细胞动粒蛋白Hec1启动子的结构及功能分析

发布时间：2018-04-23 13:15

本文选题：Hec + 动粒　；参考：《中国生物化学与分子生物学报》2006年11期

【摘要】：细胞的分裂是一个严格调控,高度有序的过程.为了将复制后的染色体均匀、准确地传递给两个子细胞,细胞在分裂中后期受到纺锤体检验点的严格监控.Hec1定位于动粒,是纺锤体检验点调控的关键蛋白之一,它通过螺旋-螺旋结构域与其他动粒蛋白相互作用调节姐妹染色体的精确分离.为研究Hec1转录水平的调控机理,采用BLAST工具,从GenBank中搜索到了人Hec1基因上游的序列,并利用在线工具http://mbs.cbrc.jp/research/db/TFSEARCH.html提供的转录因子结合位点搜索引擎,对其5′启动子调节区段进行了分析.分析结果表明:在Hec1基因上游-200～-1序列内,存在E2F、ATF4和cAMP应答元件结合蛋白(CREB)等转录因子调控元件.在结构分析的基础上,提取HeLa细胞基因组DNA,用PCR方法克隆了Hec1基因启动子,并构建了多个含启动子不同区段的pGL3荧光素酶报告基因表达质粒.瞬时转染HeLa细胞后的结果表明,-70～-63以及-155～-144之间的启动子区对维持荧光素酶活性最为关键.凝胶迁移实验证明,这两个区段分别能够和转录因子CREB以及ATF4结合.随后,采用野生型的以及含有133位磷酸化位点突变的CREB转染HeLa细胞,通过荧光定量PCR实验发现,Hec1的表达水平分别出现明显上升和下降.该结果表明,Hec1表达的调控是通过CREB的活化来完成的.
[Abstract]:Cell division is a strictly regulated and highly orderly process. In order to transfer the duplicated chromosomes homogeneously and accurately to the two daughter cells, the cells are located in the motilla under the strict monitoring of the spindle test point in the middle and late stage of division, which is one of the key proteins in the regulation of the spindle test point. It regulates the precise separation of sister chromosomes by the interaction of helical-helical domains with other motility proteins. In order to study the regulation mechanism of Hec1 transcription level, the upstream sequence of human Hec1 gene was searched from GenBank by BLAST tool, and the transcription factor binding site search engine provided by http://mbs.cbrc.jp/research/db/TFSEARCH.html was used. The regulation section of its 5 'promoter was analyzed. The results showed that in the upstream sequence of Hec1 gene, there were transcription factor regulatory elements, such as E2FG ATF4 and cAMP response element binding protein (CREB). On the basis of structural analysis, the genomic DNA of HeLa cells was extracted, the promoter of Hec1 gene was cloned by PCR method, and several pGL3 luciferase reporter gene expression plasmids containing different regions of promoter were constructed. The results of transient transfection of HeLa cells showed that the promoter region between -70-63 and -155I-144 was the most important to maintain luciferase activity. Gel migration experiments showed that the two regions could bind to transcription factor CREB and ATF4, respectively. Subsequently, HeLa cells were transfected with wild type CREB containing 133-site phosphorylation site mutation. The expression level of Hec1 was significantly increased and decreased by fluorescence quantitative PCR assay. These results suggest that the regulation of Hec1 expression is mediated by the activation of CREB.
【作者单位】：中国科学技术大学生命科学学院细胞免疫学实验室中国科学技术大学生命科学学院细胞免疫学实验室中国科学技术大学生命科学学院细胞免疫学实验室中国科学技术大学生命科学学院细胞免疫学实验室
【基金】：国家自然科学基金(No.30400078) 国家重点基础研究发展规划(973计划,No.2002CB713700) 安徽省自然科学基金资助课题(No.050430201)~~
【分类号】：Q75

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