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Isolation and characterization of full-length mouse cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome P1-450 and P3-450 |
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| Authors: | F J Gonzalez P I Mackenzie S Kimura D W Nebert |
| Institution: | Laboratory of Developmental Pharmacology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20205 U.S.A. Tel. 301-496-1016 |
| Abstract: | Polysome immuno-adsorption, with immunoglobulin G directed against two 3-methylcholanthrene-induced mouse liver cytochrome P-450 proteins, was used to enrich mRNA from 3-methylcholanthrene-treated C57BL/6N mouse liver. cDNA transcribed from the P-450-enriched mRNA was then cloned into the Okayama-Berg vector. Two cDNA classes were detected upon differential screening of the clone bank with 32P]cDNA derived from 3-methylcholanthrene-induced immuno-enriched versus control mRNA. Several representatives of these two classes were judged to be near full length by comparison with their corresponding mRNA mobilities on denaturing agarose gels. A continuous reading-frame near the 5' end of one cDNA class (P1-450) corresponds to a protein having 15 of 17 residues the same as the published N-terminal sequence of rat P-450c. A continuous reading frame near the 5' end of the other class (P3-450) corresponds exactly to the first 25 amino acids of the published N-terminal sequence of rat P-450d. The P1-450 cDNA is at least 700 bp longer than the P3-450 cDNA. Heteroduplex analysis and Southern blot hybridization demonstrate that these mRNAs share approx. 1100 bp of sequence homology. Genomic P1-450 and P3-450 clones were isolated from a gene library constructed from C57BL/6N mouse liver DNA. By heteroduplex analysis with the corresponding cDNA, the P1-450 gene spans about 6 kb and the P3-450 gene about 7 kb. The intron-exon patterns are very similar, with the second and seventh exons being much larger than the other five. The 3' terminal exon of P1-450 is about 500 bp longer than that of P3-450. These data suggest that both P1-450 and P3-450 have diverged from a common ancestral gene. |
| Keywords: | Inbred mouse genetics drug metabolism plasmid vector genomic library heteroduplex analysis polysome immunoadsorption recombinant DNA bp base pairs Buffer A 100 mM potassium phosphate buffer (pH 7 6) containing 0 6% sodium cholate 1 mM DTT 1 mM EDTA and 20% glycerol Buffer B 33 mM potassium phosphate buffer (pH 7 25) containing 0 2% Emulgen 911 0 1 mM DTT and 20% glycerol CNBr cyanogen bromide DTT dithiothreitol IgG immunoglobulin G kb kilobase pairs “P-450” denotes any or all forms of cytochrome P-450 (multisubstrate monooxygenases) SDS sodium dodecyl sulfate 1 × SSC 0 15 M NaCl To whom all correspondence and reprint requests should be addressed at Building 10 Room 6C-101 National Institutes of Health Bethesda MD 20205 U S A Tel 301-496-5128 |
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