人热休克蛋白90β（hsp90β）的转录调控
摘要
热休克蛋白90(HSP90)细胞生长、发育、分化及应急条件下对细胞的保护等生理过程中起着重要的作用。研究它的表达调控机制不仅有助于对其生理功能的进一步了解，也可为真核基因诱导表达调控机制的研究建立一个良好的模型。我们以前的研究表明人热休克蛋白90β基因受热和丝裂原的双重诱导，其表达调控主要表现在转录水平，并且对两种诱导因素表现出不同的表达动力学形式。为了深入研究hsp90β的转录调控机制，我们通过PCR分别扩增并克隆了人hsp90β上游(-1102／+68bp)和第一外显子、内含子(-4／+1530bp)片段。将它们进行删切后在荧光酶(Luc)和氯霉素乙酰基转移酶(CAT)报告基因真核表达载体的上游构建了18个亚克隆并分别转染Jurkat细胞。报告基因的酶活性检测、电泳迁移率分析及Jurkat细胞总RNA的引物延伸分析共同表明：
1．位于hsp90β上游-88／-20bp的启动子序列可以单独介导较高水平的基础转录，其转录水平明显高于相似转染条件下的其它一些启动子如IL-2、IL-2Rα、tk等，这使细胞在正常生理条件下可以维持较高水平的HSP90β的浓度，保障细胞正常生理、生化反应的进行。另外，hsp90β第一内含子的3′端存在一个启动子类似序列，它单独存在时的基础转录效率虽然远低于上游的启动子，却明显高于tk启动子，在上游启动子的协助下，它的基础转录效率可以有所升高，是hsp90β基础表达的一个组成部。热休克时它的转录效率可以被其上游的HSE提高。
Heat shock protein 90 ( HSP90 ) plays an important role in cell growth, development and differentiation, as well as protections of cells from verious kind of indury when they were exposed to stress conditions. The study of its regulation mechnism will not only lead to a better understaning of its biological functions, but also can establish a good model for regulation mechnism of induced eukaryotic gene expression.
Our previous work show the expression of human heat shock protein 90 genes( hsp90α and β ) can be induced by the dual induction of heat shock and PHA activition. Their regulation depends mainly on transcription, and shows different expression kinetics upon the two inducer. In order to study the regulation mechnism of induced expression of human hsp90P, we amplified its upstream sequence (-1102/+68bp), the first exon and intron (-4/+1503bp) by polymerase chain reaction. Truncated fragments of the amplified sequence were then subcloned into the 5' of eukaryotic expression vectors with luciferase or chloramphenicol acetyltransferase (CAT) gene as reporter genes. Reporter gene activity assay in transfected Jurkat cells, mobility shift assay and primer extension experiments show:
1．位于hsp90β上游-88／-20bp的启动子序列可以单独介导较高水平的基础转录，其转录水平明显高于相似转染条件下的其它一些启动子如IL-2、IL-2Rα、tk等，这使细胞在正常生理条件下可以维持较高水平的HSP90β的浓度，保障细胞正常生理、生化反应的进行。另外，hsp90β第一内含子的3′端存在一个启动子类似序列，它单独存在时的基础转录效率虽然远低于上游的启动子，却明显高于tk启动子，在上游启动子的协助下，它的基础转录效率可以有所升高，是hsp90β基础表达的一个组成部。热休克时它的转录效率可以被其上游的HSE提高。
Heat shock protein 90 ( HSP90 ) plays an important role in cell growth, development and differentiation, as well as protections of cells from verious kind of indury when they were exposed to stress conditions. The study of its regulation mechnism will not only lead to a better understaning of its biological functions, but also can establish a good model for regulation mechnism of induced eukaryotic gene expression.
Our previous work show the expression of human heat shock protein 90 genes( hsp90α and β ) can be induced by the dual induction of heat shock and PHA activition. Their regulation depends mainly on transcription, and shows different expression kinetics upon the two inducer. In order to study the regulation mechnism of induced expression of human hsp90P, we amplified its upstream sequence (-1102/+68bp), the first exon and intron (-4/+1503bp) by polymerase chain reaction. Truncated fragments of the amplified sequence were then subcloned into the 5' of eukaryotic expression vectors with luciferase or chloramphenicol acetyltransferase (CAT) gene as reporter genes. Reporter gene activity assay in transfected Jurkat cells, mobility shift assay and primer extension experiments show:
引文
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刘巨洪，吴宁华，沈珝琲(1995)中国医学科学院学报，已接收
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刘巨洪，吴宁华，沈珝琲(1995)中国医学科学院学报，已接收