Isolation of hem3 mutants from Candida albicans by sequential gene disruption

• Published in: Molecular & general genetics Vol. 217; no. 1; p. 47
• Main Authors: Kurtz, M B, Marrinan, J
• Format: Journal Article
• Language: English
• Published: Germany 01-05-1989
• Subjects: Alleles; Candida albicans - genetics; Candida albicans - isolation & purification; Cloning, Molecular; DNA, Fungal - genetics; DNA, Fungal - isolation & purification; Escherichia coli - genetics; Genes, Fungal; Genetic Complementation Test; Heme - biosynthesis; Heme - genetics; Hydroxymethylbilane Synthase - genetics; Hydroxymethylbilane Synthase - metabolism; Mutation; Nucleic Acid Hybridization; Plasmids; Restriction Mapping; Saccharomyces cerevisiae - genetics; Transformation, Genetic
• ISSN: 0026-8925
• DOI: 10.1007/BF00330941

Molecular methods for directed mutagenesis in Candida albicans have relied on a combination of gene disruption by transformation to inactivate one allele and UV-induced mitotic recombination or point mutation to produce lesions in the second allele. An alternate method which uses two sequential gene disruptions was developed and used to construct a C. albicans mutant defective in a gene essential for synthesizing tetrapyrrole (uroporphyrinogen I synthase). The Candida gene was cloned from a random library by complementation of the hem3 mutation in Saccharomyces cerevisiae. The complementing region was limited to a approximately 2.0 kb fragment by subcloning and a Bg/II site was determined to be within an essential region. Linear fragments containing either the Candida URA3 or LEU2 gene inserted into the Bg/II site were used to disrupt both alleles of a leu2, ura3 mutant by sequential transformation. Ura+, Leu+ heme-requiring strains were recovered and identified as hem3 mutants by Southern hybridization, transformation to heme independence by the cloned gene, and enzyme assays.