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Molecular cloning of a complementary DNA to 3-methylcholanthrene-inducible cytochrome P-450 mRNA from rat liver
Abstract:
Poly(A)+ RNA was isolated from membrane-bound polysomes of the livers of 3-methylcholanthrene (MC)-treated rats, and was partially purified by sucrose density gradient centrifugation. The mRNA was translated in an in vitro rabbit reticulocyte lysate system, and assayed for the synthesis of MC-inducible forms of cytochrome P-450 (cytochrome P-450MC) using anti-cytochrome P-450c antibody which reacted with two types of cytochrome P-450MC, P-450c, and P-450d. The mRNA activity for cytochrome P-450MC was located at around 18S, accounting for approximately 5% of total mRNA activity. The double-stranded complementary DNA which had been synthesized from the partially purified mRNA by avian myeloblastosis virus reverse transcriptase and DNA polymerase I (Klenow enzyme) was cloned in Escherichia coli X1776, using plasmid pBR322 as a cloning vector. After differential colony hybridization using [32P]cDNA's synthesized from mRNA preparation of MC-treated or untreated rat liver as a probe, a clone (3-9-1) carrying cytochrome P-450MC cDNA sequence was identified by a positive hybridization-translation assay. The specific mRNA hybridized with plasmid 3-9-1 DNA showed an enriched synthesis of a protein with apparent molecular weight of 56,000 daltons, which was immunoprecipitable with anti-P-450c antibody. In RNA blot analysis with MC-, polychlorinated biphenyls (PCB)-, and phenobarbital (PB)-induced mRNA as well as uninduced mRNA, a longer cDNA (P-34) which had been isolated by hybridization with the insertion of clone 3-9-1, and the previously isolated PB-inducible cytochrome P-450b cDNA (Fujii-Kuriyama et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2793-2797) hybridized with mRNA preparations in an inducer-specific manner.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Researchers isolated mRNA from 3-methylcholanthrene (MC)-treated rats to study cytochrome P-450MC. They successfully cloned the corresponding cDNA, identifying a specific mRNA fraction responsible for synthesizing this MC-inducible enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Cytochrome P-450 enzymes are crucial for metabolizing xenobiotics.
- 3-methylcholanthrene (MC) is a potent inducer of specific cytochrome P-450 forms.
- Understanding the molecular basis of MC-inducible cytochrome P-450 is vital for drug metabolism and toxicology studies.
Purpose of the Study:
- To isolate and characterize the messenger RNA (mRNA) encoding MC-inducible cytochrome P-450 (cytochrome P-450MC).
- To clone the complementary DNA (cDNA) for cytochrome P-450MC.
- To confirm the identity and function of the cloned cDNA through hybridization and translation assays.
Main Methods:
- Poly(A)+ RNA isolation from liver polysomes of MC-treated rats.
- In vitro translation using a rabbit reticulocyte lysate system.
- Sucrose density gradient centrifugation for mRNA purification.
- cDNA synthesis using reverse transcriptase and DNA polymerase I.
- Cloning of cDNA into Escherichia coli using plasmid pBR322.
- Differential colony hybridization and hybridization-translation assays for clone identification.
Main Results:
- MC-inducible cytochrome P-450MC mRNA activity was localized at approximately 18S, representing about 5% of total mRNA.
- A clone (3-9-1) containing the cytochrome P-450MC cDNA sequence was identified.
- Hybridization with clone 3-9-1 DNA enriched the synthesis of a 56,000-dalton protein immunoprecipitable by anti-P-450c antibody.
- RNA blot analysis demonstrated inducer-specific hybridization patterns for MC-, PCB-, and PB-induced mRNAs.
Conclusions:
- The study successfully identified and cloned the cDNA for MC-inducible cytochrome P-450MC.
- The cloned cDNA directs the synthesis of a specific MC-inducible cytochrome P-450 protein.
- This molecular tool enables further investigation into the regulation and function of cytochrome P-450 enzymes in response to chemical inducers.