Characterization of the Xenopus Rhodopsin Gene

Batni, Suchitra; Scalzetti, Lia; Moody, Sally A.; Knox, Barry E.

doi:10.1074/jbc.271.6.3179

Cited by 66 publications

(72 citation statements)

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“…In addition, we utilized transient transfections in dissected Xenopus laevis embryo heads maintained in culture ex vivo to study the ␤-PDE gene transcriptional mechanisms in the retina in situ. Although of amphibian origin, many of the photoreceptor-specific genes and their regulatory elements have been evolutionarily conserved between Xenopus and mammals (15). For in vivo studies, we generated transgenic Xenopus tadpoles carrying various lengths of ␤-PDE 5Ј-flanking region/GFP constructs.…”

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confidence: 99%

Nrl and Sp Nuclear Proteins Mediate Transcription of Rod-specific cGMP-phosphodiesterase β-Subunit Gene

Lerner

Gribanova

et al. 2001

Journal of Biological Chemistry

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cGMP-phosphodiesterase (PDE) is the key effector in rod photoreceptor signal transduction. Mutations in the gene encoding its catalytic ␤-subunit (␤-PDE) cause retinal degenerations leading to blindness. We report that the short ؊93 to ؉53 sequence in the upstream region of this gene is sufficient for ␤-PDE transcription in both Y79 human retinoblastoma cells and Xenopus embryo heads maintained ex vivo. This sequence also functions as a minimal rod-specific promoter in transgenic Xenopus tadpoles. The Nrl transcription factor binds in vitro to the ␤Ap1/NRE regulatory element located within this region and transactivates it when overexpressed in nonretinal 293 embryonic kidney cells. We also found a G/Crich activator element, ␤/GC, important for promoter activity in Y79 retinoblastoma cells and Xenopus embryos. Both the ubiquitous Sp1 and the central nervous system-specific Sp4 transcription factors are expressed in retina and interact with this element in vitro. Electrophoretic mobilities of ␤/GC-Y79 nuclear protein complexes are altered by antibodies against Sp1 and Sp4. Thus, our results implicate Nrl, Sp1, and Sp4 in transcriptional regulation of the rod-specific minimal ␤-PDE promoter. We also conclude that Xenopus laevis is an efficient system for analyzing the human ␤-PDE promoter and may be used to study other human retinal genes ex vivo and in vivo.The vertebrate retina is a highly specialized sensory extension of the central nervous system responsible for light perception and conversion into a neural stimulus, phototransduction as well as the initial visual signal integration and processing. Phototransduction occurs both in rod and cone photoreceptors. During rod phototransduction, rhodopsin-activated transducin activates cGMP-phosphodiesterase (PDE), 1 which leads to the hydrolysis of cGMP and closure of the cGMP-gated transmembrane cationic channels causing hyperpolarization of the plasma membrane and photoreceptor signaling (1).The rod photoreceptor-specific cGMP-PDE is a membraneassociated heterotetrameric enzyme composed of two catalytic ␣-and ␤-subunits and two inhibitory ␥-subunits (2). Each of these subunits is essential for normal cGMP-PDE activity required for phototransduction and the maintenance of retinal health. Mutations in the ␤-subunit of cGMP-PDE (␤-PDE) are currently the most common known cause of autosomal recessive retinitis pigmentosa (3). In addition, mutations in the ␤-PDE gene result in congenital stationary night blindness (4) and retinal degeneration in animal models (5-8). Recently, genetic defects in transcription mechanisms that control the expression of several retina-specific genes have been linked to different types of retinal disorders (9 -12). Thus, it became important to elucidate the molecular events that regulate transcription of the ␤-PDE gene, because abnormalities in these control mechanisms may be detrimental for photoreceptor function and structural integrity.The objective of this investigation was to determine the cis-acting elements and the trans-acting nucle...

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confidence: 99%

Nrl and Sp Nuclear Proteins Mediate Transcription of Rod-specific cGMP-phosphodiesterase β-Subunit Gene

Lerner

Gribanova

et al. 2001

Journal of Biological Chemistry

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“…Genomic Analysis-Southern analysis was performed as described previously (43). The blot was hybridized using a 32 P-labeled BamHIAvaI fragment (Probe 2, Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Embryo Transfections-Xenopus embryos were transfected as described previously (43), except that 10 g of DNA was mixed with 30 l of DOTAP (Roche) before transfection. Protein extracts from transfected heads or trunks were prepared in 80 -100 l of lysis buffer, and duplicate aliquots of 10-l each (equivalent to one head or trunk) were assayed for luciferase activity.…”

Section: Methodsmentioning

confidence: 99%

“…The blot was exposed using a PhosphorImager. To amplify intron 2, PCR was carried out using exon-specific primers P2 and forward primer 5Ј-tgtcatgtataagaaaacc-3Ј as described previously (43) except that 30 cycles of annealing (58°C), extension (72°C for 2 min), and denaturation (95°C for 1 min) were performed.…”

Section: Methodsmentioning

confidence: 99%

“…Xenopus is particularly well suited for studying retinal development and gene regulation; rapid histogenesis (38,39), relative abundance of rods (55%) and cones (45%) (40,41), efficient methods for gene delivery (42,43) and transgenesis (44,45) provide unique experimental strategies to address questions of cell-specific gene expression in vertebrates. We have cloned genomic fragments that contain 5Ј regulatory regions of the Xenopus rod arrestin gene and used transient transfections and transgenic approaches to study promoter function.…”

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