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Z Huang, T Fujimaki, H Kitagawa, H Sakuma, A Murakami, A Kanai, G Inana; Mutagenesis Analysis of the X-Arrestin Promoter . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1375.
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Purpose: X-arrestin is one of the new retinal genes isolated in our laboratory by a differential cloning strategy (Murakami et al. FEBS Letters 334:203-209, 1993). This gene is specifically expressed in the red-, green-, and blue-sensitive cone photoreceptors and is most likely involved in the modulation of cone phototransduction (Sakuma et al. FEBS Letters 382:105-110, 1996). The X-arrestin gene is on Xcen-q22, and consists of 17 exons with a promoter containing a TATA box and elements important for photoreceptor expression, including three CRX and one PCE-1 elements (Sakuma et al. Gene 224:87-95, 1998). We had shown that the upstream 378bp region of the gene is important for expression using a Y79 retinoblastoma assay system (Fujimaki et al. IOVS 40:4964A, 1999). In the present study, we have begun to dissect the promoter at a finer level in order to elucidate the elements responsible for pan-cone-specific expression of X-arrestin. Methods: The PCE-1 and the three CRX promoter elements of the X-arrestin gene were mutagenized as follows: all PCE-1 and CRX elements; PCE-1 only; all CRX's; one of three CRX's(3'). The 378bp promoter region containing the different mutagenized promoter elements were combined with a reporter gene (ß-galactosidase) and assayed in the Y79 system for promoter activity. Results: The 378bp upstream region with intact PCE-1 and CRX sequences showed a promoter activity 10 x greater than the SV40 early promoter-driven positive control. While mutagenesis of both PCE-1 and CRX elements reduced the promoter activity to control level, mutagenesis of the CRX elements alone, even one of three, also eliminated the promoter activity. Mutagenesis of the PCE-1 element reduced the activity to half. Conclusions: The results confirmed the importance of the CRX and PCE-1 elements in the activity of the X-arrestin promoter. The two elements appear to work cooperatively, with the CRX showing a greater control of the promoter activity.
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