RESUMO
cGMP-phosphodiesterase (PDE) is a key component in visual phototransduction. Rod and cone photoreceptors each produce their unique cGMP-PDE subunits. The alpha' catalytic subunits are believed to be cone-specific. In this study, we report that transfection of the -132 to +139 sequence in the upstream region of the human alpha'-PDE gene fused to luciferase cDNA gives the highest level of reporter gene transcription in cultured retinoblastoma Y79 cells. Transgenic Xenopus laevis carrying this sequence fused to green fluorescent protein (GFP) expressed GFP in cones, suggesting a conserved regulatory mechanism for alpha'-PDE transcription in both human and frog.
Assuntos
Regulação da Expressão Gênica , Luz , Transdução de Sinais/genética , Transcrição Gênica , Vertebrados/genética , Animais , Animais Geneticamente Modificados , Sequência de Bases , Primers do DNA , Ensaio de Desvio de Mobilidade Eletroforética , Imuno-Histoquímica , Xenopus laevisRESUMO
Retinoblastoma (Rb) is an intraocular tumor usually diagnosed in children under four years of age (1). The tumor rises when both alleles of the Rb tumor suppressor gene become inactivated in a retinal precursor cell during development (2,3). The first retinoblastoma cell line to be established in culture, Y-79 (4), has been shown to originate from neuroectodermal cells that express both neuronal and glial cell markers (3). Both Y-79 cells and Rb tumor cells produce mRNAs encoding several proteins unique to the photoreceptors (5), including different subunits of cone- and rod-specific cGMP-phosphodiesterases (6). Therefore, cultured Y-79 cells, which have a human retinal origin, could be particularly useful for studying the regulatory mechanisms of photoreceptor-specific gene expression (7).
RESUMO
A human cDNA (alpha-PDE) encoding the alpha' catalytic subunit of cone photoreceptor cGMP-phosphodiesterase has been isolated and characterized. The nucleotide sequence of 2980 bp contains an open reading frame encoding an 859-amino-acid (aa) protein with a calculated molecular mass of 99 kDa. Northern blot analysis of human retinal mRNA hybridized with the alpha'-PDE cDNA revealed a signal corresponding to a 3.2-kb transcript. Comparison of the deduced aa sequence of human cone alpha'-PDE with corresponding proteins isolated from bovine and chicken retinas shows 89 and 83% identity, respectively, and indicates that alpha'-PDE has been very well conserved in the evolutionary process. Human cone alpha'-PDE is highly homologous to rod alpha-PDE and beta-PDE, suggesting that these proteins may have a close phylogenetic relationship.
Assuntos
3',5'-GMP Cíclico Fosfodiesterases/genética , Células Fotorreceptoras Retinianas Cones/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Galinhas , DNA Complementar/genética , Expressão Gênica , Genes , Humanos , Isoenzimas/genética , Dados de Sequência Molecular , RNA Mensageiro/genética , Células Fotorreceptoras Retinianas Bastonetes/enzimologia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
The genomic organization and nucleotide structure of the human cone photoreceptor cGMP phosphodiesterase alpha'-subunit (alpha'-PDE) gene (PDEA2) as well as its chromosomal localization have been determined. This gene, which spans about 48 kb, consists of 22 exons and codes for an 858-amino-acid protein. The alpha'-PDE gene maps to human chromosome 10q24. Its coding region shows about 90% nucleotide identity and 93% amino acid identity with the corresponding region of the bovine gene. The intron-exon organization of the genes encoding human cone alpha'-PDE and rod beta-PDE are very similar, suggesting that these proteins have a close phylogenetic relationship and probably a common origin.