Single-Copy IMH3 Allele Is Sufficient To Confer Resistance to Mycophenolic Acid in Candida albicans and To Mediate Transformation of Clinical Candida Species

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Abstract
Parasexual genetic analysis of Candida albicans utilized the dominant selectable marker that conferred resistance to mycophenolic acid. We cloned and sequenced the IMH3 r gene from C. albicans strain 1006, which was previously identified as resistant to mycophenolic acid (MPA) (A. K. Goshorn and S. Scherer, Genetics 123:213–218, 1989). MPA is an inhibitor of IMP dehydrogenase, an enzyme necessary for the de novo biosynthesis of GMP. G. A. Kohler et al. (J. Bacteriol. 179:2331–2338, 1997) have shown that the wild-type IMH3 gene, when expressed in high copy number, will confer resistance to this antibiotic. We demonstrate that the IMH3 r gene from strain 1006 has three amino acid changes, two of which are nonconservative, and demonstrate that at least two of the three mutations are required to confer resistance to MPA. We used this gene as a dominant selectable marker in clinical isolates of C. albicans and Candida tropicalis . We also identified the presence of autonously replicating sequence elements that permit autonomous replication in the promoter region of this gene. Finally, we found the excision of a φ-type long terminal repeat element outside the IMH3 open reading frame of the gene in some strains. We used the IMH3 r allele to disrupt one allele of ARG4 in two clinical isolates, WO-1 and FC18, thus demonstrating that a single ectopic integration of this dominant selectable marker is sufficient to confer resistance to MPA.