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Noncoordinate regulation of the mRNAs encoding cytochromes P-450BNF/MC-B and P-450ISF/BNF-G
Abstract:
The mRNAs encoding the major polycyclic aromatic hydrocarbon-induced cytochromes P-450 from rat, P-450BNF/MC-B and P-450ISF/BNF-G, were characterized using three classes of recombinant plasmids: those complementary to (a) only P-450BNF/MC-B mRNA, (b) only P-450ISF/BNF-G mRNA, and (c) both mRNAs. These classes were identified by hybridization-selected translation and immunoprecipitation using six monoclonal and polyclonal antibodies and were later sequenced to confirm their identity and specificity. These findings indicated that the mRNAs encoding these two P-450s have regions that are unique, as well as regions that are homologous. Hybridization-selected translation also showed that the primary in vitro translation products of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs are 55 and 52 kDa, respectively, and have both unique and common structural characteristics that can be distinguished immunologically. By Northern hybridization, the P-450BNF/MC-B mRNA was found to be 2900 bases long, while the P-450ISF/BNF-G mRNA was 2100 bases long. Precursors of 3500 and 5200 bases were detected for P-450BNF/MC-B mRNA, while a 3100-base precursor was detected for P-450ISF/BNF-G mRNA. These two mRNAs were induced by beta-naphthoflavone, isosafrole, and 3-methylcholanthrene, but not by phenobarbital. In untreated rats, the P-450BNF/MC-B mRNA was consistently present at very low levels while the P-450ISF/BNF-G mRNA was present in variable amounts, suggesting that the latter mRNA can be induced by dietary or other environmental factors. The kinetics of induction of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs were measured by dot blot hybridization. P-450BNF/MC-B mRNA increased rapidly, reaching half-maximum by 4 h after treatment with 3-methylcholanthrene, while the P-450ISF/BNF-G mRNA increased more slowly, reaching half-maximum after 12 h. The levels of both mRNAs peaked at 24 h, but decreased thereafter at different rates; P-450BNF/MC-B mRNA dropped by about 20% during the next 24 h, while P-450ISF/BNF-G mRNA dropped by 50 to 70%. These differences in the kinetics of induction and the apparent stabilities of the P-450BNF/MC-B and P-450ISF/BNF-G mRNAs, in conjunction with the observed differences in their levels in untreated rats, suggested that these two mRNAs were not coordinately regulated even though they were induced by the same compounds.(ABSTRACT TRUNCATED AT 400 WORDS)
Insights
Researchers characterized two rat mRNAs for polycyclic aromatic hydrocarbon-induced cytochromes P-450. Findings reveal unique and homologous regions, distinct translation products, and differential induction kinetics, indicating non-coordinate regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Toxicology
Background:
- Cytochromes P-450 are crucial enzymes in metabolizing xenobiotics, including polycyclic aromatic hydrocarbons (PAHs).
- Understanding the regulation of specific PAH-induced P-450 mRNAs is vital for assessing toxicological responses.
Purpose of the Study:
- To characterize the mRNAs encoding rat cytochromes P-450BNF/MC-B and P-450ISF/BNF-G, which are induced by PAHs.
- To investigate the structural, translational, and regulatory differences between these two related mRNAs.
Main Methods:
- Hybridization-selected translation and immunoprecipitation using specific antibodies to identify and characterize mRNA classes.
- Sequencing of recombinant plasmids to confirm mRNA identity and specificity.
- Northern and dot blot hybridization to determine mRNA size, precursor forms, and induction kinetics.
- Analysis of in vitro translation products and their immunological characteristics.
Main Results:
- The mRNAs for P-450BNF/MC-B and P-450ISF/BNF-G share unique and homologous regions.
- Primary translation products are distinct (55 kDa for P-450BNF/MC-B, 52 kDa for P-450ISF/BNF-G) with unique and common structural features.
- mRNA sizes differ (2900 bases for P-450BNF/MC-B, 2100 bases for P-450ISF/BNF-G), with distinct precursor forms.
- Both mRNAs are induced by beta-naphthoflavone, isosafrole, and 3-methylcholanthrene, but not phenobarbital.
- Differential induction kinetics and stabilities were observed, with P-450BNF/MC-B mRNA showing faster induction and slower decay than P-450ISF/BNF-G mRNA.
- P-450ISF/BNF-G mRNA levels are variable in untreated rats, suggesting environmental influence.
Conclusions:
- The mRNAs for P-450BNF/MC-B and P-450ISF/BNF-G exhibit significant structural and regulatory differences.
- Despite induction by the same compounds, these two mRNAs are not coordinately regulated, evidenced by distinct induction/decay kinetics and basal expression levels.
- These findings provide insights into the molecular mechanisms underlying differential gene expression of PAH-induced P-450 enzymes.