2 CLOSELY LINKED BUT SEPARABLE PROMOTERS FOR HUMAN NEURONAL NITRIC-OXIDE SYNTHASE GENE-TRANSCRIPTION

In this report we demonstrate that the human cerebellum contains neuro nal nitric oxide synthase (nNOS) mRNAs with two distinct 5'-untranslat ed regions that are encoded through use of closely linked but separate promoters. nNOS cDNA clones were shown to contain different 5' termin al exons spliced to a common exon 2. Genomic cloning and sequence anal ysis demonstrate that the unique exons are positioned within 300 bp of each other but separated from exon 2 by an intron that is at least 20 kb in length. A CpG island engulfs the downstream 5'-terminal exon. I n contrast, most of the upstream exon resides outside of this CpG isla nd. Interestingly, the upstream exon includes a GT dinucleotide repeat . A fusion gene with a 414-bp nNOS genomic fragment that includes a po rtion of the upstream 5'-terminal exon and its immediate 5'-flanking D NA is expressed in transfected HeLa cells. Also expressed is a fusion gene that contains the luciferase reporter under transcriptional contr ol by a 308-bp genomic fragment that includes the region separating bo th d'-terminal exons. These results indicate that expression of these exons is subject to transcriptional control by separate promoters. How ever, the proximity of these promoters raise the possibility that comp lex interactions may be involved in regulating nNOS gene expression at these sites.