STRUCTURAL ORGANIZATION OF THE HUMAN NEURONAL NITRIC-OXIDE SYNTHASE GENE (NOS1)

Neuronal nitric oxide (NO) synthase, localized to human chromosome 12, uniquely participates in diverse biologic processes; neurotransmissio n, the regulation of body fluid homeostasis, neuroendocrine physiology , control of smooth muscle motility, sexual function, and myocyte/myob last biology, among others. Restriction enzyme mapping, subcloning, an d DNA sequence analysis of bacteriophage- and yeast artificial chromos ome-derived human genomic DNA indicated that the mRNA for neuronal NO synthase is dispersed over a minimum of 160 kilobases of human genomic DNA. Analysis of intron-exon splice junctions predicted that the open reading frame is encoded by 28 exons, with translation initiation and termination in exon 2 and exon 29, respectively. Determination of tra nscription initiation sites in brain poly(A) RNA with primer extension analysis and RNase protection revealed a major start site 28 nucleoti des downstream from a TATA box. Sequence inspection of 5'-flanking reg ions revealed potential cis-acting DNA elements: AP-2, TEF-1/MCBF, CRE B/ATF/c-Fos, NRF-1, Ets, NF-1, and NF-kappa B-like sequences. Diversit y appears to represent a major theme apparent upon analysis of human n euronal NO synthase mRNA transcripts. A microsatellite of the dinucleo tide variety was detected within the S'-untranslated region of exon 29 . Multiple alleles were evident in normal individuals indicating the e xistence of allelic mRNA sequence variation. Characterization of varia nt human neuronal NO synthase cDNAs indicated the existence of casette exon 9/10 and exon 10 deletions as examples of structural mRNA divers ity due to alternative splicing. The latter deletion of a 175-nucleoti de exon introduces a frameshift and premature stop codon indicating th e potential existence of a novel NH2 terminus protein. In summary, ana lysis of the human neuronal NO synthase locus reveals a complex genomi c organization and mRNA diversity that is both allelic and structural.