The cDNAs of the α-subunit of the pituitary gonadotrophic hormones (GTHα) of fish of the order Osteoglossiformes or the superorder Osteoglossomorpha have never been sequenced. For a better understanding the phylogenetic diversity and evolution of PGHα in fish and for future biotechnological synthesis of the gonadotrophic hormones (ag-FSH and ag-LH), of Arapaima gigas, one of the largest freshwater fishes of the world, its GTHα cDNA was synthesized by reverse transcriptase and the polymerase chain reaction starting from total pituitary RNA. The ag-GTHα-subunit was found to be encoded by 348 bp, corresponding to a protein of 115 amino acids, with a putative signal peptide of 24 amino acids and a mature peptide of 91 amino acids. Ten cysteine residues, responsible for forming 5 disulfide linkages, 2 putative N-linked glycosylation sites and 3 proline residues, were found to be conserved on the basis of the known sequences of vertebrate gonadotrophic hormones. Phylogenetic analysis, based on the amino acid sequences of 38 GTHα-subunits, revealed the highest identity of A. gigas with members of the Acipenseriformes, Anguilliformes, Siluriformes and Cypriniformes (87.1-89.5 %) and the lowest with Gadiformes and Cyprinodontiformes (55.0 %). The obtained phylogenetic tree agrees with previous analysis of teleostei, since A. gigas, of the order of Osteoglossiformes, appears as the sister group of Clupeocephala, while Elopomorpha forms the most basal group of all other teleosts.

The common gonadotrophic hormone α-subunit (GTHα) has been previously isolated by our research group from A. gigas pituitaries; in the present work the cDNA sequences encoding FSHβ and LHβ subunits have also been isolated from the same species of fish. The FSH β-subunit consists of 126 amino acids with a putative 18 amino acid signal peptide and a 108 amino acid mature peptide, while the LH β-subunit consists of 141 amino acids with a putative 24 amino acid amino acid signal peptide and a 117 amino acid mature peptide. The highest identity, based on the amino acid sequences, was found with the order of Anguilliformes (61%) for FSHβ and of Cypriniformes (76%) for LHβ, followed by Siluriformes, 53% for FSHβ and 75% for LHβ. Interestingly, the identity with the corresponding human amino acid sequences was still remarkable: 45.1% for FSHβ and 51.4% for LHβ. Three dimensional models of ag-FSH and ag-LH, generated by using the crystal structures of h-FSH and h-LH as the respective templates and carried out via comparative modeling and molecular dynamics simulations, suggested the presence of the so-called “seat-belt”, favored by a disulfide bond formed between the 3rd and 12th cysteine in both β-subunits. The sequences found will be used for the biotechnological synthesis of A. gigas gonadotrophic hormones (ag-FSH and ag-LH). In a first approach, to ascertain that the cloned transcripts allow the expression of the heterodimeric hormones, ag-FSH has been synthesized in human embryonic kidney 293 (HEK293) cells, preliminarily purified and characterized.

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Aos meus familiares por me incentivaram a não desistir no meio da jornada.Ao amigo Marcos Vinicius Nucci Capone que participou ativamente no meu processo de seleção para o ingresso nas atividades científicas.Ao amigo Flávio Sousa Silva pelas ideias trocadas.À amiga Letícia do Centro de Lasers e Aplicações (CLA) pela preciosa ajuda na disciplina de física nuclear À companheira Thais Cristina dos Anjos Sevilhano por estar sempre por perto nos momentos em que precisei. Aos amigos do Centro de Biotecnologia do Instituto de Pesquisas Energéticas eNucleares-Arlete, Dra. Beatriz, Dra. Claudia, Caroline Elias, Dr. Daniel Perez, Daniela, Eliana, Eliza, Ed, Flávia, Fernanda Arthuso, Geiza, Hugo, Ivete, José Maria, Junqueira, Dra. Kayo, Karina Corleto, Laura, Mayara, Dra. Miriam, Dra. Marina, Mosca, Dra. Nanci, Neia, Gisleine, Sandra, Natan, Dr. Nélio, Neide, Dra. Olga, Dr. Patrick, Patrícia, Rute, Dra. Natália Malavasi, Dra. Renata, Rosangela, Dr. Vincent, Tamara, pelos bons momentos, colaboração, amizade e compreensão.À todos os funcionários do Centro de Biotecnologia que colaboraram direta ou indiretamente para a realização deste trabalho.Ao Instituto de Pesquisas Energéticas e Nucleares, pela oportunidade de realizar este trabalho.Ao IPEN, FAPESP, CNPq, CAPES pelo apoio financeiro e técnico.Eu tentei 99 vezes e falhei, mas na centésima tentativa eu consegui, nunca desista de seus objetivos mesmo que esses pareçam impossíveis, a próxima tentativa pode ser a vitoriosa. (Albert Einstein)No que diz respeito ao empenho, ao compromisso, ao esforço, à dedicação, não existe meio termo. Ou você faz uma coisa bem feita ou não faz. is in danger of disappearing due to exploitation by the fishing industry and increasing human presence in its natural habitat. In the present work the cDNAs of the gonadotropin α-subunit (ag-GTH) and of follicle-stimulating hormone β subunit (ag-FSHβ) were isolated and cloned for the first time. As a consequence, a better understanding of the diversity and evolution of this glycoprotein in fish and its future biotechnological synthesis for reproductive and alimentary purposes will be possible. Both cDNAs of ag-GTHα and ag-FSHβ have been synthesized via reverse transcriptase reaction (RT-PCR) and polymerase chain reaction (PCR) using as a template total RNA extracted from A. gigas pituitary glands. Ag-GTHα-subunit has a coding sequence (open reading frame, ORF) of 348 bp, corresponding to a 115 amino acid protein, with a putative signal peptide of 24 aminoacids and a mature peptide of 91 amino acids. Ten cysteine residues, responsible for the formation of five disulfide linkages, two N-glycosylation sites and three proline residues were found highly conserved when compared to other fish species. A comparison based on the amino acid sequence of the GTHα-subunit from 38 different species of fish showed high identity of A. gigas with members of the following orders: Acipenseriformes, Siluriformes and Cypriniformes (87.1-89.5%), while the lowest identity was found with Gadiformes and Cyprinodontiformes (55...

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