Lachke Salil A, Srikantha Thyagarajan, Soll David R
Department of Biological Sciences, Rm 302 BBE, The University of Iowa, Iowa City, IA 52242, USA.
Mol Microbiol. 2003 Apr;48(2):523-36. doi: 10.1046/j.1365-2958.2003.t01-1-03448.x.
EFG1, which encodes a trans-acting factor, is expressed as a more abundant 3.2 kb transcript in the white phase and as a less abundant 2.2 kb transcript in the opaque phase of the white-opaque transition in Candida albicans. To understand how alternative phase-specific mRNAs are transcribed from the same gene locus, the 2320 bp upstream region of the gene was functionally characterized by analysing the -activity of deletion derivatives in a luciferase-based reporter system. The white phase-specific promoter contained three discrete sequences involved in white phase-specific activation, between -2022 and -1809 bp (AR1), between -1809 and -1727 bp (AR2) and between -922 and -840 bp (AR3). A higher resolution deletion and mutation analysis of AR2 revealed two regions between -1809 and -1787 bp and between -1764 and -1728 bp that are responsible for AR2 activation. Targeting of promoter constructs to the ectopic ADE2 genomic site and the 3' end of the EFG1 genomic site revealed a positional requirement for white phase-regulated activation specific for the AR2 region of the promoter. Gel mobility shift assays using AR2 revealed a white phase-specific activation complex. No discrete activation sequences were identified in the overlapping promoter of the opaque phase-specific EFG1 transcript. The strength of opaque phase activation was directly proportional to the length of the promoter. Northern analysis excluded the possibility of an opaque phase-specific repressor. These results demonstrate overlapping promoters for white and opaque phase-specific expression of the gene for the transcription factor Efg1, with distinctly different mechanisms of phase-specific activation.
EFG1编码一种反式作用因子，在白色念珠菌白色-不透明转变的白色期，它以更丰富的3.2 kb转录本形式表达，而在不透明期则以较少丰富的2.2 kb转录本形式表达。为了了解同一基因座如何转录出不同的阶段特异性mRNA，通过在基于荧光素酶的报告系统中分析缺失衍生物的活性，对该基因上游2320 bp区域进行了功能表征。白色期特异性启动子包含三个参与白色期特异性激活的离散序列，分别位于-2022至-1809 bp（AR1）、-1809至-1727 bp（AR2）和-922至-840 bp（AR3）之间。对AR2进行更高分辨率的缺失和突变分析，发现了-1809至-1787 bp以及-1764至-1728 bp之间的两个区域负责AR2的激活。将启动子构建体靶向异位的ADE2基因组位点和EFG1基因组位点的3'端，揭示了启动子AR2区域白色期调控激活的位置要求。使用AR2进行的凝胶迁移率变动分析揭示了一个白色期特异性激活复合物。在不透明期特异性EFG1转录本的重叠启动子中未鉴定到离散的激活序列。不透明期激活的强度与启动子的长度成正比。Northern分析排除了存在不透明期特异性阻遏物的可能性。这些结果表明，转录因子Efg1的基因存在白色期和不透明期特异性表达的重叠启动子，且阶段特异性激活机制明显不同。
