Isolation of hem3 mutants from Candida albicans by sequential gene disruption.
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.
Kurtz
M B
MB
Department of Microbial Biochemistry and Genetics, Squibb Institute for Medical Research, Princeton, NJ 08543-4000.
Marrinan
J
J
D000483:Alleles; D002176:Candida albicans / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics; D005800:Genes, Fungal; D005816:Genetic Complementation Test; D006418:Heme / Q000096:biosynthesis / Q000235:genetics*; D011163:Hydroxymethylbilane Synthase / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
D004271:DNA, Fungal; D006418:Heme; D011163:Hydroxymethylbilane Synthase