Expression and alternative splicing of the cytochrome P-450 CYP2A7.
In order to investigate the relative levels of expression of human cytochrome P-450 (P-450) CYP2A genes and determine how this relates to polymorphism in coumarin hydroxylase activity, cDNA clones for members of the CYP2A gene family were isolated. These clones were CYP2A6, CYP2A7 and an alternatively spliced version of CYP2A7 (CYP2A7AS). The latter clone was missing exon 2, but contained a 10 bp segment of intron 1. Translation of CYP2A7AS resulted in an in-frame deletion of 51 amino acids. The expression of these cDNAs in COS-7 cells showed that both CYP2A6 and CYP2A7 generated a protein of molecular mass 49 kDa, whereas the protein product of CYP2A7AS was about 44 kDa. Only the CYP2A6 had coumarin hydroxylase activity. The relative level of CYP2A7 and CYP2A7AS mRNA was investigated by reverse transcription followed by PCR (RT-PCR) using human liver RNAs and an RNA sample from a human skin fibroblast cell line. In one of five liver RNAs studied, the aberrantly spliced CYP2A7 mRNA was 3-4-fold more abundant than the normal mRNA. The other samples contained very low levels of this mRNA species. Interestingly, CYP2A7AS mRNA was the major CYP2A7 mRNA detected in the fibroblast cell line. In this case only a protein band of 44 kDa was observed by Western-blot analysis. The relative of mRNA encoding CYP2A6 and CYP2A7 was established in seven human liver samples by RT-PCR and found to range between 1:0.5 and 1:3. These data strength the previous findings that alternative splicing is an important factor in determining the levels of many human P-450s and that this may be subject to tissue-specific effects. Whether in this case the protein product has some function remains to be determined.
Ding
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Imperial Cancer Research Fund, Ninewells Hospital and Medical School, Dundee, Scotland.
Lake
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Friedberg
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Wolf
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CR
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000072467:Cytochrome P450 Family 2; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D005347:Fibroblasts; D015870:Gene Expression; D006801:Humans; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D054198:Precursor Cell Lymphoblastic Leukemia-Lymphoma; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D018076:DNA, Complementary; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; C423964:CYP2A7 protein, human; D000072467:Cytochrome P450 Family 2