利用同源序列确定人GIPC2核心启动子和寻找可能的顺式作用元件

Identification of human GIPC2 core promoter and potential cis-acting element using sequence homology approach

目的利用同源序列确定人GIPC2核心启动子区和寻找可能存在的基因调控区域。方法 1)利用Vista比对不同物种GIPC2基因编码区和非编码区序列,得到GIPC2同源序列;2)分别构建插入人和大鼠GIPC2翻译起始位点ATG上游不同长度截短片段的重组质粒,转染人HEK293T细胞和大鼠PC12细胞;3)根据报告基因荧光强度推断人和大鼠GIPC2的核心启动子区;4)将人GIPC2内含子区同源序列置于核心启动子前面,构建重组质粒,转染HEK293T,HT-29和PC12细胞,检测启动子活性。结果人GIPC2核心启动子区确定为+32至+190序列区域;人和大鼠GIPC2 ATG上游一段同源序列有抑制GIPC2启动子活性的功能;人和大鼠GIPC2第一个内含子内的同源序列有促进启动子活性的功能。结论利用同源序列方法确定了人GIPC2基因的核心启动子区,并且找到能抑制和促进GIPC2启动子活性的可能的顺式作用元件。

Objective To identify human GIPC2 core promoter and potential cis-acting element using sequence homology. Methods Homologous sequence of GIPC2 was identified by comparing GIPC2 coding and non-coding sequence from different species in Vista database. A series of luciferase constructs comprising human or rat GIPC2 sequences truncated at various lengths upstream of translation initiation site were tested in HEK293T and PC12 to confirm the core promoter sequence. Conserved intronic GIPC2 sequences were inserted upstream of human core promoter to test for potential cis-acting regulatory activities. Results The human GIPC2 core promoter sequence spans from ＋32 to ＋ 190 upstream of translation initiation site. A homologous sequence 1500bp upstream of human GIPC2 translation initiation sites represses GIPC2 promoter activity and a homologous sequence from the first intron promotes GIPC2 promoter activity. Conclusions We have identified human GIPC2 core promoter and potential cisacting element using sequence homology.