ISOLATION AND CHROMOSOMAL LOCALIZATION OF THE HUMAN ENDOTHELIAL NITRIC-OXIDE SYNTHASE (NOS3) GENE

Nitric oxide (NO) is an important intercellular signaling molecule syn thesized in diverse human tissues by proteins encoded by a family of N O synthase (NOS) genes. The similarity of sequence and cofactor bindin g sites has suggested that the NOS genes may also be related to cytoch rome P450 reductase, as well as to plant and bacterial oxidoreductases . Endothelial NOS activity is a major determinant of vascular tone and blood pressure, and in several important (and sometimes hereditary) d isease states, such as hypertension, diabetes, and atherosclerosis, th e endothelial NO signaling system appears to be abnormal. To explore t he relationship of the endothelial NOS gene to other similar genes, an d to delineate the genetic factors involved in regulating endothelial NOS activity, we isolated the human gene encoding the endothelial NOS. Genomic clones containing the 5' end of this gene were identified in a human genomic library by applying a polymerase chain reaction (PCR)- based approach. Identification of the human gene for endothelial NOS ( NOS3) was confirmed by nucleotide sequence analysis of the first codin g exon, which was found to be identical to its cognate cDNA. The NOS3 gene spans at least 20 kb and appears to contain multiple introns. The transcription start site and promoter region of the NOS3 gene were id entified by primer extension and ribonuclease protection assays. Seque ncing of the putative promoter revealed consensus sequences for the sh ear stress-response element, as well as cytokine-responsive cis regula tory sequences, both possibly important to the roles played by NOS3 in the normal and the diseased cardiovascular system. We also mapped the chromosomal location of the NOS3 gene. First, a chromosomal panel of human-rodent somatic cell hybrids was screened using PCR with oligonuc leotide primers derived from the NOS3 genomic clone. The specificity o f the amplified PCR product was confirmed by human and hamster genomic DNA controls, as well as by Southern blot analysis, using the NOS3 cD NA as probe. Definitive chromosomal assignment of the NOS3 gene to hum an chromosome 7 was based upon 0% discordancy; fluorescence in situ hy bridization sublocalized the NOS3 gene to 7q36. The identification and characterization of the NOS3 gene may lead to further insights into h eritable disease states associated with this gene product. (C) 1994 Ac ademic Press,Inc.