pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast.
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.
Matsuyama
Akihisa
A
Chemical Genetics Laboratory, RIKEN, Saitama 351-0198, Japan. akihisa@riken.jp
Shirai
Atsuko
A
Yashiroda
Yoko
Y
Kamata
Ayako
A
Horinouchi
Sueharu
S
Yoshida
Minoru
M
D001483:Base Sequence; D015825:Chromosomes, Fungal / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic / Q000502:physiology*; D012568:Schizosaccharomyces / Q000235:genetics* / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014170:Transformation, Genetic / Q000502:physiology
D049452:Green Fluorescent Proteins