NDLI: Identification of a cytochrome P450 gene by reverse transcription--PCR using degenerate primers containing inosine.

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Identification of a cytochrome P450 gene by reverse transcription--PCR using degenerate primers containing inosine.

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Identification of a cytochrome P450 gene by reverse transcription--PCR using degenerate primers containing inosine.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Shen, Z. Liu, J. Wells, R. L. Elkind, M. M.
Description	Author Affiliation: Shen Z ( Department of Radiological Health Sciences, Colorado State University, Fort Collins 80523.);
Abstract	A cytochrome P450-like gene, tentatively named P450CMEF, was amplified by a mixed oligonucleotide-primed amplification of cDNA from C3H mouse embryo fibroblast cells, designated 10T1/2, that had been treated with 7,12-dimethylbenz[a]anthracene (DMBA) or benz[a]anthracene (BA). A set of inosine-containing degenerate primers that were targeted to two conserved regions of known cytochrome P450 cDNAs were used. One primer was coded for the well-described and conserved heme-binding region of P450 enzymes, and the second was designed based upon other considerations of homology among P450 molecules. One of the four PCR-amplified cDNA products hybridized to two major RNA bands, 4.2 and 5.3 kb, that were induced by DMBA or BA. The amino acid sequence of the fragment deduced from the base-sequence data indicate that the amplified cDNA has a 50-55% identity with the cytochrome P450 subfamily 1A. The induction of P450CMEF mRNA preceded the induction of aryl hydrocarbon hydroxylase activity after DMBA or BA treatment, suggesting that the product of P450CMEF is involved in the metabolism of these polycyclic aromatic hydrocarbons in 10T1/2 cells. From the partial sequence of the cDNA identified by this procedure, we propose that P450CMEF is a member of the P450 superfamily, possibly in a subfamily of family 1, that is induced in 10T1/2 cells by DMBA and BA. This method should be useful in identifying additional P450 genes and genes in other gene families.
ISSN	00278424
e-ISSN	10916490
Journal	Proceedings of the National Academy of Sciences of the United States of America
Issue Number	24
Volume Number	90
Language	English
Publisher	National Academy of Sciences
Publisher Date	1993-01-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Cytochrome P-450 Enzyme System Genetics DNA Primers Inosine 9,10-Dimethyl-1,2-benzanthracene Pharmacology Amino Acid Sequence Animals Aryl Hydrocarbon Hydroxylases Biosynthesis Benz(a)Anthracenes Binding Sites Cells, Cultured Conserved Sequence Cytochrome P-450 CYP1B1 DNA, Complementary Chemistry Metabolism Enzyme Induction Drug Effects Fibroblasts Enzymology Heme Mice Mice, Inbred C3H Molecular Sequence Data Polymerase Chain Reaction RNA-Directed DNA Polymerase Research Support, U.S. Gov't, P.H.S. Multidisciplinary
Content Type	Text
Resource Type	Article
Subject	Multidisciplinary

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