The MET3 promoter:: a new tool for Candida albicans molecular genetics
The MET3 promoter:: a new tool for Candida albicans molecular genetics
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DOI：
10.1046/j.1365-2958.1999.01641.x
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发表时间：
1999-01-01
影响因子：
3.6
通讯作者：
Sudbery, PE
中科院分区：
文献类型：
--
作者：
Care, RS;Trevethick, J;Sudbery, PE
用于研究白色念珠菌基因作用的核心技术是研究该基因的两个拷贝都被删除的细胞的表型。迄今为止，只有在该基因不是必需的情况下才能进行此类研究。我们描述了使用白色念珠菌 MET3 启动子来有条件地表达必需基因，以便可以研究基因产物耗尽的后果。使用 GFP 作为报告基因，研究了环境条件对其表达的影响。根据浓度超过 1 mM 的蛋氨酸或半胱氨酸是否存在，启动子显示出大约 85 倍的表达范围。在任一氨基酸存在的情况下，表达降低至接近背景的水平。我们使用 URA3 作为模型来证明 MET3 启动子可以控制必需基因的表达，前提是使用蛋氨酸和半胱氨酸的混合物来抑制启动子。我们描述了一种可用于在 MET3 启动子控制下表达任何基因的表达载体，以及一种可用于破坏基因并同时将完整拷贝置于 MET3 启动子控制下的载体。在这些实验过程中，我们发现直接整合到 RP10 基因座中可以实现高频率的转化，为解决该领域长期存在的问题提供了一种方法。
A central technique used to investigate the role of a Candida albicans gene is to study the phenotype of a cell in which both copies of the gene have been deleted. To date, such investigations can only be undertaken if the gene is not essential. We describe the use of the Candida albicans MET3 promoter to express conditionally an essential gene, so that the consequences of depletion of the gene product may be investigated. The effects of environmental conditions on its expression were investigated, using GFP as a reporter gene. The promoter showed an approximate to 85-fold range of expression, according to the presence or absence of either methionine or cysteine in concentrations in excess of 1 mM. In the presence of either amino acid, expression was reduced to levels that were close to background. We used URA3 as a model to demonstrate that the MET3 promoter could control the expression of an essential gene, provided that a mixture of both methionine and cysteine was used to repress the promoter. We describe an expression vector that may be used to express any gene under the control of the MET3 promoter and a vector that may be used to disrupt a gene and simultaneously place an intact copy under the control of the MET3 promoter. During the course of these experiments, we discovered that directed integration into the RP10 locus gives a high frequency of transformation, providing a means to solve a long-standing problem in this field.