Lin C S, Lau A, Tu R, Lue T F
Knuppe Molecular Urology Laboratory, Department of Urology, University of California, San Francisco, California 94143-1695, USA.
Biochem Biophys Res Commun. 2000 Feb 16;268(2):596-602. doi: 10.1006/bbrc.2000.2186.
In the accompanying paper we present evidence for the existence of three PDE5A isoforms that differed only in the 5' end of the mRNAs. In this paper we present evidence that the three isoform-specific 5' ends were encoded by three alternative first exons that were arranged in the order of A1-A3-A2. Because the isoform-specific mRNAs could be transcribed from individual promoters, DNA fragments of the two intronic regions (A1-A3 and A3-A2) were tested for possible promoter activities. The intron between A1- and A3-specific exons did not exhibit any promoter activities even in smooth muscle cells that expressed the A3 isoform (see accompanying paper). In contrast, the intron between A3- and A2-specific exons had promoter activities in PDE5A2-expressing COS-7 and smooth muscle cells. This intronic promoter was bound by transcription factors AP-2 and Sp1, but not by AP-1, as shown by DNase I footprint analysis. However, the sequence bound by AP-2 (5'-GGGAAACGCTCGCGGGAGAGTTGG) is unusual in that it bears little resemblance to the consensus AP-2-binding sequence.
在随附论文中，我们提供了存在三种仅在mRNA的5'端有所不同的PDE5A亚型的证据。在本文中，我们提供证据表明这三种亚型特异性的5'端由三个按A1 - A3 - A2顺序排列的可变第一外显子编码。由于亚型特异性mRNA可能从单个启动子转录，因此对两个内含子区域（A1 - A3和A3 - A2）的DNA片段进行了可能的启动子活性测试。A1和A3特异性外显子之间的内含子即使在表达A3亚型的平滑肌细胞中也未表现出任何启动子活性（见随附论文）。相反，A3和A2特异性外显子之间的内含子在表达PDE5A2的COS - 7细胞和平滑肌细胞中具有启动子活性。如DNase I足迹分析所示，该内含子启动子与转录因子AP - 2和Sp1结合，但不与AP - 1结合。然而，AP - 2结合的序列（5'-GGGAAACGCTCGCGGGAGAGTTGG）不同寻常，因为它与AP - 2结合序列的共有序列几乎没有相似之处。
