人细胞周期相关激酶(CCRK)启动子的分析和特征研究  

Human cell cycle-related kinase(CCRK) promoter:analysis and characterization

作　　者：贾向志[1] 林李家宓[2] 何明亮[2] 马文煜[1] 黄培堂[3] 孔祥复[2] 黄翠芬[3] 

机构地区：[1]第四军医大学基础医学部微生物学教研室,西安710032 [2]香港大学分子生物学研究所 [3]军事医学科学院生物工程研究所,北京100850

出　　处：《军事医学科学院院刊》2004年第5期437-441,共5页Bulletin of the Academy of Military Medical Sciences

摘　　要：目的 :分析人CCRK基因启动子、启动子的核心序列及与其表达相关的转录因子。方法 :采用 5′ RACE技术鉴定人CCRK基因的转录起始点 ;对启动子区 3′端 3个截短片段 2 5 5bp(- 36 7/- 12 8) ,2 10bp(- 32 2 /- 12 8)和16 0bp(- 2 72 /- 12 8)进行删除分析 ,双荧光素酶分析测出的最小活性片段作为核心启动子的上游位点。通过生物软件MatInspectorV2 .2分析核心启动子区域 ,寻找并推测重要的转录因子 ,并对其核心序列进行定点突变分析 ,再通过电泳迁移率 (EMSA)实验加以鉴定。结果 :采用 5′ RACE技术 ,得到的PCR产物直接测序 ,定位 - 2 4 2“T”为转录起始点 ;通过启动子 3′端小片段删除分析 ,2 5 5 ,2 10和 16 0bp均无活性 ,由此判断 3′端 2 5 5个核苷酸不存在核心启动子序列 ,并定位一段 74bp的区域为核心启动子 (- 4 4 1/- 36 7)区域。通过MatInspector软件分析 ,发现在这段核心启动子区域内有 3个重要的结合位点 :DeltaEF 1,NF κB和Sp1。对这 3个结合位点的核心序列进行定点突变后瞬时转染U373,经双荧光素酶分析发现DeltaEF 1的活性明显升高 ,NF κB没有变化 ,Sp1的活性降低但不太明显。进一步通过电泳迁移率 (EMSA)实验鉴定 ,DeltaEF 1和NF κB能与U373核蛋白形成特异性的DNA 蛋白质复合体 。Objective: To analyze promoter, core promoter and tr anscriptional factor of human cell cycle-related kinase(CCRK). Methods: In order to identify the transcriptional start sites of the human CCRK gene, the classical 5′-RACE technique was applied. The upstream site of core promoter was located, and the s ub-deletion analysis of 255 bp,-210 bp, -160 bp was performed. Some very important binding sites within this region were sought by the bioinformatics software analysis. The core sequences of the three elements were analyzed for their mutation and confirmd by the gel-shift. Results: The product of PCR that measures the sequence directly, located position -242 “T” as the transcriptional start site of the human CCRK gene. By sub-deletion, a 74 bp was located as the c ore promoter (-441/-366) region. Three important factors, namely Delta EF-1, NF-κB and SP1 1, were found. By dual-luciferase assay the Delta EF-1 activ ity was found increased obviously after mutagenesis, the NF-κB had no change and the activity of the SP 1 decreased obviously. Two specific complexes were formed with both Delta EF-1 and NF-κB elements. Conclusion: The results are the basis for further study of the transcriptional regulation mechanisms of human CCRK in order to develop a new thinking for glioblastoma research.

关 键 词：神经胶质瘤 细胞周期相关激酶 启动子 定点突变 转录因子 

分 类 号：R346[医药卫生—基础医学]