Staab Janet F, Bahn Yong-Sun, Sundstrom Paula
Department of Molecular Virology, Immunology and Medical Genetics, The Ohio State University, Columbus, OH 43210, USA.
Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA.
Microbiology (Reading). 2003 Oct;149(Pt 10):2977-2986. doi: 10.1099/mic.0.26445-0.
The authors have engineered plasmid constructs for developmental and constitutive expression of yeast-enhanced green fluorescent protein (yEGFP3) in Candida albicans. The promoter for the hyphae-specific gene Hyphal Wall Protein 1 (HWP1) conferred developmental expression of yEGFP3 in germ tubes and hyphae but not in yeasts or pseudohyphae when targeted to the ENO1 (enolase) locus in single copy. The pHWP1GFP3 construct allows for the easy visualization of HWP1 promoter activity in individual cells expressing true hyphae without having to prepare RNA for analysis. Constitutive expression of yEGFP was seen in all cell morphologies when the HWP1 promoter was replaced with the ENO1 promoter region. The use of the plasmids for expression of genes other than yEGFP3 was examined by substituting the putative C. albicans BCY1 (SRA1) gene, a component of the cAMP signalling pathway involved in yeast to hyphae transitions, for yEGFP3. Strains overexpressing BCY1 from the ENO1 promoter were inhibited in germ tube formation and filamentation in both liquid and solid media, a phenotype consistent with keeping protein kinase A in its inactive form by association with Bcy1p. The plasmids are suitable for studies of germ tube induction or assessing germ tube formation by measuring yEGFP3 expression, for inducible expression of genes concomitant with germ tube formation by the HWP1 promoter, for constitutive expression of genes by the ENO1 promoter, and for expressing yEGFP3 using a promoter of choice.
作者构建了质粒载体，用于在白色念珠菌中实现酵母增强型绿色荧光蛋白（yEGFP3）的发育性和组成型表达。菌丝特异性基因菌丝壁蛋白1（HWP1）的启动子，在单拷贝靶向烯醇酶1（ENO1）基因座时，能使yEGFP3在芽管和菌丝中实现发育性表达，但在酵母或假菌丝中不表达。pHWP1GFP3构建体能够轻松观察到单个表达真正菌丝的细胞中HWP1启动子的活性，而无需制备RNA进行分析。当用ENO1启动子区域替换HWP1启动子时，在所有细胞形态中均可见yEGFP的组成型表达。通过用假定的白色念珠菌BCY1（SRA）基因（参与酵母向菌丝转变的cAMP信号通路的一个组成部分）替换yEGFP3，研究了这些质粒用于表达yEGFP3以外基因的情况。从ENO1启动子过表达BCY1的菌株在液体和固体培养基中的芽管形成和菌丝形成均受到抑制，这一表型与通过与Bcy1p结合使蛋白激酶A保持无活性形式一致。这些质粒适用于通过测量yEGFP3表达来研究芽管诱导或评估芽管形成，适用于通过HWP1启动子在芽管形成时诱导基因表达，适用于通过ENO1启动子组成型表达基因，以及适用于使用选择的启动子表达yEGFP3。
