RESUMEN
A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in Escherichia coli. These sequences show 96-98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.
Asunto(s)
Secuencia de Aminoácidos , Animales , Secuencia de Bases , Carpas/genética , Clonación Molecular , Cartilla de ADN/genética , ADN Complementario/genética , Escherichia coli/genética , Hormona del Crecimiento/química , India , Modelos Moleculares , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico/métodos , Hipófisis , Conformación Proteica , Análisis de Secuencia de ADNRESUMEN
A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3' region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.
Asunto(s)
Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bagres/genética , Clonación Molecular , Escherichia coli/fisiología , Biblioteca de Genes , Proteínas Fluorescentes Verdes , Hormona del Crecimiento/química , Proteínas Luminiscentes , Datos de Secuencia Molecular , Hipófisis/química , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Alineación de SecuenciaRESUMEN
As açöes do hormônio de crescimento em promover o crescimento esquelético e no metabolismo säo indiretas e diretas, respectivamente. As açöes indiretas säo mediadas através das somatomedinas ou fatores de crescimento e as diretas säo predominantemente antagonistas às da insulina. A liberaçäo do hormônio de crescimento é determinada por um equilíbrio dinâmico de peptídios hipotalâmicos inibitórios e estimulatórios que säo a somatostatina e o hormônio liberador de hormônio do crescimento. A resposta hipotálamo-hipófise da liberaçäo do hormônio de crescimento pode ser influenciada pela idade, sexo, insulina, hormônios esteróides e da tireóide e o estado nutricional. Assim, há uma importante interrelaçäo entre as açöes do hormônio de crescimento, crescimento e estado nutricional