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Arquivo3669 1.Pdf UNIVERSIDADE FEDERAL DE PERNAMBUCO CENTRO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE GENÉTICA PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E BIOLOGIA MOLECULAR DISSERTAÇÃO DE MESTRADO ANÁLISE CARIOTÍPICA, HETEROCROMATINA CONSTITUTIVA E FREQUÊNCIA DE EXPRESSÃO DAS RONs EM DUAS ESPÉCIES DE MELANOPLINAE (ORTHOPTERA: ACRIDIDAE) JÉSSICA MIRANDA DO NASCIMENTO RECIFE 2009 UNIVERSIDADE FEDERAL DE PERNAMBUCO CENTRO DE CIÊNCIAS BIOLÓGICAS DEPARTAMENTO DE GENÉTICA PROGRAMA DE PÓS-GRADUAÇÃO EM GENÉTICA E BIOLOGIA MOLECULAR DISSERTAÇÃO DE MESTRADO ANÁLISE CARIOTÍPICA, HETEROCROMATINA CONSTITUTIVA E FREQUÊNCIA DE EXPRESSÃO DAS RONs EM DUAS ESPÉCIES DE MELANOPLINAE (ORTHOPTERA: ACRIDIDAE) JÉSSICA MIRANDA DO NASCIMENTO Dissertação apresentada ao Programa de Pós-graduação em Genética e Biologia Molecular da Universidade Federal de Pernambuco como requisito para obtenção do grau de Mestre em Genética pela UFPE. Orientador: Profª. Drª. Maria José de Souza Lopes Co-orientador: Profª. Drª. Marília de França Rocha RECIFE 2009 Nascimento, Jéssica Miranda do Análise cariotípica, heterocromatina constitutiva e frequência de expressão das RONs em duas espécies de Melanoplinae (Orthoptera: Acrididae). / Jéssica Miranda do Nascimento. – Recife: A Autora, 2009. 75 folhas: il., fig., tab. Dissertação (mestrado) – Universidade Federal de Pernambuco. CCB. Departamento de Genética, 2009. Inclui bibliografia. 1. Gafanhotos - Citogenética 2. Orthoptera 3. Acrididae. I Título. 595.727 CDU (2.ed.) UFPE 595.726 CDD (22.ed.) CCB – 2009- 045 À minha família e às minhas orientadoras, Maria José e Marília, com amor dedico. “E um dia os homens descobrirão que esses discos voadores estavam apenas estudando a vida dos insetos...” Mário Quintana Agradecimentos Em primeiro lugar a Deus pelo dom da vida, por sempre estar guiando meu caminho, proporcionando alegrias e conforto nos momentos difíceis e por permitir a concretização de mais uma etapa da minha vida profissional. À minha família pelo amor, confiança, apoio, dedicação e pela torcida. Em especial, às pessoas mais importantes da minha vida: meus avós, Maria e José Miranda; minha mãe, Letícia; meus irmãos, Jonatha e Rebecca; meus tios, Betânia, Júlio, Otoniel (Tano), Kelly e Socorro; meus primos Gabriel, Túlio, Maurício e Otoniel (Taninho) e minha cunhada Diene. À Profª. Drª. Maria José de Souza Lopes pela orientação, dedicação, disponibilidade, ensinamentos e pela confiança que me fez seguir para conclusão deste objetivo. À Profª. Drª. Marília de França da Rocha pela co-orientação, amizade, carinho, paciência, disponibilidade, incentivo e por sempre ter acreditado em mim, estimulando-me nos momentos mais difíceis. Ao CNPq pelo apoio financeiro para o desenvolvimento deste trabalho (Processo 471485/2004-7). Ao professor Dr. Carlos Salvador Carbonell da Universidade de Montevideo, Uruguai, pela identificação taxonômica das espécies analisadas e pela disponibilidade. Ao Professor Dr. Marcelo Guerra e à Drª. Ana Emília pela disponibilização do sistema de captura de imagens do Laboratório de Citogenética Vegetal (Departamento de Genética/ CCB/ UFPE) e por viabilizar a realização da técnica de FISH. A Ituza e a Diogo pela ajuda com a captura das imagens de fluorescência deste trabalho. A Cirlene Maria da Silva pelo auxílio técnico, disponibilidade e, acima de tudo, pela amizade depositada nestes dois anos. A Camilla Vila Nova pela tradução e correção do texto em inglês. Aos amigos, colegas e ex-colegas do Laboratório de Genética e Citogenética Animal (UFPE): Adriana, Amanda, Angélica, Bárbara, Carolina (Carol), Cibele, Cybelle, Danielle, Diogo, Evillis, Graciela, Guilherme, Iane, Jefferson, Marcela, Martin, Myrella, Nara, Nayara, Pollyanna, Sárah, Thatiana e Tyago, minha sincera gratidão pela amizade, disponibilidade, incentivo, pelas ajudas ao decorrer do trabalho e pelas divertidas conversas no Laboratório. A Ituza Celeste pela amizade, incentivo, companheirismo e cumplicidade desde os tempos de graduação. A Merilane Calixto pela amizade, estímulo, pelas longas conversas e confidências, pelos momentos de lazer e, principalmente, por me incentivar nos momentos difíceis. Às professoras Neide Santos e Vilma Loreto pela disponibilidade, conselhos e ensinamentos. Aos amigos e ex-colegas do Laboratório de Biodiversidade e Genética de Insetos (UPE): Celso, Cristiane (Cris), Fernando, Maria Clara, Mônica, Nathália e Professora Rita Moura. Aos colegas de turma do curso de Mestrado: Betânia, Diego, Felipe, Gabriela, Geyner, Ituza, Júlia, Marília, Nara, Petra, Rodrigo, Thatiana e Will. A coordenação e secretaria do Programa de Pós-graduação em Genética (PPGG) pelo auxílio prestado durante o mestrado. Aos professores do PPGG, pela contribuição para a minha formação. Aos funcionários do Departamento de Genética, em especial, Francisca (Fran), Gilzinete (Dona Zizi) e Romildo pela atenção, disponibilidade e respeito. A Aliny Costa e a Flávia Arruda pela amizade, companheirismo, estímulo, conversas, farras e acima de tudo por nunca ter me negado ajuda nos momentos em que mais precisei. Aos amigos Camilla, Débora, Érika (Érikinha), Manuela (Manú), Márcia, Naara, Raul e a família Tiné, Maria Juliana, Fernando e Fernandinho pela amizade, paciência, incentivo, apoio e pelos maravilhosos momentos de lazer. As minhas eternas amigas Andréa, Bruna, Érica, Francimary (Mary), Leila, Ligia e Marilian pelo carinho, apoio, incentivo, confidências, farras, risadas... Amo vocês! A todos que de alguma forma contribuíram para conclusão deste trabalho. SUMÁRIO Lista de Abreviaturas i Lista de Figuras ii Lista de Tabelas iii Introdução iv Resumo vi Abstract viii 1. Revisão da Literatura 10 1.1. Considerações Gerais sobre a Família Acrididae 11 1.2. Caracterização Cariotípica em Acridoidea 13 1.2.1. Citogenética Convencional 13 1.2.1.2. Cromossomo Megamérico 15 1.2.2. Variabilidade Cariotípica 17 1.3. Heterocromatina Constitutiva (HC) 20 1.3.1. Considerações Gerais 20 1.3.2. Padrão de Distribuição e Qualificação da Heterocromatina Constitutiva em Acridoidea 24 1.4. Regiões Organizadoras de Nucléolos (RONs) 30 1.4.1. Considerações Gerais 30 1.4.2. Regiões Organizadoras de Nucléolos em Gafanhotos 32 2. Objetivos 37 2.1. Objetivo Geral 38 2.2. Objetivo Específicos 38 3. Materiais e Métodos 39 3.1. Materiais 40 3.2. Métodos 40 3.2.1. Processamento dos Espécimes 40 3.2.2. Preparações Citológicas 41 3.2.3. Coloração Convencional 41 3.2.4. Bandeamento C 41 3.2.5. Coloração CMA3/DA/DAPI 41 3.2.6. Impregnação com Nitrato de Prata (AgNO3) 42 3.2.7. Hibridização In Situ Fluorescente (FISH) 42 3.2.8. Documentação Fotográfica 43 4. Resultados 44 4.1. Baeacris punctulatus 45 4.2. Dichroplus fuscus 47 5. Discussão 57 6. Conclusões 62 7. Referências Bibliográficas 65 8. Anexo 74 8.1. Fotografias das Espécies Estudadas 75 Lista de Abreviaturas AgNO3 Nitrato de prata AT Pares de bases Adenina e Timina Ba(OH)2 Hidróxido de bário CCB Centro de Ciências Biológicas CMA3 Cromomicina A3 - CMA3 Marcação CMA3 negativa + CMA3 Marcação CMA3 positiva DAPI 4’-6-diamidino-2-fenil-indol DAPI- Marcação DAPI negativa DAPI+ Marcação DAPI positiva DNA Ácido desoxirribonucleico DNAr DNA ribossômico ER Enzimas de Restrição FISH Hibridização in situ fluorescente G Cromossomo de tamanho grande GC Pares de base Guanina e Citosina HC Heterocromatina constitutiva HCl Ácido clorídrico HF Heterocromatina facultativa M Cromossomo de tamanho médio MM Mitramicina P Cromossomo de tamanho pequeno Pb Pares de bases PEV Variegação por efeito de posição RNA Ácido ribonucleico RNAr RNA ribossômico RON Região organizadora de nucléolo SSC Solução salina citrato UFPE Universidade Federal de Pernambuco UFRPE Universidade Federal Rural de Pernambuco UPE Universidade de Pernambuco X Cromossomo X i Lista de Figuras Figura 1. Células meióticas de Baeacris punctulatus coradas convencionalmente. 49 (a) Paquíteno; (b) diplóteno; (c) metáfase I; (d) metáfase II. Barra = 5 µm. Figura 2. Bandeamento C (a, b) e tríplice coloração CMA3/DA/DAPI (c, d) em 50 cromossomos meióticos de Baeacris punctulatus. (a, b) Diplótenos; (c, d) + paquítenos. As cabeças de seta em c apontam os diminutos blocos CMA3 pericentroméricos. A seta indica o bloco intersticial adicional. Barra = 5 µm. Figura 3. Diferentes marcações das RONs ativas detectada pela impregnação 51 com AgNO3 em células meióticas de Baeacris punctulatus. (a, c, d) Zigótenos; (b) diplóteno; (e, f) paquítenos iniciais. Barra = 5 µm. Figura 4. Comparação entre as frequências de atividade das regiões 52 organizadoras de nucléolos (RONs) localizadas apenas em autossomos e em autossomos e no cromossomo X em Baeacris punctulatus. Figura 5. FISH com sonda de DNAr 45S em células meióticas de Baeacris 53 punctulatus. (a, b) Paquíteno; (c, d) metáfase II. Em a e c coloração com DAPI. Barra = 5 µm. Figura 6. Coloração convencional em células meióticas de Dichroplus fuscus. 54 (a) Paquíteno; (b) diacinese; (c) metáfase I; (d) anáfase I. Barra = 5 µm. Figura 7. Bandeamento C (a, b) e tríplice coloração (CMA3/DA/DAPI) (c, d) em 55 células paquitênicas de Dichroplus fuscus. Em c paquíteno corado com CMA3 e em d paquíteno corado com DAPI. As setas indicam os diminutos blocos pericentroméricos de HC no bivalente G2. Barra = 5 µm. Figura 8. Cromossomos meióticos de Dichroplus fuscus após impregnação com 56 AgNO3 (a, b) e FISH com sonda de DNAr 45S (c, d). (a) Zigóteno; (b, c, d) paquíteno. Em c célula corada com DAPI. As setas indicam os sítios de DNAr 45S. Barra = 5 µm. ii Lista de Tabelas Tabela 1. Número de espécies analisadas, tipos de cariótipos e mecanismos 15 sexuais em Melanoplinae. Tabela
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