Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans

We have developed a set of plasmids containing fluorescent protein cassette s for use in PCR-mediated gene tagging in Candida albicans. We engineered Y FP and CFP variants of the CFP sequence optimized for C. albicans codon usa ge. The fluorescent protein sequences, linked to C. albicans auxotrophic ma rker sequences, Ir ere amplified by PCR and transformed directly into yeast . Gene-specific sequence was incorporated into the PCR primers, such that t he tag-cassette integrates by homologous recombination at the 3'-end of the gene of interest, This technique was used to tag Cdc3 and Tub1 with GFP, Y FP and CFP, which were readily visualized by fluorescence microscopy and lo calized as expected, In addition, Tub1-YFP and Cdc3-CFP were visualized in the same cells. Thus, this technique directs one-step construction of multi ple fluorescent protein fusions, facilitating the study of protein co-expre ssion and co-localization in C. albicans cells in vivo. Copyright (C) 2001 John Wiley & Sons, Ltd.