pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast
pDUAL, a multipurpose, multicopy vector capable of chromosomal integration in fission yeast
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DOI：
10.1002/yea.1181
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发表时间：
2004-11-01
期刊：
影响因子：
2.6
通讯作者：
Yoshida, M
中科院分区：
文献类型：
--
作者：
Matsuyama, A;Shirai, A;Yoshida, M
已经开发了一系列新的质粒载体，命名为pDUAL。这些载体使人们不仅能够引入具有附加体维持的多拷贝基因，而且能够引入具有染色体整合的单拷贝基因到裂殖酵母，粟酒裂殖酵母中。通过在转化酵母细胞之前用某种限制性内切酶消化质粒，多拷贝质粒可以容易地转化为用于染色体整合的片段。所得到的片段缺乏自主复制序列，被设计用于通过同源重组靶向染色体leu 1基因座。转化体是否是质粒的附加型维持或同源基因靶向的结果可以通过它们对尿嘧啶或亮氨酸的需求或通过PCR诊断分析容易地检查。此外，我们提出使用pDUAL衍生物进行基于PCR的基因染色体标记，以采用一组引物将几个标签引入基因的5 ′-末端。使用这些一体化载体，可以选择克隆基因的合适表达模式用于个体分析，而无需任何复杂的亚克隆过程。版权所有（C）2004约翰威利父子有限公司。
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 (C) 2004 John Wiley Sons, Ltd.