Allele-specific gene targeting in Candida albicans results from heterology between alleles.
Allele-specific gene targeting in Candida albicans results from heterology between alleles.
复制标题
白色念珠菌中的等位基因特异性基因靶向源于等位基因之间的异源性。
DOI：
10.1099/00221287-146-9-2097
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发表时间：
2000
期刊：
影响因子：
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通讯作者：
Fonzi,WilliamA
中科院分区：
文献类型：
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作者：
Yesland,Kyle;Fonzi,WilliamA
条件致病真菌白色念珠菌是无性和二倍体。因此，隐性突变的引入需要两个等位基因的靶向基因置换以实现隐性表型的表达。这通常是通过回收aURA 3标记基因来实现的，该标记基因的侧翼是hisG的直接重复序列。靶向某个基因座后，重复序列之间的重组切除URA 3，在被破坏的等位基因中留下一个hisG拷贝。剩余的功能性等位基因在第二次转化中用相同的URA 3标记的构建体靶向。替换可以高度偏向于一个等位基因。在PHR 1基因座上，杂合突变体中替换破坏的等位基因与替换功能性等位基因的偏好大约是50倍。当转化的DNA缺少这些“他的伟大”时，这种偏好性降低了六到八倍。尽管如此，仍然有一个六倍的偏好，针对一个特定的等位基因ofPHR 1，这是显而易见的，即使在转化的亲本菌株含有两个野生型等位基因ofPHR 1。这两个野生型等位基因克隆和核苷酸序列比较发现24个异质性超过2 kb的区域。利用限制性酶切位点多态性来区分等位基因，观察到用等位基因PHR 1 - 1的克隆DNA转化优先靶向基因组的等位基因1。PHR 1 - 2转化具有相反的特异性。在这两种情况下，异质性存在于转化DNA的侧翼区域。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以相等的频率被靶向。可以得出结论，等位基因之间的序列异质性导致靶向重组事件中固有的等位基因特异性。
The opportunistic fungal pathogenCandida albicansis 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 aURA3marker gene that is flanked by direct repeats ofhisG. After targeting to a locus, recombination between the repeats excisesURA3leaving a single copy ofhisGin the disrupted allele. The remaining functional allele is targeted in a second transformation with the sameURA3marked construct. Replacement can be highly biased toward one allele. At thePHR1locus, 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 thehisGrepeats. Nonetheless, there remained a sixfold preference for targeting a particular allele ofPHR1and this was evident even in transformations of the parental strain containing two wild-type alleles ofPHR1. 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 allelePHR1-1preferentially targeted allele 1 of the genome. Transformations withPHR1-2exhibited 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.