Aminopeptidase-A. II. Genomic cloning and characterization of the rat promoter

Aminopeptidase-A (APA) has a wide-spread tissue distribution consistent wit h a role in the metabolism of circulating or locally produced ANG II or CCK -8. APA is also highly expressed in pre-B lymphocytes, but its role in lymp hoid cell development is unknown. To begin to;understand the basis for cell -specific regulation of APA expression, we sought to clone and characterize the rat gene promoter. Screening of a rat genomic library with a partial r at APA cDNA resulted in isolation of a 12-kb clone found: to contain the fi rst exon and >3 kb of 5'-flanking sequencer Primer extension of rat kidney mRNA indicated that the major transcription start site was 312 bp upstream of the translation start codon and 22 bp downstream from a TATA box. Constr ucts containing portions of the 5'-flanking region placed upstream of a chl oramphenicol acetyltransferase reporter gene indicated that expression was cell specific and that high activity could be obtained with constructs cont aining as little as 110 bp of 5'-flanking region sequence. We further ident ified an upstream regulatory element between -1063 and -348 that suppressed transcription in a cell-specific manner. This element (termed upstream sup pressor of APA, or USA) also suppressed transcription of a heterologous pro moter. These results indicate that the organization and regulation of the r at APA is not consistent with it being a housekeeping gene and further sugg est that rat APA gene transcription might be regulated through the presence of a novel strong upstream suppressor element.