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L.C. Macrory, P.N. Bishop; Transcriptional Regulation Of Opticin . Invest. Ophthalmol. Vis. Sci. 2004;45(13):701.
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Purpose: Opticin is a member of the small leucine–rich repeat family of extracellular matrix glycoproteins, and was originally discovered associated with the collagen fibrils of the vitreous. The functions of opticin are unknown but it may be involved in regulating matrix assembly and cellular proliferation within the vitreous cavity. In–situ hybridisation studies in the mouse eye have demonstrated that opticin is highly expressed specifically in the ciliary body region of the optic cup during development and by the non–pigmented ciliary epithelium (NPE) in the adult eye. Dot blot analysis suggested that other tissues including the brain and testis may weakly express opticin. The Purpose of this study was to investigate the unique expression pattern of opticin by defining the opticin promoter elements. Specific objectives included investigating the expression of opticin in tissues other than the eye, defining the 5' end of the gene and defining the minimal promoter(s) using transient transfection with a luciferase reporter. Methods: RT–PCR was performed using total RNA derived from mouse brain and testis to investigate opticin expression in tissues other than the eye. The 5' ends of the gene were mapped using both RT–PCR and 5' RACE. Constructs incorporating regions of the putative opticin promoter were amplified by PCR and inserted into the luciferase vector pGL3–basic and used to co–transfect rabbit NPE– and HeLa cell lines, along with the control vector, pRL–SV40. Dual luciferase assays were perfomed to measure luciferase activities 48 hours following transfection. Results: Sequence analysis of RT–PCR products suggests that opticin is expressed in the mouse brain and testis. Both 5' RACE and RT–PCR suggest the presence of at least two transcriptional start sites, which are separated by 1.5 kb. Dual luciferase assays suggest that constructs incorporating DNA from the most 5' positioned start site produced the highest levels of luciferase activities, however activity in the HeLa cell line was higher than in the rabbit NPE cell line. By comparison, the constructs incorporating regions of DNA surrounding the most 3' positioned start site produced lower levels of activity but in all constructs the activity was approximately two fold higher in the rabbit NPE cell line than in the HeLa cell line. Conclusions: Results suggest that opticin has at least two transcriptional start sites which may be used to express opticin in a tissue specific manner, whereby the most 5' positioned promoter region could be used to ‘drive’ expression of opticin in tissues other than the eye, while the most 3’ positioned start site may preferentially be used to express opticin in the eye.
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