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首页> 外文期刊>Molecular and Cellular Biology >Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants.
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Directed mutagenesis in Candida albicans: one-step gene disruption to isolate ura3 mutants.

机译：定向诱变在白色念珠菌中：一步基因破坏以分离ura3突变体。

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A method for introducing specific mutations into the diploid Candida albicans by one-step gene disruption and subsequent UV-induced recombination was developed. The cloned C. albicans URA3 gene was disrupted with the C. albicans ADE2 gene, and the linearized DNA was used for transformation of two ade2 mutants, SGY-129 and A81-Pu. Both an insertional inactivation of the URA3 gene and a disruption which results in a 4.0-kilobase deletion were made. Southern hybridization analyses demonstrated that the URA3 gene was disrupted on one of the chromosomal homologs in 15 of the 18 transformants analyzed. These analyses also revealed restriction site dimorphism of EcoRI at the URA3 locus which provides a unique marker to distinguish between chromosomal homologs. This enabled us to show that either homolog could be disrupted and that disrupted transformants of SGY-129 contained more than two copies of the URA3 locus. The A81-Pu transformants heterozygous for the ura3 mutations were rendered homozygous and Ura- by UV-induced recombination. The homozygosity of a deletion mutant and an insertion mutant was confirmed by Southern hybridization. Both mutants were transformed to Ura+ with plasmids containing the URA3 gene and in addition, were resistant to 5-fluoro-orotic acid, a characteristic of Saccharomyces cerevisiae ura3 mutants as well as of orotidine-5'-phosphate decarboxylase mutants of other organisms.
机译：开发了一种通过一步基因破坏和随后的紫外线诱导的重组将特异性突变引入二倍体白色念珠菌的方法。克隆的白色念珠菌URA3基因被白色念珠菌ADE2基因破坏，线性化的DNA用于转化两个ade2突变体SGY-129和A81-Pu。进行URA3基因的插入失活和导致4.0-碱基碱基缺失的破坏。 Southern杂交分析表明，在所分析的18个转化子中的15个中，URA3基因在一个染色体同源物中被破坏。这些分析还揭示了在URA3基因座处EcoRI的限制性位点二态性，其提供了区分染色体同源物的独特标记。这使我们能够证明，任一同源物均可能被破坏，而SGY-129的破坏转化子含有两个以上的URA3基因座拷贝。 ura3突变杂合的A81-Pu转化子通过UV诱导的重组变成纯合的，而Ura-则变成纯合的。通过Southern杂交证实了缺失突变体和插入突变体的纯合性。用含有URA3基因的质粒将两个突变体均转化为Ura +，此外，它们对5-氟乳清酸具有抗性，这是酿酒酵母ura3突变体以及其他生物体的orotidine-5'-磷酸脱羧酶突变体的特征。

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