The murine Ah locus. Comparison of the complete cytochrome P1-450 and P3-450 cDNA nucleotide and amino acid sequences.

S Kimura,F J Gonzalez,D W Nebert

doi:10.1016/s0021-9258(18)90569-7

Abstract

Mouse cytochrome P1-450 and P3-450 are most closely associated with induced aryl hydrocarbon (benzo[a]pyrene) hydroxylase (EC 1.14.14.1) and acetanilide 4-hydroxylase activity, respectively. Full-length cDNA clones of P1-450 and P3-450 were generated from mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mouse liver. P1-450 cDNA is 2620 nucleotides in length and has a coding region (base 110 to 1,675) that produces a protein with 521 residues (Mr = 58,914). P3-450 cDNA is 1,894 nucleotides in length and yields a protein with 513 residues (Mr = 58,183). P1-450 mRNA is the first reported example in mouse in which UAG is used as the termination codon. P1-450 and P3-450, both induced by polycyclic hydrocarbons and regulated by the Ah receptor, exhibit overall nucleotide and protein homology of 68, and 73%, respectively. Segments of high homology, interspersed with regions of low homology, support the hypothesis of gene conversion or unequal crossing over as possible mechanisms for divergence of these two genes. Mouse P1-450 and P3-450 cDNAs were compared with previously published data on rat P-450e cDNA and rabbit form 2 protein, corresponding to two P-450 genes from the "phenobarbital inducible" P-450 gene subfamily. Nucleotide homology between a member of either gene subfamily is about 30%, and protein homology is about 15%, suggesting that the Ah locus-associated P-450 gene subfamily diverged from the phenobarbital inducible P-450 subfamily more than 200 million years ago. An N-terminal and a C-terminal cysteinyl fragment corresponding to the regions around P1-450 Cys-158 and Cys-458, respectively, are the only two cysteinyl peptides conserved among all four proteins compared. Because of greater homology in the C-terminal conserved cysteinyl fragment between the two gene subfamilies and a greater hydrophobic pocket in the C-terminal conserved cysteinyl fragment, the data favor this cysteine as the more likely candidate for the thiolate ligand to the heme iron in the P-450 enzyme active-site.

Highlights

From the Laboratory of Developmental Phrmobgy, National Institute of Child Health and Human Development, National Institutes of Health, Bethesdu, Mayland 20205
P1-450 and Pa-450, both induced by polycyclic hydrocarbons and regulated by the Ah reand P,450 mRNA induction [10]. mRNA increases are due to transcriptional activation of the P,450 and P3-450 genes within several hours after a single doseof inducer [11].Both the P1-450 and P3-450 genes are located on mouse chromosome 9 [12] and exhibit strikingly similar intron-exon patterns [13]
Comparison of P1-450 and P3-45U cDNA with Rat P-450e differ in size become markedly elevated after 3-methylchol- cDNA-Fig. 2 represents a dot matrix analysisof PI-450 and anthrene treatmenotf C57BL/6N mice but noDt BA/2N mice P3-450cDNA compared to the ninexons reportedfor rat P

Summary

Introduction

The 5’ portion of a P3-450 partial cDNA clone was found to hybridize to the 2900-nucleotide mRNA and toa fragmentof the P1-450gene [35].These datastrongly suggest that the two induced mRNAs controlled by the Ah receptor share homology and emphasize the need for sequencing the two full-length cDNA clones. Clones from 3-methylcholanthrene-treated C57BL/6N mouse liver is consistent with the concentration differences previously noted for P3-450 and P1-450 mRNAs [11]and the P3450 and P1-450microsomal proteins [5].Three of the five PI-

Results

Conclusion