Stable transformation and regulated expression of an inducible reporter construct in Candida albicans using restriction enzyme mediated integration
Stable transformation and regulated expression of an inducible reporter construct in Candida albicans using restriction enzyme mediated integration
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DOI：
10.1007/bf02174347
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发表时间：
1996-04-24
期刊：
影响因子：
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通讯作者：
Kumamoto, CA
中科院分区：
文献类型：
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作者：
Brown, DH;Slobodkin, IV;Kumamoto, CA
为了调控克隆基因在白色念珠菌中的表达，利用白色念珠菌MAL2基因的诱导启动子构建了质粒。为了证明MAL2启动子可以调控置于其控制下的克隆基因，我们与白色念珠菌URA3基因的编码序列进行了融合构建。利用限制性内切酶介导整合（REMI）的方法将该质粒导入一株Ura（-）白色念珠菌中。该过程涉及BamHI线性化质粒在BamHI酶存在下的转化。产生的大多数转化子在染色体BamHI位点含有质粒的插入。通过生长分析和URA3基因产物活性水平测定，所有转化体均可诱导URA3表达。在非选择条件下，转化子的Ura（+）表型是稳定的。该系统在白色念珠菌中具有转化稳定、整合DNA易于恢复、基因可诱导表达等优点。
To allow the regulated expression of cloned genes in Candida albicans, a plasmid was constructed using the inducible promoter of the C. albicans MAL2 gene. To demonstrate that the MAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of the C. albicans URA3 gene. This plasmid was introduced into a Ura(-) strain of C. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of the BamHI-linearized plasmid in the presence of BamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomal BamHI sites. All transformants examined were inducible for URA3 expression, which was determined by growth analysis and by measuring the level of URA3 gene product activity. The Ura(+) phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes in C. albicans.