Alcohol dehydrogenase (ADH) is the principal enzyme responsible for ethanol metabolism. THe gene encoding the class I ADH is primarily expressed in the liver of vertebrates. Previous work indicates that transcription of the class I ADH gene is both developmentally and hormonally regulated. The identification and characterization of cellular factors that influence transcription of the class I gene are therefore essential in understanding the molecular basis of developmental and hormonal regulation of this gene. We have recently shown that two well-established mammalian transcription factors, the CCAAT/enhancer binding protein (C/EBP) and the upstream stimulatory factor (USF) interact with and activate the promoter of the rat class I ADH gene. We have also identified a third, novel cellular factor that we call EDBP (for Enhancer-site Downstream Binding Protein) which appears to play a significant role in the regulation of transcription of the rat class I gene. The GOAL of the present proposal is then, to purify and characterize EDBP in order to establish the molecular relationship between EDBP and ADH gene expression. There are three SPECIFIC AIMS planned: 1) to purify EDBP from rat liver. Two independent methods are planned. One involves biochemical purification using primarily chromatographic procedures and the other involves the screening of an expression phage complementary DNA (cDNA) library with a labeled oligonucleotide containing EDBP-binding sequences, 2) to isolate cDNA clones encoding EDBP using degenerate oligonucleotides deduced from amino acid sequences of proteolytic fragments of pure EDBP. cDNA clones will be sequenced to establish the coding sequence of EDBP, and 3) to characterize EDBP for its ability to interact with DNA and activate transcription of the class I ADH gene. Its tissue distribution will also be established.
