New cassettes for single-step drug resistance and prototrophic marker switching in fission yeast
New cassettes for single-step drug resistance and prototrophic marker switching in fission yeast
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DOI：
10.1002/yea.3097
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发表时间：
2015-12-01
期刊：
影响因子：
2.6
通讯作者：
Lorenz, Alexander
中科院分区：
文献类型：
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作者：
Lorenz, Alexander
构建用于遗传相互作用分析的多重突变菌株和在多个开放阅读框携带不同表位标签以测试蛋白质定位、丰度和蛋白质-蛋白质相互作用的菌株受到足够数量的不同选择标记的可用性的阻碍。此外，具有单基因缺失或标签的菌株通常已经存在于菌株集合中;由于历史原因，这些菌株大多携带ura 4（+）基因或G418抗性kanMX作为标记。由于使用相同的标记产生多重缺失或标记的菌株，或者用不同的标记完全重建特定菌株相当麻烦，因此标记的一步交换方案是一种节省时间的替代方案。近年来，针对clonNAT、潮霉素B和博来霉素的显性耐药标记（DDRM）已被成功地应用于粟酒裂殖酵母。相应的DDRM盒natMX、hphMX和bleMX携带来自棉阿舒囊霉的TEF启动子和终止子序列作为kanMX;这提供了侧翼同源性以使得能够通过同源基因靶向进行单步标记交换。为了扩展这个非常有用的工具集，用于单步标记交换，我构建了包含营养标记arg 3（+），his 3（+），leu 1（+）和ura 4（+）的MX盒。此外，创建了一组构建体，以实现ura 4（+）到kanMX 6、natMX 4和hphMX 4的单步交换。通过在几个基因座成功的单步标记交换证明了试剂盒的功能。这些构建体允许直接和快速地重新标记现有的ura 4（+）-和MX-缺失和标记的菌株。版权所有（C）2015约翰威利父子有限公司
Construction of multiply mutated strains for genetic interaction analysis and of strains carrying different epitope tags at multiple open reading frames for testing protein localization, abundance and protein-protein interactions is hampered by the availability of a sufficient number of different selectable markers. Moreover, strains with single gene deletions or tags often already exist in strain collections; for historical reasons these will mostly carry the ura4(+) gene or the G418-resistance kanMX as marker. Because it is rather cumbersome to producemultiply deleted or tagged strains using the same marker, or to completely reconstruct a particular strain with a different marker, single-step exchange protocols of markers are a time-saving alternative. In recent years, dominant drug resistance markers (DDRMs) against clonNAT, hygromycin B and bleomycin have been adapted and successfully used in Schizosaccharomyces pombe. The corresponding DDRM cassettes, natMX, hphMX and bleMX, carry the TEF promotor and terminator sequences from Ashbya gossypii as kanMX; this provides flanking homologies to enable single-step marker swapping by homologous gene targeting. To expand this very useful toolset for single-step marker exchange, I constructed MX cassettes containing the nutritional markers arg3(+), his3(+), leu1(+) and ura4(+). Furthermore, a set of constructs was created to enable single-step exchange of ura4(+) to kanMX6, natMX4 and hphMX4. The functionality of the cassettes is demonstrated by successful single-step marker swapping at several loci. These constructs allow straightforward and rapid remarking of existing ura4(+)-and MX-deleted and -tagged strains. Copyright (C) 2015 John Wiley & Sons, Ltd.