Protein-DNA interactions in the 5' region of the mouse alcohol dehydrogenase gene Adh-1

The mouse alcohol dehydrogenase-encoding gene, Adh-1, is expressed at high levels in adult liver. We have begun analysis of the regulation of this gene, focusing upon specific DNA-protein interactions. Preliminary deletion mapping of the 5' region indicated that a 521-bp fragment extending from nucleotide (nt) -468 to +53 (relative to the transcription start point) could direct chloramphenicol acetyl transferase synthesis in hepatoma cells. We therefore focused upon this -468 to +53 fragment. Using the gel mobility-shift assay, we detected at least four different complexes between proteins extracted from nuclei of mouse liver or hepatoma cells and regions within the -468 to +53 fragment. Two of the DNA-protein complexes can be competed with a 43-bp region from nt -90 to -48, and an oligodeoxyribonucleotide spanning this region forms two complexes. The strongest of these two DNA-protein complexes has been localized by methylation interference experiments to the palindromic sequence CACGTG located between nt -57 and -62. This region is identical in the related human ADH2 gene, and may represent a novel regulatory sequence.