Tetracycline-Inducible Gene Expression in Candida albicans
In addition to gene inactivation, the manipulation of gene expression is another highly useful tool for the analysis of gene function. Several regulatable promoters are available that enable researchers to shut off or turn on the expression of a target ge
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1. Introduction A straightforward approach for elucidating the function of a gene in an organism under study is the construction of mutants in which the gene is inactivated and the effect on the phenotype is observed. Although Candida albicans is a diploid organism without a known haploid phase, efficient methods for the generation of homozygous null mutants have been developed and are widely used for the genetic analysis of this fungal pathogen (1) (see Part 1 “Gene disruption” in this volume). However, a phenotypic effect caused by the absence of a gene can only be expected under conditions in which the gene is expressed, and the role of a gene in a biological process may also be obscured by the presence of other genes with overlapping functions. Conversely, forced expression of a gene
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_13, © Springer Science+Business Media, LLC 2012
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under conditions in which it is not normally active, or overexpression of a gene to unusually high levels, may provoke phenotypes that are not exhibited by wild-type cells under the same conditions and provide clues about the role of the gene product. Inducible gene expression is, therefore, a valuable complementary tool to gene inactivation for the functional analysis of genes. In addition, by controlling the expression of functionally characterized endogenous or heterologous genes, researchers can also manipulate the behavior of an organism in a desired way. Experimental manipulation of gene expression is facilitated by the availability of regulatable promoters that can be used to turn on or shut off the expression of a target gene at will. Several regulatable promoters are commonly used to control gene expression in C. albicans, for example the PKC1, MAL2, or MET3 promoters (2–4) (see also other chapters of the Sect. “Regulatable promoters” in this volume). These promoters are active in certain growth media and repressed in others, so that gene expression can be induced or downregulated by incubating the cells in the appropriate medium. Yet, it is often desirable to control gene expression in a growth medium-independent fashion by the simple addition of an inducing or repressing substance. Tetracycline, or its derivative doxycycline, is such a small molecule that is used to regulate gene expression in a broad variety of organisms from bacteria to mammals. The Tet system is based on the tetracycline repressor protein TetR from Escherichia coli, which binds to the tet operator (tetO) in the promoter of the tetracycline resistance operon and represses the expression of the tet genes in the absence of tetracycline. Tetracycline binds with high affinity to TetR and promotes dissociation of the repressor from its target sequence, resulting in the expression of the tetracycline resistance genes in the presence of the antibiotic (5). The bacterial Tet system has been modified for u
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