Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus
Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus
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DOI：
10.1002/yea.1815
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发表时间：
2010-12-01
期刊：
影响因子：
2.6
通讯作者：
Niki, Hironori
中科院分区：
文献类型：
--
作者：
Aoki, Keita;Nakajima, Reiko;Niki, Hironori
裂殖酵母是一种分裂酵母，最近已经开发了新的遗传工具。在这里，我们报告新的质粒载体具有高转化效率和Sz的电穿孔方法。- 是的从12166个转化子中分离出44个复制片段。通过对Sz.电池。将该序列克隆到带有Sz的ura 4（+）的pUC 19载体中。pombe（pSJU 11）或kanMX 6模块上的kan基因（pSJK 11）作为选择标记。这些质粒转化了Sz.通过电穿孔以pSJK 11的123 cfu/μ g和pSJU 11的301 cfu/μ g的频率将对数早期的细胞转染，这高于先前报道的自主复制序列。虽然有一部分质粒通过RS 1片段整合到染色体中而留在宿主细胞中，但这些质粒可以从转化体中回收。通过使用Sz. pombe ura 4（+）探针。含有ade 6（+）的质粒抑制了ade 6-domE突变体的营养缺陷型生长，表明该质粒将用于Sz. - 是的此外，pSJU 11转化了Sz. pREP 2质粒以相同的频率转染粟酒酵母细胞。本研究是一份报告，以证明实际使用的附加型质粒载体的遗传研究在Sz。- 是的RS 1已提交到DDBJ/EMBL/GenBank数据库（登录号AB 547343）。版权所有（C）2010约翰威利父子有限公司
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/mu g for pSJK11 and 301 cfu/mu g 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). Copyright (C) 2010 John Wiley & Sons, Ltd.