Araki E, Murakami T, Shirotani T, Kanai F, Shinohara Y, Shimada F, Mori M, Shichiri M, Ebina Y
Department of Metabolic Medicine, Kumamoto University Medical School, Japan.
J Biol Chem. 1991 Feb 25;266(6):3944-8.
Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.
采用改良的低背景氯霉素乙酰转移酶载体系统pSVOOCAT（荒木英树、岛田晃、志千里、森正、海老名阳，《核酸研究》16卷，1627页，1988年）转染细胞系后，在瞬时表达试验中对人胰岛素受体基因5'-侧翼序列片段进行了分析。将含有胰岛素受体基因启动子各种缺失和插入的嵌合氯霉素乙酰转移酶质粒转染到CHO和COS细胞中，结果表明，-629至-1（起始密码子ATG为+1）区域足以实现最大启动子活性。通过低背景pSVOOCAT载体系统在体内检测发现，四个G-C盒簇（-593至-618）的DNA元件增强了转录。使用部分纯化的LacZ-Sp1杂交蛋白进行的DNase I足迹实验和凝胶阻滞实验表明，转录因子Sp1能够结合到启动子的四个G-C盒簇上。因此，人胰岛素受体基因的高效表达可能需要转录因子Sp1结合到位于ATG翻译起始密码子上游-593至-618碱基对处的四个G-C盒上。
