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C. Zeitz, R. Minotti, S. Feil, W. Berger; Identification of the Promoter of the NYX Gene . Invest. Ophthalmol. Vis. Sci. 2004;45(13):672.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Identification of DNA sequences involved in the transcriptional regulation of the human and mouse NYX gene. Mutations in the NYX gene result in X–linked congenital stationary night blindness in human and mice. A common clinical feature in patients with mutations in NYX is the absence of oscillatory potentials (OPs) derived from electrophysiological tests. RNA in situ studies showed that NYX is expressed in the inner retinal cell layer in mouse and rat but not in photoreceptor cells. Initially, we began the analysis of the transcriptional regulation by identifying promoter sequences, since this information could aid in cell type–specific gene therapy approaches. Methods: Comparative sequence analysis was applied to the genomic DNA sequences of the human and mouse NYX gene to identify evolutionary conserved elements. Additionally, computer–based prediction programs have been utilized to identify a putative promoter, CpG–islands and potential binding sites for transcription factors. Corresponding DNA fragments of both, the human and mouse genomic sequences of NYX were cloned in pGL3 luciferase reporter vectors and tested for the ability to drive transcription by transient transfection studies in HeLa– and NIH 3T3 mouse fibroblast cells. Results: Comparative sequence analysis of the genomic sequence of the human and mouse NYX genes identified highly conserved regions in intron 2 and in the 5'– and 3'–UTR. About 5 kb upstream of exon 1 of the human NYX gene, an evolutionary conserved region, a CpG–island and a promoter region was predicted. The 1 kb mouse sequence containing these elements gave rise to a 3–9 fold increase of luciferase activity in different cell lines. The strongest activity was measured in NIH 3T3 mouse fibroblast cells. Deletionconstructs containing 556 and 551 bps of the mouse sequence demonstrated significant reduction in reporter gene activity, probably due to the lack of positive regulatory elements in the deleted fragments. No luciferase activity could be measured with the mouse construct containing only 375 bp of the mouse sequence. The 1 kb human putative promoter sequence cloned in the promoter– and enhancerless pGL3–Basic vector did not show regulatory activity. Conclusions: Positive regulatory elements are present approximately 4 kb upstream of exon 1 of the mouse Nyx gene. These appear to be species dependent since they function significantly stronger in mouse cell lines compared to human.
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