The broad goal of this research is to understand the specific regulation of the expression of the gene for the cytosolic form of the enzyme P-enolpyruvate carboxy-kinase (PEPCK) from the rat. This involves determining the functional organization of the promoter-regulatory region of the gene to better define the mechanisms responsible for its acute responsiveness to gene transcription to cAMP, glucocorticoids and insulin. Understanding the regulated expression of the PEPCK gene will provide insight into the patterning of metabolic processes in different tissues as well as mechanism of action of regulatory hormones and their intracellular signals. The specific goals of this research will be to: 1.Determine the function of transcriptional regulatory elements identified within the PEPCK promoter and correlate this function with the cellular effect of hormones such as glucagon (acting via cAMP), insulin and glucocorticoids. 2.Identify sequences in the promoter which are responsible for directing the expression of the gene for PEPCK to the liver and kidney in transgenic animals. This is a necessary first step toward the identification of trans- acting factors which pattern the metabolic profile characteristic of specific tissues. 3.Isolate proteins which bind to specific domains within the PEPCK promoter and establish their role in the hormonal regulation of gene expression. 4.Using an in vitro transcription assay to test the functioning of transcriptionally active proteins in order to reconstitute the regulatory process for determining the specific protein-protein and protein-DNA interactions which control transcription of the PEPCK gene. The long-term objective of our work is to provide a basis for understanding the functioning of a regulatory system critical to glucose homeostasis in mammals. This research relates directly to our knowledge of diabetes and to the design of treatments to affect the expression of a key enzyme in hepatic and renal gluconeogenesis. Progress in this area will greatly aid attempts to develop a chimeric promoter system capable of driving the regulated expression of linked structural genes specifically in the liver; a system which is of vital importance to effective gene therapy.
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