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...

The ␤-subunit of cGMP-phosphodiesterase (␤-PDE) is a key protein in phototransduction expressed exclusively in rod photoreceptors. It is necessary for visual function and for structural integrity of the retina. ␤-PDE promoter deletions showed that the ؊45/؊23 region containing a consensus Crx-response element (CRE) was necessary for low level transcriptional activity. Overexpressed Crx modestly transactivated this promoter in 293 human embryonic kidney cells; however, mutation of CRE had no significant effect on transcription either in transfected Y79 retinoblastoma cells or Xenopus embryonic heads. Thus, Crx is unlikely to be a critical ␤-PDE transcriptional regulator in vivo. Interestingly, although the ␤/GC element (؊59/؊49) binds multiple Sp transcription factors in vitro, only Sp4, but not Sp1 or Sp3, significantly enhanced ␤-PDE promoter activity. Thus, the Sp4-mediated differential activation of the ␤-PDE transcription defines the first specific Sp4 target gene reported to date and implies the importance of Sp4 for retinal function. Further extensive mutagenesis of the ␤-PDE upstream sequences showed no additional regulatory elements. Although this promoter lacks a canonical TATA box or Inr element, it has the (T/A)-rich ␤/TA sequence located within the ؊45/؊23 region. We found that it binds purified TBP and TFIIB in gel mobility shift assays with cooperative enhancement of binding affinity.One of the key components of the phototransduction cascade that takes place in rod photoreceptors is the heterotetrameric (␣␤␥ 2 ) cGMP-phosphodiesterase (1). The gene encoding the ␤-subunit of the human enzyme (␤-PDE) 1 has been well characterized and consists of 22 exons encompassing ϳ43 kb of genomic DNA (2). Genetic defects in this gene have been linked to retinal degeneration in several animal species and human (3-9). There is increasing evidence that abnormalities in transcriptional regulatory components of different genes contribute significantly to or directly cause pathological phenotypes in the retina (10 -13). Therefore, further studies on the transcriptional regulation of rod-specific ␤-PDE gene will identify additional genes important for retinal function and structural integrity and will ultimately help to establish the molecular mechanisms crucial for retina-specific expression of this and perhaps some other genes.We recently reported our initial results on the transcriptional control mechanisms that take place in the human ␤-PDE 5Ј-flanking region (14). Mutational analysis of the ␤-PDE promoter tested both in vitro and ex vivo, and confirmed by the generation of transgenic Xenopus expressing mutant ␤-PDE promoter/green fluorescent protein fusion constructs in vivo, revealed a minimal promoter region, from Ϫ93 to ϩ53, that supports high levels of rod-specific transcription (14). Two enhancer elements were localized within this minimal promoter, ␤Ap1/NRE and ␤/GC, that interact with nuclear factors and activate transcription from the ␤-PDE promoter.To continue the systematic analysis of the ␤-PDE promoter...

Phospholipase C (PLC) isoforms stimulate the hydrolysis of phosphatidyl inositol (4,5)-bisphosphate (PIP2) to produce diacylglycerol (DAG) and 1,4,5 inositol trisphosphate (IP3), with IP3 regulating the release of calcium (Ca2+) from the endoplasmic reticulum. This release of calcium is essential for oocyte activation, and a sperm-specific PLC isoform, PLCγ;, has been proposed as the primary agent that initiates the activation process. However, the oocyte contains many endogenous PLC isoforms (PLC β, γ, and δ) that could also be involved in regulating or initiating these calcium oscillations downstream of other initiating events. In order to better elucidate the involvement of endogenous PLC isoforms as well as the specific role of the sperm-specific form, small interfering RNA (siRNA) directed against the specific bovine PLC isoforms (PLCζ;, PLCγ1, PLCγ2, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3) were microinjected into bovine oocytes, and the subsequent effects on PLC mRNA levels and bovine fertilization were evaluated. Real-time PCR (qPCR) was used to quantify the levels of PLC message present in bovine oocytes at the time of injection (15 h post-maturation) and 6, 10, and 14 h post-injection. The qPCR results indicated a near-complete knockdown of mRNA levels in bovine oocytes 10 h post-injection for the isotypes PLCγ1, PLCγ2, PLCδ3, PLCδ4, PLCβ1, PLCβ3, but only partial knockdown of PLCS 1 mRNA. Oocytes microinjected with PLC siRNA were also fertilized and cultured in vitro according to our standard laboratory procedures (Reed et al. 1996 Theriogenology 45, 439-449). The oocytes microinjected with PLCζ;, PLCδ1, PLCδ3, PLCδ4, PLCβ1, PLCβ3 siRNA resulted in cleavage rates similar to the negative control siRNA, non-injected, and sham-injected treatment groups, whereas bovine oocytes microinjected with PLCγ1 and PLCγ2 siRNA had significantly lower cleavage rates compared with the controls. Additionally, complementary cRNA for each specific PLC isoform was microinjected into bovine oocytes to ascertain each isoform’s ability to induce parthenogenetic activation. Development was observed in oocytes microinjected with a variety of cRNAs, and the activating effects of the cRNA were negligible if the oocytes were microinjected with the corresponding siRNA before microinjection with cRNA. Interestingly, siRNA specific for PLCζ; failed to reduce cleavage when treated bovine oocytes were fertilized. These data illustrate the potential involvement of multiple endogenous PLC isoforms and not just the sperm-specific PLCζ; isoform in bovine oocyte activation during fertilization.