LOCALIZATION OF UPSTREAM SILENCER ELEMENTS INVOLVED IN THE EXPRESSIONOF CONE TRANSDUCIN ALPHA-SUBUNIT (GNAT2)

Purpose. To localize cis-acting elements involved in the expression of the cone-specific G-protein, cone transducin alpha-subunit (GNAT2). M ethods. In this study, the authors used a genomic clone, HGLG3, to seq uence 3139 base pairs of the upstream region of the GNAT2 gene and to localize cis-acting elements involved in the expression of GNAT2. Upst ream elements were localized functionally by transfection of chloramph enicol acetyltransferase gene constructs containing nested deletions o f this upstream region into WERI-RbI cells. Cell specificity of the lo calized elements was determined by transfection of the HeLa cells. Tra ns-acting factor-binding sites to functional cis-acting elements were determined br DNaseI footprinting. Cell specificity of protein interac tion with footprinted regions was tested by electrophoretic mobility s hifts with nuclear extracts from WERI-Rb1 and HeLa cells. Results. Tra nsfection of WERI-Rb1 and HeLa cells revealed the presence of a strong , noncell-specific silencer region between -1130 and -23, a weak, cell -specific promoter between -151 and -10, and a stronger, noncell-speci fic element between +143 and +167. DNaseI footprinting showed three ma jor footprints (S1, S2, and S3) between -807 and -176, indicating the binding sites for putative negative transacting factors. Individual fo otprinted sequences had similar electrophoretic mobility shifts when t hey were incubated with nuclear extracts from either WERI-Rb1 or HeLa cells, suggesting that these cells express the same negative factors. Conclusions. The expression of the GNAT2 gene is controlled by a stron g silencer region, a weak upstream cell-specific promoter, and a stron g downstream element. The silencer region interacts with similar prote ins from retina- and nonretina-derived cell lines.