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 approximately 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.

• PMID: 10564518
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Care RS, Trevethick J, Binley KM, Sudbery PE

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