Alpha 1抗胰蛋白酶核基质附着区增强RNA聚合酶Ⅱ依赖的转录  

Alpha 1-antitrypsin Matrix Attachment Region Enhances RNA PolymeraseⅡ-dependent Transcription

作　　者：李志艳[1] 张涌[1] 

机构地区：[1]西北农林科技大学生物工程研究所,杨陵712100

出　　处：《中国生物工程杂志》2007年第4期39-43,共5页China Biotechnology

基　　金：国家"863"计划资助项目(2004AA213072).

摘　　要：核基质附着区是一种真核生物的DNA元件,能调控染色体结构和活性。为研究体内MAR相关的染色质准入和转录调控,从人基因组中克隆了Alpha1抗胰蛋白酶核基质附着区(α1-ATMAR)并将其连入pEGFP-C1载体。分别以空载体和携带MAR的载体通过脂质体法转染人胚肾293细胞系。经G418筛选20天的细胞池用作实验分析。半定量RT-PCR及荧光显微镜观察显示此MAR能够增强临近基因的表达。进一步用染色质免疫共沉淀(ChIP)共定位CMV启动子和RNA聚合酶Ⅱ(RNAPⅡ),PCR结果显示存在MAR元件时,更多的RNA聚合酶Ⅱ被富集到启动子区。ChIP方法可用于证实MAR介导的转录激活,在实时检测方面比RT-PCR提供了更多动力学信息。这项技术为进一步研究基因表达调控提供了技术平台。Eukaryotic DNA element called Matrix Attachment Regions （MARs） can function on regulating the structure and activity of chromosome. Traditional quantitation in vitro and indirect functional analysis can not always reflect MAR-involved physiological state. In order to study transcription regulation, and make a try in methodism,or 1-antitrypsin MAR （α1-AT MAR） is cloned and incorporated into pEGFP-C1 vector. Non-MARcontaining and MAR-containing plasmids were then transfected into HEK-293 cells with LipofectamineTM 2000 respectively. Positive cell clones were assayed after 20 days of selection by G418. Semiquantitative RT-PCR and fluorescence microscope analysis show that this MAR has a positive effect on modulating nearby gene expression. Further, co-localization with newly CMV promoter and RNA polymerase Ⅱ （RNAP Ⅱ ） was detected by chromatin immunoprecipitafion （CHIP） , The PCR result demonstrates that more RNAP Ⅱ was recruited to the CMV promoter to initiate transcription in presence of MAR. ChIP can be used to confirm the MAR-mediated transcriptional activation and provide more reliable information than RT-PCR in real time. The technology is also providing a platform for our research in gene expression regulation.

关 键 词：核基质附着区 染色质免疫共沉淀 转录调控 RNA聚合酶Ⅱ 

分 类 号：Q814[生物学—生物工程]