Cassady A I, Stacey K J, Nimmo K A, Murphy K M, von der Ahe D, Pearson D, Botteri F M, Nagamine Y, Hume D A
Centre for Molecular Biology and Biotechnology, University of Queensland, Brisbane, Australia.
Nucleic Acids Res. 1991 Dec 25;19(24):6839-47. doi: 10.1093/nar/19.24.6839.
The 5' flanking regions of the mouse and pig urokinase plasminogen activator (uPA) genes were sequenced and sequence homology interrupted by repeat elements was found to extend to -4.6kb in pig and -6.6kb in mouse. A transient transfection procedure was devised for the murine macrophage cell line RAW264. Pig uPA promoter-CAT constructs were more active than mouse constructs in this assay. This contrast may involve sequence differences within 100 bp of the transcription start site. The selective deletion of distal regions of the promoter (greater than 2.6 kb upstream), and of a conserved element, 5'-AGGAGGAAATGAGG-TCA-3' around -2 kb greatly reduced the activity of reporter constructs in RAW264 cells. Electrophoretic mobility shift assays using the latter sequence identified a single nuclear protein complex. This element has been referred to as PEA3/AP1-like, but the complex did not comigrate with either AP1 or known proteins that bind polypurines (including the macrophage-specific factor PU-1) and was not competed by AP1 or polypurine oligonucleotides. uPA promoters contain multiple AP1 and AP2-like DNA sequences, which were recognised by nuclear proteins expressed constitutively in RAW264 cells. They also contain multiple binding sites for NF kappa B but activated NF kappa B was not expressed in RAW264 cells. The conserved, transcribed 5' non-coding sequences were also required for maximal gene expression. Hence, the uPA promoter contains multiple weak cis-acting elements distributed over 7.0 kb 5' to the translation start site.
对小鼠和猪尿激酶型纤溶酶原激活剂（uPA）基因的5'侧翼区域进行了测序，发现被重复元件打断的序列同源性在猪中延伸至-4.6kb，在小鼠中延伸至-6.6kb。为小鼠巨噬细胞系RAW264设计了一种瞬时转染程序。在该测定中，猪uPA启动子-CAT构建体比小鼠构建体更具活性。这种差异可能涉及转录起始位点100bp内的序列差异。启动子远端区域（上游大于2.6kb）以及-2kb左右的保守元件5'-AGGAGGAAATGAGG-TCA-3'的选择性缺失大大降低了RAW264细胞中报告基因构建体的活性。使用后一种序列进行的电泳迁移率变动分析鉴定出一种单核蛋白复合物。该元件被称为PEA3/AP1样元件，但该复合物与AP1或结合多聚嘌呤的已知蛋白质（包括巨噬细胞特异性因子PU-1）不共迁移，并且不受AP1或多聚嘌呤寡核苷酸的竞争。uPA启动子包含多个AP1和AP2样DNA序列，它们被RAW264细胞中组成性表达的核蛋白识别。它们还包含多个NF-κB结合位点，但RAW264细胞中未表达活化的NF-κB。保守的转录5'非编码序列对于最大基因表达也是必需的。因此，uPA启动子包含多个分布在翻译起始位点5'上游7.0kb的弱顺式作用元件。
