Cloning and structure of a rabbit protein inhibitor of neuronal nitric oxide synthase (PIN) gene and its pseudogene

Neuronal nitric oxide synthase (nNOS), the biosynthetic enzyme for the free radical neurotransmitter nitric oxide, was found to be inhibited by the protein inhibitor of nNOS, designated PIN (Jaffrey, S.R. and Snyder, S.H., (1996) Science 274, 774-777). The cDNA clone encoding a rabbit PIN was isolated, and the translated PIN protein was 89 amino acid long sharing 100% of its deduced amino acid sequence with rat PIN. Using a radiolabelled riboprobe derived from the rabbit PIN cDNA, the rabbit PIN gene was isolated from a rabbit genomic library and the structural organization of the gene was determined. The gene contains three exons separated by two introns spanning approx. 2.3 kb of genomic DNA. 5' RACE and primer extension analysis mapped the transcriptional initiation site 98 bp upstream of the initiator methionine codon. Characterization of the 5' flanking genomic region revealed that the rabbit PIN promoter is TATA-less, but contains a CCAAT box as well as various putative transcription factor-binding elements. The 3' untranslated region contains consensus polyadenylation signal (AATAAA). We also isolated an intronless gene with 93% nucleotide sequence similarity to the rabbit PIN cDNA. Sequence analysis indicates that the open reading frame was interrupted by a premature stop codon and frameshift which resulted in a processed pseudogene of the rabbit PIN. (C) 1998 Elsevier Science B.V. All rights reserved.