Analysis of a Candida albicans gene that encodes a novel mechanism for resistance to benomyl and methotrexate

• Published in: Molecular & general genetics Vol. 227; no. 2; pp. 318 - 329
• Main Authors: Fling, M.E, Kopf, J, Tamarkin, A, Gorman, J.A, Smith, H.A, Koltin, Y
• Format: Journal Article
• Language: English
• Published: Germany 01.06.1991
• Subjects: Amino Acid Sequence; amino acid sequences; Base Sequence; benomyl; Benomyl - pharmacology; Candida albicans; Candida albicans - drug effects; Candida albicans - genetics; candida stellatoidea; Codon - genetics; codon usage; drug resistance; Drug Resistance, Microbial - genetics; embl/x53823; Escherichia coli - drug effects; Escherichia coli - genetics; genes; Genes, Fungal; genetic code; genetic transformation; methotrexate; Methotrexate - pharmacology; Molecular Sequence Data; Mutagenesis, Site-Directed; Nucleic Acid Hybridization; nucleotide sequences; open reading frames; Open Reading Frames - genetics; Polymerase Chain Reaction; Restriction Mapping; Saccharomyces cerevisiae; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; site-directed mutagenesis
• ISSN: 0026-8925; 1432-1874
• DOI: 10.1007/BF00259685

The pathogenic yeast, Candida albicans, is insensitive to the anti-mitotic drug, benomyl, and to the dihydrofolate reductase inhibitor, methotrexate. Genes responsible for the intrinsic drug resistance were sought by transforming Saccharomyces cerevisiae, a yeast sensitive to both drugs, with genomic C. albicans libraries and screening on benomyl or methotrexate. Restriction analysis of plasmids isolated from benomyl- and methotrexate-resistant colonies indicated that both phenotypes were encoded by the same DNA fragment. Sequence analysis showed that the fragments were nearly identical and contained a long open reading frame of 1694 bp (ORF1) and a small ORF of 446 bp (ORF2) within ORF1 on the opposite strand. By site-directed mutagenesis, it was shown that ORF1 encoded both phenotypes. The protein had no sequence similarity to any known proteins, including beta-tubulin, dihydrofolate reductase, and the P-glycoprotein of the multi-drug resistance family. The resistance gene was detected in several C. albicans strains and in C. stellatoidea by DNA hybridization and by the polymerase chain reaction.