Yesland Kyle, Fonzi William A
Department of Microbiology and Immunology, Georgetown University, 3900 Reservoir Road NW, Washington, DC 20007-2197, USA1.
Microbiology (Reading). 2000 Sep;146 ( Pt 9):2097-2104. doi: 10.1099/00221287-146-9-2097.
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.
机会性真菌病原体白色念珠菌是无性二倍体。因此，隐性突变的引入需要对两个等位基因进行靶向基因替换，以实现隐性表型的表达。这通常通过回收位于hisG直接重复序列两侧的URA3标记基因来完成。靶向一个位点后，重复序列之间的重组切除URA3，在破坏的等位基因中留下hisG的单拷贝。在第二次转化中，用相同的URA3标记构建体靶向剩余的功能等位基因。替换可能高度偏向于一个等位基因。在PHR1位点，杂合突变体中破坏的等位基因与功能等位基因的替换偏好约为50倍。当转化DNA缺乏hisG重复序列时，这种偏好降低了6至8倍。尽管如此，靶向PHR1特定等位基因的偏好仍然存在6倍，甚至在含有PHR1两个野生型等位基因的亲本菌株转化中也很明显。克隆了两个野生型等位基因并进行核苷酸序列比较，发现在2 kb区域内有24个异源序列。利用限制性位点多态性来区分等位基因，观察到用等位基因PHR1-1的克隆DNA转化优先靶向基因组的等位基因1。用PHR1-2转化表现出相反的特异性。在这两种情况下，转化DNA的侧翼区域都存在异源序列。当从两个等位基因100%相同的区域选择转化DNA时，等位基因1和2被靶向的频率相等。结论是等位基因之间的序列异源性导致了靶向重组事件中固有的等位基因特异性。
