Punt P J, Greaves P A, Kuyvenhoven A, van Deutekom J C, Kinghorn J R, Pouwels P H, van den Hondel C A
TNO Medical Biological Laboratory, Rijswijk, The Netherlands.
Gene. 1991 Jul 31;104(1):119-22. doi: 10.1016/0378-1119(91)90476-r.
To analyze the promoter region(s) of divergently transcribed fungal genes, a twin-reporter vector was constructed. This vector contains two divergently oriented reported genes, encoding Escherichia coli beta-glucuronidase (uidA) and E. coli beta-galactosidase (lacZ). Terminator regions of the Aspergillus nidulans nitrate and nitrite reductase-encoding genes, niaD and niiA, respectively, have been cloned 3' to the reporter genes to ensure proficient transcription termination of the reporter genes. The reporter genes have been separated by a unique NotI restriction site, which can be used for the insertion of expression signals. A mutant argB selection marker has been introduced in order to obtain A. nidulans transformants with a single copy of the vector integrated at the argB locus. The use of the vector was demonstrated by insertion of the A. nidulans niaD-niiA intergenic region and analysis of A. nidulans transformants obtained with this construct. Control of expression of both reporter genes was found to be in accordance with previously published data on control of nitrate assimilation [Cove, Biol. Rev. 54 (1979) 291-327].
为了分析反向转录的真菌基因的启动子区域，构建了一个双报告载体。该载体包含两个反向排列的报告基因，分别编码大肠杆菌β-葡萄糖醛酸酶（uidA）和大肠杆菌β-半乳糖苷酶（lacZ）。构巢曲霉硝酸盐和亚硝酸盐还原酶编码基因niaD和niiA的终止子区域已分别克隆到报告基因的3'端，以确保报告基因的转录有效终止。报告基因被一个独特的NotI限制性酶切位点隔开，该位点可用于插入表达信号。引入了一个突变的argB选择标记，以获得在argB位点整合有单拷贝载体的构巢曲霉转化体。通过插入构巢曲霉niaD-niiA基因间区域并分析用该构建体获得的构巢曲霉转化体，证明了该载体的用途。发现两个报告基因的表达调控与先前发表的关于硝酸盐同化调控的数据一致[科夫，《生物学评论》54（1979）291 - 327]。
