CHARACTERIZATION OF THE PROMOTER REGION OF THE RAT NEPRILYSIN GENE

The neprilysin gene is composed of three distinct 5' noncoding exons w hich can be joined to the first coding exon to generate multiple mRNA species, all encoding the same protein. Genomic fragments containing u pstream sequences of each of these three noncoding exons from the rat neprilysin gene were subcloned in the promoterless vector pXp1, which contains the luciferase reporter gene. Expression was compared between a neprilysin positive human spinal cord cell line, HSC-2, and neprily sin negative lines MCF-7, a human breast adenocarcinoma cell line and Hep G2, a human liver carcinoma cell line. The first and second promot er regions showed high activity in the positive cell line, but low act ivity in the negative cell lines. An analysis of the exon 1 promoter r egion showed that the proximal 85 nucleotides exhibited basal promoter activity. An enhancer-like sequence was found to be located within a 22-bp fragment located at -136 to -115. Scanning mutagenesis of a 29-b p fragment containing the enhancer-like sequence showed that changes i n each 5- or 6-bp segment throughout this fragment decreased activity; however, mutations of the segment encompassing positions 19 to 24 eli minated >98% of the promoter activity. Binding of nuclear proteins fro m HSC-2 cells to this 29-bp fragment was observed by gel shift analysi s. The ability of mutations within the 29-bp fragment to affect enhanc er activity correlated with the ability of these mutant oligonucleotid es to compete for the wild-type sequence in gel shift assays. (C) 1998 Academic Press.