鸡局部黏着斑激酶基因5′UTR及启动子的克隆鉴定

Cloning and characterization of 5′UTR and promoter region of chick focal adhesion kinase gene

为了研究鸡局部黏着斑激酶(FAK)基因表达调控的分子机制,运用cDNA末端快速扩增技术确定了鸡胚成纤维细胞FAK基因的转录起始位点,并发现FAK基因mRNA的5′非翻译区(5′UTR)存在4种剪切形式。分析这4种剪切形式的5′端序列,发现它们都不影响蛋白的编码,但可能影响mRNA的翻译效率。另外,将启动子序列935 bp片段克隆构建到荧光素酶报告基因表达载体pGL3-Basic中,转染鸡胚成纤维细胞,测定启动子的转录活性。通过启动子序列系列缩短的克隆,将核心启动子区定位在转录起始位点上游-662至+7的669 bp片段内,该启动子的典型特征是:没有TATA盒,而富含"GC"碱基。以上结果为FAK基因的表达调控研究提供了分子基础。

To understand the mechanism of chick focal adhesion kinase gene （FAK） expression and regulation, the transcription start sites and four kinds alternatively spliced 5＇end （5＇untranslated region, 5＇UTR） of FAK mRNA in chick embryo fibroblast cells were identified by RACE. The analysis of the 5 ＇end mRNA sequences showed that they all would not affect the encoded protein sequence, they may influence the mRNA translation efficiency. The 935 bp fragment with FAK promoter was also cloned and placed upstream of the lueiferase reporter gene in pGL3-Basie vector and transfected into chick embryo fibroblast cells. Serial deletion constrncts revealed that a 669 bp region （ - 662 to ＋ 7 ） was required for the maximal FAK promoter activity. The promoter sequence showed high ＂GC＂ content and lacked TATA box. These results will provide a molecular basis for understanding the mechanism of FAK gene expression.