Immediate Upstream Sequence of Arrestin Directs Rod-specific Expression in Xenopus

Mani, Shobana S.; Besharse, Joseph C.; Knox, Barry E.

doi:10.1074/jbc.274.22.15590

Cited by 33 publications

(27 citation statements)

References 56 publications

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“…The binding was not inhibited by competition with oligonucleotides that were mutated in any part of the sequence of 5Ј-CAGACAGGC-TATA-3Ј (lanes [11][12][13][14][15][16][17][18][19][20][21][22], suggesting that this sequence is involved in the DNA-protein interaction. However, the competitors with mutations outside of the recognition site still resulted in a dosage-dependent inhibition (lanes [5][6][7][8][9][10][23][24][25], which is similar to that seen when using competitor identical to the probe.…”

Section: The Gfp Expression Pattern Is Maintained In the Transgenic Tmentioning

confidence: 65%

“…A reporter gene driven by the 70-bp promoter of the murine IRBP which contains Ret1/ PCE I and CRX binding sites is active in cultures of retinal cells and brain cells (30). A portion of the rod arrestin promoter, including CRX and AP1 binding sites, directs expression of a reporter gene only in rod cells in the transgenic X. laevis (7). In this paper we showed that the XNP(Ϫ77/Ϫ18bp) containing three repeated PCE II is sufficient to target the GFP reporter to the photoreceptors in X. laevis.…”

Section: Figmentioning

confidence: 78%

“…The Xenopus homolog of CRX has not been cloned. Therefore, the role of CRX in the regulation of nocturnin is unclear.The transgenic technique in frogs is a powerful tool to investigate the spatial expression pattern and regulation of genes of interest (7,22,25,26). GFP has been commonly used as the reporter because of its stability and ease of detectability for in vivo studies.…”

mentioning

confidence: 99%

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A Novel Promoter Element, Photoreceptor Conserved Element II, Directs Photoreceptor-specific Expression of Nocturnin in Xenopus laevis

Liu

Green

2001

Journal of Biological Chemistry

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Nocturnin is a vertebrate circadian clock-regulated gene, and in Xenopus laevis its mRNA is specifically expressed in retinal photoreceptor cells. We have investigated the transcriptional regulatory mechanism that drives this precise spatial expression pattern of the nocturnin gene. A deletion series of the nocturnin 5-flanking sequence driving the green fluorescence protein (GFP) reporter was used to generate transgenic Xenopus tadpoles. We found that a construct containing 2.6 kilobase pairs of 5-flanking sequence targeted high level GFP reporter expression specifically to photoreceptor cells, in a pattern identical to endogenous nocturnin. This photoreceptor-specific expression pattern was maintained with several further deletions of 5-upstream sequence, including a short 59-base pair fragment. Within this region of 59 base pairs, three perfect repeats of a novel protein binding site were identified by electrophoretic mobility shift assay. Competitions using varying oligonucleotide sequences demonstrated that the sequence required for protein binding is CAGA-CAGGCTATA, designated photoreceptor-conserved element II (PCE II). The protein complex that binds to this element is enriched in retinal extracts, and mutations of PCE II which fail to bind the protein complex also fail to direct GFP reporter expression to photoreceptors. These results indicate that the PCE II in the proximal promoter of the nocturnin gene is sufficient for driving the photoreceptor-specific expression of nocturnin.Precise spatial patterns of gene expression are critical for the proper function of all organisms. Within most of the central nervous system of vertebrates, the study of spatial regulation of transcription is difficult because of the vast heterogeneity of this tissue. The retina is a part of the central nervous system which is more amenable to these types of study because the cells are organized in morphologically distinct layers, and the various cell types have been well characterized (1-3).Many genes have been shown to be expressed specifically in photoreceptor cells within the retina, including those known to be involved in the phototransduction cascade or other photoreceptor processes. Biochemical studies, such as DNase I footprinting and electrophoretic mobility shift assays (EMSAs), 1 have identified a number of protein binding sites within the promoters/enhancers of several retina-specific genes, including opsin, interphotoreceptor retinoid-binding protein (IRBP), and rod arrestin (4 -7). DNA elements named Ret1 and PCE I were defined in the rat opsin promoter and the arrestin promoter, respectively, but these two elements are quite similar to each other (8,9). Further analysis of the rat opsin gene revealed two additional protein-binding elements named Ret2 and Ret3 (10). A positive acting opsin regulatory element Ret4 was identified using an in vitro transcription system from bovine retinal nuclear extracts (11). It has been demonstrated that a member of the OTD/OTX family, CRX (cone rod homeobox), binds in vitro to th...

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Section: The Gfp Expression Pattern Is Maintained In the Transgenic Tmentioning

confidence: 65%

Section: Figmentioning

confidence: 78%

mentioning

confidence: 99%

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A Novel Promoter Element, Photoreceptor Conserved Element II, Directs Photoreceptor-specific Expression of Nocturnin in Xenopus laevis

Liu

Green

2001

Journal of Biological Chemistry

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“…Several studies have shown that DNA-binding elements such as CRX, and a similar site called photoreceptor consensus element, play a pivotal role in directing cell-specific expression of genes (66). Since the mutation of the cAANAT E box eliminated 90% of reporter activity (Fig.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Chicken SerotoninN-Acetyltransferase Gene

Chong¹,

Bernard²,

Klein

2000

Journal of Biological Chemistry

133

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The abundance of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) mRNA in the chicken pineal gland exhibits a circadian rhythm, which is translated into a circadian rhythm in melatonin production. Here we have started to elucidate the molecular basis of the circadian rhythm in chicken AANAT (cAANAT). The 5-flanking region of the cAANAT gene was isolated and found to contain an E box DNA element that confers strong luciferase reporter activity. In transfection experiments using chicken pineal cells, an E box mutation dramatically decreased reporter activity. Northern blot analysis indicated that several putative clock genes (bmal1, Clock, and MOP4) are co-expressed in the chicken pineal gland. bmal1 mRNA is expressed in a rhythmic manner in the chicken pineal gland, with peak levels at early subjective night, coincident with the increase in cAANAT expression. Co-transfection experiments in COS cells demonstrated that chicken BMAL1/ CLOCK and human BMAL1/MOP4 heterodimers bound the AANAT E box element and enhanced transcription. These observations suggest that binding of clock gene heterodimers to the cAANAT E box is a critical element in the expression of the cAANAT gene in vitro.

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“…Transgenic Xenopus and CHO cells: arrestin-EGFP fusion construct The REMI method was used to create transgenic Xenopus, with EFGP and arrestin-EGFP (Arr-EGFP) fusion protein expression driven by the opsin promoter (Mani et al, 1999;Mani et al, 2001). CHO cells stably expressing EGFP were created with standard methods; the constructs are described in the supplemental data (Materials and Methods, Sections 2-5, http://jcs.biologists.org/supplemental/).…”

Section: Methodsmentioning

confidence: 99%

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors

Peet

Bragin

Calvert

et al. 2004

Journal of Cell Science

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101

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The hypothesis is tested that enhanced green fluorescent protein (EGFP) can be used to quantify the aqueous spaces of living cells, using as a model transgenic Xenopus rods. Consistent with the hypothesis, regions of rods having structures that exclude EGFP, such as the mitochondrial-rich ellipsoid and the outer segments, have highly reduced EGFP fluorescence. Over a 300-fold range of expression the average EGFP concentration in the outer segment was approximately half that in the most intensely fluorescent regions of the inner segment, in quantitative agreement with prior X-ray diffraction estimates of outer segment cytoplasmic volume. In contrast, the fluorescence of soluble arrestin-EGFP fusion protein in the dark adapted rod outer segment was approximately threefold lower than predicted by the EGFP distribution, establishing that the fusion protein is not equilibrated with the cytoplasm. Arrestin-EGFP mass was conserved during a large-scale, light-driven redistribution in which ∼40% of the protein in the inner segment moved to the outer segment in less than 30 minutes.

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Immediate Upstream Sequence of Arrestin Directs Rod-specific Expression in Xenopus

Cited by 33 publications

References 56 publications

A Novel Promoter Element, Photoreceptor Conserved Element II, Directs Photoreceptor-specific Expression of Nocturnin in Xenopus laevis

A Novel Promoter Element, Photoreceptor Conserved Element II, Directs Photoreceptor-specific Expression of Nocturnin in Xenopus laevis

Characterization of the Chicken SerotoninN-Acetyltransferase Gene

Quantification of the cytoplasmic spaces of living cells with EGFP reveals arrestin-EGFP to be in disequilibrium in dark adapted rod photoreceptors

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