RESUMO
PURPOSE: The present study examined the developmental and tissue expression of the retinitis pigmentosa GTPase regulator (RPGR) gene in Xenopus laevis. METHODS: The cDNA for X. laevis RPGR (XRPGR) was isolated from adult eye mRNA by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The deduced peptide sequence was aligned with RPGR orthologues. Gene expression was examined by whole-mount in situ hybridization and RT-PCR. The localization of XRPGR in X. laevis photoreceptor cells and XTC-2 cells was determined by immunostaining. RESULTS: The XRPGR(ex1-19) isoform encodes a protein of 727 amino acids containing an RCC1 domain and a C-terminal isoprenylation anchorage motif. It shares 33% to 41% amino acid identity with human, mouse, and dog RPGR. The C-terminal exon of the alternatively spliced RPGR(ORF15) isoform is also conserved across species. XRPGR is expressed at the earliest stages of X. laevis development and persists into adulthood, where expression is highest in the eye. XRPGR is expressed in presumptive eye fields (stages 18 to 22), becoming largely restricted to the central retina (stages 28 to 40). XRPGR protein colocalizes with beta-tubulin at the X. laevis ciliary axoneme and with gamma-tubulin at centrosomes in XTC-2 cells. CONCLUSIONS: XRPGR is widely expressed throughout development but shows highest expression after the appearance of the eye primordium and persists in the eye into adulthood. The data are consistent with XRPGR expression in a single microtubular organelle-the centriole or basal body and associated ciliary transitional zone found in modified sensory cilia of photoreceptors and motile cilia.
Assuntos
Embrião não Mamífero/metabolismo , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Fatores de Troca do Nucleotídeo Guanina/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Proteínas de Xenopus/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Centrossomo/metabolismo , Clonagem Molecular , DNA Complementar/análise , Proteínas do Olho/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Hibridização In Situ , Dados de Sequência Molecular , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Tubulina (Proteína)/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriologiaAssuntos
Metabolismo Energético , Modelos Biológicos , Retinose Pigmentar/metabolismo , Animais , Citoesqueleto/metabolismo , Dineínas , Imunofluorescência , Técnicas In Vitro , Miosina VIIa , Miosinas/metabolismo , Células Fotorreceptoras de Vertebrados/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Distribuição Tecidual , Xenopus laevisRESUMO
Because adaptation of vertebrate photoreceptors to light is mediated by changes in the level of calcium in their outer segments (OS), proteins that bind calcium are important in phototransduction. This study has used immunofluorescence to investigate the distribution of the calcium-binding protein calmodulin within photoreceptor OS dissociated from amphibian ( Xenopus laevis) retinas. The OS of rods and cones had a streak of fluorescence to calmodulin at the ciliary axoneme. The OS of rods (but not cones) also displayed regularly spaced puncta of anti-calmodulin fluorescence along longitudinal lines coinciding with their multiple incisures. This location of calmodulin immunofluorescence closely matches the known location of microtubules within the OS of amphibian rods and cones. These findings provide evidence that calmodulin is closely associated with the microtubules of both the axonemal and incisural cytoskeletal systems in OS, and suggest that this association is important for calmodulin function in photoreceptors.
