5 '-flanking sequence and promoter activity of the rabbit neuronal nitric oxide synthase (nNOS) gene

We have isolated a rabbit neuronal nitric oxide synthase (nNOS) cDNA encodi ng 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 lib rary. 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 seque nce revealed that the rabbit nNOS gene promoter lacks canonical TATA or CCA AT boxes and, instead, contains a GC-rich region and multiple Sp1 sites. Fa rther from the +1 start, various putative cis-elements including AP-1, AP-4 , NF-kappaB, STAT, CREB, C/EBP and c-Myc were observed. The functional prom oter activity of the 5'-flanking region was demonstrated by its ability to drive the expression of a P-galactosidase reporter gene in several cell typ es. Serial deletion analysis of the promoter region revealed that the -291 to -172 region, which contains two Sp1 sites, is essential for basal transc riptional activity, These results suggest that the rabbit nNOS promoter con tains characteristics of inducible genes.