人A4GNT基因5'调控区的结构和功能分析  

Characterization of the 5'-Flanking Region of Human α-N-acetylglucosaminyltransferase Gene

作　　者：张牧霞[1] 张瑶[1] 赵萌[1] 郭晓华[1] 谷玮玮[1] 

机构地区：[1]河北医科大学第三医院实验中心,石家庄050051

出　　处：《中国生物化学与分子生物学报》2010年第10期955-961,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：河北省自然科学基金项目(No.C2006000917);河北省卫生厅科研基金项目(No.08-144)~~

摘　　要：为探索人α1,4-N-乙酰葡糖胺转移酶(α1,4-N-acetylglucosaminyltransferase,A4GNT)基因表达的调控机制,应用5'cDNA末端快速扩增法和引物延伸法确定了A4GNT基因的转录起始位点.在生物信息学分析的基础上,构建了系列5'缺失荧光素酶报告基因载体和定点突变载体.瞬时转染胃癌细胞MKN45和AGS.荧光素酶活性分析表明,A4GNT基因转录的核心启动子在-141bp～+116bp区域,该区域缺乏典型的TATA盒,但含有CCAAT盒、Sp1和ETS-1等转录因子潜在结合位点.突变分析显示,-136bp～-131bp的Sp1结合位点及-93bp～-89bp正向CCAAT序列对A4GNT启动子转录激活至关重要.电泳迁移率变动分析表明,这两个顺式作用元件能够与转录因子Sp1和NF-Y结合.另外,在-1464bp～-771bp区域可能含有与基因的特异性表达相关的调控元件.To investigate the regulation of human α1,4-N-acetylglucosaminyltransferase （A4GNT） gene transcription,5＇ RACE （rapid amplification of cDNA ends） and primer extension assays were deployed to obtain the A4GNT transcription start site.A series of deletions and site-directed mutantions were constructed into the luciferase reporters based on bioinformatics analyses,and then transiently transfected into gastric cancer cells MKN45 and AGS.The luciferase assays showed that the core promoter of A4GNT gene was from-141～＋ 116 bp,which contained multiple CCAAT boxes and putative binding sites for transcription factor Sp1 and ETS-1,but lacked the consensus TATA box.The Sp1 binding site（-136～-131 bp） and the forward CCAAT （-93～-89 bp） appeared to be critical for the transcriptional activity of A4GNT as shown by mutation analyses.Electrophoretic mobility shift assays （EMSA） have demonstrated the specific binding of Sp1 and NF-Y to these elements.Our additional data also suggested that the region of -1 464～-771 bp might be responsible for the cell-specific gene expression of A4GNT.

关 键 词：α1 4-N-乙酰葡糖胺转移酶 启动子 荧光素酶报告基因 电泳迁移率变动分析 转录因子 

分 类 号：Q78[生物学—分子生物学]