RESUMO
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.
Assuntos
Sequência de Aminoácidos , Animais , Sequência de Bases , Carpas/genética , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Escherichia coli/genética , Hormônio do Crescimento/química , Índia , Modelos Moleculares , Dados de Sequência Molecular , Técnicas de Amplificação de Ácido Nucleico/métodos , Hipófise , Conformação Proteica , Análise de Sequência de DNARESUMO
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.
Assuntos
Sequência de Aminoácidos , Animais , Sequência de Bases , Peixes-Gato/genética , Clonagem Molecular , Escherichia coli/fisiologia , Biblioteca Gênica , Proteínas de Fluorescência Verde , Hormônio do Crescimento/química , Proteínas Luminescentes , Dados de Sequência Molecular , Hipófise/química , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de SequênciaRESUMO
A search for the presence of mariner-like elements in the Labeo rohita genome by polymerase chain reaction led to the amplification of a partial DNA sequence coding for a putative transmembrane domain of gonadotropin hormone receptor. The amplified DNA sequence shows a high degree of homology to the available turkey and human luteinizing and follicle stimulating hormone receptor coding sequences. This is the first report on cloning such sequences of piscine origin.
Assuntos
Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Sequência Consenso , Peixes/genética , Humanos , Dados de Sequência Molecular , Receptores da Gonadotropina/genéticaRESUMO
Recombinant transformation vectors (ZPPypGH and ZpprtGH) consisting of fish growth hormone cDNA, and a reporter gene p-galactosidase driven by fish promoter (Zp) were constructed. Freshly fertilized eggs of zebrafish (Brachydanio rerio) were electroporated at optimum conditions (0.07 kV voltage; 25 J.1F capacitance; 00 ohm resistance and 2 pulses) in the presence of one of these transformation vectors (100 J.1g circular DNNml). In either cases 72% of the electroporated eggs successfully hatched, in comparison to the 85% hatchability of the control eggs. Genomic DNA extracted from fins of randomly chosen Fo individuals was screened (by Southern blot hybridization); the transgenes were retained in the host-. genome of all the randomly chosen adult transformants. Fin-positive presumptive founder parents were crossed with control counterparts and the DNA of randomly chosen F] progenies was screened for germ-line transformation. Southern analysis of chosen F1 progenies revealed the persistence of ZPPypGH or ZpPrtGH in 53% of the F1 progenies. Southern analyses of chosen F1 progenies and the frequency (53% of F1 ZpPrtGH and 53% of F1 ZPPypGH) of transmission revealed the degree of mosaicism i!1 F 0 transformants. Expression was confirmed from the 3-4 times elevated levels of activity of the reporter gene and 30-40% accelerated growth of transgenic Fo and F1 progenies.
RESUMO
P25 protein was extracted from cocoons of the silkworm Bombyx mori by alkali solubilization and purified by gel elution. The purity and authenticity of the protein were confirmed by SDS-PAGE, 2-dimensional gel electrophoresis and peptide mapping. Polyclonal anti-P25 sera were raised in rabbit and mice. The relative abundance of P25 protein present in the larva during different developmental stages was analysed by SDS-PAGE, and quantified by sandwich ELISA. The minimum level (0·2 μg/animal) of this protein was recorded at the beginning of the first instar and maximum (16·7mg/pair of silkgland) on the final day of the V instar. During each moult period, P25 protein level was suppressed; the level increased with the initiation of feeding and reached maximum on the 3rd day of each instar except the final instar where the maximum was recorded prior to pupal moult. Western blot analysis also confirmed the developmental stage-specific accumulation of P25 protein in the silkworm Bombyx mori.