al. 1996). Similarly, insects haveatleastsixactingenes(Fyrberg etal .1980).Theactingenefamilyofplants ismuchlarger,comprising844genes,dependingonthetaxa(Reece etal .1992).

35

Thus,theactinmultigenefamilyprovidesanexcellentmodelforthestudyofthe molecularmechanismsofgeneregulationandexpressionandforinsightsintoits evolutionandfunction.

The multipleactinisoforms are expressedin differentstagesand/ortissues andseemtobeencodedbyasetofstructurallyrelatedgenesthatwereoriginated bygeneduplicationfollowedbyfunctionaldivergence(Hightower&Meagher1986,

Miwa et al. 1991, Venkatesh et al. 1996). Thecontroversy betweentheactinhigh structure conservation and several biophysical properties suggests that its several isoformsresultfromaveryfewaminoacidsubstitutionsinkeyfunctionalpositions.

Smalldifferencesrelatedtonucleotideoraminoacidsubstitutionscanconstitutethe structuralbaseforchangesintheconformationoftheactinproteinwhich,inturns, couldmodifyitsaffinitytonumerousbindingsfoundindistinctcellulartypes(Ooi&

Soematsu2007).

Although there are several data on mammalian actins, the evolutionary origin, pattern of organization, and their diversity in lower vertebrates, especially fishes, remain to be better investigated. Moreover, data on the organization and expressionofthemaincomponentsofthemuscletissuescangivenewinsightson themoleculareventsunderlyingmusclecontractionand/orgrowth.Therefore,the purpose of the present study was the isolation and characterization of skeletal muscleactingenesof Leporinusmacrocephalus ,afishspeciesthatrepresentsan important food resource that has been intensively exploited in the subtropical aquaculture(Takahashi etal. 2004).Theanalysesresultedinthefirstdescriptionof whiteandredskeletalmuscleactingenesthatmaybegeneticallyandfunctionally distinguishableinfishspecies.

36

Material and Methods

Animalsamples

Ten adult specimens (five males and five females) of Leporinus macrocephalus (piauçu) (Characiformes, Anostomidae) were obtained from a private fishery station (Piscicultura Água Milagrosa, Itajobi, São Paulo State,

Brazil), where they were maintained under the same conditions. Animals with at least 40 days were selected since they already present gonadal differentiation

(Toriyama 2001) which permits the sex identification under stereomicroscopy.

White and red muscle samples from the dorsal region of the animals were collectedandimmediatelystoredat80 oCuntilRNAextraction.Itwasnotpossible toobtainmusclesamplesfromtheintermediatelayerbetweentheredandwhite musculatureduetothedifficultytocorrectlyisolatethistissue.Kidneytissuewas alsocollectedandimmediatelyusedforchromosomepreparations.

RNAisolation

Whiteandredmusclefrozensamplesweremechanicallyhomogenizedwith

TRizolReagent(Invitrogen)andtotalRNAextractionfollowedthemanufacturer’s protocol.RNAsampleswereelutedinRNasefreewaterandquantified(NanoDrop

1000 Spectrophotometer) by measuring the opticaldensity(OD) at260nm. RNA puritywasensuredbyobtaininga260/280nmODratio≥1.80.

37

RTPCR andcDNAamplification

Each RNA sample was reverse transcribed with the commercial kit

SuperScript FirstStrand Synthesis System for RTPCR (Invitrogen) using an oligonucleotide (dT) 1218 as an anchor primer, according to the manufacturer’s instructions. cDNA (2 L) was amplified using forward AactF (5’

AGGCCAACAGGGAGAAGATT3’) and reverse AactR (5’

TCCATACCGATGAAGGAAGG3’) primers, designed based on alphaactin gene sequence GenBank databases of the fish Takifugu rubripes

(http://www.ncbi.nlm.nih.gov ), in order to amplify a segment of this gene. Each

PCR consisted of 2 L of cDNA, 0.2mM of each primer, 1x 25mM MgCl 2 PCR buffer,0.2mMofdNTPs,and1unitofPlatinum Taq DNApolimerase(Invitrogen), in a final volume of 25 L. PCR amplifications were carried out with an initial denaturation step at 95 oC for 2 minutes, followed by 34 cycles at 95 oC for 30 seconds,55 oCfor30seconds,and72 oCfor1minute,withanadditionalextension stepat 72 oCfor 5minutes.PCRproductswerefractionatedon1%agarosegel, stained with ethidium bromide, and visualized under UV light using Eagle Eye II imagedocumentationsystem(Strategene).

SemiquantitativeRTPCR andstatisticalanalysis

Four pools of RNA samples were prepared in order to minimize the detectionofsimplevariationsamong Leporinusmacrocephalus individuals(poolof

RNA samples from white muscles of males; pool of RNA samples from white musclesoffemales;poolofRNAsamplesfromredmusclesofmales;poolofRNA samples from red muscles of females). Each RNA pool was reverse transcribed individually as described above. cDNA amplification followed the methodology

38 describedinMarone etal. (2001)using2 LoftheobtainedcDNA,andPCRwas carriedoutat95 oCfor2minutes,followedby30cycleswithdenaturationat95 oC

(30 seconds), annealing at 55 oC (30 seconds), and extension at 72 oC (1minute and15seconds),followedbyanadditional5minuteextensionstepat72 oC.The

PCR signals were normalized to the housekeeping 18S rRNA gene. A 245 bp segment of the 18S rRNA gene was amplified using the four RNA pools individually and the primers 18S1 (5’TACCACATCCAAAGAAGGCAG3’) and

18S2 (5’TCGATCCCGAGATCCAACTAC3’), designed based on consensus sequences of this gene described for several fish species. Preliminary PCR experiments,using28303234cycles,wereconductedtodeterminethenumber of cycles that represented the linear amplification range for analysis (Tom et al.

2004).PCRproductswerefractionatedon1%agarosegel,stainedwithethidium bromide, and visualized under UV light using Eagle Eye IIimagedocumentation system (Strategene). The bands corresponding to each gene were quantified by densitometry as Integrated Optical Density (IOD) using Imaging Master VDS

Softwareversion3.2(GELifeSciences).

QuantitativePCRandstatisticalanalysis

Quantitative RTPCR was performed using Power SYBER Green PCR

MasterMixKit(AppliedBiosystems),accordingtothemanufacturer’sinstructions.

Standardreactions(25 L)wereassembledusing12.5 LofPowerSYBERGreen

PCR Master Mix 2x, 2 L of forward primer AactF2 (5 M) (5’

CCATCTATGAGGGTTACGCTCTTC3’),2 LofreverseprimerAactR2(5 M),(5’

CGGTTGTGACGAAAGAGTAGCC3’), 2 L of template (10ng/ L), and 6.5 L of ultrapure water. Primers were designed with the software Primer Express v.2.0

39

(Applied Biosystems), based on the actin gene sequences previously obtained from white and red muscles of L. macrocephalus . Templates cDNA were 1:10 diluted,andcDNAsampleswerereplacedbyDEPCwaterinthenegativecontrols.

AllrealtimeassayswerecarriedoutinduplicateusinganAppliedBiosystem7300

HTRealTimePCRsystem(AppliedBiosystems).Fortyamplificationcycleswere performedandeachcycleconsistedof94 oCfor15secondsfollowedby60 oCfor1 minute. Amplification and dissociation curves generated by the 7300

System/SequenceDetectionSoftwareversion4.0(AppliedBiosystems)wereused forgeneexpressionanalysis.Asperformedforthesemiquantitativeanalysis,the qRTPCR signalswerenormalizedtoasegmentofthe18SrRNAhousekeeping geneusing the primers 18S3(5’CGGAATGAGCGTATCCTAAACC3’)and18S4

(5’GCTGCTGGCACC AGACTTG3’), also designed based on consensus sequencesofthisgenedescribedforseveralfishspecies.Followingtheremoval of outliers, raw fluorescence data were exported to the online program QPCR

Standard Curve Slope to Efficiency Calculator

(http://www.stratagene.com/techtoolbox/calc/qpcr_slope_eff.aspx) to determine thePCRamplificationefficiency.TheCtvalueswereusedtocalculatearelative gene expression value for each transcript, according to 2 ∆Ct method (Livak &

Schimittgen 2001). The relative gene expression values were submitted to statistical analysis usingStudent’s unpairedttest (p<0.05) tonormaldistribution, or MannWhitney T value (MannWh) (p<0.05) to data which did not display a normal distribution (Zar 1999). Differences were considered significant when p<0.05anda95%confidencelevelofthedifferencewasused.

Cloning,sequencing,andsequenceanalysis

40

PCR products were cloned into pGEMT (Promega) vector and used to transform competent cells of the E. coli strain DH5 α (Invitrogen), following the manufacturers’ instructions. Clones were submitted to automated sequencing on an ABI 377 Automated DNA Sequencer (Applied Biosystems) with a DYEnamic

ETTerminatorCycleSequencingkit(GEHealthcareLifeSciences),followingthe manufacturer’s instructions, and using primers complementary to vector arms.

Nucleicacidandaminoacidsequencedatabasesearcheswereperformedusing

BLAST/N (Altschul et al. 1990) at the National Center for Biotechnology

Information (NCBI) website (http://www.ncbi.nlm.nih.gov/blast). Sequence alignments wereobtainedbyClustalW function(Thompson etal. 1994) and the consensus sequences were manually determined. The deduced amino acid sequences were subjected to the bioinformatics computer program CLC Main

Workbench 4.0.1 to generate protein secondary structure data and hydropathy plotsusingtheKyteDoolittlescalewithawindowsizedefaultvalueof9(Kyte&

Doolitle1982).

Chromosomeanalyses

Mitotic chromosomes were obtained from a suspension of anterior kidney cells using direct preparations(Bertollo etal .1978).A cDNA actinproduct(300

1.000ng), obtained using primers AactF and AactR, was isolated from agarose gels using a commercial kit (Sephaglas TM BandPrep Kit, GE Healthcare Life

Sciences).ASCOMP(singlecellcomparativegenomichybridization)methodology forDNAamplificationwasperformed.Briefly,thecDNAwasdigestedusing24Uof

Mse I(NewEnglandBiolabs)at37 oCfor3hours.Inaseparatetube,theadaptor oligonucleotides LIB1 (5’AGTGGGATTCCTGCTGTCAGT3’) and ddMseI (5’

41

TAACTGACAGCdd3’)wereincubatedat65ºCto15ºC,usingaslopeof1min/ºC.

Further,1LofATP(10mM)and1LofT4DNAligase(5U/L)wereadded.The digestedcDNAwasmixedtothissolutionandincubatedat15ºCovernight.Thekit

Elongase Amplification System (Invitrogen) was used for the primary DNA amplification,followingthemanufacturer’sinstructions.Theamplifiedproductwas used as atemplate forasecondamplificationreaction,usingtheQIAquickPCR

Amplification Kit (QIAGEN), following its instructions. The oligonucleotide LIB1 wasremovedbya Tru Itreatmentat65ºCfor3hours.TheDNAprobe(8 g)was furtherprecipitatedinthepresenceofthecompetitorHumanCot1DNA(50x)and salmon sperm DNA (50x), and labeled with biotin11dUTP by nick translation usingthekitBionick TM LabelingSystem(Gibco),accordingtothemanufacturer’s instructions.Afterchromosometreatmentwith10%pepsinefor5minutesat37 oC,

FISH (Fluorescent in situ hybridization) was performed as detailed in Wasko &

Galetti (2000). Metaphases were examined with an Olympus BX50 epifluorescencemicroscope.

42

Results and Discussion

Thealphaskeletalactinplaysakeyroleinbiologicalmusclemovementand represents the major protein in muscles alongwithmyosin. Thisproteinhasbeen intensively studied in mammals and its functions include polymerization in neutral saltandbindingtoCa 2+ ,Mg 2+ ,adeninenucleotides,tropomyosin,andmyosin(Estes et al. 1992, Reisler1993).These multiplefunctionsmakeitoneofthestructurally bestconservedcellproteins.Althoughmusclesofmostvertebratescontainone α skeletalactin,thepresenceoftwoisoformsinthemusclesoffishspecieshasbeen reported. Venkatesh et al. (1996) identified two αskeletal actins in the pufferfish

Takifugurubripes ,whichhave98.7%ofidentityattheaminoacidsequencelevel.

Twoother fishspecies Coryphaenoidesacrolepis and C.cinereus alsopresent twodistinct αskeletalmuscleactinisoforms(Morita2000).However,thefunctional differenceofthesetwoactinsinfishmusclewasnotelucidated.

Distinct muscle actin isoforms, with tissue and/or stagespecific patterns, couldberelatedtothedifferentstructureofthemuscletissuesoffishspecies.Asso, theaimofthepresentstudyexploitstheisolationandcharacterizationofactingenes of white and red skeletal muscles of the neotropical fish species Leporinus macrocephalus , through the idea that it should be feasible to also find distinct α skeketalactingenes,asalreadyidentifiedinotherfishspecies.Moreover,itshould bepossibletoidentifythegeneticbasesthatcouldbebeyondthespecificfunctions ofdifferentskeletalactinisoforms.

TheamplificationofthecDNAsamplesof L.macrocephalus fromwhiteand redmuscle,usingthetwoselectedactinprimers,revealedfragmentsofapproximate sizes of 450 bp. Cloning and sequencing of the PCR products lead to the characterization of segments composed by 472 bp (Figure 1). Database

43

DDBJ/EMBL/GenBank searches for nucleotide and amino acid similarity index indicated that the isolated fragments of L. macrocephalus correspond to partial α skeletalmuscleactingenes,containingthecompleteregionsofexons2,3,and4, and a fragment of exon 5 (Figure 1). These results were confirmed through comparisonswithdataonvertebrateskeletalmuscleactingenesthatarecomposed by 6 exons, whose proteins present 375 amino acid residues (Venkatesh et al.

1996).

The results evidenced no nucleotide differences between the isolated actin genesfromwhitemusclesofmalesandfemalesof L.macrocephalus .Inthesame manner,nodifferencescouldbedetectedbetweensexeswhencomparingtheactin gene from red muscle. However, several base substitutions were identified when comparing the isolated αskeletal muscle actin genes from white and red muscle tissues 7 and 12 nucleotide substitutions were found in the exons 3 and 4, respectively(Figure1).Despitethesedifferencesbetweenthewhiteandredmuscle actin gene sequences of L. macrocephalus, their deduced aminoacidsequences, referring to the residues 113 to 269, are very conserved (Figure 2), since most nucleotide variations correspond to synonymous substitutions and just three nucleotidedifferencesarerelatedtoaminoacidsubstitutions.

Availabledataonfishactinsindicatetheoccurrenceoftwodifferentskeletal muscle actin genes, firstly denominated αSk1 and αSk2 by Venkatesh et al.

(1996).Comparisonswithaminoacidsequencesofdatabasebanksindicatedthat theαskeletalmuscleactingeneisolatedfromwhitemuscleof L.macrocephalus presentsahigheridentitywiththeactingenetype2identifiedinthefishspecies

Takifugu rubripes (Venkatesh et al. 1996), Coryphaenoides acrolepis , and C. cinereus (Morita 2000). On the other hand, the characterized αskeletal muscle

44 actingenefromredmuscleof L.macrocephalus ismoresimilartotheactingene type1alsocharacterizedinthesesameotherfishspecies.

The precursors of vertebrate muscle actins (musclelike actins) are found only in urochordates and represent a transition from nonmuscle like actins to vertebrate skeletal muscle actins (Kovilur et al . 1993). Actins have undergone a rapid diversification during the emergence of vertebrates to give rise to several muscletype actins, as seen in fish species. Distinct αskeletal muscle actin isoforms were not evidencedin other vertebrate groups.Therefore,it ispossible that some of the actins that have been characterized in fish species are either specifictotheteleostlineageorareyettobeidentifiedinhighervertebrates.

The actin monomer structure, already characterized in several species, presents two domains, originally termed large and small, although they are now known to be nearly identical in size. The small domain comprehends the subdomain1(a.a.residues132,70144,and338375)andthesubdomain2(a.a residues 3369), while the large domain comprises the subdomains 3 (a.a. residues145180and270337)and4(a.a.residues181269)(Kabsch etal. 1990)

(Figure 3a). The structure and rotations of these domains with respect to one another are important in many biological processes, such as enzyme catalysis, ligand binding, and oligomerization (Page et al. 1998). Amino acid substitutions could lead to conformational changes of the protein which in turn would modify chemicalaffinitiesfornumerousligandsfoundinsidethecells(Mounier&Sparrow

1997).Thestrongconservationofactinisoformsindicatesagreatrestrictioninthe rate and nature of amino acid changes in this protein during evolution. Only substitutions compatible with the folded structure and protein function will have

45 been retained. As so, no extreme change on protein conformation is thus expected,but,rather,smallandlocalmodifications(Mounier&Sparrow1997).

One of the actin ligands is the ATP molecule (Figure 3a), which is hydrolysed to ADP.Pi, playing a role in force generation by the actomyosin complexand accompaniesassembly ofactinmonomers(Gactin) intofilaments

(Factin)(Chen etal. 2000).Aminoacidresidues1217and154161comprehend two protruding loops that form a hydrogen bond with the phosphate of ATP

(Kabsch etal. 1990,Schüler etal. 1999,Morita2000)sincetheiraminenitrogen can be hydrogen bounded to oxygen atoms of the nucleotide phosphate tail

(Schüler et al. 1999). This binding bridges the two loops between the small and the large domains, and prevents the unfolding of the protein and stabilizes its structure (Kabsch et al 1990, Schüler et al. 1999). Any disturbance in the interdomaincouplingisexpectedtoresultinadecreasedstability.

An amino acid difference observed between the αskeletal muscle actins from white muscle and from red muscle of L. macrocephalus corresponds to a substitutionofaSerine(whitemuscle)foranAlanine(redmuscle)attheresidue

155 (Figure2) thatlies inside oneof theATPbinding siteto the actin molecule, found at the subdomain 3. This amino acid substitutioncaninfluence theactin’s affinity for ATP, a feature that has been described in actin isoforms of some vertebrate species, including fish, and in Sacharomyces cervisae (Table 1), an yeastthathasoneessentialactingenethatencodesaproteinthatsharesahigh identity degree (87%) to vertebrate skeletal muscle actins (Geeves et al. 2005).

Similarly,ithasbeenevidencedthataSer/Alasubstitutionattheaminoacid14,at theactinsubdomain1,resultsina40to60folddecreaseinactin’saffinityforATP

(Chen et al. 1995) (Table 1), since Alanine has a lower hydrogen bonding

46 capacity. Ser 14 hydroxyl forms a polar bridge between the γphosphate ATP and theamide nitrogen ofGly 74 , thusconferringadditionalstabilityontheactinsmall domain (Morita 2000, Ooi & Soematsu 2007). Therefore, the Ser/Ala 155 substitution found in the αskeletal actin isoform of red muscle of L. macrocephalus could also influence the actinATP interaction and, consequently, alterthisproteinstability,structure,orfunction.

Infish,thepresenceofaSerine 155 hasbeenalsoidentifiedinanαskeletal muscleactintypeofsomespecies,as Takifugurubripes (Venkatesh etal. 1996),

Coryphaenoidesacrolepis and C.cinereus (Morita2000),andinαcardiacmuscle actins,asidentifiedin T.rubripes (Venkatesh etal. 1996), Oreochromisniloticus and O.mossambicus (Wasko etal. 2007),and Salmotrutta (Mudalige etal. 2007).

Ontheotherhand,Alaninewasalsoidentifiedasthe155 th aminoacidinasecond

αskeletal muscle actin isoform in the fish Takifugu rubripes (Venkatesh et al.

1996), Coryphaenoidesacrelepis , C.cinereus ,and Cyprinuscarpio (Morita2000).

Therefore, it is reasonable to suppose that the occurrence of a Serine or an

AlanineinthevicinityoftheactinATPbindingsite,attheaminoacidresidue155, could be related to the type of contraction of the muscle fibers (fast vs. slow contraction),sinceforcegenerationduringmusclecontractioncanbeunderstood in terms of cyclical length changes in segments of actin thin filaments moving trough the treedimensional lattice of myosin filaments (Schutt & Lindberg 1998,

Sieck & Regnier 2001) and any disturbance in these proteins could alter the chemomechanicaltransduction(work,force,andshortening)inmuscle.

Therefore,theresidueSer 155 ,thatseemstoleadtoahigheraffinitytoATP molecules, could be related to the contractile performance of white skeletal and cardiacmuscletissues(fastcontractionfibers).Ontheotherhand,thepresenceof

47 anAla 155 mightresultinadecreaseinactin’saffinityforATP,whichcouldalsobe related to the slow contraction fibers of the red muscle tissue. Moreover, it is possiblethatthegeneticbasedifferencesbetweenwhiteandredskeletalmuscle actins of fish species may have been hidden since previous analyses probably haveusedbothmuscletissuestogetherandnotindividually,asperformedinthe present study. Therefore, some of the αskeletal muscle actin isoforms already described for other fish species, as the denominated Sk1 actin of Takifugu rubripes (Venkatesh et al. 1996), actin1 of Coryphaenoides acrolepis and C. cinereus (Morita 2000), and the actin2b of Coryphaenoides yaquinae and C. armatus (Morita 2003), that also present an Ala 155 may represent a red skeletal muscleactintype,whiletheotherskeletalactinisoformswithaSer 155 identifiedin thesesamefishspeciesmayberelatedtoawhitemuscletype.Asso,theamino acid155mightbeadiagnosticpositioninαskeletalactinsofredandwhitemuscle tissues of fish species. In contrast, other vertebrates,asrabbit,chicken, mouse, human, and frog, synthesize a single αskeletal actin isoform with Ser 155 . This differentpatternmayreflectthatfishskeletalmusculatureislayeredorganizedand largely confined to locomotion demands rather than bundled as in the other vertebrates(Luther etal. 1995).

Comparisonsbetweenthetwodistinctαskeletalmuscleactinisoformsof L. macrocephalus alsoevidencedanotheraminoacidsubstitution(Phe/Ile 262 )(Figure

2).Althoughthereisnoavailabledataregardingtoactinaminoacidsubstitutions at this residue, analyses of the protein structure show that residues Phe 262 to

Gly 273 form an outer loop which buttresses both subdomains 3 and 4, and stabilizes the extensive intersubdomains interface (Page et al. 1998). Therefore, the identified Phe/Ile 262 amino acid variation could also influence the Factin

48 formation, nucleotide binding and exchange, and interactions with various actin bindingproteins.

Theproposalthatthedifferentαskeletalactinisoformsalreadydescribedin some fish species may be correlated to white and red muscle tissues led us to predicttheirproteinsecondarystructure,sinceaminoaciddifferencescanleadto a weak aggregation of these residues which could strongly influence the chain’s motifs as the alpha helices (coiled conformation in which every backbone NH group donates a hydrogen bond to the backbone C=O group of the amino acid fourresiduesearlier)andthebetastrands(typically510aminoacidlongwhose peptidebackbonesarealmostfullyextended)anditsfolding(Kurgan2008).As thepresentstudycharacterizedapartialregionoftwodifferentαskeletalactinsof

Leporinus macrocephalus , a protein diagram representation (Figure 3b) was achievedbasedonthecompleteaminoacidsequenceoftheskeletalactin1and skeletal actin2 described for Coryphaenoides acrolepis and C. cinereus (Morita

2000), since they present a identity level of 99.6% and 100% with the partial deduced actin amino acid sequences (residues 113269) of the actin isoforms from red and white muscles of L. macrocephalus , respectively. Comparisons between the predicted secondary protein structures evidenced no differences between the two analyzed actin isoforms, since their alpha helices and beta strands configurations were identical, even at the 155 th and 262 th amino acid residuesthatevidenceaSer/AlaandaPhe/Ilesubstitution,respectively.

However, a comparison of the amino acids hydropathy of the two actin isoformsevidencedthataSerineinsteadofanAlanineasthe155 th residueresults in a small decrease in the protein hydrophobicity. Similarly, the presence of an

Isoleucine instead of a Phenylalanine as the 262 th amino acid also leads to a

49 reduction in the actin hydrophobicity. Although the Ser 155 found in the white muscleactinisoformof L.macrocephalus probablynotaltertheproteinstabilityor function,theIle 262 identifiedintheredmuscleactinisoformofthisfishspeciescan altertheproteinsinceaminoacids262to274formanactinhydrophobicplug(H plug)thatisimportantfortheglobularactinmonomertopolymerizereversiblyinto doublestranded actin filaments and also important for the protein stability

(Shvetsov et al. 2008). Actin stability problems due to hydrophobicity variations have been shown to be related to amino acid substitutions along the 262274 residues,evenwhenthesesubstitutionscausednosignificantchangesinoverall secondarystructure(Kuang&Rubenstein1997).

Geneticanalysesofactinsinfishspeciesaremainlyrestrictedtonucleotide and amino acid sequences descriptions and there is no data concerning the chromosome localization of these genes throughout in situ hybridization, probably duetothedifficultyinobtainingagoodresolutionlevel,speciallyusingPCRproducts directly or in plasmid vectors as hybridization probes. However, FISH mapping of actingenescouldbeareasonablyefficientandstraightforwardwayofimprovingthe resolutionofcomparativemaps.AcDNAamplificationproductof L.macrocephalus , related to actin genes, was used as a probe in chromosome fluorescence in situ hybridization analyses. Preliminary results evidenced that the characterized α skeletalmuscleactingeneswerelocalizedatthepericentromericregionoftheshort armofalargesubmetacentricchromosomepair(Figure4).Noadditionalfluorescent signalsweredetectedandgeneralytwosignalswereobservedininterphasecells.

Data on vertebrate chromosome in situ localization of muscle actin genes were mostly obtained for human cells, indicating that the αcardiac, αskeletal, α vascular, βcytoplasmic, and γcytoplasmic genes are located on chromosomes

50

15q14,1q42.1,10q23.3,7p22,and17q25,respectively(Ueyama etal. 1995,1996).

Thisdistributionpatternofeachactingenetoaparticularchromosomepairseemsto becommoninothermammalspecies.However,actinlociinthegenus Drosophila are dispersed over all chromosomal elements (Bondinas et al. 2002). Linkage mapping data on fish species, as Danio rerio

(http://www.ncbi.nlm.nih.gov/genome/guide/zebrafish/index.html), also suggest that in this vertebrate group actin loci can be spread through several chromosomes.

Therefore, even though the two different isolated αskeletal actin genes of L. macrocephalus seemstobelocalizedinamajorgroupalongachromosomepair, othersmallactincistronsmightnotbeidentifiedduetotheFISHtechnicalprocedure.

Although no variations were identified in the nucleotide sequences of the actin genes isolated from males and females of L. macrocephalus , quantitative

PCR analyses were performed in the αskeletal actin genes of white and red muscletissuesfromeachsexinordertoverifytheeventualpossibilityoffindinga genderassociated gene expression in this fish species. The occurrence of actin genes associated to a sexwasalreadyobservedinsomeeukaryotes, as Aedes aegypti (Muñoz etal .2004,Vyazunova&Lan2004), Schistosomamansoni (Davis et al. 1986), and Schistosoma japonicum (Fitzpatrick et al . 2004), a feature that seemstobeassociatedtomusclesthatarerequiredtosustainspecificactivitiesin each sex, as feeding, reproduction, or particular movements (Fitzpatrick et al.

2004).

The semiquantitative analysis performed using RNA sample pools from white and red muscles of males and females of L. macrocephalus separately, evidencedanestimatedactinmRNAleveldecreaseinfemalescomparedtomales

(IODratiovaluesofwhitemuscle:males0.675vs.females0.492;IODratiovalues

51 ofredmuscle:males0.873vs.females0.642)(Figure5).Furthergeneexpression analyseswerealsoperformed,throughoutRealTimePCR,usingRNAsamplesof white and red muscles of each male and each female separately, in order to confirm the previous semiquantitative gene expression results. Surprisingly, individual results indicated that the characterized αskeletal muscle actin genes had a higher relative expression in most females than in males (white muscle: females 0.01175±0.00247 vs. males 0.01082±0.004447; red muscle: females

0.000847±0.000613 vs. males 0.000754±0.000374) (Figure 6a, b). However, statistical analysis revealed no significantdifference in gene expression between sexes,asvaluesoft=0.479,p=0.641,andt=0.343,p=0.738wereobtainedwhen comparing the αskeletal actin genes from white and red muscles of males and females,respectively.

The apparently different results obtained through semiquantitative and quantitativegeneexpressionanalysescouldberelatedtotheuseofamixtureof

RNA samples and the use of individual RNA samples in each of these two analyses, respectively. Moreover, although there was no significant difference whencomparingallmalesandallfemalestogether,therewasarelevantvariation on gene expression when comparing individuals within each sex (Figure 6a, b).

Although the biological significance of these differences is unknown, it may be related to the analysis of animals in different developmental stages, even these individualswereinadultphaseandpresentedgonadaldifferentiation.Asso,future gene expression analyses should be done in order to verify the occurrence of a variationinαskeletalmuscleactingeneexpressionthroughoutthedevelopmental stagesof L.macrocephalus .

52

Gene expression comparisons between the two characterized αskeletal muscleactingenesof L.macrocephalus ,throughRealTimePCR,alsorevealeda significanthigheractinmRNAlevelinwhitemusclewhencomparedtoredmuscle

(T=105 MannWh; p<0.001) (Figure 6c). This finding could be related to the energetic demands of the white muscle tissue, with fast contraction fibers and glycolyticmetabolismforenergysupply (Driedzic&Hochachka1976,Luther etal.

1995,Sänger&Stoiber2001).

Duetoitsconservedandubiquitousnature,actingenesareoftenusedas internal controls in gene expression analyses based on data that they are expressed at a constant level across all samples. However, the present results andalsodataonβactingenesoffishspecies(e.g.Tang etal. 2007)indicatethat theseconstitutivegenescanexhibitvariableexpressionandshouldbeusedwith careful as internal standards in order to normalize each particular set of experimentalRNAsamples.

Takentogether,thepresentdataproposethatαskeletalactinsfromwhite and red muscle tissues appear to be somewhat genetically and functionally distinguishableinfishspeciesandshouldbeunderdifferentevolutionaryselective pressures,afeaturethatwasnotfoundinothervertebrategroupssofar.Further analyses comparing the organization and expression pattern of distinct actin isoforms in several vertebrate species would be useful to better understand the molecular evolution and the function of these genes and to verify if the variable muscleactinisoformsoffishthatarenotfoundinotheranimalgroupsareeither specifictothislineageorareyettobeidentifiedinhighervertebrates.

53

Acknowledgements

TheauthorsthankMr.OdvalS.Stradioti(PisciculturaÁguaMilagrosa,Itajobi,

SP)forfishsupplysamples,andDr.MaeliDalPaiSilva,Dr.JoãoPessoaandDr.

DéboraColombifortechnicalassistanceontheRealTimeanalyses.Thisworkwas supportedbygrantsfromFAPESP(FundaçãodaAmparoàPesquisadoEstadode

São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e

Tecnológico).

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60

Table, Figures and Legends

1

Table 1: Actin amino acid residues as adenosine triphosphate binding sites and actin’s affinity for ATP related to some actin isoforms of distinctorganisms.Thevertebratedatawerebasedoncomparisonswiththechickenαskeletalmuscleactin,andtheyeastdatawerebased on Saccharomycescerevisae wildtypeactin,whichwereusedascontrols. Actin isoform Amino acid residue ATP binding Reference carp( Cyprinuscarpio )αskeletalmuscleactin* a.a.155Ser→Ala weak Ooi&Soematsu(2007) browntrout( Salmotrutta )αskeletalmuscleactin a.a.155Ser→Ala weak Mudalige etal. (2007) Atlanticsalmon( Salmosalar )αskeletalmuscleactin a.a.155Ser sameascontrol Mudalige etal. (2007) Pacificgrenadier (Coryphaenoidesacrolepis )αskeletalmuscleactin1 a.a.155Ser→Ala weak Morita(2003) Pacificgrenadier (Coryphaenoides.acrolepis )αskeletalmuscleactin2a a.a.155Ser sameascontrol Morita(2003) Loosescale grenadier (Coryphaenoides yaquinae ) αskeletal muscle a.a.155Ser→Ala weak Morita(2003) actin2b Abyssalgrenadier (Coryphaenoidesarmatus )αskeletalmuscleactin2b a.a.155Ser→Ala weak Morita(2003) Saccharomycescerevisae actin* a.a.14Ser→Ala weak Chen etal. (1995);Chen&Rubenstein(1995) Saccharomycescerevisae actin* a.a.157Asp→Ala sameascontrol Schüler etal. (1999) Saccharomycescerevisae actin* a.a177Arg→Asp sameascontrol Schüler etal. (2000) Saccharomycescerevisae actin* † a.a.177Arg→His sameascontrol Wen&Rubenstein(2003) Saccharomycescerevisae actin* a.a.38Pro→Ala sameascontrol Aspenström etal. (1993) Saccharomycescerevisae actin* a.a.374Cys→Ser sameascontrol Aspenström etal. (1993) chickensmoothactin a.a.17Val→Cys weak StrezeleckaGotlaszewska etal. (1985) a.a.89Thr→Ser bovinesmoothactin a.a.17Val→Cys weak StrezeleckaGotlaszewska etal. (1985) a.a.89Thr→Ser *refertoinducedactinaminoacidsubstitutions † equivalent to the not induced zebrafish (Danio rerio ) cardiofunk actin mutation and to the not induced human cardiomyopathy mutation that cause abnormal heart development (Wong et al. 2001)

62

Figure 1: Consensus nucleotide sequences of partial áskeletal muscle actin genes isolated from white muscle (WM) and red muscle (RM) of Leporinus macrocephalus . Red lette rs indicate nucleotide variations. Regions ofbeginning andendingoftheexonsareindicatedbyarrows.

63

113155 167 WMKANREKMTQIMFETFNVPAMYVAIQAVLSLYASGRTTGIVLD SGDGVTHNVPIYE RM...... A...... 222 WMGYALPHAIMRLDLAGRDLTDYLMKILTERGYSFVTTAEREIVRDIKEKLCYVALD RM...... 262 269 WMFENEMATAASSSSLEKSYELPDGQVITIGNERFRCPETL FQPSFIGM RM...... I......

Figure 2: Deducedaminoacidsequences(residues113269)ofthepartial αactin skeletalmusclegenesisolatedfromwhitemuscle(WM)andredmuscle(RM)of Leporinusmacrocephalus . Dotsindicateidentical aminoacid residues.The155 th andthe262 th residuesareindicatedbyboldletters.

64

Figure 3: Actin monomer (Gactin) tridimensional structure. (a) the small subdomain (composed of subdomains 1 and 2) and the large subdomain (composedofsubdomains 3and4),andtheATPbindingsiteatthesubdomain 3. TheNandCterminiarelocatedinsubdomain 1. (b) predictedsecondarystructure of the αskeletal muscle actin2 of Coryphaenoides acrolepis and C. cinereus (based on amino acid data described by Morita 2000), evidencing the alpha helicesandthebetastrandsconfigurations.

65

Figure 4: In situ hybridization results of the actin genes in metaphase chromosomesof Leporinus macrocephalus .Arrowsindicatetheactin loci. Scalebar=3 m.

66

L 1.000 850 RMWM RMWM

650 MFMF MFMF 500 396 220

(a) (b)

1 0.9 0.8 0.7 0.6 Males 0.5 IOD Females 0.4 0.3 0.2 0.1 0 WM RM

Figure 5: Alphaskeletal muscle actin representative semiquantitative RTPCR resultsfromwhitemuscle(WM)andredmuscle(RM)ofmales(M)andfemales(F)of Leporinus macrocephalus . 1% agarose gel evidencing the expression of the constitutive18SrRNA geneusedascontrol (a) ,andtheexpressionoftheαskeletal muscle actin genes of white and red muscle tissues (b) , statistical data on gene expressionlevelsoftheαskeletalmuscleactin genesofwhiteandredmuscletissues (c) . Quantification of PCR signals was obtained by densitometric analysis of the productasIntegratedOpticalDensity(IOD). L,1KbDNAmolecularmarker.

67

0,018 0,0016 0,016 0,0014 0,014 0,0012

0,012 0,001 0,01 Male Male 0,0008 0,008 Female Female 0,0006 0,006 0,0004 Relative Expression Relative Relative Expression Relative 0,004 0,0002 0,002 0 0 WhiteMuscle RedMuscle

1,6 1,4 1,2 1 RedMuscle 0,8 WhiteMuscle 0,6 0,4 Relative Expression Relative 0,2 0 1 Muscle Tissues

Figure 6: Relative gene expression analysis (RealTime PCR) of the αskeletal muscleactin genesofwhiteandred (c) muscletissuesofmalesandfemales (a, b) of Leporinus macrocephalus relativetotheconstitutive18SrRNA gene(18S)used ascontrol.Datawereruninduplicate.Normalizeddatawereexpressedasmeans± SD.

68

CAPÍTULO 2:

Identificação de expressão gênica dimórfica relacionada ao sexo em tecido

cerebral da espécie de peixe Leporinus macrocephalus através de análises

de display diferencial e PCR em tempo real

69

Identification of sexually dimorphic gene expression in brain tissue of the fish Leporinus macrocephalus through mRNA differential display and real time PCR analyses

Abstract

Differentially expressed genes in males and females of vertebrate species generally have been investigated in gonads and, to a lesser extent, in other tissues.Therefore,weattemptedtoidentifysexuallydimorphicgeneexpressionin the brains of adult males and females of Leporinus macrocephalus , a gonochoristic fish species that presents a ZZ/ZW sex determination system, throughout a comparative analysis using differential display reverse transcriptase

PCR and realtime PCR. Four cDNA fragments were characterized, representing candidate genes with differential expression between the samples. Two of these fragmentspresentednosignificantidentitywithpreviouslyreportedgenesequences.

The other two fragments, isolated from male specimens, were associated to the gene that codes for the protein APBA2 (amyloid beta (A4) precursor protein binding, family A, member 2) and to the Rab 37 gene , a member of the Ras oncogene family. The overexpression of these genes has been associated to a greaterproductionoftheβamyloidproteinwhich,inturns,isthemajorfactorthat leads to Alzheimer’s disease, and to the development of braintumors, respectively. Quantitative RTPCR analyses revealed a higher Apba2 gene expression in males, thus validating the previous data on differential display. L. macrocephalus mayrepresentaninterestinganimalmodeltotheunderstandingof the function of several vertebrate genes, including those involved in neurodegenerativeandcancerdiseases.

70

Introduction

Thedevelopmentofthesexualphenotypeinseveralvertebratesinvolvesa complexcascadeofevents,initiatedatconceptionbythefusionoftwogametesand involvingtheactionofregulatorysexdeterminationgenesofteninaresponsetoa karyotypesignalandendingintheproductionofsexspecificmolecules,anatomy, and behaviors (Marin & Baker 1999, Eriksson et al. 1999). Sex determination pathwaysarehighlyvariableamongdifferentvertebrategroups,althoughtheyoften includethepresenceorabsenceofatestisdeterminingfactor(SRY),leadingtothe formation of ovaries or testis, which in turns determine several sexspecific expression of gene networks in somatic tissues (Zarkower 2001, Wilhelm et al.

2007).Thislackofconservationisintriguingitself,butmeansthatthepathwaysof genderspecificgenesmustbedissectedataspecieslevel.

Mammals and birds present a relative stability in the sexual determination process, due to the presence of a XX/XY or a ZZ/ZW sex chromosome system, respectively. However, the presence and role of sex chromosomes in sex determination and differentiation might not be always clear in all vertebrates, particularlyinfishspecies.Teleostfishcanhavedifferentsexdeterminationsystems, including chromosomes that are not morphologically differentiated between males and females and sex chromosomes thatarecompletely differentbetween thetwo sexes.Moreover,males(XY)orfemales(ZW)canrepresenttheheterogameticsex in different species. It has been also reported that some teleost fish have sex determination systems composed by multiple chromosomes (Tave 1993, Moreira

Filho et al. 1993, AlmeidaToledo & Foresti 2001). Despite this, the sexual differentiation in fish can also be determined by environmental factors and,

71 depending on the species, different hermaphroditic and gonochoristic modes of reproductioncanoccur(Yamamoto1969,Conover etal. 1992).

The chromosomal, gonads, and behavioral differences observed between males and females of vertebrate species represent the major factors that lead to several efforts in the isolation and characterization of genes that are differentially expressed,especiallyingonads.Infish,sexspecificgeneshavebeenisolatedfrom maleandfemalesgonadsgenerallyinordertounderstandthesexualdetermination, andalsosexualreversion,hermaphroditism,andunisexualityprocesses(Liu etal.

1996, Kanamori 2000, Guan et al. 2000, D´Cotta et al. 2001, Denslow et al.

2001a,b, Xie etal. 2001,Matsuda et al. 2002,Kondo etal. 2003,Lutfalla etal.

2003).

Despite the relative extensive data on genes with differential expression in vertebrate’stestisandovaries,dataonsexuallydifferentialexpressedgenesinother organs are not so frequent, probably because these transcripts seem to be less commoninsomatictissues(Santos etal. 2008).Althoughgenderassociatedgenes have been described in heart, liver, kidney, adipose tissue, and muscles of some vertebrate species (Hutchison 1997, Mayer et al. 1998, Eriksson et al. 1999,

Grossmann etal. 2004,Rinn&Snyder2005,Yang etal. 2006,Isensee etal. 2008), dataonsexuallydimorphicgenesinbrainarestillscarceawellcharacterizationof thesetranscriptswasonlyperformedinmouse(Eriksson etal. 1999,Xu etal. 2001,

Yang etal. 2006,Yuge etal. 2007),speciallyduetothebrainstructuredimorphism between males and females. However, brain represents an extremely interesting tissue in order to analyze sexually dimorphic gene expression. Hundreds of sex differences have been identified in human and animal brains, ranging from structure and behaviors to molecules. Many of these differences have been

72 proventobeduetohormonesanditsmetabolites,actinginthedevelopingbrain and permanently wiring it in a sex specific fashion (McCarthy & Konkle 2005,

Becker etal .2005,Morris etal .2004,Xu&Disteche2006).

Geneexpressiondifferencesinbrainassociatedtomalesandfemaleshave only been described in few fish species, as Onchorhynchos nerka (Jadhao et al.

2001), Danio rerio (GotoKazeto et al. 2004, Kallivretaki et al. 2007, Santos et al.

2008), Fundulusheteroclitus (Lauer etal. 2006), Pimephalespromelas (Filby&Tyler

2007), Oreochromisniloticus (Davis etal. 2008),and Dicentrarchuslabrax (Moles et al. 2007). There is no data on differentially expressed genes on brain tissues of neotropical fish species. Analyses in the brain of lower vertebrates represent an importantsourcefortheidentificationandcharacterizationoftargetgenesthatcould berelatedtodifferencesinneurophysiologybetweenmalesandfemalesandforthe understandingofthemolecularpathwaysthatareresponsibleforthesedifferences.

Inanattempttoisolatesexuallydimorphicgenesexpressedinadultfishbrain weusedaDDRTPCRapproachformRNAscreeningin Leporinusmacrocephalus

(piauçu), a gonochoristic fish species that represents an important food resource thathasbeenintensivelyexploitedinthesubtropicalaquaculture(Takahashi etal.

2004)andthatpresentsaZZ/ZWsexdeterminationsystem(Galetti&Foresti1987).

FurtherquantitativePCRanalysis,throughrealtimePCR,permittedtoidentifythe expressionlevelsoftheisolatedtranscripts.

73

Material and Methods

Animalsamples

Adult specimens of Leporinus macrocephalus (Characiformes,

Anostomidae) were obtained from a private fishery station in São Paulo State,

Brazil(PisciculturaKabeyamunicipalityofGlicério),wheretheyweremaintained underthesameconditions.Animalswithatleast40dayswereselectedsincethey already present gonadal differentiation (Toriyama 2001) which permits the sex identificationunderstereomicroscopy.Braintissuesamples(includingthemedulla, cerebellum, optic lobes, pineal gland, pituitary gland, cerebral hemispheres, and olfactorylobes)oftheanimalswerecollectedandimmediatelystoredat80 oCuntil

RNAextraction.

RNAisolation

Approximately 100mg of the brain samples were mechanically homogenized with 1mL of TRizol Reagent (Invitrogen) and total RNA extraction followed the manufacturer’s protocol. RNA samples were eluted in RNasefree water and quantified (NanoDrop 1000 Spectrophotometer) by measuring the opticaldensity(OD)at260nm.RNApuritywasensuredbyobtaininga260/280nm

ODratio≥1.80.

DDRTPCR

Approximately2goffiveRNAsamplesfrommalesandfemaleswereused to prepare RNA pools of each sex which were reverse transcribed (RTPCR), separately,withthecommercialkitSuperScriptFirstStrandSynthesisSystemfor

74

RTPCR (Invitrogen) using an oligonucleotide (dT) 1218 as an anchor primer, according to the manufacturer’s instructions. The cDNA samples were amplified using assingleprimerssome oligonucleotides with 1524bpthat weredesigned basedonVNTRcoresequences(Table1),andalsoRAPD(RandomlyAmplified

Polymorphic DNA) decamer primers (OPP series Operon Technologies Inc.)

(Table 2). Each cDNA amplification reaction consisted of 2L of cDNA (10%),

0.2mMofprimer,1x25mMMgCl 2 PCRbuffer,0.2mMofdNTPs,and0.2unitof

Platinum Taq DNApolymerase(Invitrogen),inafinalvolumeof50 L.Reactions using the 1524 bp primers were carried out with an initial denaturation step at

94 oC for 2 minutes, followed by 40 cycles at 94 oC for 50 seconds, 55 oC for 2 minutes,and72 oCfor50seconds,withanadditionalextensionstepat72 oCfor5 minutes. Reactions using the 10mer primers were carried out with an initial denaturation step at 95 oC for 5 minutes, followed by 45 cycles at 94 oC for 1 minute, 36 oC for 1 minute, and 72 oC for 2minutes,following the manufacturer’s instructions of the Kit ReadyToGo RAPD Analysis Beads (GE Healthcare

Biosciences).DDRTPCRproducts(10 L)werefractionatedon6%polyacrilamide gel, stained with 0.17% silver nitrate (AgNO 3) (Sambrook & Russel 2001), and visualizedunderwhitelight.Themolecularweighoftheamplifiedfragmentswas assignedthroughcomparisonwith1KbDNAladder(Invitrogen).

DNAisolationandreamplification

Selected DNA fragments that seemed to correspond to genes with differentialexpressionbetweentheanalyzedsampleswerereamplifiedinorderto obtain a higher DNA content to be used on cloning and nucleotide sequencing.

The fragments were cut from the polyacrilamide matrix and purified using a

75

QIAquick® Gel Extraction Kit (Qiagen), following the manufacturer’s instructions.

The purified fragments were used on reamplification reactions, as described in

Dakis & Kouretas (2002). The amplification reactions followed the conditions described above. PCR products were analyzed through 6% polyacrilamide gel electrophoresis.

Cloning,sequencing,andsequenceanalysis

ProductsofreamplificationwereclonedintopGEMT(Promega)vectorand usedtotransformcompetentcellsofthe E.coli strainDH5 α(Invitrogen).Clones were submitted to automated sequencing on an ABI 377 Automated DNA

Sequencer (Applied Biosystems) with a DYEnamic ET Terminator Cycle

Sequencing kit (GE Healthcare Life Sciences), following the manufacturer’s instructions, and using primers complementary to vector arms. Nucleic acid sequence database searches were performed using BLAST/N (Altschul et al.

1990) at the National Center for Biotechnology Information (NCBI) website

(http://www.ncbi.nlm.nih.gov/blast). Sequence alignments were obtained by

ClustalW function (Thompson et al. 1994) and the consensus sequences were manuallydetermined.

RealtimePCRandstatisticalanalysis

Quantitative RTPCR was performed using Power SYBER Green PCR

MasterMixKit(AppliedBiosystems),accordingtothemanufacturer’sinstructions.

Standardreactions(25 L)wereassembledusing12.5 LofPowerSYBERGreen

PCRMasterMix2x,2 Lofeachprimer(5 M),2 LoftemplatecDNAtreatedwith

76

DNase I (Invitrogen) (10ng/ L), and 6.5 L of ultrapure water. Primers were designedwiththesoftwarePrimerExpressv.2.0(AppliedBiosystems),basedon the DNA sequences previously obtained (Table 3). Templates cDNA were 1:10 diluted,andcDNAsampleswerereplacedbyDEPCwaterinthenegativecontrols.

AllrealtimeassayswerecarriedoutinduplicateusinganAppliedBiosystem7300

HTRealTimePCRsystem(AppliedBiosystems).Fortyamplificationcycleswere performedandeachcycleconsistedof94 oCfor15secondsfollowedby60 oCfor1 minute. Amplification and dissociation curves generated by the 7300

System/SequenceDetectionSoftwareversion4.0(AppliedBiosystems)wereused for gene expression analysis. The qRTPCR signals were normalized to a segment of the 18S rRNA housekeeping gene using the primers 18S3 (5’

CGGAATGAGCGTATCCTAAACC3’) and 18S4 (5’

GCTGCTGGCACCAGACTTG3’),alsodesignedbasedonconsensussequences ofthisgenedescribedforseveralfishspecies.Followingtheremovalofoutliers, rawfluorescencedatawereexportedtotheonlineprogramQPCRStandardCurve

Slope to Efficiency Calculator

(http://www.stratagene.com/techtoolbox/calc/qpcr_slope_eff.aspx) to determine thePCRamplificationefficiency.TheCtvalueswereusedtocalculatearelative gene expression value for each transcript, according to 2 ∆Ct method (Livak &

Schimittgen 2001). The relative gene expression values were submitted to statistical analysis usingStudent’s unpairedttest (p<0.05) tonormaldistribution, or MannWhitney T value (MannWh) (p<0.05) to data which did not display a normal distribution (Zar 1999). Differences were considered significant when p<0.05anda95%confidencelevelofthedifferencewasused.

77

Results and Discussion

Polymerase chain reaction (PCR)based mRNA differential display has beenwidelyusedforidentifyingdifferentiallyexpressedtranscriptsinavarietyof species (e.g. Liang 2002). The choice of applying a DDRTPCR approach in

Leporinusmacrocephalus inordertoidentifygeneswithdifferentexpressionlevels inmalesandfemaleswasmainlyduetotheprinciplethatthismethodologydoes notneed a previous knowledge on themRNAsequencesofthetargetbiological samples. Moreover, the differential display (DD) technique can simultaneously visualize increased or decreased expression of numerous mRNAs from many samples, and requires relatively small amounts of starting material (Pardee &

McClelland1999).AlthoughDDalsohassomedisadvantages,asthepossibilityof isolation “falsepositive”transcripts(PCRproducts that appear tobedifferentially expressed on the gel, but that cannot be verified when subsequent expression analyses are performed) (McClelland et al. 1995, Liang 1998, Pardee &

McClelland 1999), technical improvements have been increasing its successful application. Modifications which have been proposed to improve differential displayincludetheuseofprimerstailoredtoamplifymembersofaparticulargene family (Jurecic 1996), the optimization of annealing temperature (Malhotra et al.

1998),andoptimizationofprimerdesign(Graf etal. 1997).

In order to improve the isolation of differentially expressed genes in L. macrocephalus ,totalRNAsampleswereincubatedwithDNaseItoremoveDNA contamination which represents a further source of falsepositive bandsthat can beobservedondifferentialdisplaygels.AfterDNasetreatment,smallaliquotsof each sample were loaded onto an agarose gel to check for the RNA integrity.

Subsequently, good quality RNA samples were used to prepare RNA pools of

78 males and females, separately, in order to avoid the detection of interindividual variationsnotrelatedtodifferencesbetweensexes.

Further, preliminary analyses were performed to determine primers that give good and reproducible amplification results. As so, cDNA amplification was achievedusingdecameroligonucleotidescommonlyusedinRAPDanalysis,and also oligonucleotides that were designed based on VNTR (Variable Number of

TandemRepeats)coresequencesthatcorrespondtominisatelliteshortandhighly conserved regions (Jeffreys et al. 1985). The use of the 10mer primers usually resulted on several faint and diffuse bands, and the results were generally not reproducible(datanotshown).Thesefaintbandsoftenmakeinterpretationofthe data difficult. Moreover, just a reduced number of candidate differentially expressedtranscriptscouldbeidentifiedwhencomparingmalesandfemalesof L. macrocephalus throughtheuseoftheseRAPDprimers.Theuseoflongerprimers on differential display strategies can also lead to detection of DNA polymorphism through RAPDlikeresults. Somestudiesevidencedthat primerswith13basesor longerhavebettereffectsintheDDefficiency(Zhao etal. 1995,Liang1998,Motlik etal. 1998,Huang etal. 2001).AstheoligonucleotidesofVNTRcoresequences

(1424 bp), used as single primers to amplify the cDNA samples of L. macrocephalus ,arelongerthanRAPDprimers,themethodologycouldbeeffectively carriedoutatarelativelyhighstringency,thusyieldingbetterresults–thegenerated bands were more distinct and the results of cDNA amplification were consistently repeatable.

The DD methodology generally leads to the visualization of 5 to 100 amplificationproductsonpolyacrilamidegel(McClelland etal. 1995).Theobtained results indicated that, even using longer primers, it was not possible to achieve

79 complexamplificationpatternswithahighnumberofbands,afactthatcouldbe duetothehighstringencyofthePCRconditions,troughanannealingtemperature of55 oC,andduetotheuseofRNAsamplepoolsthatcanavoidthedetectionof individual variations. Despite the relative reduced number of amplified fragments

(around 5 to 15 bands) visualized on polyacrilamide gel, some of them were identified only in males or females, which indicate that they correspond to presumptivedifferentiallyexpressedtranscriptsbetweenthetwosamples.Thirtyone cDNAsappearedtobedifferentiallyexpressedbetweenmalesandfemalesof L. macrocephalus .

Duringthecourseoftheexperiments,weexperiencedafailuretoreconfirm differencesinmRNAexpression,duetodifficultiesinreamplifyingand/orcloning theselectedtranscripts,aproblemthatisrelativelycommonindifferentialdisplay strategies(Liang etal. 1993,Pardee&McClelland1999).Therefore,justthreeof the detected candidate fragments (Figure 1) were reamplified and cloned into a plasmid vector. The primer INS leaded to the identification of a conspicuous fragment of approximately 400 bp only in male samples of L. macrocephalus

(Figure1a).Afragmentofaround300bpwasalsoidentifiedonlyinmalesamples throughtheuseoftheprimerHBV5(Figure1b).TheprimerOPP11permittedthe visualizationofabandofapproximately400bp,uniquetofemalesofthespecies

(Figure 1c). Since different cDNA species can be contained theoretically within one differentially expressed fragment (Liang & Pardee 1995), at least five white colonieswereselectedandanalyzedfromeachclonedfragment.

Realtime PCR was used to validate the differential display results. The indication that some of the isolated genes do not present a sexually dimorphic expression between males and females of L. macrocephalus , as previously

80 deducedthroughthedifferentialdisplaydata,canbeassociatedtoseveralfactors that can affect results. Not only the experimental designs and the amplification reaction conditions, but also thesystems being compared can alter theobtained data. Comparisons between the same type of cells,as performed in thepresent study,leadstoahigherrateoffalsepositivessimplyduetothefewerdifferences ingeneexpression(Liang1998).

DDRTPCRresultsusingprimerINS

Cloning and sequencing of the selected band from males of L. macrocephalus , obtained with the primer INS, permitted the characterization of twodistinctfragmentswith265bpand231bp(Figure2a,b).

Database searches for nucleotide similarity evidenced that the 265 bp fragmenthasahighidentitylevelwithapartialregionofthegenethatcodesfor the protein APBA2 or amyloid beta (A4) precursor proteinbinding, family A, member 2, of several vertebrates. A high identity level was evidenced with the apba2 gene described for the species Danio rerio (85%) and Xenopus tropicalis

(77%), in relation to a 95% query coverage of the analyzed sequence. Gene expression analyses were performed throughout RealTime PCR using primers denominated Bami (F) and Bami (R) (Table 3), leading to the amplification of a fragment of 126 bp. Further cloning and sequencing of this fragment confirmed thatitrepresentsasegmentofthe apba2 gene.TheqRTPCRanalysis(reaction efficiency above 99%; R2 correlation coefficients ≥0.97) revealed a significant differencebetweenmalesandfemales,asahighergeneexpressionwasdetected in malesof L.macrocephalus (Figure3a,d;Table4)thusvalidatingtheisolated

81 fragment as a differentially expressed transcript. The mRNA level of males was almostthreefoldhigherthaninfemales.

TheAPBA2correspondstoatransmembranecellsurfaceglycoproteinthat ismainlyexpressedinbrain(Sutcliffe etal. 2003,Taru&Suzuki2004,Sano etal .

2006)andthatcanmodulatetheAβPP(amyloidβproteinprecursor)expression, leading to its decrease or increase. Consecutive cleavages of the AβPP by secretasesgeneratebetaamyloidproteins(βA)(Taru&Suzuki2004,Sano etal .

2006) that, at normal physiological levels, appear to be involved in neurotrophic events,i.e.thegrowthandsurvivalofdevelopingneuronsandthemaintenanceof mature neurons (Deister & Schimidt 2006). However, an overexpression of the aβpp gene can lead toanincreased production ofthebetaamyloid protein(e.g.

Mattson et al. 1993), a feature that has been related to Alzheimer’s disease, a frequent neurodegenerative disorder of the central nervous system that causes mental deterioration and progressive dementia due to neuropathologic lesions including the presence of senile plaques mainly composed by the betaamyloid protein (e.g. Selkoe 1994). Furthermore, it also seems that mutations along the aβpp gene or mutations in the enzymes that cleave the AβPP into betaamyloid proteinscanalsoleadtothedevelopmentofAlzheimer’sdisease(Harrison etal.

1991,Hendriks&VanBroeckhoven1996,Hartmann etal. 1996,Panegyres1997,

Cole etal. 2001,Yamamoto&Behl2001,Xie etal. 2003).

Data on Alzheimer’s disease indicate a prevalence of this neurodegenerative disorder in females of some human populations, a resultthat seem to be correlated not only to hormones, as estrogen and gonadotropins

(Casadesus et al. 2008), but also to age and education level (Letenneur et al.

2000). The identification of a higher apba2 gene expression in males of L.

82 macrocephalus can be correlated to an increase in amyloid βprotein precursor which,inturns,wouldleadtoahighercontentofbetaamyloidproteininthebrain of animals of this sex. Even though the functional significance of the increased apba2 expression in males than in females of L. macrocephalus is unclear, this fishspecies,similarlytoothervertebratesasmouse(Fisher etal. 1991), Xenopus laevis (VandenHurk etal. 2001)andchicken(Carrodeguas etal .2005),canalso beusedasamodelinordertostudyingAlzheimer’sdiseasedevelopmentoreven diseasetherapeuticagents.

The second characterized 231 bp fragment of L. macrocephalus , isolated throughtheuseoftheprimerINS,evidencedahighsimilaritywithapartialregion ofthe Rab37 gene,amemberofthe Ras oncogenefamily.Thisisolatedtranscript of L.macrocephalus has87%and85%identity,atnucleotidelevel,tothe Rab37 gene described for Danio rerio and with the variant transcript 3 of this gene of

Homo sapiens , respectively. The quantitative gene expression analysis (reaction efficiency above 99%; R2 correlation coefficients ≥0.97) was performed using designedprimersdenominatedRab37(F)andRab37(R)(Table3),leadingtothe amplification of a fragment of 170 bp. Further cloning and sequencing of this fragmentconfirmedthatitrepresentsasegmentofthe Rab37 gene.Althoughthe statisticalanalysesrevealedahigherexpressionlevelinmalesthaninfemalesof

L. macrocephalus , the differences were not statistically significant (Figure 3b, d;

Table4).

The Rab37 gene is amember ofthemost commononcogenes relatedto humantumorsthe Ras oncogenefamily.TheRasproteinscorrespondtosmall

GTPbinding proteins that are involved in signal transduction pathway of several normalcellularfunctions,ascellproliferation,differentiation,adhesion,apoptosis,

83 and migration (Masuda et al. 2000, Stenmark & Olkkonen 2001). In brain, the

Rab37 gene is mainly related to the differentiation of neuronal cells (BarSagi &

Feramisco1985,Noda etal. 1985,Hagag etal. 1986).Cancerinductionseemsto be related to overexpression of Ras genes or alterations in their proteins, which lead to an abnormal cellular proliferation (Capon et al. 1983, Brown et al. 1984,

Feramisco etal. 1984,Stacey&Kung1984).

Higherexpressionlevelsof Ras oncogeneshavebeendescribedinhuman braintumors (Zimmer et al. 1987, Lee et al. 2008). However, there is no conclusive dataonasexuallydifferentialexpressionofthesegenes,sincesome genes of the Ras family present a higher expression in women while others are highly expressed in men, a feature that can also be related to the type of the cancerandage (Stenmark&Olkkonen2001).Therefore,theobtainedresultsthat suggest a higher expression of the Rab37 gene in males of L. macrocepalus remaintobeelucidated.Comprehendingthewaysinwhichtheregulatoryactions of Rab proteins intertwine with cellsignaling cascades and developmental processesremainstobeclarified.

DDRTPCRresultsusingprimerHBV5

AftercloningandsequencingthecDNAfragmentthatwasamplifiedthrough the use of primer HBV5, observed in male samples of L. macrocephalus ,it was possible to characterize a 194 bp segment (Figure 2c). Data base searches revealednosignificantsimilaritywithanyknowngene,despitea~30bpregionthat showed an identity level of 93% with a region of the gene encoding protein C

(PROC) of Gallus gallus that functions as an antiinflammatory and also as an inhibitor of the coagulation factors Va and VIIIa, subsequently blocking the

84 generation of thrombin (Jijo et al . 2007). Although the isolated transcript did not revealasignificantsimilaritytothe proc gene(databasesearchesevidenceda15% querycoverageoftheanalyzedsequence),itisalsopossiblethatthecharacterized segment represents an untranslated region of the mRNA which is commonly less conserved than the coding regions between homologous genes in different organisms(ColonaRomano etal. 1998).

AnewsetofprimersHBV5(F)andHBV5(R)wasdesignedinorderto be used onqRTPCR(reaction efficiencyabove99%;R2correlationcoefficients

≥0.96), leading to the amplification of a fragment of 108 bp. Cloning and sequencing of this fragment confirmed a high identity with the previous isolated fragmenttroughthedifferentialdisplaystrategy.Althoughmostmalespresenteda higher expression level of this transcript, the observed difference between both sexeswasnotstatisticallysignificant(Figure3c,d;Table4).

DDRTPCRresultsusingprimerOPP11

The cDNA amplification using primer OPP11 generated a femalespecific bandofapproximately400bp(Figure1c).Cloningandsequencingofthisfragment permitted the characterization of a 414 bp segment that revealed no significant identitylevelwithpreviouslyreportedgenesequences.An89%identitylevelcould be observed betweena36bpof the isolated fragment of L. macrocephalus and theBACcloneCH251315P9of Pantroglodytes andtheBACcloneRP1154F22of

Homosapiens thatweremappedtotheXchromosomeofthesespecies.Despiteno information on the sequences contained in these BACs, the database searches evidencedextremelyreducedquerycoverage(9%)oftheanalyzedsequence.

85

Thecharacterizednucleotidesequenceoftheisolatedtranscriptwasused todesignasetofprimers–primers11P(F)and11P(R)(Table3)–tobeusedon the real time PCR assay. However, there was no success in obtaining a single amplification product. Primer design and PCR conditions could have lead to the observed results, a feature that seem to be common in quantitative RTPCR analyses(Roling etal .2004).Althoughtheobtaineddatadonotproviderelevant information about the nature of the isolated fragment, its putative dimorphic expressioninmalesandfemalesof L.macrocephalus indicatesthatthistranscript shouldalsobebetterinvestigated.

Finalconsiderations

Sexuallydimorphic geneshave beendescribed fordifferentvertebrates in somatic tissues, a feature that has been proven to be due to sexual hormones, speciallyestrogensandandrogensanditsmetabolites(Dewing etal. 2003,Morris et al. 2004, McCarthy & Konkle 2005, Becker et al. 2005, Tomassini & Pozzilli

2006, Verma & Shapiro 2006). Recentevidences indicate thatdifferences in the expression of some genes in brain also arise under the influence of the sex chromosome complement, independent of gonadal sex steroids levels (Arnold

2004).However,thereisstillseverallacksinthecomprehensionoftheregulation of these genes in brain, especially in lower vertebrates. Improving data on the isolationandcharacterizationofthesetranscriptsinfishspecies,thatcomprisethe basalvertebrategroup,couldgivenewinsightsintogenesstructure,functionand evolution.

To get better insights into several genes that can present a different expressionleveldependingonthesex,theavailabilityofapropermodelsystem may be crucial. The zebrafish Danio rerio is actually one of the few lower

86 vertebrate models in which genomewide methods can be employed effectively.

However,thefundamentalprocesscontrollingsexassignmentinthisspecieshas yettobeclarified,sincenoheteromorphicsexchromosomeshavebeendetected

(Sola &Gornung2001). Leporinusmacrocephalus isagonochoristicspeciesthat presentsaZZ/ZW sexdeterminationsystem(Galetti&Foresti1987).Moreover,it hasbeenintensivelyexploitedinthesubtropicalaquacultureduetocharacteristics as the facility to adaptation to captive breeding conditions, good reproductive performance and excellent growth rate (Takahashi et al. 2004). Therefore, it may represent an interesting fish model tothe understandingof thefunctionofseveral vertebrategenesassociatedtosexdeterminationanddifferentiation.Inaddition,as evidencedbythepresentresultsonthe apba2 and Rab37 genes ,itcouldalsobe usedasanaturalmodelfortheassayofdrugsthatcanregulateseveralprocesses relatedtogenesinvolvedinneurodegenerativeandcancerdiseases.

Acknowledgements

TheauthorsthankDr.MaeliDalPaiSilva,Dr.JoãoPessoa,andDr.Débora

Colombi for technical assistance on the Real Time analyses. This work was supportedbygrantsfromFAPESP(FundaçãodaAmparoàPesquisadoEstadode

São Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e

Tecnológico).

87

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Tables, Figures and Legends

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Table 1: Oligonucleotides that were designed based on VNTR core sequences and used as single primers for the amplification of the cDNA samples of Leporinus macrocephalus . Primer Sequence (5’ - 3’) Reference INS ACAGGGGTGTGGGG Nakamura etal. (1987) YNZ22 CTCTGGGTGTCGTGC Nakamura etal. (1987) HBV5 GGTGTAGAGAGGGGT Nakamura etal. (1987) HBV3 GGTGAAGCACAGGTG Nakamura etal. (1987) FvIIex8 ATGCACACACACAGG Murray etal. (1988) EMBL AGAGCTTCAGGCTGGGCAGCTAAG Harris&Wright(1995)

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Table 2: Decamer oligonucleotides used as single primers for the amplification of the cDNA samples of Leporinusmacrocephalus . Primer Sequence (5’ - 3’) OPP1 GTAGCACTCC OPP2 TCGGCACGCA OPP3 CTGATACGCC OPP4 GTGTCTCAGG OPP5 CCCCGGTAAC OPP6 GTGGGCTGAC OPP7 GTCCATGCCA OPP8 ACATCGCCCA OPP9 GTGGTCCGCA OPP10 TCCCGCCTAC OPP11 AACGCGTCGG OPP12 AAGGGCGAGT OPP13 GGAGTGCCTC OPP14 CCAGCCGAAC OPP15 GGAAGCCAAC OPP16 CCAAGCTGCC OPP17 TGACCCGCCT OPP18 GGCTTGGCCT OPP19 GGGAAGGACA

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Table 3: Set of primers that were designed based on the nucleotide sequences of the isolated DNA fragments from Leporinus macrocephalus, inordertobeusedonRealTimePCRassays. Primer (5’ - 3’) Bami(F) CATTCTTACGCTCTGCCATG Bami(R) TAGTAGCCCGTGATAAGGAC Rab37(F) GTGGGAGTTCAAATGCTGGG Rab37(R) GGAAATTCATCCCTGGCTCC HBV5(F) CCCTGTTACTGCGCATGAG HBV5(R) CACGCGGTATCAGGATCA P11(F) GCTACATTTCAGAGTATAATAG P11(R) CATATGAAAGCTTAGAATCTCT F=forward;R=reverse

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Table 4 : Data on the relative expression of the isolated genes from brain tissue of Leporinusmacrocephalus .

Designed set of primers Mean* ± SD “t” value “P” values 5 6 M=2,53 x10 ±3,47 x10 Bami 6 6 14,27 <0,001 F =8,16 x10 ±1,53 x10 7 7 M=8,44 x10 ±4,89 x10 Rab37 7 7 98** 0,623** F =7,03 x10 ±4,33 x10 7 8 M=1,92 x10 ±8,52 x10 HBV5 7 8 1,568 0,134 F=1,4 x10 ±5,85 x10 *Meanvaluereferingtothevaluesobtainedfor2 ∆Ct **Refersto“T”valueobtainedforthesesamplesthroughtheMannWhitneystatisticaltest M,malesamples;F,femalesamples

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L M F L M F L F M

3.054 3.054 3.054 2.036 2.036 2.036 1.636 1.636 1.636 1.018 1.018 1.018

517/506 517/506 517/506 396 396 396 344 344 344 298 298 298 220/201 220/201 220/201 154 154 154 134 134 134 75 75 75 (a) (b) (c)

Figure 1: cDNA amplification patterns of Leporinus macrocephalus , visualized in 6% polyacrilamide gel,obtained through the useof primers INS (a) ,HBV5 (b) ,and OPP11 (c) .Sex associated fragments areindicated by arrows.L,1KbDNAladder;M,males;F,females.

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5’ ACAGGGGTGTGGGG AACTCTGCTTGCAGTGGGACGTCGAGTCCTGCGTG.....49 5’GGTGTAGAGAGGGGT GCTGGAGCTGTGATACCACACGCGGTATCAGGATC.....50 GCTCCTGTCCTGCAGCGATCCTCCAGCCATAGCTTTGGCATTCTTACGCTC T....101 AGAATTCTGGTATCACGACAGACGCACAGCTGCAGTAGCGACCCTGCTGATA....102 GCCATGATCCGAGAGCAGATCTCATGCAGACTAGTGGCGATGGCTTTGGCAG....153 ACGGGTACTCGGTTACAGCACTCATGCGCAGTAACAGGGTCTCCGGGCTTTC....154 GCGTGTCACAGCGGAAAACGTGACATTTTAATATCCGAGTGTCCTTATCAC G....205 TGGTTCTCCTGCTGACCCGATGATGACCCCTCTCTACACC 3 ’...... 194 GGCTACATACGCAAAATCCCTCTCTCTGCCGTTGTCCCGGCCTACGCCCCAC ....257 ACCCCTGT 3’...... 265 (c) (a) 5’AACGCGTCGG GCCAGGAATCAAAAATAAATATATTTAAGGATATATCTGA....50 AGATGTNCAGTACCACCAAAGACAATCAAAGTACCATATGAAAGCTTAGAAT...102 CTCTACTTTTCGGTCGTTTTGCCATTTTTTCTTTATTTTCATTCAGAAAATA...154 5’ ACAGGGGTGTGGGG GGGTGGGAGTTCAAATGCTGGGTACTATAGTGAAA.....49 AATATTTTGTTCAAAAACTAGCGTTAGTATGACAAAATATTAGGTACACTAC...206 GATCAATCATGGAAAGGCAAAAAAAGATGAACTGTGTATTCTGCTCTACGCA....101 GAACATTTTTATTTTTCGTTAAGCCATCTTTTGGCCAATCTGGACATATTAC...258 ACGCTGCAAATAACATATTAAATGCTTGTTTTATTCGTGAAGCCGATGCCCA....153 ACATCAAACGAATGGGGAGACCCTCAGGATTACGATGGTGTAAGCTATTATA...310 CAGTAGCAGAAAAGGAGCCAGGGATGAATTTCCCCTGATCAAACTGGACGA G....205 CTCTGAAATGTAGCAGTATTTAAATAAAAAATAAAGAATGGCTTTTCCAAAC...362 CAGGGACGTTTTCCCCACACCCCTGT 3 ’...... 231 AGTAACACGCCTCCTAGCGCCGCCACTACTTCTGCGTTTCTCCGACGCGTT 3 ’.414 (b) (d)

Figure 2: Consensus nucleotide sequences of cDNA fragments of Leporinus macrocephalus, corresponding to candidate differentiallyexpressedgenesbetweenmalesandfemales,isolatedfrombraintissueusingprimersINS (a and b) ,HBV5 (c) , andOPP11 (d) .Theunderlinedregionsarehomologoustotheprimersequences.

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35 0,3 30 0,25 25 0,2 20 Male Male 0,15 15 Female Female 0,1 10 Relative Expression Relative Relative Expression Relative 5 0,05

0 0 Apba2 HBV5

(a) (c) 3,5 1,6 3 1,4 2,5 1,2 2 1 Male Male 1,5 Female 0,8 Female 1 0,6 Relative Expression Relative 0,5 0,4 Relative Expression Relative 0 0,2 Apba2 HBV5 Rab37 0 Genes Rab37

(b) (d)

Figure 3: Relative gene expression analysis (RealTime PCR) of the candidate sexually dimorphic expressed genes isolatedthroughDDRTPCRfrombraintissueof Leporinusmacrocephalus ,relativetotheconst itutive18SrRNAgeneused ascontrol. (a) , (b) ,and (c) representdataonmalesandfemales,separately,usingsetofprimers(denominatedasBami, Rab37, and HBV5) designed based on the previous nucleotide sequences of the isolated transcripts; (d) repre sents a comparative analysis of the relative expression of the three isolated genes – the females were assigned by a reference value=1,andthemaleswererepresentedbyaratiooftheirvaluesinrelationtothereferencefemalevalue.Datawererun induplicate.Normalizeddatawereexpressedasmeans ±SD.

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CAPÍTULO 3*

Distribuição espaço temporal de transcritos do gene de fatty acid-binding

protein 6 ( fabp6 ) em zebrafish ( Danio rerio ) adulto e em desenvolvimento

* Artigopublicado: FEBSJournal 275:33253334(2008).

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Spatio-temporal distribution of fatty acid-binding protein 6 ( fabp6 ) gene transcripts in the developing and adult zebrafish (Danio rerio )

Abstract

Wehavedeterminedthestructureofthefattyacidbindingprotein6( fabp6 )gene and the tissuespecific distribution of its transcripts in embryos, larvae and adult zebrafish ( Danio rerio ). Like most members of the vertebrate FABP multigene family, the zebrafish fabp6 gene contains four exons separated by three introns.

The coding region of the gene and expressed sequence tags code for a polypeptideof131aminoacids(14kDa,pIof6.59).TheputativezebrafishFabp6 proteinshared greatest sequenceidentitywithhumanFABP6(55.3%)compared to other orthologous mammalian FABPs and paralogous zebrafish Fabps.

Phylogenetic analysis showed that the zebrafish Fabp6 formed a distinct clade withthemammalianFABP6s.Thezebrafish fabp6 genewasassignedtolinkage group (chromosome) 21 by radiation hybrid mapping. Conserved gene synteny was evident between the zebrafish fabp6 gene on chromosome 21 and the

FABP6/Fabp6 genes on human chromosome 5, rat chromosome 10 and mouse chromosome11.Zebrafish fabp6 transcriptswerefirstdetectedinthedistalregion of the intestine of embryos at 72 h postfertilization. This spatial distribution remained constant to 7dayold larvae, the last stage assayed during larval development. In adult zebrafish, fabp6 transcripts were detected by RTPCR in

RNA extracted from liver, heart, intestine, ovary and kidney (most likely adrenal tissue),butnotinRNAfromskin,brain,gill,eyeormuscle. Insitu hybridizationof a fabp6 riboprobetoadultzebrafishsectionsrevealedintensehybridizationsignals in the adrenal homolog of the kidney and distal region of the intestine, and to a

107 lesserextentintheovaryandliver,atranscriptdistributionthatissimilar,butnot identical,tothatseenforthemammalian FABP6/Fabp6 gene.

Introduction

Intracellular lipidbinding proteins (iLBPs) are encoded by a highly conservedmultigenefamily,andincludefattyacidbindingproteins(FABP/Fabps), cellular retinolbinding proteins (CRBPs) and the cellular retinoic acidbinding proteins (CRABPs) [1, 2]. Currently, 16 paralogous iLBP genes have been identified in animals, but no member of this multigene family has thus far been identifiedinplantsandfungi.Schaap etal. [2]havethereforesuggestedthatthe first iLBP gene emerged after the divergence of animals from plants and fungi approximately 930 million years ago. This ancestral iLBP gene presumably then underwentaseriesofduplicationeventsfollowedbysequencedivergence,giving risetotheextantiLBPmultigenefamily.

To date, 11 isoforms of FABP/Fabps or their genes, or both, have been identified in vertebrate species [3]. Originally, these proteins were named accordingtothetissuefromwhichtheywereinitiallyisolated, e.g., livertypefatty acidbinding protein (LFABP), braintype fatty acidbinding protein (BFABP), intestinaltypefattyacidbindingprotein(IFABP), etc. However,thisnomenclature hasbecomeconfusingbecausedifferenttypesofFABPshavebeenisolatedfrom the same tissue, and some orthologous FABPs from different species exhibit distinctly different tissuespecific patterns of distribution [4, 5]. Furthermore, two socalled livertype fabp genes, fabp1a and fabp1b (based on phylogenetic analysis and conserved gene synteny), are not expressed in the liver of teleost

108 fishes [6]. In this paper, we have used analternativenomenclature proposedby

Hertzel and Bernlohr [4], which uses numerals to distinguish the FABP proteins and genes corresponding to the chronological order of their discovery, e.g.,

FABP1 (livertype FABP), FABP2 (intestinaltype FABP), FABP3 (hearttype

FABP), etc. We have also followed the gene and protein designations for mammalianandteleostfishgenesandproteinsaccordingtotherecommendations of the Zebrafish Model Organism Database ( http://zfin.org ), in which zebrafish genes and proteins are represented as fabp6 and Fabp6, respectively, human genes and proteins are given in uppercase letters, e.g., FABP6 and FABP6, respectively,andthemousegeneisdesignated Fabp6 anditsproteinFABP6.

Phylogenetic studies have identified three main groups for the FABPs: group 1 includes FABP1, FABP6 and Fabp10 (Fabp10 has only been found in nonmammalian vertebrates), group 2 consists of a single protein, FABP2, and group3consistsofFABP4,FABP5,FABP8,FABP9andFabp11(Fabp11maybe uniquetoteleostfishes[3]).Schaap etal. [2]estimatethattheFABPsfromgroup

1divergedfromthelastcommonancestralFABPgeneapproximately679million yearsago.

Although the first FABP, FABP1, was described almost four decades ago

[7], and extensive studies have focused on the tissue distribution and binding activities and regulation of FABP genes, including FABP gene knockout experiments [8], our understanding of the physiological function(s) of these proteinsremainslimitedor,inmanycases,unknown.However,sufficientevidence existstostronglysuggestthefollowingrolesforFABPs:(a)uptakeoffattyacids acrosstheplasmamembraneandtransporttovarioussubcellularorganelles;(b) modulationofactivityofenzymesinvolvedinfattyacidmetabolism;(c)protection

109 of enzymes and membranes from the detergent effects of excess fatty acids by sequestering them, and (d) modulation of cell growth and differentiation by transport of fatty acids to the nucleus where they activate specific gene transcription[4,8,9].

Here we report studies on the fabp6 gene from zebrafish, the first fabp6 gene described for nonmammalian vertebrates. Previous work has reported the cloningandsequencingofmammalian FABP6/Fabp6 genesandcDNAs,andtheir expression in mammalian species including human [10], rat [1113], mouse [14,

15]andpig[16].Overtheyears,FABP6hasbeengivenavarietyofnames,such as the ileal lipidbinding protein, intestinal 15kDa protein, ileal bile acidbinding protein and gastrotropin, reflecting the speculationsof authorsonits intracellular function(s). In vitro binding assays revealed a surprisingly low affinity of recombinantderivedhumanFABP6andratFabp6forlongchainfattyacids,such as palmitate and oleate, despite these proteins having a common three dimensionalstructuralmotifwithotherFABP/Fabpsknowntobindlongchainfatty acids[10,17].WorkbyGong etal. [12]suggeststhattheligandsofFABP6are bile salts and that FABP6 is involved in their uptake from the ileal epithelium.

However,otherstudieshavedetectedmammalian FABP6/Fabp6 genetranscripts andencodedproteinintheovaryandsteroidendocrinecellsoftheadrenalgland, leading to speculation that FABP6 may also function in steroid metabolism [11].

Comparative studies of FABP6/Fabp6/fabp6 gene expression in mammals and teleostfishesmayprovideadditionalevidencefortheroleofthisproteinincellular physiology.Inthispaper,wedescribethestructureofthezebrafish fabp6 gene,its linkage group (chromosome) assignment, conserved gene synteny with

110 mammalian orthologs, and the tissuespecific distribution of the fabp6 gene transcriptsinembryos,larvaeandadults.

Material and Methods

Husbandryofzebrafish

The ABstrain of zebrafish was used throughout this work andmaintained according to established procedures [27]. Experimental protocols were reviewed by the Animal Care Committee of Dalhousie University in accordance with guidelinessetdownbytheCanadianCommitteeonAnimalCare.

Nucleotidesequenceofzebrafishfabp6cDNAandgene

We retrieved a previously uncharacterized Ensembl gene

(ENSDAR00000044566)byaBLASTnsearchofthezebrafishgenomesequence database at the Wellcome Trust Sanger Institute (version Zv7, scaffold 296.3, http://www.ensembl.org/Danio_rerio/index.html) using NM_001002076 (GenBank accession number) as the query sequence. This sequence was also used in

BLASTn searches for other ESTs coding for zebrafish Fabp6. Based on the

NM_001002076 sequence, primers were designed for RTPCR amplification of thistranscriptfromtotalRNAextractedfromawholeadultzebrafishofstrainAB

(forwardprimer,5'CTCTTCTTCTCCGCTCAA3';reverseprimer,5'ATCAGT

TTA GCT CGT ACA3'). Theresultingproduct ofexpected size as estimated by agarose gel electrophoresis was cloned into the pGEMT vector (Promega,

Madison, WI, USA) and six clones sequenced. To identify SNPs, the cDNA sequences obtained by us were compared to the coding sequence of zebrafish fabp6 gene retrieved from the Zebrafish Genomic Sequence Database at the

Wellcome Trust Sanger Institute by alignment using CLUSTALW [28]. The

111 molecular massandisoelectricpointoftheFabp6polypeptideencodedbyclone

NM_001002076 was determined using the program at http://ca.expasy.org/tools/pi_tool.html.

Phylogeneticanalysis

Sequencealignmentanddeterminationofpercentageaminoacidsequence identity of FABP/Fabp sequences from zebrafish and other vertebrates was performed using BIOEDIT (v.7.0.9) [29]. Phylogenetic analysis was performed using CLUSTALW [28] to generate a neighbourjoining tree. Bootstrap values werebasedon100replicates.

Linkage group (chromosome) assignment by radiation hybrid mapping of the zebrafishfabp6gene

RadiationhybridsoftheLN54panelwereusedtoassignthe fabp6 geneto aspecificzebrafishlinkagegroupaccordingtotheprotocoldescribedbyHukriede et al. [21]. Two primers were designed (forward primer, 5'TAG GCA AAG AGA

GCCACATGCAGA3';reverseprimer,5'TGCTCAAATCCTGACACCATG

GAC3')toPCRamplifyaportionofthezebrafish fabp6 genefromgenomicDNA samples isolated from the LN54 hybrid panel using Platinum PCR Super Mix

(Invitrogen,Burlington,Ontario,Canada).

Wholemountinsituhybridizationtozebrafishembryosandlarvae

Wholemount in situ hybridization using a cloned fabp6 cDNAto generate an antisense riboprobe was performed according to the methods described previously[30].

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Detectionoffabp6transcriptsinadultzebrafishtissuesbyRTPCR

RTPCRwasusedtodeterminethetissuedistributionof fabp6 transcriptsin

RNA extracted from tissues of adult zebrafish. RNA was extracted from tissue usingTrizolreagent(Invitrogen).FollowingsynthesisofcDNAusingtheOmnicript

RT kit (Qiagen, Mississauga, Ontario, Canada), the zebrafish fabp6 transcripts were amplified by PCR from total RNA extracted from various tissues using the forwardprimer,5'TAGGCAAAGAGAGCCACATGGAGA3',andthereverse primer,5'GCGGTTAAACCTTCTTGCTTGTGC3',accordingtotheprotocol describedbyLiu etal. [23].Theconstitutivelyexpressedgeneforelongationfactor

1α ( ef1α) was usedas a positivecontrol toassaythe integrityof RNAextracted from each tissue. The primers and RTPCR conditions employed have been describedpreviously[31].

Detectionoffabp6transcriptinadultsectionsofzebrafishbyinsituhybridization

Asyntheticantisenseprobe,5'GTACTGGGTCCATGTGAAGTCATC

TCC GTT C3', was used for in situ hybridizations to detect fabp6 transcripts in sectionsofadultzebrafishaccordingtothemethoddescribedbyDenovanWright etal. [32].

Results and Discussion

IdentificationofzebrafishcDNAandgenomicfabp6sequences

Following BLAST searches of GenBank at the National Center for

BiotechnologyInformation(NCBI),weidentifiedanexpressedsequencetag(EST)

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(GenBank accession number: NM_001002076) [3] for which the deduced amino acid sequence showed highest percentage sequence identity to the amino acid sequencesofmammalianFABP6/Fabp6s(seebelow).UsingthisESTsequence as a query, we retrieved numerous other ESTs coding for zebrafish Fabp6 from

NCBI and the sequence for the zebrafish fabp6 gene from the genomic DNA assembly Zv7, scaffold 296.3 (ENSDARG00000044566), at the Wellcome Trust

Sanger Institute (http://www.ensembl.org/Danio_rerio/index.html). In order to generateahybridizationprobeforfurtherstudyofthetissuespecificdistributionof fabp6 transcripts in zebrafish embryos, we amplified the fabp6 transcript by RT

PCRoftotalRNAextractedfromawholeadultzebrafish.TheresultingDNAofthe expectedsizewascloned,andsixindependentclonesweresequencedandfound tobeidentical(GenbankaccessionnumbersEU665309–EU665314).Fivesingle nucleotide polymorphisms (SNP) were seen between the sequence of the fabp6 transcriptsclonedbyusandthecodingsequenceofthe fabp6 gene(Fig.1).Only oneSNPinthecodingsequence,locatedatnucleotide(nt)position+1633inFig.

1,changedthededucedaminoacidsequenceofFabp6,achangeofvaline(GTC) toisoleucine(ATC).WeattributethefiveSNPstodifferencesbetweenestablished strains in zebrafish. The coding sequence derived from the fabp6 gene at the

Wellcome Trust Sanger Institute was derived from the Tuebingen strain of zebrafish, while the sequences for the fabp6 cDNA generated in this studywere derivedfromtheABstrainofzebrafish(seehttp://zfin.orgforstraindetails).

Thecodingsequenceofthe fabp6 genecontainedanopenreadingframe of393bp(notincludingthestopcodon),with5'and3'untranslatedregionsof50 and74bp,respectively(Fig.1). The openreadingframecodesforapolypeptide of131 aminoacidswithamolecularmassof14406Daandanisoelectricpointof

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6.59. WiththeexceptionofsomeFabp10s,whichhaveanisoelectricpointof8.8

9.0,allotherFABPshaveisoelectricpointsofapproximately6[18].

Thezebrafish fabp6 gene consists offourexons of112,174,89and141 bp, coding for 22, 59, 30 and 20 amino acids, respectively. The intron/exon structureofthe fabp6 genefromzebrafishisconsistentwiththatofallother fabp genesstudied todate[1]withtheexceptionofthemuscletypeFABP(MFABP) genes from desert locust [19], which lacks intron II, and the fabp1b gene from zebrafish, which contains an additional intron in the 5'untranslated region [6].

Eachoftheintron/exonsplicejunctionsinthezebrafish fabp6 geneconformtothe

GT/AGruleproposedbyBreathnachandChambon[20].

Alignment of the zebrafish Fabp6 sequence with mammalian FABP6 sequences(human,rat,mouseandpig)andtoparalogsofotherzebrafishFabps and human FABPs showed that zebrafish Fabp6 shared greatest sequence identity with human (55.3%), mouse (50%), rat (50%) and pig (49.2%) FABP6 sequences (Fig. 2). Sequence identity of the zebrafish Fabp6 with paralogous zebrafish Fabps and human FABPs varied from 42.1% to 21.8%. Phylogenetic analysisrevealedtheinclusionofzebrafishFabp6withhuman,rat,mouseandpig

FABP6s in a distinct clade with a robust bootstrap value of 99/100(Fig. 3). The phylogenetic tree indicates a closer evolutionary relationship between FABP6s and FABP1s, and a more distant relationship between FABP6s and FABP7s, a finding consistent with early phylogenetic studies of rat and human FABP6 and

FABP1[10,12].Thesequencealignment(Fig.2)andphylogeneticanalysis(Fig.

3) strongly suggests that the ESTs and genomic sequence retrieved from DNA assemblyZv7,scaffold296.3(WellcomeTrustSangerInstitutezebrafishgenome sequence)describedabove,codeforFabp6inzebrafish.

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Linkage group assignment of the zebrafish fabp6 gene by radiation hybrid mappinganditsconservedgenesyntenywithmammalianFABP6/Fabp6genes

ToprovideadditionalevidencethatthegenelocatedontheDNAassembly

Zv.7,scaffold296.3,indeedcodesforzebrafishFabp6,wedeterminedthelinkage group (chromosome) assignment of the zebrafish fabp6 gene and examined its conserved gene synteny with the human, rat and mouse FABP6/Fabp6 genes.

UsingtheLN54panelofradiationhybrids[21]andspecificprimerstoexon2and introns 2, respectively (see Fig. 1 and experimental procedures), the zebrafish fabp6 genewasmappedtolinkagegroup(chromosome)21atadistanceof26.79 cRfromthemarkerfc08c06withaLOD(logarithmoftheodds[tothebase10])of

10.8(mappingdataavailableathttp://dir.nichd.nih.gov/Img/devb.htm).Thisresult isconsistentwiththechromosomallocationof fabp6 onZv6attheWellcomeTrust

Sanger Institute database, but not with the latest version, Zv7, which places the zebrafish fabp6 gene on chromosome 3. We have previously observed incompatibilities between radiation hybrid mapping data for other zebrafish fabp genesandtheirchromosomalassignmentintheWellcomeTrustSangerInstitute genome database for the zebrafish. Later, versions of the zebrafish genome sequencehavebeencorrectedinagreementwiththechromosomalassignmentof fabp genesbyradiationhybridmapping.

The conserved gene synteny between the zebrafish fabp6 gene on chromosome 21 and human FABP6 geneonchromosome5 is extensive(Table

1). Conservedgene synteny was alsoevidentbetweenthezebrafish fabp6 gene andthe Fabp6 genesonratchromosome10andmousechromosome11.Notall thegenesthatshowconservedgenesyntenybetweenzebrafishchromosome21

116 and human chromosome 5 are located on rat chromosome 10 and mouse chromosome11.Othergenesarelocatedonratchromosomes2,17,18and20, and mouse chromosome, 13, 15 and 18, suggesting chromosomal rearrangementsor translocationsintheseregionsafterdivergenceofthehuman androdentlineages.Despitethesechromosomalrearrangements,theconserved gene synteny shown in Table 1 strongly indicates that a common linkage group containing the FABP6/Fabp6/fabp6 gene wasinherited froma commonancestor offishesandmammals.Theconservedgenesynteny(Table1),sequenceidentity

(Fig. 2) and phylogenetic analysis (Fig. 3) provide compelling evidence that the putative zebrafish fabp6 gene described hereand themammalian FABP6/Fabp6 genes areorthologs.

Distributionoffabp6genetranscriptsinzebrafishembryosandlarvae

To determine the spatiotemporal distribution of fabp6 transcripts during zebrafish embryonic and larval development, we performed wholemount in situ hybridization to zebrafish embryos and larvae at various developmental stages

(Fig. 4). fabp6 transcripts were not detected in embryos at 48 h postfertilization

(hpf),butaverystronghybridizationsignalwasdetectedinthedistalregionofthe zebrafish intestine at 72 hpf (Fig. 4A) indicating that initiation of fapb6 gene transcriptionoccurredbetween48and72hfp.Thedistributionof fabp6 transcripts remainedconstantinthedistalregionoftheintestineofzebrafishlarvaefrom3to

7 days postfertilization (Fig. 4AC). A transverse section of a 4dayold larva showedthepresenceof fabp6 transcriptslocatedpredominatelyinepithelialcells oftheintestine(Fig.4B).

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To our knowledge, only two studies have investigated the tissuespecific distribution of FABP6/Fabp6 transcripts during embryogenesis [14,15]. In the mouse, Sacchettini et al. [14] showed by dotblot hybridization that no Fabp6 transcriptsweredetectedinanytissuesduringfetallife,orthroughoutthesuckling period of 112 postnatal days. Mouse Fabp6 transcripts were first detected and restricted to the ileum at the beginning of the suckling/weaning transition at postnatal days 1214. In contrast, Crossman et al. [15] did detect Fabp6 transcripts in mouse embryos. They used Northern blot analysis and quantified mRNA steadystate levels by scanning autoradiograms of RNA extracted from total intestine and sections along the entire length of the intestine ( i.e. , from the gastroduodenal junction to the rectum). Fabp6 transcripts were first detected in

RNAfromtotalintestineatE18whichisthestageatwhichthe‘proximaltodistal wave of cytodifferentiation of the pseudostratified gut epithelium to a monolayer had reached the ileum’ [14]. During postnatal development, Fabp6 transcripts were restricted to the distal third of the small intestine and cecum. No Fabp6 transcripts were detected in the duodenum, jejunum or 12 other extraintestinal tissues (the latter tissues were not specified). The transcriptional initiation of the zebrafish fabp6 geneinthedistalregionoftheintestineataround72hpf,priorto hatching (Fig. 5), occurs at approximately the same developmental stage as the transcriptioninitiationofthemouse Fabp6 geneintheileumatE18[14].

Tissuespecificdistributionoffabp6genetranscriptinadultzebrafish

We explored the tissuespecific distribution of fabp6 transcripts in adult zebrafishbyRTPCRamplificationfromtotalRNAextractedfromvarioustissues andby insitu hybridizationofa fabp6 specificantisenseoligonucleotideprobeto

118 sectionsofadultzebrafish.A fabp6 specificRTPCRproductofexpectedsizewas amplified from total RNA extracted from liver, heart, intestine, ovary and kidney

(Fig. 5, top panel). No fabp6 specific RTPCR product was amplified from total

RNA extracted from skin, brain, gill, eye or muscle. As a positive control to determine theintegrity of theRNAsamplesusedintheseassays,transcriptsfor the constitutively expressed elongation factor 1 α ( ef1 α) gene were amplified by

RTPCR.AproductoftheexpectedsizewasgeneratedfromRNAextractedfrom alltissuesassayed(Fig.5,bottompanel).

In situ hybridization of an antisense fabp6 oligonucleotide probe to adult zebrafish sections revealed an intense hybridization signal in the distal region of the intestine (Fig. 6, 1A) and in the adrenal homolog of the kidney (Fig. 6, 1D).

Lessintensehybridizationsignalswereobservedintheliver(Fig.6,1B)andthe ovary (Fig. 6, 1C). Despite the difference in sensitivity of the two methods employed,thetissuedistributionof fabp6 transcriptsinadultzebrafishassayedby

RTPCRandby insitu hybridizationwasidentical.

In adult mammals, the reported tissuedistributions of FABP6/Fabp6 gene transcriptsanditsproteinhavevaried,probablyduetotheassaytechniquesused.

Forexample,Fujita etal. [10]usedNorthernblotanalysistodetectasinglesized

FABP6 transcriptinRNAextractedfromtheterminalregionofthehumanileum, whereasRTPCRgeneratedanabundant FABP6 specificproductfromtotalRNA extractedfromtheileum,andtoamuchlesserextentfromRNAextractedfromthe humanovaryandplacenta.Unfortunately,theauthorsdonotstatewhetherother tissueswereassayedbyRTPCRinwhich FABP6 transcriptswerenotdetected.

Rat Fabp6 transcripts were detected by Northern blot analysis in RNA extracted from the ileum and ovary, but not in RNA extracted from the stomach, jejunum,

119 colon, adrenal, brain, heart, or liver [12]. Iseki et al. [11] used immunocytochemistrytolocalizetheratFabp6proteinand insitu hybridizationto localize Fabp6 transcriptstotheenterocytesoftheileum,lutealcellsoftheovary andasubpopulationofsteroidendocrinecellsoftheadrenalgland.Sato etal. [13] also detected rat FABP6 in theadrenal gland and ovary.In adultmouse, Fabp6 transcriptswereonlydetectedbyblothybridizationintheintestine,andnotinthe liver,stomach,pancreas,kidney,spleen,testis,skeletalmuscle,heartorlung[14].

Withtheexceptionofonereport[12],thesestudiesconsistentlyshowthat the FABP6/Fabp6 genetranscriptsareexpressedathighlevelsintheileumandto alesserextentintheovaryandadrenalglandofadultmammals.Inzebrafish,we showed by RTPCR and by in situ hybridization that fabp6 transcripts were detectedathighlevelsinthedistalregionoftheintestine,thetissuehomologous to the mammalianileum.Thepresenceoffabp6 transcriptssuggeststhatFabp6 maywellplayaroleintheuptakeoflipidsfromthedistalregionofthezebrafish intestine, which is similar to the suggested role for FABP6 in the uptake of bile saltsfromthemammalianileum[11,12].Zebrafishfabp6 transcriptswereshown byRTPCRassay(Fig. 5)and by insitu hybridization (Fig.6) tobeabundantin the ovary and kidney of adult zebrafish, similar to the distribution of mammalian

FABP6/Fabp6 transcripts,whicharealsogenerallyfoundintheovaryandadrenal gland.Infishes,theadrenalhomologisnotascompactastheadrenalglandfound in mammals. In fishes, adrenal tissue exists as aminergic chromaffin and inter regnalcellsmostlyinsidetheheadkidneywiththetwotissuesbeingeithermixed, adjacent,orcompletelyseparated[22].Thedistributionofthehybridizationsignal forzebrafish fabp6 transcriptsintheadrenalhomologofthekidney(Fig.6,1D)is consistent with the structure of the adrenal homolog in teleost fishes. With the

120 exception of the zebrafish liver and heart, the overall pattern of adult tissue distribution of zebrafish fapb6 and mammalian FABP6/Fabp6 gene transcripts, and the transcriptional initiation of these genes at similar embryonic stages of development,issurprisinglyconcordant,incontrasttosomeothermembersofthe multigene family of iLBP genes ( e.g., fabp1a/b , fabp10, fabp11, rbp2 )

[3,6,16,23,24].AsFABP6hasbeenimplicatedinhumancolorectalcancer[25]and type2diabetes[26],zebrafishmayserveasausefulmodelexperimentalsystem toinvestigatetheroleofFABP6inthesediseasestates.

Acknowledgements

The authors wish to thank Santhosh Karanth (Department of Biology,

Dalhousie University, Halifax, Canada) and Paul Wright (University of Virginia

HealthSciencesCenter,Charlottesville,VA,USA)fortechnicalassistanceduring the course ofthesestudies.Thisworkwassupported by funds fromthe Natural

SciencesandEngineeringResearchCouncilofCanada(toJ.M.W.),theCanadian

InstitutesofHealthResearch(toE.D.W.),andtheNationalInstituteofHealth,the

European Commission as part of the ZFModels integrated project in the 6 th

Framework Program (to B.T. and C.T.). F.AC. was a recipient of a scholarship from FAPESP, “Fundação de Amparo à Pesquisa do Estado de São Paulo”,

Brazil.

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Table, Figures and Legends

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Table 1 :Conservedgenesyntenybetweenzebrafishlinkagegroup(chromosome)21andhumanchromosome 5,ratchromosome10andmousechromosome11.

Chromosomal Position Genes Zebrafish Human Rat Mouse LG 5 2 10 17 /20 18 11 13 15 18

c6 21 5p13 2q16 153.0cM c7 21 5p13 2q16 153.0cM rpl37 21 5p13 2q16 15A1 fgf10 21 5p13p12 2q15q16 1375.0cM taf9 21 5q11.2q13.1 2q12 13D1 f2rl1 21 5q13 2q12 1375.0cM thbs4 21 5q13 2q12 1346.99cM bhmt 21 5q13.1q15 2q12 13D1 glrx 21 5q14 2q11 1344.0cM ell2 21 5q15 2q11 13C1 pcsk1 21 5q15q21 2q11q12 1344.0cM rnf14 21 5q23.3q31 18p11 1817.0cM cdc23 21 5q31 18p12 1817.0cM pou4f3 21 5q31 18p11 1824.0cM sept8 21 5q31 10q22 1128.5cM skp1a 21 5q31 10q22 1131.0cM vdac1 21 5q31 10q22 1129.0cM cnot8 21 5q31q33 10q22 11B1.3 ddx46 21 5q31.1 17p14 13B2 pdlim4 21 5q31.1 1128.5cM rapgef6 21 5q31.1 10q22 11B1.3 sara2 21 5q31.1 10q22 11B1.3 tcf7 21 5q31.1 10q22 1128.0cM zcchc10 21 5q31.1 10q22 1128.5cM spry4 21 5q31.3 18p11 1818.0cM zmat2 21 5q31.3 18p11 18B2 rbm22 21 5q33.1 18q12.1 18D2 larp1 21 5q33.2 10q22 11B2 sap30l 21 5q33.2 10q22 11B2 rnf145 21 5q33.3 11B1.1 fabp6 21 5q33.3-q34 10q21 11 24.0cM sgcd 21 5q33.3q34 10q21 11B1.2 mat2b 21 5q34q35 10q12 11A5 drd1 21 5q35.1 20p12 1332.0cM fgfr4 21 5q35.1 17p14 1333.0cM rars 21 5q35.1 10q12 11A4 ubtd2 21 5q35.1 11A4 cnot6 21 5q35.3 10q22 11B1.2 nola2 21 5q35.3 10q22 1128.5cM

126

tccaaacatatcatgaccctgagcatgctgccaaatta -560 gttaaaatgtactttaaggacaacagagtgaatgttttggagtggccatcacaaagccctgagctcaatcctatagaaa -480 atttgtgggcagagttgaaaaagcttgtgcgagcaaaacagccaacaaatttgacttagttacaccaattccgtcagga -400 ggaatgagccaaaattcgttcaaactattgtgagaagcttgtggaaggatacccaaaacatttgaccaaagttatacag -320 tttaaaatcaaagctaaaaaattcttatggtaatttttttgtattgtattataattgtattgcacgattataaagtgat -240 tttgcccatgtatggcatatcatcaaccatgtattttgcccatgtaaggcatatcatcactacccaggtgagcagccgt -160 gcggtcatgcactttatgacttactctattttgtggggccatcataaatgtttatggcttgattatgatgctggtgtac -80 cactaaggccatgagaaagagtcagagttgaggaacagttgggta TATAA acaccgcatacttcacccatcagtacctc -1

AGCTCTCAACCCGCTCTTCTTCTCCGCTCAATCAACACCAAAACC ATG GCT TTC AAC GGC AAG TGG GAA A +70 M A F N G K W E CC GAA TCT CAG GAG GGA TAT GAA CCA TTC TGC AAA CTG ATC G +112 T E S Q E G Y E P F C K L I

gt gaggct... INTRON I ... cacctc ag (+113 - +1576)

A GT ATC CCT GAT GAT GTC ATC GCA AAG GGC CGT GAC TTC AAG CTT GTG ACA GAG ATC GTC +1635 G I P D D V I A K G R D F K L V T E I V/I CAG AAC GGA GAT GAC TTC ACA TGG ACC CAG TAC TAC CCC AAT AAC CAT GTT GTG ACC AAC +1694 Q N G D D F T W T Q Y Y P N N H V V T N A AAA TTC ATC GTA GGC AAA GAG AGC GAC ATG GAG ACT GTA GGA GGG AAG AAA TTT AAG +1751 K F I V G K E S D M E T V G G K K F K

gt gtggca... INTRON II ... ttcacc ag (+1752 – +3729)

C GGC ATA GTT TCC ATG GAA GGA GGC AAG CTG ACC ATA AGC TTC CCC AAA TAT CAA CAA ACA +3789 G I V S M E G G K L T I S F P K Y Q Q T ACT GAG ATC AGC GGT GGA AAG CTG GTG GAG +3819 T E I S G G K L V E

gt gagata... INTRON III ... ttttc ag (+3820 – +4140)

ACC TCC ACA GCC AGT GGC GCC CAG GGT ACC GCT GTT CTT GTG CGC ACA AGC AAG AAG GTT +4200 T S T A S G A Q G T A V L V R T S K K V G T TAA CCGCATTGTTGAGATGAATGACCCTTTTCTGCTCTGACACTCAATTTACTG AATAAA GTGTACGAGCTAAACTGA +4278 * TAAA +4282

Figure 1. Thenucleotidesequenceofthezebrafish fabp6 gene.Thecodingsequence isshowninuppercaseletterswiththededucedaminoacidsequenceindicatedbelow. The size of each intron is shown with each exon/intron splice junctions (gtag) highlightedinboldfontandunderlinedandthe5'upstreamsequenceofthe fabp6 gene is presented by lowercase letters with a putative TATA box in uppercase letters, underlined and in bold font. Single nucleotide polymorphisms (SNPs) based on differencesbetweencDNAsequencesfromtheTuebingenandABstrainsofzebrafish arepositionedabovethegenomicsequenceandinboldfont.Apolyadenylationsignal sequence,“AATAAA”,isunderlinedandinboldfont.

127 ZF FABP6 M--AFNGKWETESQEGYEPFCKLIGIPDDVIAKGRDFKLVTEIVQNGDDFTWTQYYPNNHVVTNKFIVGK HU FABP6 .--..T..F.M..EKN.DE.M..L..SS...E.A.N..I...VQ.D.Q....S.H.SGG.TM....T... MO FABP6 .--..S..Y.F..EKN.DE.M.RL.L.G...ER..N..II..VQ.D.Q....S.S.SGGNIMS...TI.. RA FABP6 .--..T..Y.F..EKN.DE.M.RL.L.E...ER..N..II..VQ.D.EN...S.S.SGGNIMS...TI.. PI FABP6 .--..T..Y.I..EKN.DE.M.RLAL.S.A.D.A.NL.IIS.VK.D.QN...S.Q..GG.SI..T.TI.. ZF FABP1B .--S.T..YQL.....F.E.M.AV.L...M.E..K.I.S.S..EE..NQ.KV.VT-TGSK.L..S.TI.Q HU FABP1 .--S.S..YQLQ...NF.A.M.A..L.EEL.Q..K.I.G.S...... KH.KF.IT-AGSK.IQ.E.T..E ZF FABP1A .--..T..YQL..H.NF.A.M.AV.V...EVE..K.I.SIS..H.D.K..KV.VT-AGTK.ILYS.T..E HU FABP3 .VD..L.T.KLVDSKNFDDYM.SL.VGFATRQVASMT.PT.I.EK...IL.LKTH-STFKNTEIS.KL.V ZF FABP7A .VD..CAT.KLVDSQNFDEYM.SL.VGFATRQV.NVT.PTIV.SHE..KVVIKTL-STFKNTEIS.KL.E HU FABP2 .--..DST.KVDRS.N.DK.MEKM.VNIVKRKLAAHDN.KLT.T.E.NK..VKES-SAFRNIEVV.EL.V HU FABP7 .VE..CAT.KLTNSQNFDEYM.AL.VGFATRQV.NVT.PTVI.S.E..KVVIRTL-STFKNTEIS.QL.E ZF FABP3 .AD..I.T.NLKESKNFDEYM.G..VGFATRQVANMT.PT.I.SKE..V..LKTV-STFKSTEIN.KL.E HU FABP4 .CD..V.T.KLV.S.NFDDYM.EV.VGFATRKVAGMA.PNMI.SV...VI.IKSE-STFKNTEIS..L.Q ZF FABP2 .--T...T.KVDRN.N..K.MEQM.VNMVKRKLAAHDN.KITLE.T..K.NVKEV-STFRTLEIN.TL.V ZF FABP7B .VD..C.T.KLV.SDNFDEYM.SL..GFATRQV.NVT.PTLV.SKE.EKVVIKTQ-STFKNTEIS.TL.E

ZF FABP6 ESDMETVGGKKFKGIVSMEG-GKLTISFPK------YQQTTEISG-GKLVETSTASGAQGTAVLVRTSKKV- 100.0% HU FABP6 ..NIQ.M...T..AT.Q...-...VVN..N------.H..S..V.-D....V..IG.VT----YE.V..RLA 55.3% MO FABP6 .CE.Q.M...... AT.K...-..VVAE..N------.H..S.VV.-D....I..IGDVT----YE.V..RLA 50.0% RA FABP6 .CE.Q.M...... AT.K...-..VVAD..N------.H..S.VV.-D....I..IGDVT----YE.V..R.A 50.0% PI FABP6 .C.I..I...... AT.Q...-..VVVNS.N------.HH.A..VD-.....V..VG.VT----YE.V...LA 49.2% ZF FABP1B .A.I..LT.E.V.TT.NR..-N..KVVLNR------ITSI..LVDTNT..N.L.LG.LV----YK.I..R.A 42.1% HU FABP1 .CEL..MT.E.V.TV.QL..DN..VTT.KN------IKSV..LN.-DIITN.M.LGDIV----FK.I..RI- 39.3% ZF FABP1A .CEL..FT.DRA.TV.Q.D.-N...AFVKG------IESV..LD.-DTISN.LSFN.IV----YK.I.RRIS 36.3% HU FABP3 .F.ET.ADDR.V.S..TLD.-...VHLQKWD--GQETTLVR.LID-...IL.L.HGT.V----CT..YE.EA 26.8% ZF FABP7A .F.ET.ADDRHV.ST..L..-DN.VQVQRWD--GKETKFVR..KD-..M.M.L.FE.V.----A...YE.A- 25.5% HU FABP2 TFNYNLAD.TELR.TW.L..-N..IGK.KRTDNGNELNTVR..I.-DE..Q.YVYE.VE----AK.IF..D- 24.8% HU FABP7 .F.ET.ADDRNC.SV..LD.-D..VHIQKWD--GKETNFVR..KD-..M.M.L.FGDVV----A..HYE.A- 24.0% ZF FABP3 .F.ET.ADDR.V.SVITLD.-...LHVQKWD--GKETTLLR.V.D-NN.TL.L.LGDIV----ST.HYV.AE 23.9% HU FABP4 .F.EV.ADDR.V.STITLD.-.V.VHVQKWD--GKSTTIKRKRED-D...VECVMK.VT----ST.VYERA- 22.6% ZF FABP2 TF.YSLAD.TELT.SWVI..-DT.KGT.TRKDNGKVLTTVRT.VN-.E..QSYSYD.VE----AK.IF.RA- 22.6% ZF FABP7B .FEET.ADDRHC.ST.LLK.-NQ.VHVQKWD--GKETTFIR..KD-..M.MKL.FGDVE----AL..YE.A- 21.8%

Figure 2. SequencealignmentandaminoacidsequenceidentityofzebrafishFabp6andFABP6fromdifferentspecies,andparaloguesofotherzebrafish FabpsandhumanFABPs. Thededuced aminoacidssequenceofthezebrafishFabp6(EnsemblPeptideID:ENSDARP00000065447)wascomparedto sequences of FABP6s from human (HU FABP6; U19869), mouse (MO FABP6; CAI24826), rat (RA FABP6; NP_058794), pig (PI FABP6; P10289), zebrafishFabpparalogues,FABP1A(ZFFABP1A;DQ062095),FABP1B(ZFFABP1B;DQ062096),FABP2(ZFFABP2;AAH75970),FABP3(ZFFABP3; NP_694493),FABP7A(ZFFABP7A;NP_571680),FABP7B(ZFFABP7B;AAQ92970),andhumanFABPs,FABP1(HuFABP1;M10617),FABP2(HU FABP2;M18079),FABP3(HUFABP3;X56549),FABP4(HUFABP4;NP_00133)andFABP7(HUFABP7;CAI15449).Dotsindicateaminoacidsidentity. Dasheshavebeenintroducedtomaximizealignment.ThepercentageaminoacidsequencesimilaritiesbetweenthezebrafishFabp6andotherFABPsare shownattheendofeachsequence.

128

98 MO FABP6 67 RA FABP6 89 HU FABP6 99 PI FABP6 100 ZF FABP6 ZF FABP1b HU FABP1 95 87 ZF FABP1a 100 HU FABP2 ZF FABP2 52 HU FABP3 59 ZF FABP3 HU FABP4 99 HU FABP7 ZF FABP7a 99 48 ZF FABP7b

0.2

Figure 3. A neighbourjoining tree showing the phylogenetic relationship of zebrafish fabp6 with selected paralog ous and orthologous FABP/Fabps from zebrafish and mammals.Thebootstrapvalues(based100duplicates)areindicatedatthenodes.The sequencesusedwere:zebrafishFABP6(EnsemblPeptideID:ENSDARP00000065447); mammalian sequences for FABP6 from human ( HU FABP6, U19869), mouse (MO FABP6,CAI24826),rat(RAFABP6,NP_058794)andpig(PIFABP6,P10289);zebrafish FABP1A (ZF Fabp1A, DQ062095), FABP1B (ZF Fabp1B, DQ062096), FABP2 (ZF Fabp2,AAH75970),FABP3(ZFFabp3,NP_694493),FABP7A(ZFFabp7A,NP_571680 ) andFABP7B (ZFFabp7B, AAQ92970); humanFABP1 (HuFABP1,M10617),FABP2 (HU FABP2, M18079), FABP3 (HU FABP3, X56549), FABP4(HU FABP4, NP_00133) and FABP7 (HU FABP7, CAI15449). The distinct clade of FABP6/Fabp6s is boxed in grey.Scalebar=0.2substitutionspersite.

129

AB

C

Figure 4 .Spatiotemporaldistributionof fabp6 transcriptsduringzebrafishembryonic and larval development was determined by wholemount in situ hybridization. fabp6 transcriptswerefirstdetectedat72hourspostfertilization(hpf)inthedistalregionof theintestine(Fig.4A)andremainedconfinedtothisregionoftheintestineupto7day postfertilization(Fig.4C),thelasttimepointassayed.Fig.4B showsthedistributionof fabp6 transcriptsthroughouttheenterocytesoftheintestineinatransversesectionat4 dayspostfertilization.

130

S B L GHIOEM K _

fabp6

ef1 ααα

Figure 5. RTPCR detectionof fabp6 transcriptsin RNA extractedfromtissuesofadultzebrafish.RTPCR generateda fabp 6mRNAspecificproductfromRNAextractedfromadultzebrafishliver(L),heart(H),intestine (I),ovary(O)andkidney(K).No fabp 6mRNAspecificproductwasgeneratedbyRTPCRofRNAextracted fromadultzebrafishskin(S),brain(B),gills(G),eyes(E),muscle(M),orthenegativecontrol()lackingtotal RNAderivedfromawholefish.AsapositivecontrolfortheintegrityofeachRNAtemplate,an ef1 ámRNA specificproductwasgeneratedfromalltheadultzebrafishtissuesanalyzed.

131

1 2

B

C B

A C 3 A D C D

Figure 6 .Tissuespecificdetectionof fabp6 genetranscriptsby insitu hybridizationofan antisenseriboprobetosectionsofadultzebrafish.Panel 1showsthedistributionofthe fapb6 transcript in the distal region of the intestine (A), liver (B), ovary (C) and the adrenalhomologinthefishkidney(D).Panel 2showsthelocationofdistalregionofthe intestine (A), liver (B), ovary (C) andtheadrenal homologin the fish kidney (D) inan adjacenttissuesectionstainedwithcresylviolet.

132

CAPÍTULO 4

Análise comparativa de expressão de transcritos gênicos em tecido cerebral de zebrafish ( Danio rerio ) selvagem e transgênico para o gene do hormônio do crescimento (GH), utilizando uma abordagem de DDRT- PCR

133

A comparative expression analysis of gene transcripts in brain tissue of wild-type and GH-transgenic zebrafish ( Danio rerio ) using a DDRT-PCR approach

Abstract

Previous data on homozygote individuals of the GHtransgenic zebrafish

F0104line indicatedthat,despite expressingahigheramountofexogenous growth hormone in relation to hemizygote animals, they presented a significant reduced growth rate (similar to nontransgenic individuals). In the present study, hemizygote (HE) and homozygote (HO) samples of this GH transgenic fish line were compared to nontransgenic (NT) samples of the species through a DDRTPCR ( Differential Display Reverse Transcriptase

PolymeraseChainReaction )approach,withthegoalofidentifyingcandidate differentially expressed transcripts in brain tissue that could be involved in growth process. Two isolated fragments evidenced a high identity level with the genes that code for the proteins p300 (E1A binding protein p300) and

ADCY2 (adenylate cyclase 2). Densitometric analyses of the p300 amplification product pointed to a significant lower gene expression in the transgenic genotypes, specially in HE (17.41±5.32) than in HO (32.25±3.21) samples, when compared to NT samples (49.35±12.32). Inferences on the

ADCY2 transcript suggested a higher expression level in NT animals

(22.73±3.80)thaninHE(14.05±4.95)andHOindividuals(10.63±0.67).These results corroborate previous data that evidenced that p300 and adenylate cyclase are involved in muscle formation/growth and in GH release, respectively.Theobtaineddatacangivesupporttofurtheranalysesintothe

134 elucidationofthemechanismsbeyondvertebrategrowthandGHexpression genecontrol.

Introduction

Zebrafish ( Danio rerio ) is a wellestablished model organism in scientificresearch(Kimmel1989,Westerfield1995),especiallyinbehavioral, physiologic,anddevelopmentalanalysesduetoseveralcharacteristicsofthe species.Thelarge,robustandtransparentembryosandeggs,andtherapid early development are traits that facilitate experimental manipulation and observation. Moreover, the continuous reproduction, external development, relatively short generation time, large number of progeny, and small size adults, represent important features that allow for largescale experiments, even in reduced spaces (Kimmel 1989, Westerfield 1995, Higashijima et al.

1997, Detrich et al. 1998, Dahm et al. 2007, Bass & Gerlai 2008, Fadool &

Dowling2008).

The facility to manipulate this species permitted gene transfer experiments, leading to the development of several transgenic lines, which contribute to new research on vertebrate gene expression (Sprague et al.

2003).Growthhormone(GH)transgeniczebrafishrepresentsoneofthemost explored lines of the species with an exogenous gene. There is a great interestinGHgeneoverexpressioninfishspecies,sincegenerallyitcanbe directlyrelatedtoahighgrowthperformanceoftheanimalsinashortperiod oftime,animportanttraitinaquaculture(Fletcher&Davies1991,Hew etal.

135

1995,Zbikowska2003,Devlin etal. 2004,Devlin etal. 2006,Figueiredo etal .

2007a,Rosa etal. 2008).

Even though some studies have shown that only a 20% increase in growth rate can be achieved by an exogenous GH application in zebrafish

(Simpson et al. 2000, Morales et al. 2001, Biga & Goetz 2006), contrasting with other growth hormonetransgenic fish, as coho salmon, that are extremelyfastgrowing(Devlin etal. 1994),GHtransgenic Daniorerio canbe used as an experimental model on basic research analyses to understand vertebrate growth mechanisms and GH expression gene control. Moreover, thegrowthhormoneisalsoinvolvedinotherphysiologicalprocesses,sinceit can regulate the metabolism of several molecules, as lipids, proteins, and carbohydrates (Moller & Norrelund 2003), improve the function and maintenance ofallmajorimmunecelltypesmaintenance(Jeay etal. 2002), andinfluencethestressresponseandbehavior(Yoshizato etal .1998).

A growth hormonetransgenic line of Danio rerio , denominated F0104 lineage,iscomposedbygeneticallymodifiedanimalswithanexogenousGH gene from the marine fish Odonthestes argentinensis (Figueiredo et al.

2007a). Asignificanthighgrowthperformancewasevidencedinhemizygote

GHtransgenic animals of this line, although individuals of the homozygote genotype did not present a differential growth rate when compared to wild type zebrafish. However, homozygote animals presented a higher GH expression than hemizygote and nontransgenic animals, leading to the assumption that a GH excess do not correspond to a higher growth performanceinzebrafish(Figueiredo etal. 2007b).

136

Duetogrowthdifferencesamongnontransgenic,andhomozygoteand hemizygotezebrafishoftheF0104line,itisexpectedthatgenesinvolvedin growth process are differentially expressed in these animals. Therefore, the present study intended to identify candidate DETs (Differentially Expressed

Transcripts)inthesethreedifferentsamplesusingaDDRTPCR(Differential

Display Reverse Transcription Polymerase Chain Reaction) approach. The resultscangivenewinsightsintotheelucidationofthemechanismsbeyond vertebrategrowthandoftheGHexpressiongenecontrol.

Material and Methods

Animalsamples

Adult individuals of zebrafish ( Danio rerio ) were obtained from

Fundação UniversidadeFederaldoRioGrande(RioGrande,RS),usingthe transgenic line F0104 that has the carp ( Cyprinus carpio) βactin promoter driving the expression of two exogenous genes the marine silverside fish

Odonthestes argentinensis growth hormone (msGH) gene and the jellyfish

(Aequorea victoria ) GFP (Green Fluorescent Protein) as a reporter gene to monitorfortransformedzygotes(Figueiredo etal. 2007a).Differentgenotypes wereobtainedthroughcrossingGHtransgenichemizygoteindividualsleading to a progeny with a proportion of 25% homozygote (HO), 50% hemizygote

(HE), and 25% nontransgenic (NT) animals. Genotypes were determined through UV fluorescence analysis, due to the GFP expression, with an epifluorescence microscope (excitation = 485 nm; emission = 520 nm). HO individualswereidentifiedbypresentingahigherfluorescencelevel.

137

RNAisolation

Braintissuewascollectedfromtwoanimalsofeachgenotype(HO,HE, andNT)andimmediatelystoredat80 oCuntilRNAextraction.Approximately

100mg of the brain samples were mechanically homogenized with 1mL of

TRizol Reagent (Invitrogen) and total RNA extraction followed the manufacturer’s protocol.RNAsampleswereelutedinRNasefreewaterand quantified (NanoDrop 1000 Spectrophotometer) by measuring the optical density (OD) at 260nm. RNA purity was ensured by obtaining a 260/280nm

ODratio≥1.80.

RTPCR andcDNAamplification

Each RNA sample (5 g) was reverse transcribed (RTPCR) with the commercial kit SuperScript III RT (Invitrogen) using the oligonucleotide AP

(5´GGCCACGCGTCGACTAGTAC(T) 17 3´), according to the manufacturer’s instructions.ADDRTPCRapproachwasusedinordertoamplifythecDNA samples.Oligonucleotideswith1524bpweredesignedbasedonVNTRcore sequences(Table1)andusedassingleprimersforcDNAamplification.Each cDNAamplification reactionconsistedof 1L of cDNA,0.2mM ofprimer,1x

25mMMgCl 2 PCRbuffer,0.2mMofdNTPs,and0.2unitofPlatinum Taq DNA polymerase(Invitrogen),inafinalvolumeof25 L.Reactionswerecarriedout withaninitialdenaturationstepat95 oCfor2minutes,followedby40cyclesat

94 oCfor50seconds,55 oCfor2minutes,and72 oCfor50seconds,withan additionalextensionstepat72 oCfor5minutes.DDRTPCRproducts(10 L)

138 werefractionatedon6%polyacrilamidegel,stainedwith0.17%silvernitrate

(AgNO 3) (Sambrook & Russel 2001), and visualized under white light. The molecular weight of the amplified fragments were assigned through comparisonwith1KbDNAladder(Invitrogen).

Densitometricanalysis

The amplified fragments corresponding to putative differentially expressed transcripts were quantified by densitometry as Integrated Optical

Density (IOD) using Imaging Master VDS Software version 3.2 (GE

Healthcare Life Sciences). Mean and standard deviations (SD) were determinedusingMacrosoftExcelSoftware.

DNAisolationandreamplification

Selected DNA fragments that seemed to correspond to genes with differential expression among the analyzed zebrafish samples were reamplifiedinordertoobtainahigherDNAcontenttobeusedoncloningand nucleotidesequencing.Thefragmentswerecutfromthepolyacrilamidematrix anduseddirectly(withoutanyDNApurification)onreamplificationreactions, as described in and Dakis & Kouretas (2002). The amplification reactions followed the conditions described above. PCR products were analyzed through6%polyacrilamidegelelectrophoresis.

139

Cloning,sequencing,andsequenceanalysis

PCRproductswereclonedintopGEMT(Promega)vectorandusedto transformcompetentcellsofthe E.coli strainDH5 α(Invitrogen),followingthe manufacturers’instructions.Clonesweresubmittedtoautomatedsequencing on an ABI 377 Automated DNA Sequencer (Applied Biosystems) with a

DYEnamic ET Terminator Cycle Sequencing kit (GE Healthcare Life

Sciences), following the manufacturer’s instructions, and using primers complementary to vector arms. Nucleic acid sequence database searches wereperformedusingBLAST/N(Altschul etal. 1990)attheNationalCenter for Biotechnology Information (NCBI) website

(http://www.ncbi.nlm.nih.gov/blast). Sequence alignments were obtained by

ClustalW function (Thompson et al. 1994) and the consensus sequences weremanuallydetermined.

Results and Discussion

Muscledevelopmentandgrowtharedynamicprocessescontrolledby a number of factors including growth hormone, insulinlike growth factors

(IGFs), anabolic steroids, thyroid hormone, cytokines and cyclins. The GH, thatplays amajor roleinsomaticgrowth,ismainlyproducedattheanterior pituitary gland, under the control of hypothalamic factors as GHRH (GH release hormone) and somatostatin (GHIH growth hormone inhibiting hormone)andisfurtherreleasedincirculationblood,actinginspecificorgans throughitsassociationwithdifferentreceptorspresentatthesurfaceoftarget cells(Florini etal. 1996,LeRoith etal. 2001).Nowadays,severalanalysesin

140 disorders that result from under or overproduction of GH, and in transgenic animals that overexpress this hormone have been conducted in order to understandthesehormone’sinteractionswithotherproteins.

Regardless the mechanisms responsible for the actions of GH, the resultingeffectofitsoverexpressionisgenerallyanincreaseinmusclemass, as observed for some fish species (Nam et al. 2001, Devlin et al. 2006).

However, a higher GH transgene expression level is not related to an enhancement of growth rate in other fish, as observed in the tilapia

Oreochromisniloticus and O.hornorum (Rahman etal. 1998,Martínez etal.

1999). Homozygotes of the GHtransgenic Danio rerio F0104 line also demonstrated a reduced growth, similar to nontransgenic animals, even thoughtheypresentedahigherGHexpression(Figueiredo etal. 2007b).The different growth performance between hemizygote (HE) and homozygote

(HO)GHtransgeniczebrafishofthislineleadtoanalysesongenesthatcould be involved in growth process, including IGFI (insulinlike growth factor I),

GHG (GH receptor), and SOCS (suppressor of cytokine signaling). The results permitted to identifydistinctexpression levelsof these genesamong the different GHtransgenic genotypes and the nontransgenic (NT) animals

(Figueiredo etal. 2007b,Studzinski2008).

InordertoimplementdataondifferentiallyexpressedgenesinNT,HE, andHOzebrafish,totalRNAofbraintissueofthesethreedifferentsamples wereobtainedandusedonaDDRTPCRapproach.cDNAamplificationwas achievedusingoligonucleotidesthatweredesignedbasedonVNTR(Variable

NumberofTandemRepeats)coresequencesthatcorrespondtominisatellite short and highly conserved regions (Jeffreys et al. 1985). The use of longer

141 primers(1424pb)ondifferentialdisplaystrategiescanleadtodetectionofDNA polymorphismthroughRAPDlikeresults.However,sincetheseoligonucleotides arelongerthanRAPDprimers,thismethodologycanalsobeeffectivelycarried out at relatively high stringencies, thus yielding more reproducible results

(Wasko&AlvesCosta*).Twooftheprimers(INSandHBV3)usedtoamplify the cDNA templates lead to the identification of presumptive differentially expressedtranscriptsamongtheanalyzedsamples.

DDRTPCRusingprimerINS

cDNA amplifications using primer INS lead to the identification of an intensestainedfragmentofapproximately1.000bp,presentinNT,andinGH transgenic HE and HO individuals of zebrafish (Figure 1a), that seemed to correspond to a transcript with differential gene expression among the three samplesthepolyacrilamideelectrophoresisresultspermittedthevisualization of a fragment with distinctive stain intensities among the different samples

(Figure 1a). Densitometric analyses of this amplified product, through IOD values,suggestedasignificantlowergeneexpressionlevelinthetransgenic genotypes, specially in HE (17.41±5.32) than in HO (32.25±3.21) samples, when compared to NT samples (49.35±12.32) (Figure 2a). The suggested expressionlevelsoftheanalyzedtranscriptinHEandHOsamplesrepresent approximately35%and65%oftheIODvalueobtainedforthenontransgenic samples,respectively.

CloningandsequencingofthetranscriptobtainedwiththeprimerINS permittedthecharacterizationofa941bpDNAfragment(Figure3).Database searchesfornucleotidesimilarityindicateda95%identitylevelwithapartial region ofthegenethatcodesforthep300proteinof Daniorerio (accession

142 number XM_001332682, NM_001510744) and also a high identity with the p300 gene of several other vertebrates, as Ctenopharyngodon idella (81%),

Gallusgallus (81%),and Musmusculus (78%).Thep300,alsoknownasE1A binding protein p300 , is involved in several processes, as cellular differentiation and proliferation, cycle cell regulation, growth, apoptosis, and histoneacetylation(Huret2000,Vleugel etal. 2006).It hasbeendescribedas a transcriptional coactivator that interacts with other proteins, leading to the activation of the transcriptional process of several genes (Huret 2000, Tu &

Luo 2007), including those related to cancer (Vachtenheim et al . 2006,

Vleugel et al . 2006, Zhao et al . 2006) and neural pathologies development

(Renoult etal .2007,Francis etal .2007),andtoimmuneresponses(Zhang et al .2005).

Recently, it was proposed that the p300 is also involved in muscle tissue formationand growth. Thep300proteincanregulatethetranscription of the myogenic regulatory factor MyoD (Ji et al. 2003) and of the gene

Cugbp1 (Huichalaf et al. 2007). The CUGBP1 protein (CUG triplet repeat,

RNAbindingprotein1)isessentialforthemuscledifferentiationprocessand any disturbance in the Cugbp gene expression leads to a delay in the myogenesisprocessortoamuscledystrophy(Bhagwati etal. 1996).

Moreover, the interaction between p300 and GH was already evidenced for some genes, as the RhoA gene ( Ras homolog gene family member A) and the early response cfos gene, whose proteins regulate the actin cytoskeleton in the formation of fibers and the cell proliferation and differentiation,respectively(Ling&Lobie2004,Cui etal. 2005).Itseemsthat the growth hormone acts in the regulation of these genes, since GH can

143 stimulatethep300proteintooccupythepromoterofthesegenesleadingto theiractivation(Cui etal. 2005).

Thepresentresultsindicatethatthep300gene,isolatedfromthebrain ofzebrafish,mayhavedifferentexpressionlevelsinnontransgenicandGH transgenicanimalsHEindividualsseemtopresentalowerexpression,while anintermediaryandahigherexpressionlevelscouldbeassociatedtoHOand

NTanimals,respectively.Thesedatareinforcethep300roleinmusclegrowth anditsinteractionwiththegrowthhormone.

DDRTPCRusingprimerHBV3

TheuseoftheprimerHBV3toamplifythecDNAsamplesresultedon several faint and diffuse bands (Figure 1b). However, a fragment of approximately900bpwasclearlyvisibleintheNT,andintheGHtransgenic

HEandHOsamplesof Daniorerio (Figure1b).Althoughthedifferenceinthe stainintensityofthisfragmentamongthethreeanalyzedsampleswasnotso evident,itwaspossibletonoticeadarkerstainingintheNTsamples,whichled ustoinferthatthisfragmentcouldalsocorrespondtoadifferentiallyexpressed transcript. The densitometric analyses of this transcript suggested a higher expressionlevelinNTanimals(22.73±3.80)thaninHE(14.05±4.95)andHO samples(10.63±0.67)(Figure2b).IntheHEandHOsamples,theexpression level of the analyzed transcript represented approximately 62% and 47% of the IOD value obtained fortheNTanimals,respectively.Therefore,theIOD valuedecreaseobservedinHEandHOindividualscanbedirectlyrelatedto theincreasedexogenousGHlevelinthesetwosamplesofzebrafish.

Nucleotide sequencing analysis of the transcript obtained with the primer HBV3 evidenced a DNA fragment with 831 bp (Figure 4). BLAST

144 database searches for nucleotide similarity indicated a high identity level

(94%)withthe3’endofthegenethatcodesfortheproteinadenylatecyclase

2(ADCY2)foundinbraintissueof Daniorerio (accessionnumbersCR759914 and GI6270118). The Adenylate cyclase (AC) catalyses the conversion of

ATPintocAMPthatthenactstoregulateawidevarietyofcellularprocesses.

TheACactivitycanberegulatedbyhormones,neurotransmittersinteractions withtheirreceptors,andcalmodulin(Aikawa etal. 2000,Niewiadomski etal.

2002,Zawilska etal. 2003).

Recently, it was evidenced that the neuropeptide PACAP (pituitary adenylate cyclaseactivating polypeptide) can also modulate the AC activity, leadingnotonlytotheformationofcAMP,butalsoplayingaroleinmediating growth hormone release in the pituitary gland of vertebrates (Matsuda et al.

2008,Mitchell etal. 2008),includingfishspecies,asgrasscarp(Wong etal.

2005),common carp(Xiao etal. 2002),sockeysalmon(Parker etal. 1997), and European eel (Montero et al. 1998). Wong et al. (2005) verified that increased concentrations of PACAP and/or higher concentrations of cAMP wereeffectiveininducingGHrelease.Therefore,theperformeddensitometric data suggest that the lower adenylate cyclase expression level in HO zebrafishcouldresultinadecreaseinGHreleasethusleadingtothereduced growth performance of these animals. Since Figueiredo et al. (2007b) evidenced that these HO animals, despite presenting a higher level of exogenous GH, were expressing approximately only one third of the endogenous GH observed in nontransgenic animals, the p300 should be involvedintheendogenousgrowthhormonereleasecontrol.

145

Finalconsiderations

Thegrowthhormonehasphysiologicalactionsinseveralorgans,since growth hormone receptors (GHR) have been identified in different tissues

(Hughes&Friesen1985,Kelly etal. 1991).AstheGHisamultiplefunction hormone,itsexcesscancauseseveralphysiologicalandbehavioralcollateral effects in the organism, as decrease in fertility (Cecim et al. 1995a, b), increase in oxygen consumption (Cook et al. 2000, Mackenzie et al. 2003), premature aging (Wolf et al. 1993, Bartke et al. 1998), decrease in learning and memory (Meliska et al. 1997), and oxidative stress (Rollo et al. 1996,

BrownBorg etal. 2001,Rosa etal. 2008).Therefore,thepresenceofahigher level of exogenous GH in transgenic fish could also leads to metabolic alterations that could influence not only the animal growth but also several othertraits.

PreviousanalysesevidencedthatHEGHtransgeniczebrafishreached a finalaveragemasshigherthanHOandNTanimalsandthat,surprisingly,

HOfishpresentedagrowthperformancesimilartoNTanimals(Figueiredo et al. 2007b). Taking into account the amount of endogenous and exogenous growth hormone present in HO and HE animals, both transgenic samples producehigherlevelsofbiologicalactiveGHthanNTindividuals.Therefore, thehormoneexcesscouldbeinducinganegativefeedbacksystemoverthe endogenous GH expression (Figueiredo et al. 2007b). These data could be related to regulation systems of intracellular GH that involves IGFI (insulin likegrowthfactorI),GHR(GHreceptor),andSOCSproteins(suppressorof cytokine signaling) (Figueiredo et al. 2007b, Studzinski 2008). The present dataalsoindicatethatthep300andADCY2arealsoinvolvedinaregulation

146 system for GH in zebrafish in the case of high circulating hormone levels.

Therefore,itseemthatthereducedgrowthperformanceobservednotonlyin homozygote GHtransgenic Danio rerio but also in other vertebrates with an exogenousGHgeneisaresultofthebiologicalactionofseveralproteinsand theirinteraction.Furtherresearchisneededtocharacterizethesignificanceof theseproteinsincontrollingvertebratemusclegrowth.

Acknowledgements

ThisworkwassupportedbygrantsfromFAPESP(FundaçãodaAmparo

à Pesquisa do Estado de São Paulo), CNPq (Conselho Nacional de

Desenvolvimento Científico e Tecnológico), and CAPES (Coordenação de

AperfeiçoamentodePessoaldeNívelSuperior).

147

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155

Table, Figures and Legends

156

Table 1: Oligonucleotides that were designed based on VNTR core sequences and used as single primers for the amplification of the cDNA samples of Danio rerio .

Primer Sequence (5’ - 3’) Reference INS ACAGGGGTGTGGGG Nakamura etal. (1987) YNZ22 CTCTGGGTGTCGTGC Nakamura etal. (1987) HBV5 GGTGTAGAGAGGGGT Nakamura etal. (1987) HBV3 GGTGAAGCACAGGTG Nakamura etal. (1987) FvIIex8 ATGCACACACACAGG Murray etal. (1988) EMBL AGAGCTTCAGGCTGGGCAGCTAAG Harris&Wright(1995)

157

INS HBV3

L NT NT He He Ho Ho L NT NT He He Ho Ho

2.036 1.636 3.054 2.036 1.018 1.636

1.018 517 506 396 344 (a) (b)

Figure 1: DDRTPCRpatternsofnontransgenic(NT)andofGHtransgenichemizygote(HE) and homozygote (HO) zebrafish of the F0104 line, visualized trough 6% polyacrilamide gel electrophoresis. (a) brain cDNA amplification using primer INS; (b) brain cDNA amplification using primer HBV3. L, 1 Kb DNA molecular marker. Arrows indicate putative differentially expressedtranscriptsamongthethreeanalyzedsamp les.

158

30

25

70 20 60 50 15 IOD 40 10 IOD 30 20 5 10 0 0 NT HE HO NT HE HO Genótipos (a) (b) Figure 2: Densitometric analyses of the fragments that correspond to putative differentially expressedtranscriptsidentifiedthroughDDRTPCRinnontransgenic(NT)andinGHtransgenic hemizygote(HE)andhomozygote(HO)zebrafishofth eF0104line. (a) estimatedexpressionlevel ofthefragmentamplifiedwiththeprimerINSthatcorrespondstothegenethatcodesforprotein p300(E1Abindingproteinp300); (b) estimatedexpressionlevelofthefragmentamplifiedwiththe primer HBV3 that corresponds to the gene that codes for protein ADCY2 (adenylate cyclase 2).

159

5’acaggggtgtcatg angaanatctggcatgangacgtnacccacgatc.....48 tgcgcaaccanctgntgaagaagctagtccaggctatttttcccacacca.....98 gaccctgctgcactgaaagaccgtcggatggagaatctagtggcctatgc....148 acgtaaagttgagggtgacatgtatgagtctgctaacagcagggcggagt....198 attatcactttctggcagagaagatctataaaattcagaaagaactggaa....248 gagaagcgaaggacacggttacagaagcagggtataatgccctcccagga....298 tggcatgaacccctgtggcctgcancaagcgaacgctgggattggtcagc....348 cctggccacccaacaggactgctttataatggcccactattagatccatt....398 cagtagtggtcccaactggcccaaaccagatgatgaacagaatgcaagaa....448 tactgcctgggatgaattcatttgggaatcatatgggaatgcagtccatg....498 ggccagagatcaacaccacctcttaaccagggaagcatggtgcctgggag....548 gatgccacagccgaatgttgcacagatgcaaaatcaatacatgcaaactg....598 gaccgtttcaagcttcaagtcctgttcgtagtgctggtcctgttgaccta....648 gtacacggaggaaacgatggtgctgccactcaacagggacaaatgccgtt....698 atcatctttaccagtcgggagtcctttagcccaacctgggtctgctggcg....748 gggcaggcagcgggtcatctgtgggctccttgggtcccagcagtatgagt....798 gctgttcctccatcatccacccccacccactccatcagcctcagtcattg....848 ccgacctgtacatcagcattcactttcaactgntgatagccggacgcgca....898 gacccatgccaggttaccagacgcctcatccccacacccctgt 3’...... 941 Figure 3: Consensus nucleotide sequence of a partial region of the gene that codes for proteinp300,isolatedfrombraintissueof Daniorerio throughaDDRTPCRapproachusing primerINS.Theunderlinedregionsarehomologoustotheprimersequence.

160

5’ggtgaagcacangtg caaacagtattatgtgggcaatcctaaaagaga...... 48 tgtcaaaaacaaagaggtcggtccaggtggctagcaccagaaaacaataa...... 98 acacanatgatctaacatggcagagtaaggcaaggagaaatgctttgtaa...... 148 aggctgatttatactcctgcgtcaaacactggcgtatgctacggcgctga...... 198 cgcatagcccttcgccctggccgtcgccgtcactgacgtgcaccactcta...... 248 aaaattggaactacacatgggaacgacgcgtatcgcaaagtctgtgattg...... 298 gtcggcttggtagcgttgacgagtcggggcaggaccgagacccgtgcgaa...... 348 tggtgcaaacccaatgtagcgattgtttacaagtgtggagtcccgtgaag...... 398 gagcttcagatggaaagttttgttttgtgtttacctcatagttaaagttg...... 448 ctgcatgtccgccggttgctgcctcaaaatgagcgagtttcagccacttg...... 498 tacattccggaagtgttcaggaaaagcaaaaaagcagcgaagaaactcaa...... 548 cacaaaggaacatttacacctcactgccaactagcatttcggaagtgtta...... 598 atccagaccaacagagacagcgcgcagaagtataaatgcacagccacgtg...... 648 cgttgcatgcaccgtgggttacgccggtcacttgacgcagaagtattaat...... 698 caggcttaatgtgtccaggtaacaacaagactcagtaattgcatgtatgt...... 748 atgtgtctgtgagtgtgtgtgtgtgagcgttgcatgaatagtccatgtaa...... 798 tgagtgcttaaccagcttcacctgtgcttcacc 3’...... 831

Figure 4: Consensus nucleotide sequence of a partial region of the gene that codes for protein ADCY2, isolated from brain tissue of Danio rerio through a DDRTPCR approach usingprimerHBV3.Theunderlinedregionsarehomol ogoustotheprimersequence.

161

Considerações Finais e Conclusões

Oobjetivoprincipaldopresentetrabalho,especificadoinicialmente,de isolarecaracterizartranscritosdiferencialmenteexpressosemduasespécies de peixes Leporinus macrocephalus e Danio rerio foi alcançado com sucesso, dada a obtenção de resultados referentes às diferentes sub propostasdepesquisa.

Assim,asanálisesdesenvolvidasnopresenteestudoevidenciaram: • Duas isoformas de αactina esquelética na espécie Leporinus

macrocephalus , geneticamente e provavelmente funcionalmente

distintas, cujas características podem estar relacionadas à demanda

energéticadostecidosmuscularesesqueléticosbrancoevermelho. • Um genecomexpressãodiferencial em tecido cerebral de machose

fêmeas de Leporinus macrocephalus , relacionado à proteína APBA2

(amyloidbeta(A4)precursorproteinbinding,familyA,member2 ),cuja

expressão está associada à produção da proteína βamilóide a qual,

porsuavez,relacionaseàdoençadeAlzheimer. • Aestruturadogene fabp6 ( fattyacidbindingprotein6 )de Daniorerio ,

similar a grande parte dos demais membros da família multigênica

FABP deoutrosvertebrados,esuadistribuiçãotecidualemembriões,

larvaseadultos,tambémsimilaràevidenciadaemoutrosorganismos,

especialmentemamíferos.

162

• Dois genes candidatos diferencialmente expressos, relacionados à

proteína p300 ( E1A binding protein p300 ) e à proteína ADCY2

(adenylatecyclase2 )envolvidasnaformação/crescimentomusculare

naliberaçãodeGH,respectivamente,entreexemplaresde Daniorerio

nãotransgênicosetransgênicoshomozigotosehemizigotosparagene

dohormôniodecrescimento.

Dadaaimportânciadaobtençãoeampliaçãodedadosgenéticosem peixes,queconstituemumgrupoextremamentediversificadoebasalentreos vertebrados, os resultados obtidos contribuem para a melhor compreensão da evolução, função e organização molecular de diversos genes e podem servirdebaseafuturosestudos.Alémdisso,osdadosobtidosconfirmama utilidade de Danio rerio como espécie modelo em estudos genéticos e indicam que Leporinus macrocephalus representa um potencial modelo experimental em análises direcionadas a genes envolvidos no desenvolvimentodedoençashumanas,comoalteraçõesneurodegenerativas, distrofiasmuscularesecâncer.

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SÚMULA CURRICULAR (Outras atividades vinculadas e desenvolvidas no Doutorado) 1.Trabalhosapresentadosemcongressos:

ALVES,F.A.; FORESTI,F.,OLIVEIRA,C.,MARTINS,C.2005.Usode seqüênciasdeDNAr5ScomomarcadormolecularemtilápiadoNilo (Oreochromis niloticus ). In : “ XVI Encontro Brasileiro de Ictiologia ”, JoãoPessoa,Paraíba,Brasil. ALVES, F.A.; MARTINS, C., WASKO, A.P. 2005. Caracterização molecular de genes de actina isolados de músculos branco e vermelho de Leporinus macrocephalus (Pisces, Anostomidae). In : “51 o.CongressoBrasileirodeGenética”, ÁguasdeLindóia/SP,Brasil . GeneticsandMolecularBiology(Suppl.)26,GA123,p.123. MATOS,F.D.C.;ALVES,F.A.; MARTINS,C.;ALVES,A.L.;FORESTI, F.;OLIVEIRA,C.;WASKO,A.P.2005.CaracterizaçãodoDNAr5S emespéciesdafamíliaSciaenidae(Pisces,Perciformes). In : “ 51 o. Congresso Brasileiro de Genética”, Águas de Lindóia/ SP, Brasil . GeneticsandMolecularBiology(Suppl.)26,GA199,p.199 . WONG,M.S.L.;ALVES,F.A.; WASKO,A.P.2005.Identificaçãodeum marcador RAPD associado a fêmeas de Brycon cephalus (Pisces, Bryconidae). In : “ 51 o Congresso Brasileiro de Genética”, Águas de Lindóia/ SP, Brasil . Genetics and Molecular Biology (Suppl.) 26 , GA229,p.229 . ALVESCOSTA,F.A.; MARTINS,C.;WASKO,A.P.2006.Identificação de transcritos sexoespecíficos em Leporinus macrocephalus (Pisces, Anostomidae) através da metodologia de DDRTPCR. In : “XXVICongressoBrasileirodeZoologia”, UniversidadeEstadualde Londrina–UEL,Londrina/PR,Brasil.N o.858_1791.

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WONG,M.S.L.;ALVESCOSTA,F.A.; WASKO,A.P.2006.Prospecção de fragmentos sexoespecíficos em Brycon cephalus (Pisces, Bryconidae)atravésdametodologiadeRAPD. In :“ XXVICongresso Brasileiro de Zoologia”, Universidade Estadual de Londrina – UEL, Londrina/PR,Brasil . No.2123_1791. ALVESCOSTA,F.A.; MARTINS,C.;WASKO,A.P.2006.Identificaçãoe caracterizaçãodetranscritoscompotencialexpressãodiferencialentreos sexos de Leporinus macrocephalus (Pisces, Anostomidae). In : “ 52 o Congresso Brasileiro de Genética” and “ 12 o. Congresso de la Associación Latinoamericana de Genética”, Bourbon Cataratas Resort&ConventionCenter,FozdoIguaçu/PR,Brasil .p.187. . WONG, M.S.L.; ALVESCOSTA, F.A.; WASKO, A.P. 2006. IdentificaçãoecaracterizaçãodemarcadoresRAPDassociados ao sexo em Brycon cephalus (Pisces,Bryconidae). In : “ 52 o Congresso Brasileiro de Genética” and “ 12 o Congresso de la Associación Latinoamericana de Genética”, Bourbon Cataratas Resort & ConventionCenter,FozdoIguaçu/PR,Brasil . p.235. GOMES, M.L.; ALVESCOSTA, F.A.; NISHIDA, S.M.; WASKO, A.P. 2006. Análise de locos microssatélites em codorna japonesa Coturnix japonica (Galliformes, Phasianidae) para identificação de polimorfismos, visando a elaboração de testes de paternidade. In : “52 o Congresso Brasileiro de Genética” and “ 12 o. Congresso de la Associación Latinoamericana de Genética”, Bourbon Cataratas Resort&ConventionCenter,FozdoIguaçu/PR,Brasil . p.264. ALVESCOSTA,F.A.; MARTINS,C.;WASKO,A.P.2006.Prospecçãode DETS (Differentially Expressed Transcripts) em machos e fêmeas de Leporinus macrocephalus (Anostomidae). In : “ XI Brazilian Symposium on Fish Cytogenetics and Genetics and I International Congress of Fish Genetics” , Universidade Federal de São Carlos, São Carlos/SP, Brasil. p. 277.

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WONG, M.S.L.; ALVESCOSTA, F.A.; WASKO, A.P. 2006. Identificação e caracterização de fragmentos de DNA sexo específicos em Brycon cephalus (Characidae) através da metodologia de RAPD. In : “ XI Brazilian Symposium on Fish CytogeneticsandGeneticsandIInternationalCongressofFishGenetics” , UniversidadeFederaldeSãoCarlos,SãoCarlos/SP,Brasil.p.210. ALVESCOSTA, F.A.; MARTINS, C.; WASKO, A.P. 2006. Uso de seqüênciasdeDNAr5Scomomarcadoresmolecularesemespécies marinhas da família Sciaenidae (Pisces, Perciformes). In : “ VI JornadasdeCienciasdelMarandXIVColoquiodeOceanografía”, PuertoMadryn,Chubut,Argentina,p.158. TONIATO, J.; MARTINS, C.; ALVESCOSTA, F.A.; ROCHA, K.K.H.R. 2007. Diferenciação molecular da tilápia do Nilo com base em marcadoresmolecularesdebaixocusto. In :“ XXIIReuniãoAnualda FESBE” , Águas de Lindóia/SP, Brasil. Métodos, 49.011 EquipamentoseEnsino. ALVESCOSTA,F.A.; FIGUEIREDO,M.A.;LANES,C.F.C.;ALMEIDA,D.V.; MARINS,L.F.; WASKO, A.P. 2007. Isolamento e caracterização de DETs(“DifferentiallyExpressedTranscripts”)emtecidocerebralde uma linhagem de de peixe transgênico ( Danio rerio ) superexpressando o gene do hormônio do crescimento (GH). In : “53 o. Congresso Brasileiro de Genética”, Hotel Monte Real Resort, ÁguasdeLindóia/SP,Brasil.p.168. . GOMES, M.L.; ALVESCOSTA, F.A.; NISHIDA, S.M.; WASKO, A.P. 2007. Elaboração de testes de paternidade em codorna japonesa Coturnix japonica (Galliformes, Phasianidae). In : “ 53 o. Congresso Brasileiro de Genética”, Hotel Monte Real Resort, Águas de Lindóia/SP,Brasil.p.257.

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TONIATO, J.;MARTINS,C.;ALVESCOSTA,F.A. 2007. Aplicaçãode marcadores RFLPPCR na identificação de espécies de tilápias (Perciformes: Cichlidae). In : “ 53 o. Congresso Brasileiro de Genética”, Hotel Monte Real Resort, Águas de Lindóia/SP, Brasil . p.91 GOMES, M.L.; ALVESCOSTA, F.A.; NISHIDA, S.M.; WASKO, A.P. 2007 Testes de paternidade para a codorna japonesa Coturnix japonica (Galliformes, Phasianidae): ferramenta para evidenciar a competição póscópula. In : “ XXV Encontro Anual de Etologia”, UNESP/SãoJosédoRioPreto,SãoJosédoRioPreto,SP,Brasil. Oralpresentation. 2.Participaçõesemcongressos: XVIEncontroBrasileirodeIctiologia ,realizadoemJoãoPessoa.Paraíba,no períodode24a28dejaneirode2005. 51 o. CongressoBrasileirodeGenética realizadoemÁguasdeLindóia, SP,noperíodode7a10desetembrode2005. XXVI Congresso Brasileiro de Zoologia , realizado na Universidade Estadual de Londrina e Centro Universitário Filadélfia, Londrina, PR,de12a17defevereirode2006 . 52 o. Congresso Brasileiro de Genética e 12 o. Congresso de la Associación Latinoamericana de Genética, realizado no Bourbon CataratasResort&ConventionCenter,FozdoIguaçu,PR,de3a6 desetembrode2006 . XIBrazilianSymposiumonFishCytogeneticsandGeneticsandIInternational CongressofFishGenetics ,realizadonaUniversidadeFederaldeSãoCarlos, SãoCarlos,SP,de10a13deoutubrode2006.

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VI Jornadas de Ciencias del Mar y XIV Coloquio de Oceanografía, realizadanacidadedePuertoMadryn,Chubut,Argentina,de4a8 dedezembrode2006. 3.Cursosassistidos: Evolução de proteínas através de embaralhamento de DNA , realizado durante o 51 o. Congresso Brasileiro de Genética, em Águas de Lindóia,SP,noperíodode7a10desetembrode2005,totalizando 3horas. ExpressãoGênica:novasformasdeanáliseeabordagens ”,realizadodurante o52 o.CongressoBrasileirodeGenética ,BourbonCataratasResort &ConventionCenter,FozdoIguaçu,PR,noperíodo de3a6de setembrode2006,totalizando3horas . Melhoramento Genético em Peixes ”, realizado durante o XI Brazilian Symposium on Fish Cytogenetics and Genetics and I International CongressofFishGenetics,naUniversidadeFederaldeSãoCarlos,São Carlos, SP, no período de 10 a 13 de outubro de 2006, totalizando 6 horas. 4.Cursosministrados:

Curso de extensão Universitária “ IntroduçãoàBiologia Molecular ”, realizado noLaboratóriodeGenéticadePeixes,juntoaoDepartamentodeCiências Biológicas,UNESP,Bauru,SP,durante operíodode16a19deagosto de2005,comduraçãode24horas.

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Curso “ Marcadores Citogenéticos e Moleculares: conceitos e aplicações ”, ministrado durante o VI Workshop de Genética, no Instituto de Biociências, UNESP, Botucatu, SP, no período de 20 a 21 de maio de 2006,apresentandocargahoráriaequivalentea8horas. Curso “ Marcadores Moleculares e Sua Aplicação em Teste de Paternidade: uma abordagem sobre conservação genética ”, ministrado durante o VII WorkshopdeGenética,noInstitutodeBiociências,UNESP,Botucatu,SP, no período de 20 a 21 de maio de 2007, apresentando carga horária equivalentea8horas. 5.Monitoriaemcursosdeextensão: Monitoria na disciplina “Métodos de Estudo em Biologia Molecular”, sob a responsabilidade do Prof. Cesar Martins, no curso de pós graduação em Ciências Biológicas (Genética), ministrada de 10 a 19deoutubrode2005. Colaboraçãocomomonitoraduranteocursodeextensãouniversitária (I Curso de Inverno) “Manipulação de Ácidos Nucleicos: PCR,RT PCRePCRemtemporeal”,realizadonoInstitutode Biociências, UNESP,Botucatu,SP,noperíodode10a14dejulhode2006 Colaboração como monitora da oficina “Experimentando Genética”, caracterizada como parte do Prpgrama de Extensão “Difundindo e Popularizando a Ciência”, realizada no Instituto de Biociências da UNESP,Botucatu,SP,noperíodode15a20dejaneiro de 2007, totalizando48horasdeatividade. Colaboraçãocomomonitoradurantecursodeextensãouniversitária(II Curso de Inverno “Manipulação de Ácidos Nucleicos: PCR, RT PCRePCRemtemporeal”,realizadonoInstitutode Biociências, UNESP,Botucatu,SP,noperíodode16a20dejulhode2007.

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6.Palestrasproferidas: “Expressão de Proteínas Recombinantes”, realizada durante curso de extensão universitária “Introdução à Biologia Molecular”, Instituto de Biociências de Botucatu, Universidade Estadual Paulista UNESP (Botucatu,SP),nodia13dejulhode2007. “Brazilian Conservation Genetics Issues”, realizada na Acadia University, Wolfville,NovaScotia,Canadá,nodia10deoutubrode2007. 7.Publicações:

AlvesCosta FA , Wasko AP, Oliveira C, Foresti F, Martins C (2006) Genomic organization and evolution of the 5S ribosomal DNA in Tilapiinifishes. Genetica 127:243252. AlvesCosta FA, Martins C (2006) DNAr 5S um novo marcador molecular para análise genética de tilápias. Revista Biotecnologia, KL3PublicaçõesLtda 35:2227. Wasko AP, Bento AP, Ferreira DC, Souza DB, AlvesCosta FA , Rossetto FO, Gatinho IA, Maia IG, Mazzuchelli J, Paiva LRS, Ribolla PEM, Almeida RA, Barbetta SR, Salles VP, Teixeira WG, Martins C (2007) Aliando conceitos e criatividade: proposta de dramatizaçãonaáreadegenéticaebiologiacelularparaalunosdo ensinomédio. GenéticanaEscola 4:3438. AlvesCosta FA , Matos FDC, Foresti F, Oliveira C, Martins C, Wasko AP (2008) 5S rDNA characterization in twelve Sciaenidae fish species (Teleostei: Perciformes):depicting genediversity and molecularmarkers. GeneticsandMolecularBiology 31,1(suppl):303307.

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AlvesCosta FA, DenovanWright EM, Thisse C, Thisse B, Wright JM (2008) Spatialtemporaldistributionoffattyacidbindingprotein6( fabp6) genetranscriptsinthedevelopingandadultzebrafish( Daniorerio ). FEBS Journal 275:33253334. 8.Estágiosrealizados: EstágiosupervisionadopeloProf.DrLuisFernandoMarins,noLaboratóriode Biologia Molecular do Departamento de Ciências Fisiológicas, FURG (FundaçãoUniversidadedoRioGrande),realizadode14a26deoutubro de 2006, com participação no desenvolvimento da tecnologia de DDRT PCR ( Differential Display Transcriptase Reverse – Polymerase Chain Reaction ) dentro do projeto ”Peixes Geneticamente Modificados para o GenedoHormôniodeCrescimento(GH)”,sobacoordenaçãodomesmo professor. Estágio supervisionado pelo Prof. Dr. Jonathan M. Wright, Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada, no período de30dejulhoa07dedezembrode2007,desenvolvendootrabalhode pesquisa “Caracterização da organização e da expressão gênica da proteínaFABP6(“FattyAcidbindingProtein6”)naespéciedepeixe Danio rerio (zebrafish)”.

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9.Participaçãoemprojetosdepesquisa:

“Isolamento e caracterização de transcritos sexoespecíficos em peixes utilizando a técnica de DDRTPCR (“Differential Display Reverse Transcriptase”): modelo inicial para implementação de linha de pesquisa em expressão gênica diferencial”. Projeto de Jovem Pesquisador Supervisor(a): Dra. Adriane Pinto Wasko) – FAPESP no. 03/067611. Laboratório de Biologia Molecular e do Desenvolvimento, Departamento deMorfologia,InstitutodeBiociências,UNESP(Botucatu,SP).Períodode Dezembrode2003aNovembrode2006.

“Identificação de marcadoressexoespecíficosem Leporinusmacrocephalus (Pisces,Anostomidae)utilizandoatécnicadeRAPD”.ProjetodeIniciação Científica (Aluna: Marina Seik Lien Wong) – FAPESP n o. 04/103284,. Departamento de Morfologia, Instituto de Biociências, UNESP(Botucatu, SP).Dezembrode2004àNovembrode2005.

“Identificação de marcadores sexoespecíficos em Brycon cephalus (Pisces, Bryconinae)utilizandoatécnicadeRAPD”.ProjetodeIniciaçãoCientífica (Aluna: Fernanda Del Campos de Matos) – FAPESP n o. 04/109111, Departamento de Morfologia, Instituto de Biociências, UNESP(Botucatu, SP).PeríododeDezembrode2004aNovembrode2006.

“Comparação e aperfeiçoamento de técnicas para isolamento de DNA de avesatravésdeamostragemnãoinvasiva”.ProjetodeIniciaçãoCientífica (Aluno:TiagoJoséBeneditoEugênio).LaboratóriodeBiologiaMoleculare do Desenvolvimento, Departamento de Morfologia, Instituto de Biociências,UNESP(Botucatu,SP).PeríododeAbrilde2005aMarçode 2006.

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“Identificação de marcadores RAPD sexoespecíficos e DNAs satélites em Brycon cephalus (Pisces, Bryconinae): contribuições a caracterização genética da espécie” Projeto de Iniciação Científica (Aluna: Marina Sek Lien Wong) –FAPESP n o. 04/103284, Departamento de Genética, InstitutodeBiociências,UNESP(Botucatu,SP).PeríododeDezembrode 2006aNovembrode2007.

“Isolamento e caracterização de transcritos sexoespecíficos em Leporinus macrocephalus (Pisces, Characiformes), utilizando a técnica de DDRT PCR( DifferentialDisplayReverseTranscriptase )”–ProjetodeDoutorado (Alunadepósgraduação:FernandaAntunesAlvesdaCosta)FAPESPn o. 04/100245,. Laboratório de BiologiaMolecularAnimal, Departamentode Genética, Instituto de Biociências, UNESP (Botucatu, SP). Período de Fevereirode2005aAgostode2008. “Determinação das relações entre o comportamento de acasalamento e o sucesso reprodutivo na codorna japonesa ( Coturnix japonica ): uma abordagem multidisciplinar através de análises etológicas e genéticas”. Projeto de Mestrado (Aluna de pósgraduação Magali Lira Gomes) – FAPESP n o.05/579964, Laboratório de Biologia Molecular Animal, Departamento de Genética, Instituto de Biociências, UNESP (Botucatu, SP).PeríododeAbrilde2005aAgostode2008.

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10.Disciplinascursadas: Disciplinas Créditos Conceito

GenéticaMolecularemAnimaisdeProdução(mamíferos) 03 A

FundamentosemGenética 06 A

Tópicos especiaisemgenética:expressão,purificação e 03 A caracterizaçãodeproteínasrecombinantes

Desenvolvimento embrionário comparado: modelos de 03 A estudo

SistemáticaMolecular 03 A

Análises cromossôm icas moleculares: técnicas de 03 A hibridação insitu

Organizaçãoeevoluçãodosgenomas 04 A

Difundindo Genética: popularização da ciência pelos 04 A genes

Mecanismos de Regulaçào e Métodos de Análise da 03 A ExpressãoGênica

ConservationGenetics 03 A Pro fessores Responsáveis: Adriane Pinto Wasko (Departamento de Genética, Instituto de Biociências, UNESP, Botucatu, SP) e Stephen Mockford (Acadia University,Wolfville,NovaScotia,Canada)

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