Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata.
The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination.
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5402, USA. bcormack@jhmi.edu
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D001483:Base Sequence; D015139:Blotting, Southern; D002175:Candida / Q000235:genetics* / Q000472:pathogenicity*; D003433:Crosses, Genetic; D017931:DNA Primers; D005800:Genes, Fungal; D005838:Genotype; D008954:Models, Biological; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014170:Transformation, Genetic
D017931:DNA Primers