Developmetn of a method for cloning of cDNAs by phenotypic complementation of mammalian or fission yeast cells

开发通过哺乳动物或裂殖酵母细胞表型互补来克隆 cDNA 的方法

• 批准号: 01890009
• 负责人: OKAYAMA Hiroto
• 金额: $ 14.98万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Developmental Scientific Research
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-01890009/
• 关键词: cDNA; Expression Cloning; Schizosacchromyces Pombe; Heterologous Genetic Complementation; Cell Cycle; cdc25; Cdc2; Weel; Schizosaccharomyces pombe; 動物細胞; 遺伝子相補; 酵母ベクタ-; 酵母トランスフェクション法

We have developed a method that permits cloning of CDNAs based on. phenotypic complementation of mammalian or fission yeast cells. This method is composed of 1)a high-efficiency full-length CDNA cloning method ; 2)the pcD2 expression vector ; 3)a high-frequency mammalian cell transfection method ; 4)a high-efficiency yeast transfection method ; 5)highly-stable yeast vectors for transducing CDNA libraries into fission yeast.Using this method, we have isolated two distinct human and rat homologs of the cdc25^+ gene, a fission yeast mitotic inducer, and human and rat homologs of the weel^+ gene, a fission yeast mitotic inhibitor. In addition we have isolated several human genes that suppress temperal'ure-sensitive cdc2 mutants or/and a temperature-sensitive sexual differentiation-proficient patl mutant. Some of them encodes a protein with RNA-bindng domains and some others a protein which. might associate with cdc2 kinase.This CDNA cloning system will be useful for the islation of a wide variety of mammalian genes.

我们已经开发了一种基于哺乳动物或裂殖酵母细胞的表型互补来克隆cDNA的方法。该方法由1）高效全长cDNA克隆法; 2）pcD 2表达载体; 3）高频哺乳动物细胞转染法; 4）高效酵母转染法; 5）用于将cDNA文库转导到裂殖酵母中的高稳定性酵母载体。使用这种方法，我们分离了cdc 25 ^+基因的两种不同的人类和大鼠同源物，一种裂变酵母有丝分裂诱导剂，以及人类和大鼠的Weel^+基因同源物，一种裂变酵母有丝分裂抑制剂。此外，我们已经分离出几个抑制温度敏感性cdc 2突变体或/和温度敏感性性分化熟练的patl突变体的人类基因。它们中的一些编码具有RNA结合结构域的蛋白质，而另一些编码具有RNA结合结构域的蛋白质。该cDNA克隆系统的建立将为哺乳动物基因的克隆提供一个新的途径。

项目成果

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Inoue,H.：“用质粒高效转化大肠杆菌。”

Tanaka, K.: "A cDNA sequence of xeroderma pigmentosum group A gene encoding a protein with zinc finger structure." Nature. 348. 73-76 (1990)

Tanaka, K.：“着色性干皮病 A 组基因的 cDNA 序列，编码具有锌指结构的蛋白质。”

Okazaki,N.: "High-frequency transformation method and library transducing vectors for cloning mammalian cDNAs by trans-complementation of Schizosaccharomyces pombe." Nucl.Acid.Res.18. 6485-6489 (1990)

Okazaki,N.：“通过裂殖酵母反式互补克隆哺乳动物 cDNA 的高频转化方法和文库转导载体。”

Igarashi, M.: "Weel^+-like gene in human cells." Nature. 353. 80-83 (1991)

Igarashi, M.：“人类细胞中的 Weel^ 样基因。”

Hiroto Okayama: "An improved method for high-efficiency transformation of Echerichiacoli with plasmids."

Hiroto Okama：“一种用质粒高效转化大肠杆菌的改进方法。”