1887

Abstract

The opportunistic fungal pathogen 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 marker gene that is flanked by direct repeats of . After targeting to a locus, recombination between the repeats excises leaving a single copy of in the disrupted allele. The remaining functional allele is targeted in a second transformation with the same marked construct. Replacement can be highly biased toward one allele. At the 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 repeats. Nonetheless, there remained a sixfold preference for targeting a particular allele of and this was evident even in transformations of the parental strain containing two wild-type alleles of . 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 - preferentially targeted allele 1 of the genome. Transformations with - 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.

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2000-09-01
2019-10-14
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