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

点击翻译按钮获取中文摘要

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.