Analysis of protein function in clinical C. albicans isolates
Analysis of protein function in clinical C. albicans isolates
复制标题
DOI：
10.1002/yea.2910
复制
发表时间：
2012-08-01
期刊：
影响因子：
2.6
通讯作者：
Berman, Judith
中科院分区：
文献类型：
--
作者：
Gerami-Nejad, Maryam;Forche, Anja;Berman, Judith
临床分离株是原养型的，因此不适合使用营养标记进行遗传操作。在此，我们描述了一组携带NAT1（诺尔丝菌素）耐药性标记的新质粒（Shen et al.，2005），其可用于临床分离株和实验室菌株。我们构建了含有HA-NAT1或MYC-NAT1盒的新型质粒，以促进PCR介导的具有C-末端表位标记蛋白的菌株的构建，以及NAT1-pMet3-GFP质粒，以使具有或不具有融合在N-末端的绿色荧光蛋白的蛋白的条件表达成为可能。此外，对于需要内源性N-和C-末端来发挥功能的蛋白质，我们构建了携带截短等位基因的GF-NAT1-FP盒，所述截短等位基因有助于将完整的单拷贝GFP插入编码序列内部。此外，构建了GFP-NAT1、RFP-NAT1和M-Cherry-NAT1质粒，表达两种不同标记的基因产物，用于蛋白质共表达和体内共定位的研究。总之，这些载体提供了一套有用的遗传工具，用于研究临床和实验室菌株的基因功能的各个方面。白色念珠菌版权所有（C）2012约翰威利父子有限公司
Clinical isolates are prototrophic and hence are not amenable to genetic manipulation using nutritional markers. Here we describe a new set of plasmids carrying the NAT1 (nourseothricin) drug resistance marker (Shen et al., 2005), which can be used both in clinical isolates and in laboratory strains. We constructed novel plasmids containing HA-NAT1 or MYC-NAT1 cassettes to facilitate PCR-mediated construction of strains with C-terminal epitope-tagged proteins and a NAT1-pMet3-GFP plasmid to enable conditional expression of proteins with or without the green fluorescent protein fused at the N-terminus. Furthermore, for proteins that require both the endogenous N- and C-termini for function, we have constructed a GF-NAT1-FP cassette carrying truncated alleles that facilitate insertion of an intact, single copy of GFP internal to the coding sequence. In addition, GFP-NAT1, RFP-NAT1 and M-Cherry-NAT1 plasmids were constructed, expressing two differently labelled gene products for the study of protein co-expression and co-localization in vivo. Together, these vectors provide a useful set of genetic tools for studying diverse aspects of gene function in both clinical and laboratory strains of C. albicans. Copyright (C) 2012 John Wiley & Sons, Ltd.