Functional analysis of the human recoverin gene promoter

Abstract

Purpose. Following photoactivation of rhodopsin, recoverin inhibits rhodopsin kinase activity in retinal photoreceptors by reducing the binding of arrestin to rhodopsin and therefore prolonging the termination of the phototransduction cascade. To identify potential cis -elements that may be involved in understanding the mechanisms that determine the cellspecific expression of recoverin in retinal and cancerous cells, the promoter region of the human recoverin gene was studied in cultured human Y79 retinoblastoma cells. Methods. A 2.5 kb EcoRI fragment of a 9.4 kb cosmid that contains the 5' non-coding region of the human recoverin gene was sequenced and cloned into an expression vector upstream of a luciferase gene. Cultured Y79 human retinoblastoma cells were transfected with DNA constructs using lipofection. Deletion mutants were generated by site-directed mutagenesis, cloned into the expression vector, and transfected into Y79 cells. Reporter gene activity was measured with a luciferase assay, and normalized to ß-beta-galactosidase activity resulting from a co-transfected SV-ß-galactosidase SV40 vector. Results. Reporter gene expression in the transfected Y79 cells demonstrated an increase in activity between 232 and 620 bp from the translational start site of the recoverin gene. There was a decrease in the reporter gene expression between 900 and 1200 bp from the start site, followed by an increase between 1200 and 1440 bp. Conclusions. This analysis suggests that there are cis -acting elements in the 5' non-coding region of the recoverin gene that are involved in the activation and suppression of gene expression.