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MR Verardo, NI Piriev, BE Knox, DB Farber; Two Sequences in the 3'UTR of ß-PDE Control Gene Expression in Human Cells and Xenopus Laevis . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3654.
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Purpose: Previously, we reported that in Y-79 retinoblastoma cells two 100 nt fragments from the 3'untranslated region (3'UTR) of rod cGMP-phosphodiesterase ß-subunit (ß-PDE) enhance luciferase reporter gene expression above that produced by the complete 3'UTRs of ß-PDE, SV-40, and other photoreceptor specific genes. The purpose of our current work was to determine if this effect was specific to Y-79 cells and the SV-40 promoter that was used to drive luciferase expression of the constructs. Further, we wanted to find out if the effect of these 3'UTR fragments on gene expression was conserved across species. Methods: The two 100 nt fragments from the 3'UTR of ß-PDE were cloned downstream of the luciferase gene in a modified pGL3 vector under control of the ß-PDE promoter. These constructs were transfected ex-vivo in Xenopus laevis tadpole heads and also in Y-79 cells. HEK 293, HeLa, and SY5Y cells were transfected with the SV-40 promoter constructs. Luciferase assays were performed on harvested cell and tissue lysates. Results: With constructs driven by the SV-40 promoter, the entire 3'UTR of ß-PDE decreased luciferase expression in all cell lines tested, but both 100 nt fragments increased it in HEK293 and had no effect in HeLa cells. Only one 3'UTR fragment increased luciferase expression in SY5Y cells. Interestingly, constructs driven by the ß-PDE promoter transfected ex-vivo in Xenopus heads reproduced the observations obtained in vitro with Y-79 retinoblastoma cells. Conclusion: Although the complete 3'UTR of ß-PDE decreased luciferase expression, the two 100 nt fragments increased it differently, depending on the cell lines used for transfection. This implies cell specific mechanisms may control expression by elements of the ß-PDE 3'UTR. Our results also show that the effect of the 3'UTR fragments is conserved from human to amphibians, suggesting that these sequences may have an important role in gene regulation in vertebrate retinas.
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