Localization of upstream silencer elements involved in the expression of cone transducin alpha-subunit (GNAT2).
OBJECTIVE
To localize cis-acting elements involved in the expression of the cone-specific G-protein, cone transducin alpha-subunit (GNAT2).


METHODS
In this study, the authors used a genomic clone, HGLG3, to sequence 3139 base pairs of the upstream region of the GNAT2 gene and to localize cis-acting elements involved in the expression of GNAT2. Upstream elements were localized functionally by transfection of chloramphenicol acetyltransferase gene constructs containing nested deletions of this upstream region into WERI-Rb1 cells. Cell specificity of the localized elements was determined by transfection of the HeLa cells. Trans-acting factor-binding sites to functional cis-acting elements were determined by DNasel footprinting. Cell specificity of protein interaction with footprinted regions was tested by electrophoretic mobility shifts with nuclear extracts from WERI-Rb1 and HeLa cells.


RESULTS
Transfection 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-specific element between +143 and +167. DNaseI footprinting showed three major footprints (S1, S2, and S3) between -807 and -176, indicating the binding sites for putative negative trans-acting factors. Individual footprinted sequences had similar electrophoretic mobility shifts when they 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 strong silencer region, a weak upstream cell-specific promoter, and a strong downstream element. The silencer region interacts with similar proteins from retina- and nonretina-derived cell lines.
Morris
T A
TA
Department of Ophthalmology, Indiana University, Indianapolis 46202, USA.
Fong
W B
WB
Ward
M J
MJ
Hu
H
H
Fong
S L
SL
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005134:Eye Neoplasms / Q000378:metabolism; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D012175:Retinoblastoma / Q000378:metabolism; D015293:Transducin / Q000096:biosynthesis / Q000235:genetics*; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured
D005136:Eye Proteins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D015293:Transducin