The rate of biosynthesis of the catecholamines dopamine, norepinephrine and epinephrine, can be modulated by numerous neuronal, hormonal and environmental influences.
The aim of the experiments described in this proposal is to define and characterize the genetic regulatory elements for two catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH), the initial and rate-limiting enzyme, and dopamine beta- hydroxylase (DBH), the third enzyme of the pathway. In many tissues, including the adrenal medulla, sympathetic ganglia and locus ceruleus, TH and DBH are co-expressed and coordinately regulated by induction stimuli. The major focus of this project is to determine whether individual effectors which stimulate transcription from each gene do so through interactions at common or unique sites on that gene, and also whether the coordinated expression and modulation of activity of the TH and DBH genes is due to shared cis-acting genetic regulatory sites which are recognized by similar or identical trans-acting factors. Both cDNA and genomic clones for rat TH are already available, and will be used to define and characterize the regulatory elements of the TH gene required for stimulation of TH transcription by cyclic AMP, epidermal growth factor and cell density in pheochromocytoma cells. The 5' flanking sequences of the TH gene are ligated to a heterologous """"""""reporter"""""""" gene and the TH sequences are assayed for the ability to confer inducer responsiveness onto the reporter gene. The required sequence boundaries will be defined, and the distance and orientation specifities of these genetic elements will be further analyzed. A cDNA for rat DBH will be isolated by immunoselection from a cDNA library derived from a tumor of the adrenal medulla. The cDNA will be used to assay the levels of DBH RNA transcripts under conditions known to induce DBH enzyme activity in both tissue culture and intact animals. The cDNA will also be used to isolate genomic clones for DBH, and the genetic regulatory elements important for the specificity and modulation of DBH gene expression will be defined. The regulatory elements of the TH and DBH genes will be compared by DNA sequence analysis and also by determining whether these genetic elements interact with, and can complete for, similar trans-acting control factors.
