Alcohol dehydrogenase (ADH) is the rate-limiting step in the oxidation of alcohol by mammals. ADH gene expression occurs primarily in the liver and is regulated by steroid hormones. Human ADH is composed of numerous isozymes encoded by five genes which display differential patterns of tissue-specific gene expression. Since human ADH shows a strong preference for expression in the liver (high levels) and kidney/gut/lung (moderate levels) it is of great interest to my laboratory to study the mechanism of this tissue-specificity. Regulation at the level of transcription initiation will be analyzed in detail to ascertain the cis-acting DNA elements and ultimately the transacting protein factors necessary to produce the pattern of ADH gene expression seen in humans. The involvement of steroid hormones in regulating ADH transcription will also be analyzed. This knowledge will be important for understanding the overall effects of alcohol abuse upon humans since the ability of individuals to alter ADH levels during times of ethanol-induced stress may play a role in determining the extent to which they suffer from this disease. The broader beenfit of biochemical studies of this kind is that it will eventually lead to an understanding of how higher eukaryotes establish a specific level of expression for a given gene, and knowing this it will be possible to more fully comprehend disease states in which normal gene expression is altered. Methods: (a) DNA fragments containing regulatory information for human ADH genes will be fused to the gene encoding chloramphenicol acetyl transferase (CAT). Mutations which effect transcriptional activity of these ADH-CAT fusions will be generated to localize important cis-acting DNA elements. (b) Wild-type and mutant ADH-CAT fusions will be assayed for transcriptional activity in vivo by transfection of tissue culture cells (+/- steroid hormones) and measurement of CAT activity. (c) ADH-CAT fusions will also be assayed by in vitro transcription in nuclear extracts derived from liver and kidney. (d) ADH DNA fragments implicated as cis-acting regulatory elements will be characterized by footprint analysis using nuclear extracts from liver and kidney. (e) Nuclear extracts will be fractionated and assayged by footprinting to begin chracterization of trans-acting protein factors implicated in ADH transcription initiation. (f) Chromosome walking will be performed to link the alpha, beta, and gamma ADH genes and the pi ADH gene will be cloned.
Showing the most recent 10 out of 21 publications
