Premium
pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast
Author(s) -
Matsuyama Akihisa,
Shirai Atsuko,
Yashiroda Yoko,
Kamata Ayako,
Horinouchi Sueharu,
Yoshida Minoru
Publication year - 2004
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1181
Subject(s) - biology , plasmid , subcloning , homologous recombination , genetics , flp frt recombination , schizosaccharomyces pombe , gene , transformation (genetics) , shuttle vector , schizosaccharomyces , restriction enzyme , autonomously replicating sequence , t dna binary system , microbiology and biotechnology , origin of replication , vector (molecular biology) , genetic recombination , recombinant dna , recombination , saccharomyces cerevisiae
A novel series of plasmid vectors named pDUAL have been developed. These vectors enable one to introduce not only multicopies of genes with episomal maintenance but also a single copy with chromosomal integration into the fission yeast, Schizosaccharomyces pombe . The multicopy plasmids can be easily converted to fragments for chromosomal integration by digestion of the plasmids with a certain restriction endonuclease before transformation of the yeast cells. The resultant fragments, lacking the autonomously replicating sequence, are designed for targeting into the chromosomal leu1 locus by homologous recombination. Whether the transformants are the results of episomal maintenance of the plasmid or homologous gene targeting can be readily checked by their requirement for uracil or leucine, or by the PCR diagnostic analysis. Furthermore, we propose the use of pDUAL derivatives for PCR‐based chromosomal tagging of a gene to introduce several tags into 5′‐terminus of a gene, employing a set of primers. Using these all‐in‐one vectors, a suitable mode of expression of a cloned gene can be selected for individual analysis without any complicated subcloning processes. Copyright © 2004 John Wiley & Sons, Ltd.