Nature Biotechnology
19, 235 - 241 (2001)
doi:10.1038/85677
An antisense-based functional genomics approach for identification of genes critical for growth of Candida albicansMarianne D. De Backer1, Bart Nelissen1, Marc Logghe3, Jasmine Viaene3, Inge Loonen1, Sandy Vandoninck1, Ronald de Hoogt1, Sylviane Dewaele3, Fermin A. Simons1, Peter Verhasselt2, Greet Vanhoof2, Roland Contreras3
& Walter H.M.L. Luyten11
Department of Advanced Bio-Technologies, Janssen Pharmaceutica, Turnhoutseweg 30, B2340 Beerse, Belgium. 2
Department of Biotechnology and High Throughput Screening, Janssen Pharmaceutica, Turnhoutseweg 30, B2340 Beerse, Belgium. 3
Department of Molecular Biology, Subunit Fundamental and Applied Molecular Biology, University Gent and Vlaams Instituut voor de bevordering van het Wetenschappelijk Technologisch onderzoek in de industrie (IWT/VIB), K.L. Ledeganckstraat 35, B9000 Gent, Belgium.
Correspondence should be addressed to Marianne D. De Backer mdebacke@prius.jnj.comConverting the complete genome sequence of Candida albicans into meaningful biological information will require comprehensive screens for identifying functional classes of genes. Most systems described so far are not applicable to C. albicans because of its difficulty with mating, its diploid nature, and the lack of functional random insertional mutagenesis methods. We examined artificial gene suppression as a means to identify gene products critical for growth of this pathogen; these represent new antifungal drug targets. To achieve gene suppression we combined antisense RNA inhibition and promoter interference. After cloning antisense complementary DNA (cDNA) fragments under control of an inducible GAL1 promoter, we transferred the resulting libraries to C. albicans. Over 2,000 transformant colonies were screened for a promoter-induced diminished-growth phenotype. After recovery of the plasmids, sequence determination of their inserts revealed the messenger RNA (mRNA) they inhibited or the gene they disrupted. Eighty-six genes critical for growth were identified, 45 with unknown function. When used in high-throughput screening for antifungals, the crippled C. albicans strains generated in this study showed enhanced sensitivity to specific drugs.
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