IDENTIFICATION OF MULTIPLE DNA ELEMENTS REGULATING BASAL AND PROTEIN-KINASE A-INDUCED TRANSCRIPTIONAL EXPRESSION OF THE RAT PRODYNORPHIN GENE

The prodynorphin gene encodes the precursor molecule from which the dy norphin family of opioid peptides is generated. The gene is transcript ionally active in a wide variety of brain regions and endocrine tissue s. Much is known regarding the physiological and receptor-mediated eve nts that regulate prodynorphin gene expression in vivo. However, the m olecular mechanisms by which specific cis- and trans-acting factors co ntrol activity of the prodynorphin promoter are not as clearly defined . In the study described here, transient transfection of prodynorphin promoter-chloramphenicol acetyl transferase plasmid constructs into CV 1 cells served to identify three nucleotide sequence elements conformi ng to cAMP regulatory element motifs which regulate both basal and pro tein kinase A (PKA)-induced transcription. The three elements are clus tered at positions -1543, -1627, and -1659 relative to the RNA cap sit e. Site-specific mutagenesis further reveals that although the sites c an act independently to positively regulate transcription from the pro dynorphin promoter, they can also act combinatorially to produce maxim al transcriptional efficacy. Gel retention analysis employing rat brai n protein extracts also describes the ability of these sequence elemen ts to form sequence-specific DNA/protein complexes.