Development of cell-free transcription system for a study of tissue-specific transcriptional regulation in plant gene

开发用于研究植物基因组织特异性转录调控的无细胞转录系统

• 批准号: 03554027
• 负责人: YAMAZAKI Ken-ichi
• 金额: $ 8.7万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research (B)
• 财政年份: 1991
• 资助国家: 日本
• 起止时间: 1991 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03554027/
• 关键词: plant; gene expression; in vitro transcription; nuclear extract; RNA polymerase; transcription system; RNAポリメラーゼII; RNAポリメラ-ゼ

An assay system that allows the rapid, direct, and quantitative detection of promoter-dependent transcription in vitro with nuclear extracts from tobacco cells has now been developed. The transcriptional machinery from tobacco cell nuclei effectively and accurately transcribes specific genes, and the optimum conditions for such a system are described. The system consists of a protein raction extracted from the nuclei of suspension-cultured tobacco cells (TBY-2), substrates, and an exogenously added plasmid DNA template. Specific initiation at the promoter was determined by analysis of the sizes of transcripts on a polyacrylamide gel that contained 8 M urea. Our system using a plasmid DNA template, based essentially on a G-free sequence system, was developed to elucidate the function of plant trans-acting factors that influence the activity of RNA polymerase II.However, further modifications that allow us to elucidate tissue-specific transcriptional activation may be required from now on.

现在已经开发出一种检测系统，可以使用烟草细胞的核提取物在体外快速、直接和定量检测启动子依赖性转录。烟草细胞核的转录机制有效且准确地转录特定基因，并描述了该系统的最佳条件。该系统由从悬浮培养的烟草细胞 (TBY-2) 细胞核中提取的蛋白质组分、底物和外源添加的质粒 DNA 模板组成。通过分析含有 8 M 尿素的聚丙烯酰胺凝胶上转录物的大小来确定启动子处的特异性起始。我们的系统使用质粒 DNA 模板，基本上基于无 G 序列系统，旨在阐明影响 RNA 聚合酶 II 活性的植物反式作用因子的功能。然而，进一步的修改使我们能够阐明组织-从现在开始可能需要特定的转录激活。

项目成果

山口 淳二: "Isolation and characterization of nuclei from rice embryos." Cell Structure & Function. 17. 87-92 (1992)

Junji Yamaguchi：“水稻胚胎细胞核的分离和表征”，17. 87-92 (1992)。

山崎 健一: "植物遺伝子のインビトロ転写法,植物細胞工学" 秀潤社, 88 (1990)

山崎健一：“植物基因的体外转录方法、植物细胞工程”《书俊社》，88（1990）

山崎 健一: "TGAla,a tobacco DNA-binding protein,increases the rate of initiation in a plant in vitro transcription system." Proceedings of Natinal Academie of Science. 87. 7035-7039 (1990)

Kenichi Yamazaki：“TGAla 是一种烟草 DNA 结合蛋白，可提高植物体外转录系统的起始速率。” 国家科学院院刊 87. 7035-7039 (1990)。

山崎 健一: "Selective and accurate initiation of transcription at the T-DNA promoter in a soluble chromatin extract from wheat germ." Molecular & General Genetics. 209. 445-452 (1987)

Kenichi Yamazaki：“小麦胚芽可溶性染色质提取物中 T-DNA 启动子的选择性且准确的转录起始。” 209. 445-452 (1987)

山口 雄記: "Preparation of an in vitro Transcription System of Plant Origin,with Methods and Templates for Assessing Its Fiderity." Plant Mol Biol Manual. (印刷中). (1994)

Yuki Yamaguchi：“植物来源体外转录系统的制备，以及评估其可靠性的方法和模板”（植物分子生物学手册）（1994 年）。