Gene structure and nucleotide sequence for rat cytochrome P-450c

doi:10.1016/0003-9861(85)90300-5

Archives of Biochemistry and Biophysics

Volume 237, Issue 2, March 1985, Pages 465-476

https://doi.org/10.1016/0003-9861(85)90300-5 Get rights and content

Abstract

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. λMC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from λMC4, the EcoR1, A fragment, hybridized to a partial cDNA clone for the 3′ end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res., 12, 2929–2938]revealed that the EcoR1, A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, λMC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5′ regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by $GT AG$ . Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5′ to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P₃-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.

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^☆: This study was supported in part by grants from the NIH (ES-01974) and the Council for Tobacco Research (No. 1369).

³: Present address: Rockfeller University, New York, N. Y. 10021.

¹: J. Levy was supported under a training grant from the National Cancer Institute (CA-09286).

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Gene structure and nucleotide sequence for rat cytochrome P-450c☆

Abstract

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