A novel negative element in the promoter of the mouse alcohol dehydrogenase gene Adh-1.
The mouse alcohol dehydrogenase gene, Adh-1, is expressed in a tissue-specific manner. We examined the promoter activity of a series of 5' deletions extending from bp -473 to -47 and demonstrated that there are positive regulatory elements between bp -229 and +54 and a negative regulatory element between bp -323 and -229. To identify the sequence of the negative regulatory element, gel retardation and DNase I footprint assays were performed using nuclear proteins from mouse liver and from a hepatoma cell line, H4IIE-C3. A specific protein-binding site covered bp -324 and -297. Within this region, we identified sites of close protein-DNA contact by methylation interference assays, located in the sequence TGGAAGTTTCAGGTT (nt -316 to -302). Site-directed mutagenesis of four protein-DNA contact sites within this sequence eliminated the specific protein-DNA binding, assayed by gel retardation, and restored expression of Adh-1 in transient transfection assays to the levels seen when the entire region containing the negative element was deleted. Thus, we have identified a negative regulatory element within the Adh-1 promoter. No homology was found between this negative element and other regulatory elements, suggesting that this is a novel negative element.
Lin
Z
Z
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Edenberg
H J
HJ
Carr
L G
LG
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015342:DNA Probes; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D003850:Deoxyribonuclease I; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008745:Methylation; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D004247:DNA; D000426:Alcohol Dehydrogenase; D008156:Luciferases; D003850:Deoxyribonuclease I