Modular Gene Over-expression Strategies for Candida albicans
Over-expression is a valid functional genomics approach to characterise genes of unknown function on a genome-wide scale. Strains are engineered to over-express a specific gene and the resulting gain-of-function phenotype assessed. Here, we describe the s
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. Introduction There has been exponential growth in the number of available fungal genome sequences over recent years. Despite this wealth of sequence information, a significant number of fungal genes remain uncharacterised and the functions assigned to the majority of genes are based on sequence homology alone. The classical method of elucidating gene function is by disrupting or deleting each gene and examining the resulting null mutant for measurable phenotypes. In fungi such as the human pathogen, Candida albicans, which is constitutively diploid and lacks a full sexual cycle, gene knockouts can be problematic and time consuming. One approach that circumvents the need to generate null mutants is to engineer strains that over-express the gene of interest by placing the gene under the control of a strong promoter. An over-expression strategy
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_15, © Springer Science+Business Media, LLC 2012
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has been used, for example, in the model yeast Saccharomyces cerevisiae and has successfully led to the discovery of new signalling pathways (1) and new functions and target genes of transcription factors (2). This strategy is particularly valuable in the analysis of the function of genes that are members of multigene families. Classical gene deletion of one member of a gene family may not confer a demonstrable phenotype due to functional redundancy within the family or compensatory activation of other family members. However, over-expression of one family member may result in altered cellular properties. The choice of promoter, i.e. a strong constitutive promoter versus a regulatable promoter, is extremely important and there are advantages and disadvantages to both. In addition, it is crucial that the level of over-expression itself does not frequently confer a fitness disability to the cells. The environmental conditions that activate the promoter of choice should also be considered. Are they relevant to in vitro growth of the fungus and, in human pathogens, will the promoter be active and drive strong levels of expression in vivo. Several promoters have been utilised to over-express a selected number of genes in C. albicans, for example ADH1 (3), PCK1 (4), TEF1 (5), TDH3 (6, 7), and a tetracycline-inducible promoter (8). Over-expression strategies have already been applied on a small scale to investigate C. albicans gene function, interactions with the host and virulence attributes such as biofilm formation and morphogenesis. Over-expression using the TEF1 and TDH3 promoters has been utilised to identify the target genes of regulators of biofilm formation (Bcr1 and Zap1) and host interactions (Rim101) and to study their roles in those processes (5–7). Fu et al. (9) placed a number of cell wall protein genes under the control of a tetracycline-inducible promoter. Over-expression of IFF4 increased adhesion to epithelial cells and to
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