Abstract
Neuronal nitric oxide (NO) synthase, localized to human chromosome 12, uniquely participates in diverse biologic processes; neurotransmission, the regulation of body fluid homeostasis, neuroendocrine physiology, control of smooth muscle motility, sexual function, and myocyte/myoblast biology, among others. Restriction enzyme mapping, subcloning, and DNA sequence analysis of bacteriophage- and yeast artificial chromosome-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 transcription initiation sites in brain poly(A) RNA with primer extension analysis and RNase protection revealed a major start site 28 nucleotides downstream from a TATA box. Sequence inspection of 5'-flanking regions revealed potential cis-acting DNA elements: AP-2, TEF-1/MCBF, CREB/ATF/c-Fos, NRF-1, Ets, NF-1, and NF-kappa B-like sequences. Diversity appears to represent a major theme apparent upon analysis of human neuronal NO synthase mRNA transcripts. A microsatellite of the dinucleotide variety was detected within the 3'-untranslated region of exon 29. Multiple alleles were evident in normal individuals indicating the existence of allelic mRNA sequence variation. Characterization of variant human neuronal NO synthase cDNAs indicated the existence of casette exon 9/10 and exon 10 deletions as examples of structural mRNA diversity due to alternative splicing. The latter deletion of a 175-nucleotide exon introduces a frame-shift and premature stop codon indicating the potential existence of a novel NH2 terminus protein. In summary, analysis of the human neuronal NO synthase locus reveals a complex genomic organization and mRNA diversity that is both allelic and structural.
Highlights
From the Renal Division and Department of Medicine, St
Restriction enzyme mapping, subcloning,and DNA sequence analysis of bacteriophage- and yeast artificial chromosomederived human genomic DNA indicated that the mRNA for neuronal nitric oxide (NO) synthase is dispersed over a minimum of 160kilobases of human genomic DNA
Neuronal Nitric Oxide Synthase 3'-cDNA-A recombinant bacteriophage clone was isolated by plaque hybridization from a n oligo(dT)
Summary
Neuronal Nitric Oxide Synthase 3'-cDNA-A recombinant bacteriophage clone was isolated by plaque hybridization from a n oligo(dT)-. Heteroduplexes were digested with RNase Ai"1 a [a32PldCTP-labeled990-nt genomic BamHIIEcoRI restriction enzyme (Ambion Inc., Austin,TX)at 37 "C for 30 min. RNase-digested products fragment of bacteriophage clone NOSl A50. This restriction fragment were heat-denatured analyzed on 4% denaturing polyacrylamide corresponded to3'-UTR sequences fromexon 29 of human neuronalNO gels adjacent to sequencing ladders derived from the genomic DNA synthase. 7.0), 65 "C with final post-hybridization washes performeadt 0.5 x SSC, [a3'P]UTP-labeled RNase-protected fragment VCS M13 helper phage allowed in vitro phagemid rescue of a GGT CGT-3') This allowed size comparison of RNase-protected frag-.
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