Allele-specific gene targeting in Candida albicans results from heterology between alleles The GenBank accession numbers for the sequences reported in this paper are AF247189 and AF247190 for PHR1-1 and PHR1-2, respectively.

Abstract

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.

Allelic variation in the contiguous loci encoding Candida albicans ALS5, ALS1 and ALS9

“…In this work, nucleotide changes led to a premature stop codon in one allele and three amino acid changes in another that rendered both incapable of producing functional C-5 sterol desaturase. Allelic sequence variation has been detected outside coding regions as demonstrated by Yesland & Fonzi (2000) who showed that nucleotide differences in regions flanking PHR1 were responsible for biased targeting of disruption cassettes during mutant construction. Staib et al (2002) documented allelic sequence variation in the promoter region of SAP2 and showed that the divergent sequences resulted in differential regulation of the alleles.…”

The Candida albicans 14‐3‐3 gene, BMH1, is essential for growth

“…In all cases the recombination event occurred in the already disrupted allele or at an unexplained location. Since small nucleotide differences can significantly lower the efficiency of homologous recombination in C. albicans (Yesland and Fonzi, 2000), a second disruption cassette, pDC4-G, was constructed, which was directed against the remaining wild-type allele. Sequence analysis identified seven nucleotide differences between pDC1 and pDC4 and all were within the Figure 1.…”

The ARO4 gene of Candida albicans encodes a tyrosine-sensitive DAHP synthase: evolution, functional conservation and phenotype of Aro3p-, Aro4p-deficient mutants The GenBank accession number for the sequence reported in this paper is U53216.

“…Nearly 1000 Ura + transformants were screened by PCR, and in all cases the disruption cassette was found to have integrated either at the previously disrupted (∆aro4 ::hisG) allele, or at a much lower frequency, at some other site in the genome (data not shown). This type of event may have been favoured over gene replacements at the wild-type allele because of more extensive homology between the hisG sequences in the transforming DNA fragment and the ∆aro4 ::hisG allele in the chromosome, resulting in more relaxed recombination intermediates, or may be due to recently revealed allele preferences during targeting integration as described by Yesland & Fonzi (2000).…”