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Evaluation of the CaMAL2 promoter for regulated expression of genes in Candida albicans

机译：CaMAL2启动子在白色念珠菌中调控基因表达的评估

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摘要

An expression vector (CIpI0-MAL2p) for Use in Candida albicans has been constructed in which a gene of interest can be placed under the control of the CaMAL2 maltase promoter and stably integrated at the CaRPIO locus. Using this vector to express the Candida URA3 gene from the CaMAL2 promoter, we have demonstrated tight regulation of CaURA3 expression by carbon source. Thus under conditions when the CaMAL2 promoter is not induced, expression of Candida URA3 was unable either to complement a C. albicans ura3 mutation or to confer sensitivity to 5-fluoroorotic acid, a compound which is highly toxic to URA3 strains. Since Candida albicans is an obligate diploid organism, analysis of gene function requires manipulation of both copies of any gene of interest. Our expression vector provides a strategy by which the remaining copy of a gene of interest can be placed under CaMAL2 promoter control in a strain where the first copy has been deleted, permitting analysis of gene function by manipulation of carbon source. Clpl0-MAL2p should therefore provide a useful means for functional analysis of genes in C. albicans. We have used this strategy with' C. albicans DPB2 to demonstrate that the gene is essential and that loss of function leads cells to adopt a hypha-like morphology as they cease proliferation.
机译：已经构建了用于白色念珠菌的表达载体（CIp10-MAL2p），其中可以将感兴趣的基因置于CaMAL2麦芽糖酶启动子的控制下，并在CaRPIO基因座处稳定整合。使用该载体从CaMAL2启动子表达假丝酵母URA3基因，我们证明了碳源对CaURA3表达的严格调控。因此，在不诱导CaMAL2启动子的条件下，假丝酵母​​URA3的表达不能补充白色念珠菌ura3突变或不能赋予对5-氟乳清酸的敏感性，该化合物对URA3菌株具有高毒性。由于白色念珠菌是专性二倍体生物，对基因功能的分析需要操纵任何目的基因的两个拷贝。我们的表达载体提供了一种策略，通过该策略，可以将感兴趣的基因的其余副本置于已删除第一个副本的菌株中的CaMAL2启动子控制下，从而允许通过操纵碳源来分析基因功能。因此，C1010-MAL2p应该提供用于白念珠菌基因功能分析的有用手段。我们已将此策略与白色念珠菌DPB2一起使用以证明该基因是必不可少的，并且功能丧失会导致细胞在停止增殖时采取类似菌丝的形态。

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