apoAⅠ启动子-93 bp～-63 bp片段及-75 bp位点突变对基因转录调控作用的研究

The Role of Oligonucleotides -93～-63 bp of ApoA-Ⅰ Promoter and -75 Mutant on Gene Transcription Regulation

目的:研究载脂蛋白AⅠ(apoAⅠ)启动子区域-93bp～-63bp对基因转录活性的影响,探讨-75bp位点G→A置换在基因转录中的作用。方法:在人类基因组数据库中截取并下载apoAⅠ基因启动子序列,对照基因图谱人工合成apoAⅠ启动子区域-93bp～-63bp片段,-75bp位点分别为突变型(A)或野生型(G),进行生物素标记。培养HepG2细胞,提取核蛋白混合物。将核蛋白与标记好的两条DNA链结合,进行电泳迁移率(EMSA)实验,观察两者结合情况。结果:apoAⅠ启动子-93bp～-63bp区域可与某种特异性核蛋白转录因子结合。-75bp位点为G等位基因的探针与含A的探针相比,核蛋白的结合明显增多。同时,竞争抑制实验表明,含G的探针与核蛋白的亲和力高于含A的探针。结论:apoAⅠ基因启动子-93bp～-63bp区域通过与核蛋白转录因子的结合参与基因调控,-75bp位点G→A置换降低与转录因子的亲和力,降低apoAI转录活性从而影响基因表达。

Objective： To study the effects of oligonucleotides -93 bp--63 bp of apoA- Ⅰ promoter and -75 bp mutant on gene transcription regulation, and to explore the effects of nuclear proteins that bind to the -75 region of apoA I promoter on gene transcription. Methods： Nuclear proteins were prepared from HepG2 cells and challenged with two labeled oligonucleotides corresponding to the region -93 bp and -63 bp from the transcription start site of apoA Ⅰ gene in the A or G configuration. Results： DNA-protein complexes were formed by two DNA oligonucleotides with nuclear proteins from HepG2 cells. Three retarded bands were visible. Competition with the homologous oligonucleotide efficiently reduced the intensity of such complexes. When oligonucleotide A was used as competitor versus labeled oligonucleotide G, unlabeled oligonucleotide G competed more efficiently than oligonucleotide A. Conclusion： The nuclear factor could mediate the activation of transcription of promoters observed in the experiment. The G→A transition decreases its binding affinity to the -75 position and represses the apoAI gene transcription.