Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Abstract

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. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343).

Genome-wide identification and characterization of replication origins by deep sequencing

“…(c) The replication profile of a region of S. octosporus dataset So1, as in (a). (d) The replication profile of a region of S. japonicus dataset Sj2 containing two cloned ARSs [28], as in (a). ORF, open reading frame.…”

“…For S. japonicus , we compared our data to 11 ARSs isolated from a genomic library [28]. Six of the ARSs encompass a replication peak and four others were within 2.5 kb of a replication peak ( P = 0.012); for one ARS, we found no associated replication peak (Figure 1d; Table S3 in Additional file 2).…”

Non-model model organisms

“…S. japonicus can be used as a valuable system on its own to study phenomena not apparent in the established yeast models. Importantly, it has all the advantages of the simple experimental system, including straightforward culturing, short cell cycle, and the ease of genetic manipulations—the latter owing largely to Hironori Niki whose group developed S. japonicus genetic tools [110, 111] and Nick Rhind who spearheaded the fission yeast clade genomes project. Beyond its utility studying mitotic NE dynamics and other aspects of mitotic division, S. japonicus could become a great system for investigating the cell biology of hyphal transition [112, 113], energy metabolism [114], and centromere biology [95, 115].…”

Transformation of Schizosaccharomyces japonicus

“…Plasmids specific for transformation of S. japonicus (Aoki et al 2010) were constructed using an autonomously replicating sequence (ARS) isolated from the S. japonicus genome.…”