RESUMO
Five clones were isolated from a human retina cDNA library whose cDNA inserts allowed reconstruction of the total sequence of the human clone cGMP phosphodiesterase alpha'-subunit cDNA comprising 3455 bp. The protein's deduced sequence contains 858 amino acids residues with molecular mass 99,169 Da. A substantial homology was revealed between the amino acid sequence of the human cones cGMP-phosphodiesterase alpha'-subunit and the corresponding sequences of alpha, beta, and alpha' subunits of visual cGMP-phosphodiesterase of bovine, murine, chicken and human retinas. Four recombinant bacteriophages were isolated from a genomic library whose inserts made it possible to reconstruct a 32-kb fragment of the human cones cGMP-phosphodiesterase alpha'-subunit gene. 5'-Flanking region of the gene and first 14 exons, encoding an N-terminal segment of the protein, along with the adjacent intron segments were sequenced.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , DNA Complementar/genética , Células Fotorreceptoras Retinianas Cones/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Sequências Repetitivas de Ácido NucleicoRESUMO
Two recombinant bacteriophage lambda clones encoding a 27.8-kb fragment of the human phosphodiesterase beta-subunit gene were isolated from a human genomic library. The nucleotide sequences of 19 exons (from the 4th to 22nd), 18 introns, and the 3'-flanking region were determined. The analysis of the nucleotide sequence of the phosphodiesterase beta-subunit gene revealed four Alu repeats and four minisatellite regions.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Diester Fosfórico Hidrolases , Células Fotorreceptoras/enzimologia , Sequência de Aminoácidos , Bacteriófago lambda/genética , Sequência de Bases , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , DNA Complementar , DNA Satélite , Escherichia coli/genética , Éxons , Humanos , Íntrons , Dados de Sequência Molecular , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Retina/enzimologiaRESUMO
Four independent phage clones containing the fragments of cone-specific cGMP phosphodiesterase (PDE) alpha' subunit (PDE-alpha') cDNA were isolated from the human cDNA library. The screening of the genomic library resulted in isolation of four independent phage clones with the fragments of human cone PDEalpha' gene including 5'-flanking region and exons ranged from 1 to 14 (overall 32 kilobases). Structural studies of the clones made it possible to establish the complete human cone PDEalpha' cDNA structure (3455 base pairs). The encoding polypeptide consists of 858 amino acid residues with a calculated molecular mass of 99169 Da. The deduced amino acid sequence displays high homology to the earlier analyzed catalytic alpha, beta and alpha' subunits of bovine, human, chicken and mouse photoreceptor PDEs.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Células Fotorreceptoras Retinianas Cones/enzimologia , 3',5'-GMP Cíclico Fosfodiesterases/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Galinhas , Clonagem Molecular , DNA Complementar/química , Éxons , Biblioteca Gênica , Biblioteca Genômica , Humanos , Íntrons , Substâncias Macromoleculares , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos , Células Fotorreceptoras/enzimologia , Mapeamento por Restrição , Retina/enzimologia , Homologia de Sequência de AminoácidosRESUMO
cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase (PDE) were isolated from a human retina library and their sequence was determined. The encoded polypeptide consists of 854 amino acid residues with a calculated molecular mass of 98,416 Da. Alignment of the deduced amino acid sequence with the earlier analysed alpha-, beta- and alpha'-subunits of bovine and mouse PDEs demonstrates a high homology. Two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene were isolated from the genomic library. A total nucleotide sequence of exons 4-22 of the PDE beta-subunit gene was established which completely corresponded to the cDNA structure. According to sequence analysis no potential possibility for alternative splicing of the beta-subunit gene was observed between exons 20 and 21 which led to the formation of the beta'-subunit as described for mouse PDE. Polymerase chain reaction (PCR) experiments also confirm the absence of the PDE beta'-subunit in human retina.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , DNA , Diester Fosfórico Hidrolases , Células Fotorreceptoras/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Nucleotídeo Cíclico Fosfodiesterase do Tipo 6 , Humanos , Dados de Sequência Molecular , Homologia de Sequência do Ácido NucleicoRESUMO
cDNA clones encoding the beta-subunit of the photoreceptor cGMP phosphodiesterase-(PDE) were isolated from a human retinal library. The encoded polypeptide has 854 amino acid residues with calculated molecular mass of 98416 Da. Alignment of the deduced amino acid sequence with the previously analysed alpha-, beta- and alpha'-subunits of the bovine and mouse PDEs demonstrates highly significant similarities. We have also isolated, from a genomic library, two overlapping recombinant lambda phage clones containing 26 kb of the human PDE beta-subunit gene. The cloning of the human gene and the knowledge of its genomic organization will allow the rapid assessment of the role of this gene in the causation of human retinopathies.
