Ethanol metabolism in the liver is mediated by class I alcohol dehydrogenase (ADH) and cytochrome P4502E1 (CYP2E1). Class I ADH in mice is encoded by the Adh-1 gene which is under complex hormonal and developmental control. Transgenic mice will be used to determine the role of intragenic and 5'- and 3'-flanking sequences in control of the total expression phenotype. A region extending to -10kb attached tot an ADH minigene controls proper expression in kidney, adrenal, and other tissues; however, other cis-acting elements are required for liver expression. Cosmid and lambda genomic clones containing all intragenic sequences and different 5'- and 3'-flanking distance are now isolated and will be used in transgenic analysis to identify sequences necessary for liver expression. The same transgenic approach, using alternative promoters and cosmid clones, will be used to produce transgenic mice over expressing ADH and/or CYP2E1. A unique approach to analysis will allow RNA and protein produced from the transgene to be distinguished from production from the endogenous gene. For Adh-1, the unique genetic variant to be used will enable proper expression at the level of cell type within a tissue to be determined. Over expressing mice will be use to test major hypotheses about ethanol-induced liver injury including free radical formation, lipid peroxidation and production of fatty-liver. In addition, a genetic variant in Adh-3 expression identified among inbred strains will be defined at the molecular level and will provide a mechanism for differential tissue expression of this gene.
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