Analysis of Factors Regulating Expression of Human Thymidylate Synthase Gene

人胸苷酸合酶基因表达调控因素分析

• 批准号: 01571221
• 负责人: TAKEISHI Keiichi
• 金额: $ 1.34万
• 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: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01571221/
• 关键词: Thymidylate synthase; Human genomic DNA; Promotor; CAT vector; Regulatory factor; Differentiation; DNA binding protein; 転写制御因子; ミニ遺伝子

In this research project we studied factors involved in the control of expression of human thymidylate synthase (TS) gene.First, we attempted to identify an unusual promotor sequence of human TS gene utilizing two kinds of CAT (chloramphenicol acetyltransferase) vectors with an enhancer sequence. Using these vectors the promoter activity was first successfully detected and it was concluded that the promotor existed within about 400 base pairs upstream from the transcriptional initiation site. This success opened the way to identify the promotor sequence at the level of a base.Secondly, we searched for nuclear protein factors that bind to several DNA fragments derived from the 5'-terminal region of human TS gene. For this purpose, we examined nuclear extracts from human cultured cells by the gel mobility shift assay method. As the result, three kinds of nuclear factors were found. One of these factors was confirmed to be a factor which binds to an octamer sequence. The other two factors showed the change in the quantity which depended on the stage of differentiation of human promyelocytic leukemia HL-60 cells, i. e., one decreased during the cellular differentiation, whereas the other increased. More interestingly, it was revealed that both the factors bind to the same DNA region around the ATG initiation codon of human TS gene.We are planning to purify the nuclear factors for the preparation of antibodies against them and for the determination of their partial amino acid sequences. Utilizing these materials or data, we are going to clone the genes for the factors and to elucidate their role in the regulation of the expression of human TS gene.

本研究首先利用两种带有增强子序列的CAT（chloramphenicol acetyltransferase）载体，对人胸苷酸合成酶（TS）基因的启动子序列进行了鉴定，并对两种CAT载体的启动子序列进行了比较。使用这些载体，启动子活性首次被成功地检测到，并得出结论，启动子存在于转录起始位点上游约400个碱基对内。这一成功为在碱基水平上鉴定启动子序列开辟了道路。其次，我们从人TS基因5 '端的DNA片段中寻找了与之结合的核蛋白因子。为此，我们研究了从人培养细胞的核提取物的凝胶迁移率变动分析方法。结果，发现了三种核因子，其中一种因子被证实是与八聚体序列结合的因子。另外两个因子则表现出随人早幼粒白血病HL-60细胞分化阶段而变化的量，即：例如，一种在细胞分化过程中下降，而另一种则上升。更有趣的是，这两种因子与人TS基因ATG起始密码子周围的相同DNA区域结合，我们计划纯化核因子，用于制备抗它们的抗体和测定它们的部分氨基酸序列。利用这些材料或数据，我们将克隆这些因子的基因，并阐明它们在人TS基因表达调控中的作用。

项目成果

N. Horie, J. Nalbantoglu S. Kaneda, D. Ayusawa, T. Seno, and K. Takeishi: "Identification and Characterization of an L1 Family Sequence with a Very Long Open Reading Frame in the Third Intron of the Human Thymidylate Synthase Gene" J. Biochem.106. 1-4 (19

N. Horie、J. Nalbantoglu S. Kaneda、D. Ayusawa、T. Seno 和 K. Takeishi：“人类胸苷酸合酶基因第三内含子中具有超长开放阅读框的 L1 家族序列的识别和表征

Keiichi Takeishi: "Molecular Cloning and Molecular Biological Studies of Human Thymidylate Synthase Gene" Yakugaku Zasshi. 110. 891-907 (1990)

Keiichi Takeishi：“人胸苷酸合酶基因的分子克隆和分子生物学研究”Yakugaku Zasshi。

N. Horie, R. Nozawa, and K. Takeishi: "Identification of Cellular Differentiation-Dependent Nuclear Factors That Bind to Human Thymidylate Synthase Gene" Nucleic Acids Res.

N. Horie、R. Nozawa 和 K. Takeishi：“与人胸苷酸合酶基因结合的细胞分化依赖性核因子的鉴定”核酸研究。

K. Takeishi: "Molecular Cloning and Molecular Biological Studies of Human Thymidylate Synthase Gene" Yakugaku Zasshi. 110. 891-907 (1990)

K. Takeishi：“人胸苷酸合酶基因的分子克隆和分子生物学研究”Yakugaku Zasshi。

Nobuyuki Horie: "Identification and characterization of an LI family sequence with a very long open readeing frame in the third intron of the human thymidylate synthase gene" J.Biochem.106. 1-4 (1989)

Nobuyuki Horie：“人胸苷酸合酶基因第三个内含子中具有非常长的开放阅读框的 LI 家族序列的鉴定和表征”J.Biochem.106。