Isolation and characterization of a cloned DNA sequence associated with the murine Ah locus and a 3-methylcholanthrene-induced form of cytochrome P-450*
- †Developmental Pharmacology Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205
- ‡Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20014
- §Laboratory of Molecular Virology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
Abstract
Using partially purified mouse liver 23S mRNA known to be associated with the Ah locus and 3-methylcholanthrene-induced cytochrome P 1-450, we synthesized double-stranded cDNA by the successive action of reverse transcriptase (RNA-directed DNA nucleotidyltransferase) and the Klenow A fragment of Escherichia coli DNA polymerase I. The double-stranded cDNA was inserted into pBR322 plasmid DNA by Pst I cleavage and homopolymeric “tailing” and cloned in E. coli LE392. Clone 46 hybridized with [32P]cDNA made from 23S mRNA from “Ah-responsive” C57BL/6N mice but did not hybridize with similarly prepared [32P]cDNA from “Ah-nonresponsive” DBA/2N mice. Clone 30 was positive, and clone 7 was negative, with both C57BL/6N and DBA/2N [32P]cDNA probes; these two clones were therefore used as “positive” and “negative” control clones, respectively. By translation-arrest experiments, clone 46 DNA and clone 30 DNA were shown to be associated with anti-P 1-450- and anti-albumin-precipitable material, respectively. By agarose gel electrophoresis of Pst I digests, the clone 46 DNA insert was shown to be 1100 base pairs in total length and to contain one internal Pst I site. The cDNA made from total mRNA isolated from 3-methylcholanthrene-treated C57BL/6N mice hybridized to the two fragments of Pst I-digested DNA from clone 46, whereas similarly prepared cDNA from 3-methylcholanthrene-treated DBA/2N and control C57BL/6N and DBA/2N mice did not. Of 11 restriction endonucleases used, two (Pst I and Xba I) had sites within the clone 46 DNA insert. After hybridization of clone 46 32P-labeled nick-translated DNA to EcoRI fragments from A/HeJ mouse genomic DNA and fractionation by RPC-5 chromatography and gel electrophoresis, only one positive band (3-4 kilobase pairs appeared. These data demonstrate conclusively that pBR322 clone 46 DNA is associated with mRNA controlled by the murine Ah locus, presumably the structural gene encoding 3-methylcholanthrene-induced P 1-450.
Footnotes
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↵ * Portions of this work were presented at the Third International Congress on the Biochemistry, Biophysics, and Regulation of Cytochrome P-450, Saltsjöbaden, Sweden, June 1980 (34).
