人FAM33A基因启动子的克隆与鉴定  

Cloning and Identification of Human FAM33A Promoter

作　　者：杜刚[1,2] 卜友泉[1,2] 杨正梅[3] 兰欢[1,2] 崔涛[1,2] 镇磊[1,2] 刘革力[1] 宋方洲[1,2] 

机构地区：[1]重庆医科大学分子医学与肿瘤研究中心,重庆市400016 [2]重庆医科大学生物化学与分子生物学教研室,重庆市400016 [3]重庆医科大学实验动物中心,重庆市400016

出　　处：《医学分子生物学杂志》2009年第3期219-224,共6页Journal of Medical Molecular Biology

基　　金：国家自然科学基金（No.30801356、30871237、30872758）,重庆市科委自然科学基金（No.2007BB5302）

摘　　要：目的鉴定人FAM33A基因的启动子，为进一步研究其转录调控机制奠定基础。方法采用5’RACE技术（5’端cDNA快速扩增）鉴定FAM33A的转录起始位点。采用PCR定向克隆、酶切亚克隆等策略，构建FAM33A启动子荧光素酶报告基因。采用Lipofeetamine^TM2000转染H1299细胞，并通过Dual-Lu-ciferase@ Reporter Assay System进行荧光素酶报告基因活性检测。结果确定了FAM33A的转录起始位点，构建了覆盖FAM33A 5’端ATG附近约2kb区域的一系列FAM33A启动子荧光素酶报告基因。启动子活性分析表明，这些重组体均具有较高的启动子活性，同时含有典型的GC盒以及Sp1、E2F和GATA-1等潜在的转录因子结合位点。结论FAM33A启动子区域主要定位于转录起始位点附近约590bp的区域内。Objective To identify the promoter region of human FAM33A gene for further investigation of its transcriptional regulatory mechanism. Methods 5＇-RACE was used to identify the transcriptional start sites for FAM33A gene. PCR method and DNA blunting method were used to construct FAM33A promoter reporters. Cells were transiently transfected by Lipofectamine^TM 2000. Promoter activity was measured by Dual Luciferase Reporter Assay. Results The transcriptional start sites for FAM33A gene were identified by 5＇-RACE. Several overlapping genomic fragments from the 5＇-flanking region of FAM33A gene were cloned into pGL3-basic vector to construct FAM33A promoter reporters. Luciferase reporter assay indicated that all these FAM33A promoter reporters have relatively high activity. FAM33A promoter contains GC box, as well as other putative transcriptional factor binding sites such as Spl, E2F, and GATA-1. Conclusion FAM33A promoter region is mainly located in a 590-bp region upstream transcriptional start site.

关 键 词：FAM33A基因 启动子 转录调控 细胞周期 

分 类 号：Q784[生物学—分子生物学]