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Analysis of the Regulatory Region with Unusual structural Features of the Human Thymidylate Synthase Gene

人胸苷酸合酶基因异常结构特征调节区的分析

基本信息

批准号：
05671835
负责人：
TAKEISHI Keiichi
金额：
$ 1.41万
依托单位：
University of Shizuoka
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05671835/
关键词：
Thymidylate synthase CAT assay DNA element DNA binding protein RNA binding protein DNA polymorphism Differentiation Nuclear factor 転写制御転写後制御ヒト遺伝子プロモーターサイレンサー

项目摘要

In this research project, we mainly analyzed in detail regulatory elements and cellular protein factors involved in the regulation of the expression of human thymidylate synthase (TS) gene. It must have unique control features, since it is required for highly regulated expression in a cell.First of all, we prepared many mutant DNA fragments from a specific DNA fragment derived from the region around the transcriptional initiation site of the human TS gene and analyzed promoter and cis-acting elements using the mutant DNA fragments. As the result, we identified promoter elements and several other regulatory elements in the region upstream from the major cap site. An unusual inverted repeat including a triple tandem repeat present in the region immediately upstream from an initiation codon ATG was also found to be necessary for the efficient expression of the human TS gene. In addition, the triple tandem repeat was revealed to be polymorphic with respect to the number of a repeat unit and the polymorphic DNAs were shown to differ in the expression activity of the human TS gene. Furthermore, a protein factor that bindsto an RNA fragment which corresponds to the region containing the inverted repeat was found to be present both in nuclear and cytoplasmic extracts. Finally, when human leukemia HL-60 cells differentiated into granulocytes by the retinoic acid treatment, a nuclear factor NF-TS3, that was found in our previous study and that binds to the region around the initiation codon ATG of the human TS gene was shown to increase soon after the cells started to differentiate.We think that it is important to identify additional regulatory elements and protein factors involved in the regulation of the human TS gene expression and to reveal their roles, especially the quantitative change of the positive and negative regulatory factors in response to the alteration of the cellular environment.

本研究项目主要对参与人胸苷酸合酶（TS）基因表达调控的调控元件和细胞蛋白因子进行了详细分析。它必须具有独特的控制特征，因为它是在细胞中高度调控表达所必需的。首先，我们从源自人类TS基因转录起始位点周围区域的特定DNA片段制备了许多突变DNA片段，并使用突变DNA片段分析了启动子和顺式作用元件。结果，我们在主要帽位点上游的区域中鉴定了启动子元件和其他几个调控元件。还发现一个不寻常的反向重复序列（包括存在于起始密码子 ATG 紧上游区域的三重串联重复序列）对于人 TS 基因的有效表达是必需的。另外，揭示了三重串联重复在重复单元的数量方面具有多态性，并且多态性DNA在人TS基因的表达活性方面表现出不同。此外，还发现与含有反向重复序列的区域相对应的RNA片段结合的蛋白质因子存在于细胞核和细胞质提取物中。最后，当人白血病HL-60细胞通过视黄酸处理分化为粒细胞时，我们在之前的研究中发现的与人TS基因的起始密码子ATG周围区域结合的核因子NF-TS3在细胞开始分化后很快就增加了。我们认为，识别参与调节人TS基因表达的其他调节元件和蛋白质因子并揭示它们的作用非常重要。作用，特别是响应细胞环境变化的正负调节因子的数量变化。