A single-transformation gene function test in diploid Candida albicans
A single-transformation gene function test in diploid Candida albicans
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DOI：
10.1128/jb.182.20.5730-5736.2000
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发表时间：
2000-10-01
影响因子：
3.2
通讯作者：
Mitchell, AP
中科院分区：
文献类型：
--
作者：
Enloe, B;Diamond, A;Mitchell, AP
真菌病原体白色念珠菌是天然的二倍体，目前的基因破坏策略需要两次连续的转化。这里我们描述了一种可选择两个拷贝的遗传结构(UAU1)。在单次转化后，将UAU1插入到一个基因组位置，允许选择具有两个插入副本的分离物。主要类型的分离体是那些携带纯合插入突变和等位基因三倍体的分离体，它们有两个插入等位基因和一个野生型等位基因。因此，通过纯合子和三倍体的聚合酶链式反应测试，可以快速区分非必需和必需基因图谱。我们发现，在ADE2、RIM20和YGR189这三个非必需基因座上可以分离到纯合子突变，而在两个基本基因座(SNF1和CDC28)上只发现等位基因三倍体。我们出人意料地分离到了CDC25处突变的纯合突变株；它们是有活力的，但在含血清的培养液中有缺陷。因此，UAU1盒对于快速评估白色念珠菌基因的重要性是有用的。
The fungal pathogen Candida albicans is naturally diploid, and current gene disruption strategies require two successive transformations. We describe here a genetic construct (UAU1) for which two copies may be selected. Insertion of UAU1 into one genomic site, after a single transformation, allows selection for segregants with two copies of the insertion. Major classes of segregants are those carrying homozygous insertion mutations and allelic triplications, which have two insertion alleles and a wild-type allele. Thus nonessential and essential genes map be distinguished rapidly through PCR tests for homozygosis and triplication. We find that homozygous mutations may be isolated at three nonessential loci (ADE2, RIM20, and YGR189), while only allelic triplications were found at two essential loci (SNF1 and CDC28). We have unexpectedly isolated homozygous mutants with mutations at CDC25; they are viable but defective in filamentation on serum-containing medium. The UAU1 cassette is thus useful to assess rapidly the essentiality of C. albicans genes.