New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe
New drug-resistant cassettes for gene disruption and epitope tagging in Schizosaccharomyces pombe
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作者：
Sato, M;Dhut, S;Toda, T
我们在分裂酵母裂糖酵母中描述了新的基于pcr的基因靶向的异源模块。两个细菌基因，hph和nat，显示显性耐药表型，被用作这些模块的新的选择标记。这两种基因都已成功地应用于出芽酵母酿酒酵母，其中hph赋予对湿霉素B的抗性，而nph赋予对食糖丙氨酸的抗性（Goldstein和McCusker， 1999）。使用先前构建的pfa6a - mx6衍生质粒构建用于基因破坏和3HA、13Myc和GFP(S65T) c端标记的载体模块（Bahler等，1998；Wach等，1997）。结合现有的基于g418抗性基因（kan）的系统，可以构建三重基因缺失或标签。此外，还开发了一种将单个RFP （mRFP）一步整合到感兴趣的蛋白质c端的载体。最后，寡核苷酸允许一个简单的标记切换从kan到hph或nat，反之亦然，被描述。在这里开发的新结构应该有助于裂变酵母中蛋白功能的后基因组分子分析。版权所有(c) 2005 John Wiley & Sons, Ltd。
We describe new heterologous modules for PCR-based gene targeting in the fission yeast Schizosaccharomyces pombe. Two bacterial genes, hph and nat, which display dominant drug-resistance phenotypes, are used as new selectable markers in these modules. Both genes have been used successfully in the budding yeast Saccharomyces cerevisiae, in which hph confers resistance to hygromycin B, while nat confers nourseothricin resistance (Goldstein and McCusker, 1999). Vector modules for gene disruption and C-terminal tagging with 3HA, 13Myc and GFP(S65T) are constructed using previously constructed pFA6a-MX6-derived plasmids (Bahler et al., 1998; Wach et al., 1997). In combination with the existing systems that are based upon the G418-resistance gene (kan), triple gene deletions or tags could be constructed. In addition a vector for one-step integration of a monomeric RFP (mRFP) to the C-terminus of proteins of interest is developed. Finally, oligonucleotides that allow a simple marker switch from kan to hph or nat, and vice versa, are described. The new constructs developed here should facilitate post-genomic molecular analysis of proyein functions in fission yeast. Copyright (c) 2005 John Wiley & Sons, Ltd.