Vitamin A, the collective name for a group of related retinoids, is essential for normal development and growth in animals. Too little of too much results in a wide variety of biological defects, and in addition, increases toxicity caused by alcohol and various environmental toxins. The biologically active retinoid is retinoic acid (RA), and its concentration within cells is critical to health. Previous studies have indicated that a cellular retinoic acid-binding protein (CRABP) plays a major role in maintaining intracellular RA at a constant concentration. However, because CRABP is so vital in this regard, mutation and other manipulation that greatly reduce or eliminate this protein are generally lethal to the organism, and hence cannot be used to study CRABP function. The major goal of this proposal is to understand the positive and negative regulatory elements of CRABP gene and to create specific changes in this gene so its expression is altered but not eliminated in animals. Specifically, we will 1. determine the region of the gene essential for its regulation, by systematically deleting portions of the 5' region of the gene fused to a LacZ reporter, and 2. create transgenic mice in which CRABP is expressed in tissues normally not expressing this protein, as well as animals in which normal CRABP expression is blocked. Taken together, these studies should illustrate the interaction of RA and CRABP and the mechanisms that regulate intracellular RA concentration, and ultimately lead to better preventive/therapeutic application of vitamin A in humans.
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