Vectors designed for efficient molecular manipulation in Candida albicans

Abstract

Functional studies on genes of Candida albicans have been hampered by the fact that few vectors are available for efficient cloning and expression in C. albicans, in contrast to Saccharomyces cerevisiae. Here we report that six vectors were constructed for molecular manipulation in C. albicans. All of them contained the autonomous replicating sequence ARS2 and the uracil gene as a selective marker. Introduction of multicloning site (MCS) facilitated directional cloning into various convenient restriction sites is discussed. Distal to the MCS, the additions of sequences encoding yeast-enhanced green fluorescent protein 3 (yEGFP3) and the terminator of chitin synthase 2 (TCHS2) enabled us to express an open reading frame (ORF) with its own promoter as a GFP fusion protein, so that its intracellular localization could be easily determined. A vector of 7.4 kb was also constructed to express a cloned ORF as a GFP fusion protein under the control of an inducible MET3 promoter (PMET3) located proximal to the MCS. Since this vector was relatively large in size for expressing ORFs, two additional vectors of 6.7 kb were constructed by inserting PMET3 and TCHS2 proximal and distal to the MCS of the above vector containing MCS only, respectively. These six vectors made it possible to study C. albicans in greater detail. They can be used in identification of a promoter, intracellular localization of a protein, and in the induction of lethal genes. Copyright © 2002 John Wiley & Sons, Ltd.