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.

我们开发了一种方法，可以基于 cDNA 进行克隆。哺乳动物或裂殖酵母细胞的表型互补。该方法由1）高效全长cDNA克隆方法组成； 2)pcD2表达载体； 3）高频哺乳动物细胞转染方法； 4）高效酵母转染方法； 5)高度稳定的酵母载体，用于将cDNA文库转导到裂变酵母中。利用这种方法，我们分离出了cdc25^+基因的两种不同的人类和大鼠同源物，裂变酵母有丝分裂诱导剂，以及人类和大鼠的cdc25^+基因同源物^ + 基因，裂殖酵母有丝分裂抑制剂。此外，我们还分离了几个抑制温度敏感的cdc2突变体或/和温度敏感的性分化熟练的pat1突变体的人类基因。其中一些编码具有 RNA 结合结构域的蛋白质，另一些则编码具有 RNA 结合结构域的蛋白质。可能与 cdc2 激酶有关。该 cDNA 克隆系统可用于分离多种哺乳动物基因。

项目成果

Inoue,H.: "High efficiency transformation of Escherichea coli with plasmids." Gene. 96. 23-28 (1990)

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。”

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。”

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。”

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。”