Xenopus Rhodopsin Promoter

Abstract

To understand the mechanisms that control the cellspecific visual pigment gene transcription, the Xenopus rhodopsin 5 regulatory region has been characterized in vivo using transient transfection of Xenopus embryos and transgenesis. The principal control sequences were located within ؊233/؉41, a region with significant conservation with mammalian rhodopsin genes. DNase footprinting indicated seven distinct regions that contain potential cis-acting elements. Sequences near the initiation site (؊45/؉41, basal region) were essential, but not sufficient, for rod-specific transcription. Two negative regulatory regions were found, one between ؊233 to ؊202, with no apparent similarity to known elements, and a second Ret-1-like CAAT (؊136/؊122) motif. Deletion of either sequence led to a 2-3-fold increase in expression levels, without a change in rod specificity. Sequences between ؊170 to ؊146, which contain an E-box motif, were necessary for high level expression in transgenic tadpoles but not in transient transfections. Sequences between ؊84 and ؊58, which contained an NRE-like consensus were found to be necessary for high level expression in both assays. Although expression levels were modulated by various proximal sequences in the rhodopsin promoter, none of the tested sequences were found to be necessary for rod specificity. Promoter constructs with a consensus BAT-1 sequence in conjunction with an NRE-like element upstream of the basal promoter directed low level green fluorescent protein expression in the central nervous system in transgenic tadpoles. These results suggest that rod cell-specific expression of rhodopsin is controlled by redundant elements in the proximal promoter.Phototransduction occurs in the photoreceptor layer of the vertebrate retina, which is composed of distinct cell types: rods and cones (1). These cells express a number of specific proteins that regulate the light-dependent currents mediating vision (2, 3). Among these cell-specific proteins are the visual pigments, which combine with 11-cis-retinal to form the light-sensitive component of the transduction cascade. The visual pigments are a large family of genes, which contain rod-specific rhodopsins and at least four classes of cone-specific opsins (4). Rhodopsin, required for nocturnal vision, is the most abundant opsin in many vertebrate retinae by virtue of the size of the rod outer segments, abundance of the rod cells, and the high level of transcription. As such, the regulation of rhodopsin expression has been a focus for understanding mechanisms of cellspecific gene expression in the retina (5).Transcription initiation has been identified as the major control point for rhodopsin gene expression (6, 7). A variety of studies using different approaches have demonstrated that important transcriptional control sequences lie within the 5Ј upstream regions of various rhodopsin genes. Functional assays using transgenic mice have shown that 2-4 kb 1 of upstream sequences from the mouse and bovine rhodopsin genes direct reporter gene express...

Characterization of Human Cone Phosphodiesterase-6 Ectopically Expressed in Xenopus laevis Rods

“…To prepare the transgene construct (Fig. 1A), the pXOP(Ϫ508/ϩ41)EGFP vector (19) was first modified to eliminate the stop codon after the EGFP sequence and introduce a new restriction site (XmaI) downstream of EGFP. Human PDE6C was cloned into the vector using NotI and XmaI sites to obtain the pXOP(Ϫ508/ϩ41)EGFP-PDE6C vector.…”

“…Rods-Transgenic expression of the EGFP fusion protein of human cone PDE6C in rods was directed by the well characterized Xenopus opsin promoter (19) (Fig. 1A).…”

Conserved Transcriptional Activators of the Xenopus Rhodopsin Gene

“…Another highly conserved feature is the Nrl response element (NRE), 1 TGCTGA. The NRE is necessary for high levels of rhodopsin expression in both transfected mammalian cells and in transgenic Xenopus (20,23,25,26). Given the functional and sequence similarities in rhodopsin promoters, it appears likely that homologous transcription factors would mediate transcriptional activation in vertebrates.…”

“…Two of the most highly conserved sequences are the Ret-1 and BAT-1 elements, each of which contain a core ATTA, and are recognized by Otx family homeodomain proteins in vitro (i.e. Crx (23,24)). The functional role these elements play in vivo is not fully understood (23).…”

“…Crx (23,24)). The functional role these elements play in vivo is not fully understood (23). Another highly conserved feature is the Nrl response element (NRE), 1 TGCTGA.…”