Punt P J, Dingemanse M A, Kuyvenhoven A, Soede R D, Pouwels P H, van den Hondel C A
TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
Gene. 1990 Sep 1;93(1):101-9. doi: 10.1016/0378-1119(90)90142-e.
Analysis of the promoter region of the highly expressed Aspergillus nidulans gpdA gene is described. The nucleotide (nt) sequence of a 1.3-kb region upstream from the ATG was determined. Comparison with promoter regions of other Aspergillus and Neurospora genes revealed several regions of similar sequence. Both random and site-specific mutations were introduced into the promoter region of the gpdA gene, and the resulting mutant promoters were fused to the Escherichia coli lacZ gene. The constructed fusions were introduced into A. nidulans and transformants that contained one copy of these fusions at the argB locus were analysed. beta-Galactosidase assays and primer extension experiments were used to identify sequence elements involved in transcription activation and transcription initiation. Two elements, located around 650 and 250 nt upstream from the major transcription start point (tsp), were identified as transcription activation elements. These elements coincide with regions of putative secondary structure (direct or inverted repeats). A third element, a C + T-rich region directly upstream from the major tsp, was shown to be involved in correct initiation of transcription.
本文描述了对高表达的构巢曲霉gpdA基因启动子区域的分析。测定了ATG上游1.3kb区域的核苷酸（nt）序列。与其他曲霉和脉孢菌基因的启动子区域比较，发现了几个序列相似的区域。将随机突变和位点特异性突变引入gpdA基因的启动子区域，并将所得的突变启动子与大肠杆菌lacZ基因融合。将构建的融合体导入构巢曲霉，并分析在argB位点含有一份这些融合体的转化体。使用β-半乳糖苷酶测定和引物延伸实验来鉴定参与转录激活和转录起始的序列元件。在主要转录起始点（tsp）上游约650和250nt处发现的两个元件被鉴定为转录激活元件。这些元件与推定的二级结构区域（直接或反向重复）一致。第三个元件，即主要tsp直接上游的富含C + T的区域，被证明参与转录的正确起始。
