Abstract
alpha A-crystallin is an abundant soluble protein of the vertebrate eye lens. In addition to the TATA box, four positive cis-regulatory elements of the chicken alpha A-crystallin gene have been identified by linker scanning mutagenesis, DNase I footprinting, and gel mobility shift experiments. The regulatory elements described here have been named DE2A (at positions -144 to -134), DE2B (at positions -128 to -118), and DE1A (at positions -114 to -103). DE2A and DE2B form a dyad of symmetry between positions -141 and -118 (5'-AGACTGTCAT....AGGTCAGTCT-3'), consistent with the close similarity in the mobility of complexes formed with lens nuclear proteins by these two elements. Mutations in DE2A, DE2B, and DE1A leading to loss of promoter activity using the bacterial chloramphenicol acetyltransferase reporter gene transfected into primary embryonic chicken lens epithelial cells resulted in a corresponding loss in the ability to compete for complex formation with lens nuclear proteins in gel mobility shift assays. Mutation of the alpha A-CRYBP1-like site (-67/-57), necessary for function of the mouse alpha A-crystallin promoter, did not affect the activity of the chicken promoter. The DNase I footprinting and gel mobility shift experiments confirmed the previously noted binding of nuclear proteins to a dyad of symmetry at positions -153 to -140. In contrast to DE2A, DE2B, and DE1A, mutagenesis and gel mobility shift experiments failed to correlate function and protein binding for the -153/-140 dyad. DE2A, DE2B, and DE1A agree well with the regulatory elements alpha CE1 (-162/-134), alpha CE3 (-135/-121), and alpha CE2 (-119/-99) (Matsuo, I., and Yasuda, K. (1992) Nucleic Acids Res. 20, 3701-3712) for this gene. The present results suggest, however, that the lens enhancer activity of alpha CE1 is due to the sequence -141/-134, which forms the upper half of the DE2A/DE2B dyad of symmetry, rather than the -153/-140 dyad as previously suspected.
Highlights
AA-crystallin is an abundant soluble protein of the dance and characteristic pattern of expression during lens vertebrate eye lens
The DNase I foot- We have been investigating the molecular basis for lens printing andgel mobility shift experimentsconfirmed expression of the murine andchicken aA-crystallin gene (see the previously noted binding of nuclear proteins to a Piatigorsky (1989,1992)).The murine aA-crystallin promoter dyad of symmetry at positions -153 to -140
DNase I Footprinting-The -242/+77 chicken aA-crystallin promoter probe was examined by in uitro DNase I footprinting using both crude and partially purified nuclear extracts prepared from 13-15-day-old embryonic lenses
Summary
DNase I Footprinting-The -242/+77 chicken aA-crystallin promoter probe was examined by in uitro DNase I footprinting using both crude and partially purified nuclear extracts prepared from 13-15-day-old embryonic lenses. Adistinct footprint was found between positions -30 and -27 on the upper strand andbetween positions -32 and -22 on the lower strand;this region includes the TATA box (reviewed by Sawadogo and Sentenac(1990) and Pugh and Tjian (1992)). The data collected from the transfection experiments and footprints aresummarized in Fig. 3.Three of four functionally important elements (namely DEZA, DE2B, and the TATA box) were identified as potential protein binding sites using the DNase Ifootprinting assay. TheDElAsite was not footprinted.Proteinbinding sites were identified a t -loo/-93 (called DE1B) and -67/-57 Electrophoretic Mobility Shift Assays-Electrophoretic mo- We examined oligonucleotide -142/-127, which conbility shift assays were performed using radioactively labeled tains theDE2A element and a few nucleotides from DE3
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