Mangé A, Julien E, Prudhomme J C, Couble P
Université Claude Bernard Lyon I, Centre de Génétique Moléculaire et Cellulaire, CNRS-UMR 5534 43, Villeurbanne, France.
J Mol Biol. 1997 Jan 24;265(3):266-74. doi: 10.1006/jmbi.1996.0734.
To identify the functional regulatory elements of the promoter of the cytoplasmic actin A3 gene in Bombyx mori, transient expression of A3-LacZ mutants was assayed in cultured Lepidoptera cells. This led to the recognition of two proximal and contiguous domains exerting strong negative and positive effects, respectively on promoter activity. The negative region contains a ten-base-pair sequence that binds Bombyx silk gland cell nuclear proteins in vitro. The positive regulatory element was identified as a serum response element (SRE) by its sequence, and its in vitro binding properties. Moreover, structural analysis of posterior and median silk gland cell chromatin by dimethyl sulfate-aided LMPCR revealed that SRE is bound to its cognate factor in situ, in most, if not all, the approximately 100,000 A3 copies of the polyploid DNA stock. The regulation of the A3 promoter in the silk gland would thus result from the combined action of these two antagonist factors.
为了鉴定家蚕细胞质肌动蛋白A3基因启动子的功能调控元件，我们在培养的鳞翅目细胞中检测了A3-LacZ突变体的瞬时表达。这使得我们识别出两个相邻的近端结构域，它们分别对启动子活性产生强烈的负向和正向影响。负向区域包含一个十碱基对序列，该序列在体外能与家蚕丝腺细胞核蛋白结合。通过其序列和体外结合特性，正向调控元件被鉴定为血清反应元件（SRE）。此外，硫酸二甲酯辅助的LMPCR对后部和中部丝腺细胞染色质的结构分析表明，在多倍体DNA库中大约100,000个A3拷贝中，即使不是全部，大多数情况下SRE在原位都与其同源因子结合。因此，丝腺中A3启动子的调控将源于这两种拮抗因子的共同作用。
