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Alternative splicing of mRNA encoding rat liver cytochrome P450e (P450IIB2)
D Lacroix1, M Desrochers, M Lambert
1Centre de Recherche en Cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, Canada.
Abstract:
Cytochrome P450e (P450IIB2) is a phenobarbital(PB)-inducible member of the rat liver P450IIB subfamily. Among P450 cDNA clones previously isolated from a cDNA library made from the liver of a single rat were several that contained P450e inserts, including PB13, PB16, and PB22. By nucleotide sequence analysis, the PB16 and PB22 inserts have now been found to contain an additional 24-bp segment not present in the PB13 insert or in previously reported P450e-coding sequences. According to the published P450e genomic sequence, the 24-bp segment is exactly at the junction of the fifth and the sixth exons and its sequence is identical to the first 24 bp of the fifth intron. Translation of this segment would add 8 amino acid residues to the P450e protein. To detect the alternatively spliced P450e mRNA, a synthetic oligodeoxyribonucleotide (oligo) corresponding to 18 of the 24 bp of the intronic sequence found in the PB16 and PB22 inserts was made. This oligo hybridized with a 2.1-kb RNA on Northern blots of liver RNA from PB- or Aroclor 1254-treated rats. Taken together, these results indicate that individual rats can possess both forms of P450e mRNA and that an alternative splicing mechanism is responsible for their formation.
Insights
Rats possess two forms of Cytochrome P450e mRNA, generated by alternative splicing. This molecular mechanism influences the expression of this important liver enzyme.
Area of Science:
- Biochemistry
- Molecular Biology
- Genetics
Background:
- Cytochrome P450e (P450IIB2) is a phenobarbital-inducible enzyme in the rat liver P450IIB subfamily.
- Previous studies identified P450e cDNA clones, including PB13, PB16, and PB22, from rat liver cDNA libraries.
Purpose of the Study:
- To investigate the structural differences in P450e cDNA clones.
- To identify the mechanism responsible for the generation of different P450e mRNA forms.
Main Methods:
- Nucleotide sequence analysis of P450e cDNA clones (PB13, PB16, PB22).
- Comparison of cDNA sequences with the published P450e genomic sequence.
- Northern blot analysis using a synthetic oligodeoxyribonucleotide probe.
Main Results:
- PB16 and PB22 cDNA inserts contain an additional 24-bp segment not present in PB13 or previously reported sequences.
- This 24-bp segment corresponds to the junction of the fifth and sixth exons and the beginning of the fifth intron.
- A synthetic oligonucleotide probe hybridized to a 2.1-kb RNA, indicating the presence of alternatively spliced P450e mRNA in rat liver.
Conclusions:
- Individual rats can express both forms of P450e mRNA.
- Alternative splicing is the mechanism responsible for generating these distinct P450e mRNA variants.
- This finding contributes to understanding the regulation of P450 enzyme expression.