Martin Ronny, Hellwig Daniela, Schaub Yvonne, Bauer Janine, Walther Andrea, Wendland Jürgen
Department of Microbiology, Friedrich-Schiller-University, Jena, Germany.
Yeast. 2007 Jun;24(6):511-22. doi: 10.1002/yea.1489.
PCR-based techniques for directed gene alterations have become standard tools in Candida albicans. To help to increase the speed of functional analysis of Candida albicans genes, we previously constructed and updated a modular set of pFA-plasmid vectors for PCR-based gene targeting in C. albicans. Here we report the functional analyses of C. albicans ORFs whose homologues in S. cerevisiae are involved in endocytosis, to explore their potential involvement in polarized cell growth. Three C. albicans genes, ABP1, BZZ1 and EDE1, were found to be non-essential. Yeast and hyphal morphogenesis were not affected by the individual deletions and the mutant strains appeared wild-type-like under the different growth conditions tested. On the other hand, deletion of both alleles of the C. albicans PAN1 homologue was not feasible. Promoter shut-down experiments using a MET3p-PAN1/pan1 strain indicated severe growth defects and abolished endocytosis, indicating that PAN1 is an essential gene. Subcellular distribution of CaAbp1 and CaPan1 was analysed via GFP-tagged proteins. Both proteins were found to localize at the cortex and at hyphal tips in a patch-like manner, supporting their role in endocytosis. Localization patterns of Abp1 and Pan1, however, were distinct from that of the FM4-64 stained Spitzenkörper.
基于PCR的定向基因改造技术已成为白色念珠菌中的标准工具。为了帮助提高白色念珠菌基因功能分析的速度，我们之前构建并更新了一组模块化的pFA质粒载体，用于白色念珠菌中基于PCR的基因靶向。在此，我们报告了白色念珠菌中开放阅读框（ORF）的功能分析，其在酿酒酵母中的同源物参与内吞作用，以探索它们在极化细胞生长中的潜在作用。发现白色念珠菌的三个基因ABP1、BZZ1和EDE1是非必需的。单个基因缺失不影响酵母和菌丝形态发生，并且在测试的不同生长条件下突变菌株表现出野生型样。另一方面，白色念珠菌PAN1同源物的两个等位基因缺失是不可行的。使用MET3p - PAN1/pan1菌株进行的启动子关闭实验表明存在严重的生长缺陷并消除了内吞作用，表明PAN1是一个必需基因。通过绿色荧光蛋白（GFP）标记的蛋白分析了CaAbp1和CaPan1的亚细胞分布。发现这两种蛋白均以斑块状方式定位于皮质和菌丝尖端，支持它们在内吞作用中的作用。然而，Abp1和Pan1的定位模式与FM4 - 64染色的纺锤体极体不同。
