Bile salts are maintained in an enterohepatic circulation whose pool size is determined by specific transport processes located on liver and ileal plasma membranes. Although the basic characteristics of bile salt uptake, translocation and secretion are known, the specific protein(s) involved in sinusoidal and canalicular membrane transport of bile salts have not been isolated, their molecular structure deduced, or their regulatory sequences identified. The long term goals of this study are to characterize the structural features of sodium-coupled organic anion transporters, determine whether their adaptive regulation is due to transcriptional or translational processes, and if regulation is under transcriptional control, to isolate and characterize the cis and trans-regulatory factors. The short term goal of this project is to isolate the cDNA(s) for hepatic bile salt carrier(s) using a functional expression assay for the sodium coupled transport of bile salts. Our cloning strategy is to prepare a cDNA-eukaryotic expression library. This library will then be assayed for the functional expression of bile acid transport in transfected cells. By using a functional-expression cloning strategy, we hope to avoid possible problems associated with other cloning protocols which rely on antibody or sequences recognition, especially when these screening reagents are derived from """"""""purified"""""""" materials which may contain small amounts of contaminants. RNA will be isolated from human liver and mRNA purified on oligo-dT cellulose. The cDNA library will be constructed (Invitrogen) in the plasmid expression vector pCDNA/1. The library will be plated in pools of 500 clones and the DNA extracted. These DNA pools will be transfected into COS cells (which allow the episomal replication of the transfected DNA) and screened for expression of bile acid transport. COS cells do not transport bile acids. The expression of functional activity will be assayed using a synthetic bile acid, [125I]tyrosine-glycocholate. At physiological concentrations approximately 50% of this bile acid is transported in rat hepatocytes by the sodium dependent bile acid transporter. Positive pools will be subdivided and re-screened until individual clones have been identified. These sequences will be tested for the ability of taurocholate to inhibit transport and the sodium dependence of transport. Positive clones will then be sequenced.
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