Gene structure of a phenobarbital-inducible cytochrome P-450 in rat liver.

Abstract

The gene structure of a phenobarbital-inducible rat liver cytochrome P-450 was elucidated by sequence analysis of two isolated genomic clones. The total length of the gene was approximately 14 kilobases and was separated into nine exons by eight intervening sequences. Nucleotide sequences of all exon/intron boundaries follow the G-T/A-G rule. A putative transcription initiation site was assigned to an A, 30 base pairs upstream from the initiation codon by SI nuclease protection mapping. A possible "TATA" equivalent sequence, C-A-T-A-A-A, was found 27 base pairs further upstream from this initiation site. A poly(A) attachment site was determined to be 386 or 387 base pairs downstream from the termination codon by comparison with the cDNA sequence. Detailed comparison with two cDNA sequences determined previously showed the coding nucleotide sequence of the genomic clones to concur with that of the pcP-450pb2 cDNA clone coding for cytochrome P-450e except for three neutral base substitutions. Therefore, we conclude that the gene sequence determined here is for the cytochrome P-450e gene or a similar gene. On the other hand, 40 base substitutions were found in about 1,900 base pairs compared between the sequences of the genomic clone and the other cDNA clones (pcP-450pbl and 4) coding for cytochrome P-450b, and 15 of them result in 14 amino acid replacements in the total 491 amino acid residues. These base substitutions occur in relatively limited regions of the sequences. Most of them are found in exons 6, 7, 8, and 9; most frequently in exon 7.Cytochrome P-450 is a component of the microsomal monooxygenase system which metabolizes endogenous substrates such as steroids and fatty acids as well as exogenous substrates such as many kinds of drugs and other lipophilic xenobiotics. This unusual metabolic versatility of the monooxygenase system results from the participation of multiple forms of cytochrome P-450, each of which exhibits a different but, in some cases, overlapping substrate specificity (1-3). It is known, in addition, that most of these species of cytochrome P-450 are inducible enzymes; administration of drugs and other xenobiotics to animals induces the synthesis of specific forms of cytochrome P-450 (2, 3). The molecular multiplicity and mechanisms of induction of cytochrome P-450 can be best understood by using recombinant DNA technology to perform studies at the gene or DNA level.We previously reported deduced primary amino acid sequences of phenobarbital-inducible cytochrome P-450 from sequence analysis of the cDNA clones (4). By using these cDNAs as probes, we have isolated several genomic clones of the cvtochrome P-450 which are independent of one another on the basis of their restriction analyses (5).Here we report the structural analysis of one of the isolated genes for phenobarbital-inducible rat liver cytochrome P-450 and a comparison of the nucleotide sequences of the genomic and cDNA clones. MATERIALS AND METHODS.Restriction endonucleases were obtained from Takara Shuzo (Kyoto, Japa...

Mouse cytochrome P3-450: complete cDNA and amino acid sequence

“…Three of the five predicted COOterminal amino acids for mouse P3-450 (Fig. 2) correspond to those of the rat P-450d protein, whereas no such correspondence exists with the COOterminal amino acids for rat P-450c protein (41 (10,16). A 39-nucleotide sequence associated with the "highly conserved tridecapeptide" (9-11, 16, 18, 42) has been described in every P-450 protein or cDNA sequence studied to date and is known to be located in the center of exon 7 in the P-450b (10) and P-450e (11) genes.…”

“…One cysteinyl peptide, in exon 3 of P-450b (10), centers around the cysteine 150 in rabbit form 2 (8). This region (Fig.…”

“…There are two highly conserved cysteinyl peptides in rabbit form 2 (8,9), rat P-450b (10,11), and bacterial P-450cam (12) that are under consideration for the source of the thiolate ligand to the heme iron atom near the enzyme active-site. One cysteinyl peptide, in exon 3 of P-450b (10), centers around the cysteine 150 in rabbit form 2 (8).…”

“…The recent cloning of near-full length cDNA's for rat P-450b (10) and P-450e (11) has allowed the first comparison for both nucleotide and amino acid sequence of two closely related phenobarbital-inducible forms. The predicted rat P-450b and P-450e protein sequences have been compared with the published rabbit form 2 P-450 (8,9) and Pseudomonas putida P-450cam (12) protein sequences.…”

Effects of the induction of hepatic microsomal metabolism on the toxicity of cyclophosphamide

“…During recent years ample evidence has accumulated to demonstrate the existence in hepatic microsomes of multiple forms of cytochrome P-450, the terminal enzyme of the oxidase system (Guengerich, 1979;Guengerich et al, 1982). This evidence has resulted from the application of gel electrophoresis, immunological, and recombinant DNA techniques in the investigation of cytochrome P-450 multiplicity in hepatic microsomes from several species of animals treated with different types of microsomal enzyme inducers (Guengerich, 1979;Guengerich et al, 1982;Fujii-Kuriyama et al, 1982;Mizukami et al, 1983). Phenobarbital, a commonly used prototype inducer, has been shown to induce at least four different forms of cytochrome P-450 in rats with overlapping but different substrate specificities (Guengerich 1979;Guengerich et al, 1982).…”