Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus.
Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4(+) of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early-log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4(+) probe. The plasmid containing ade6(+) suppressed the auxotrophic growth of the ade6-domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus.
Aoki
Keita
K
Microbial Genetics Laboratory, Genetic Strains Research Center, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan.
Nakajima
Reiko
R
Furuya
Kanji
K
Niki
Hironori
H
D005821:Genetic Techniques; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D012568:Schizosaccharomyces / Q000235:genetics* / Q000254:growth & development; D014170:Transformation, Genetic