Allele-specific gene targeting in Candida albicans results from heterology between alleles.
The opportunistic fungal pathogen Candida albicans is asexual and diploid. Thus, introduction of recessive mutations requires targeted gene replacement of two alleles to effect expression of a recessive phenotype. This is often performed by recycling of a URA3 marker gene that is flanked by direct repeats of hisG. After targeting to a locus, recombination between the repeats excises URA3 leaving a single copy of hisG in the disrupted allele. The remaining functional allele is targeted in a second transformation with the same URA3 marked construct. Replacement can be highly biased toward one allele. At the PHR1 locus, there was an approximately 50-fold preference for replacement of the disrupted versus the functional allele in a heterozygous mutant. This preference was reduced six- to eightfold when the transforming DNA lacked the hisG repeats. Nonetheless, there remained a sixfold preference for targeting a particular allele of PHR1 and this was evident even in transformations of the parental strain containing two wild-type alleles of PHR1. Both wild-type alleles were cloned and nucleotide sequence comparison revealed 24 heterologies over a 2 kb region. Using restriction site polymorphisms to distinguish alleles, it was observed that transformation with the cloned DNA of allele PHR1-1 preferentially targeted allele 1 of the genome. Transformations with PHR1-2 exhibited the reciprocal specificity. In both these instances, heterology was present in the flanking regions of the transforming DNA. When the transforming DNA was chosen from a region 100% identical in both alleles, alleles 1 and 2 were targeted with equal frequency. It is concluded that sequence heterology between alleles results in an inherent allele specificity in targeted recombination events.
Yesland
Kyle
K
Department of Microbiology and Immunology, Georgetown University, 3900 Reservoir Road NW, Washington, DC 20007-2197, USA1.
Fonzi
William A
WA
Department of Microbiology and Immunology, Georgetown University, 3900 Reservoir Road NW, Washington, DC 20007-2197, USA1.
D000483:Alleles; D001051:Apoenzymes / Q000235:genetics; D002176:Candida albicans / Q000235:genetics* / Q000254:growth & development; D003001:Cloning, Molecular; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000235:genetics; D005656:Fungal Proteins; D018390:Gene Targeting; D005800:Genes, Fungal; D006579:Heterozygote; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D014170:Transformation, Genetic
D001051:Apoenzymes; D005656:Fungal Proteins; D008562:Membrane Glycoproteins; C091515:PHR1 protein, Candida albicans; D004255:Deoxyribodipyrimidine Photo-Lyase