通过具有短同源区域的基因破坏对白色念珠菌进行快速假设检验。
Rapid hypothesis testing with Candida albicans through gene disruption with short homology regions.
作者信息
Wilson R B, Davis D, Mitchell A P
机构信息
Department of Microbiology and Institute of Cancer Research, Columbia University, New York, New York 10032, USA.
出版信息
J Bacteriol. 1999 Mar;181(6):1868-74. doi: 10.1128/JB.181.6.1868-1874.1999.
Abstract
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.
摘要
破坏病原体白色念珠菌中新鉴定出的基因是确定基因功能的关键步骤。先前描述的几种基因破坏方法使用位于选择标记两侧的长同源区域。在此，我们描述了用PCR产物破坏白色念珠菌基因的方法，该PCR产物在选择标记的两端与基因组序列具有50至60 bp的同源性。我们使用该方法破坏了两个已知基因ARG5和ADE2，以及通过念珠菌基因组计划新鉴定出的两个序列HRM101和ENX3。HRM101和ENX3与酿酒酵母和构巢曲霉保守的RIM101（以前称为RIM1）和PacC途径中的基因同源。我们表明，三个独立的hrm101/hrm101突变体和两个独立的enx3/enx3突变体在Spider培养基上的丝状化过程中存在缺陷。这些观察结果表明，HRM101和ENX3序列确实是基因的一部分，并且各自的基因产物具有相关功能。
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