Characterization of the 5'-flanking sequence of rat class I alcohol dehydrogenase gene.
Expression of the rat class I alcohol dehydrogenase (ADH) gene is highest in the liver and regions of the intestine. We characterized over 3 kilobases of the gene's 5'-flanking region by sequencing and transient transfection. Alignment of the flanking sequence of the rat gene with those of the mouse and human class I genes revealed a cis-acting element, known to be a functional glucocorticoid response element in the human gene and conserved in the mouse, is interrupted in the rat promoter by a 490-base pair processed retropseudogene of the ribosomal protein S25. Southern analysis indicated that this inserted element is present in the class I ADH promoters of multiple strains of rat. Transfection analysis of the rat and mouse promoters showed that the mouse, but not the rat promoter, is inducible by dexamethasone. Electrophoretic mobility shift assays using nuclear extracts from dexamethasone-treated cells confirmed that the mouse's element interacts with the glucocorticoid receptor. Transient transfection of the 5'-flanking region of the rat gene linked to a human growth hormone reporter demonstrated the liver and intestinal specificity of the rat promoter. Two positive elements, one from nucleotides -1,327 to -977 and the other from -241 to -12, were shown to support high levels of reporter activity. In addition, a suppressive element was localized between nucleotides -403 and -241, a region of DNA situated within the domain of the S25 ribosomal protein pseudogene.
Cortese
J F
JF
Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Majewski
J L
JL
Crabb
D W
DW
Edenberg
H J
HJ
Yang
V W
VW
D000426:Alcohol Dehydrogenase / Q000096:biosynthesis / Q000145:classification / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004251:DNA Transposable Elements / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D005938:Glucocorticoids / Q000494:pharmacology*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011544:Pseudogenes / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012269:Ribosomal Proteins / Q000235:genetics; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D013045:Species Specificity
D004251:DNA Transposable Elements; D005938:Glucocorticoids; D007527:Isoenzymes; C489168:RPS25 protein, human; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; C056212:Rps25 protein, rat; D000426:Alcohol Dehydrogenase