人γ-谷氨酰半胱氨酸合成酶催化亚单位基因的一个新转录调控区的实验研究

New transcriptional regulatory region of human γ-glutamylcysteine synthase catalyzed subunit gene：an experimental study

目的 研究人γ-GCS催化亚单位（GCLC）基因转录起始位点上游-810--769 bp调控区的功能及转录调控元件.方法 克隆人GCLC基因调控区片段,构建野生型质粒.缺失突变-770--766间的GATAAG核苷酸及定点突变-765,-764的GC为TA,-755,-754的GG为TA,构建突变型表达质粒.通过质粒转染表达方法研究转录调控功能,EMSA＋super-shift方法鉴定GCLC基因转录起始位点上游-810--769 bp的转录调控元件.结果 人GCLC基因始位点上游-810--769 bp为正性调控区,这一区间有NF-κB、AP-2转录调控元件,转录因子AP-2能抑制转录因子NF-κB对人GCLC基因转录起始位点上游-790--766 DNA序列的结合.结论 人GCLC基因转录调控区转录起始位点上游-810--769 bp为新发现的正性调控区,其内存在NF-κB、AP-2调控元件.

Objective To determine the function and transcriptional regulatory elements of the regulatory region of-810--769 bp upstream from the transcription initiation site of the human glutamate-cysteine ligase catalytic subunit （GCLC） gene. Methods The human GCLC gene regulatory region fragments were cloned and the wild-type plasmids were constructed. The mutant expression plasmids of deletion mutant-770--766 GATAAG nucleotides and the site-directed mutant-765 and-764 GC to TA,-755 and-754 GG to TA were constructed. The transcriptional regulatory was determined by plasmid transfection. EMSA＋super-shift method was used to identify transcriptional regulatory elements of-810--769 bp upstream from the transcription initiation site of GCLC gene. Results The-810--769 bp upstream of the transcription initiation site of human GCLC gene is the positive regulatory region,including NF-κB and AP-2 transcriptional elements. The transcription factor AP-2 could inhibit the binding of transcription factor NF-κB to the upstream-790--766 DNA sequence of human GCLC gene transcription initiation site. Conclusion The regulatory region of-810--769 bp upstream from the transcription initiation site of the human GCLC gene is a newly discovered positive regulatory region,which contains NF-κB and AP-2 regulatory elements.