The consequences of excessive ethanol metabolism lead to various states of liver pathology. Major routes of ethanol metabolism are mediated by alcohol dehydrogenase (ADH) or the ethanol-inducible form of cytochrome P-450 (P-450alc). This research is to study the regulation of the expression of these genes in genetically defined mouse and deermouse systems. Transgenic mice will be produced harboring mouse Adh-1 minigenes linked to various Adh-1 5'-flanking regions in order to determine the cis acting sites necessary for normal expression in a tissue-specific, and hormonally responsive manner. Sequences necessary for the completely normal phenotypic expression will be delineated by testing various 5'-deletion constructs. Lines will be analyzed for total Adh-1 phenotype by assaying for transgene expression using Northern blot analysis. Transgenic lines will be established with elevated levels of ADH-1 protein and enzyme activity using an albumin gene enhancer/promoter fused to an Adh-1 hybrid gene composed of genomic and cDNA sequences. These animals are viewed as potentially relevant models for the study of alcohol induced fatty liver and cirrhosis. Other transgenic lines harboring albumin gene enhancer/promoter sequences linked to P-450alc genomic and cDNA fusion sequences will also be produced to look at the physiological and pathological consequence of the elevation of this pathway. Studies on the mechanism of kidney P-450alc induction by testosterone will be performed to determine if transcriptional activation occurs in response to hormone, or alternatively if mRNA stability is affected. Possible translational control of P-450alc production by ethanol and other inducers in liver will also be studied. At the molecular and genetic level, the genes for mouse P-450alc and deermouse Adh-1 and Adh-2 genes will be mapped. Molecular organization of the deermouse genes and several mouse Adh-like genes will be studied in genomic clones. Molecular organization of the Adh-1 gene in the chromosome will be studied during development and hormonal induction by assaying DNase hypersensitive sites. Trans acting factors at distal sites in the Adh-1 gene will be studied using gel mobility shift and DNase protection assays.
