5′-Flanking Sequence and Promoter Activity of the Rabbit Neuronal Nitric Oxide Synthase (nNOS) Gene

Abstract

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 + 1 start, various putative cis-elements including AP-1, AP-4, NF-KB, 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 β-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.