Jeong Y, Won J, Kim C, Yim J
National Creative Research Initiative Center for Genetic Reprogramming, Institute for Molecular Biology and Genetics, School of Biological Sciences, Seoul National University, Korea.
Mol Cells. 2000 Oct 31;10(5):566-74. doi: 10.1007/s10059-000-0566-7.
We have isolated a rabbit neuronal nitric oxide synthase (nNOS) cDNA encoding a protein of 1,435 amino acids. Using the cDNA clones as probes, the 5'-flanking region of the nNOS gene was isolated from a rabbit genomic DNA library. 5'RACE and primer extension analysis of rabbit brain total RNA mapped multiple transcription initiation sites localized 474-487 bp upstream from the translation start codon. Analysis of 5,197 bp of the 5'-flanking sequence revealed that the rabbit nNOS gene promoter lacks canonical TATA or CCAAT boxes and, instead, contains a GC-rich region and multiple Sp1 sites. Farther from the +1start, various putative cis-elements including AP-1, AP-4, NF-kappaB, STAT, CREB, C/EBP and c-Myc were observed. The functional promoter activity of the 5'-flanking region was demonstrated by its ability to drive the expression of a beta-galactosidase reporter gene in several cell types. Serial deletion analysis of the promoter region revealed that the -291 to -172 region, which contains two Sp1 sites, is essential for basal transcriptional activity. These results suggest that the rabbit nNOS promoter contains characteristics of inducible genes.
我们分离出了一个编码1435个氨基酸蛋白质的兔神经元型一氧化氮合酶（nNOS）cDNA。以该cDNA克隆为探针，从兔基因组DNA文库中分离出nNOS基因的5'侧翼区。对兔脑总RNA进行5'RACE和引物延伸分析，确定了多个转录起始位点，这些位点位于翻译起始密码子上游474 - 487 bp处。对5197 bp的5'侧翼序列分析表明，兔nNOS基因启动子缺乏典型的TATA或CCAAT框，取而代之的是一个富含GC的区域和多个Sp1位点。在离+1起始位点更远的地方，观察到了包括AP - 1、AP - 4、NF - κB、STAT、CREB、C/EBP和c - Myc等各种假定的顺式作用元件。5'侧翼区的功能性启动子活性通过其在几种细胞类型中驱动β - 半乳糖苷酶报告基因表达的能力得以证明。对启动子区域进行系列缺失分析表明，包含两个Sp1位点的 - 291至 - 172区域对于基础转录活性至关重要。这些结果表明兔nNOS启动子具有可诱导基因的特征。
