Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions
Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions
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DOI：
10.1128/jb.181.6.1868-1874.1999
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发表时间：
1999-03-01
影响因子：
3.2
通讯作者：
Mitchell, AP
中科院分区：
文献类型：
--
作者：
Wilson, RB;Davis, D;Mitchell, AP
破坏病原体白色念珠菌中新鉴定的基因是确定基因功能的重要步骤。先前描述的几种基因破坏方法采用选择标记侧翼的长同源区。在这里，我们描述了C的中断。白色念珠菌基因，其PCR产物与选择标记的每一端上的基因组序列具有50至60 bp的同源性。我们使用该方法破坏了两个已知基因ARG 5和ADE 2，以及通过念珠菌基因组计划新鉴定的两个序列HRM 101和ENX 3，HRM 101和ENX 3与酿酒酵母和构巢曲霉的保守RIM 101（以前称为RIM 1）和PacC途径中的基因同源。我们发现三个独立的hrm 101/hrm 101突变体和两个独立的enx 3/enx 3突变体在Spider培养基上表达缺陷。这些观察结果表明，HRM 101和ENX 3序列确实是基因的一部分，并且各自的基因产物具有相关的功能。
Disruption of newly identified genes in the pathogen Candida albicans is a vital step in determination of gene function. Several gene disruption methods described previously employ long regions of homology flanking a selectable marker. Here, we describe disruption of C. albicans genes with PCR products that have 50 to 60 bp of homology to a genomic sequence on each end of a selectable marker. We used the method to disrupt two known genes, ARG5 and ADE2, and two sequences newly identified through the Candida genome project, HRM101 and ENX3, HRM101 and ENX3 are homologous to genes in the conserved RIM101 (previously called RIM1) and PacC pathways of Saccharomyces cerevisiae and Aspergillus nidulans. We show that three independent hrm101/hrm101 mutants and two independent enx3/enx3 mutants are defective in filamentation on Spider medium. These observations argue that HRM101 and ENX3 sequences are indeed portions of genes and that the respective gene products have related functions.