A barbiturate-regulated protein binding to a common sequence in the cytochrome P450 genes of rodents and bacteria.
Analyses of the 5' regulatory sequences of genes encoding barbiturate-inducible cytochromes P450BM-1 and P450BM-3 from Bacillus megaterium and of the 5' sequences of genes for barbiturate-inducible P450b and P450e of the rat revealed a string of 17 base pairs in each of the genes that shared a high degree of sequence identity. Labeled oligonucleotide probes of each of these four sequences were tested in gel retardation assays with protein obtained from B. megaterium grown either in the presence or absence of barbiturates or with protein from nuclear extracts from livers of rats left untreated or injected with phenobarbital. Each of the four 17-mers bound strongly to a single protein from bacteria grown in the absence of barbiturates, but this binding was dramatically reduced with protein from pentobarbital- or phenobarbital-grown cells. Conversely, the probes complexed weakly to one protein band from nuclear extracts from untreated rats but much more strongly with protein from phenobarbital-treated rats. Similar effects could be obtained by prolonged incubation with phenobarbital of either soluble protein from the bacteria grown in the absence of barbiturates or nuclear extract protein from untreated rats. Deletion analysis of the 5'-flanking region of the P450BM-1 gene of B. megaterium revealed a putative repressor binding site located within a 24-base pair DNA segment that included the 17-base pair sequence involved in barbiturate-regulated protein binding.
He
J S
JS
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90024-1737.
Fulco
A J
AJ
D000818:Animals; D001410:Bacillus megaterium / Q000201:enzymology*; D001463:Barbiturates / Q000494:pharmacology*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D004790:Enzyme Induction; D015971:Gene Expression Regulation, Enzymologic; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D010957:Plasmids; D051381:Rats; D011919:Rats, Inbred Strains; D012045:Regulatory Sequences, Nucleic Acid
D001463:Barbiturates; D003577:Cytochrome P-450 Enzyme System; D015500:Chloramphenicol O-Acetyltransferase