A novel cAMP-response element, CRE1, modulates expression of nor-1 in Aspergillus parasiticus
A novel cAMP-response element, CRE1, modulates expression of nor-1 in Aspergillus parasiticus
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DOI：
10.1074/jbc.m400075200
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发表时间：
2004-06-25
影响因子：
4.8
通讯作者：
Linz, JE
中科院分区：
文献类型：
--
作者：
Roze, LV;Miller, MJ;Linz, JE
丝状真菌寄生曲霉中黄曲霉毒素的积累水平受到多种环境因素的调节。液体和固体葡萄糖最低盐（GMS）生长培养基中存在的葡萄糖（优选的碳源）强烈刺激黄曲霉毒素积累。蛋白胨（非首选碳源）在蛋白胨最低盐（PMS）培养基刺激只有低水平的黄曲霉毒素积累。葡萄糖刺激的黄曲霉毒素结构基因ver-1和nor-1的转录类似的中间水平，在液体GMS，而在固体培养基上，ver-1的转录被刺激到20倍以上的水平比nor-1。PMS液体和固体培养基刺激非常低或不可检测水平的两种基因的转录。电泳迁移率变动分析使用nor-1启动子片段（norR）和A.寄生虫细胞蛋白提取物揭示了不同的GMS和PMS固体和液体培养基上的流动性的特定的DNA-蛋白质复合物。一个不完善的cAMP反应元件，CRE 1，被确定在norR介导的特定的DNA-蛋白质复合物的形成。CRE 1或AflR 1（AflR顺式作用位点）的突变导致cAMP介导的nor-1启动子活性在GMS琼脂上的刺激降低高达3倍。South-Western印迹分析确定了一个32 kDa的蛋白，特异性结合norR。p32可以被抗AflR抗体免疫共沉淀，并与AflR-麦芽糖结合蛋白融合物共纯化，证明AflR和p32之间在体外的物理相互作用。我们假设，p32协助AflR在绑定到nor-1启动子，从而调节nor-1基因表达响应环境的线索。
The level of aflatoxin accumulation in the filamentous fungus Aspergillus parasiticus is modulated by a variety of environmental cues. The presence of glucose ( a preferred carbon source) in liquid and solid glucose minimal salts (GMS) growth media strongly stimulated aflatoxin accumulation. Peptone ( a non-preferred carbon source) in peptone minimal salts (PMS) media stimulated only low levels of aflatoxin accumulation. Glucose stimulated transcription of the aflatoxin structural genes ver-1 and nor-1 to similar intermediate levels in liquid GMS, while on solid media, ver-1 transcription was stimulated to 20-fold higher levels than nor-1. PMS liquid and solid media stimulated very low or non-detectable levels of transcription of both genes. Electrophoretic mobility shift analysis using a nor-1 promoter fragment (norR) and A. parasiticus cell protein extracts revealed specific DNA-protein complexes of different mobility on GMS and PMS solid and liquid media. An imperfect cAMP-response element, CRE1, was identified in norR that mediated formation of the specific DNA-protein complexes. Mutation in CRE1 or AflR1 (AflR cis-acting site) caused up to a 3-fold decrease in cAMP-mediated stimulation of nor-1 promoter activity on GMS agar. South-Western blot analysis identified a 32-kDa protein that specifically bound to norR. p32 could be co-immunoprecipitated by anti-AflR antibody and co-purified with an AflR-maltose-binding protein fusion demonstrating a physical interaction between AflR and p32 in vitro. We hypothesize that p32 assists AflR in binding to the nor-1 promoter, thereby modulating nor-1 gene expression in response to environmental cues.