"An Antisense-Based Functional Genomics Approach for Identification of Genes Critical for Growth of Candida albicans".
Marianne D. De Backer 1, Bart Nelissen 1, Marc Logghe 3, Jasmine Viaene 3, Inge Loonen 1, Sandy Vandoninck 1, Ronald de Hoogt 1, Sylviane Dewaele 3, Fermin A. Simons 1, Peter Verhasselt 2, Greet Vanhoof 2, Roland Contreras 3 & Walter H.M.L. Luyten 1
1 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 M D De Backer. e-mail: mdebacke@prius.jnj.com
Converting 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.
Additional References:
1. "Oncogenes as Molecular Targets within Active Chromatin".