Structurally and functionally conserved regions of cytochrome P-450 reductase as targets for DNA amplification by the polymerase chain reaction. Cloning and nucleotide sequence of the Schizosaccharomyces pombe cDNA.
1. Alignments of the available cytochrome P-450 reductase amino acid sequences, and comparison with the crystal structure of ferredoxin-NADP reductase, indicate that two highly conserved regions are of functional importance. 2. Degenerate oligonucleotide primers, based on these sequences, were used in the polymerase chain reaction to amplify a 309 bp fragment of the cytochrome P-450 reductase gene from Schizosaccharomyces pombe for use as an homologous probe. 3. A 2.6 kb cDNA was cloned from a lambda library, and sequencing revealed an open-reading frame of 2034 bp encoding a protein of M(r) 76774. This protein shares 38-41% identity with other eukaryotic cytochrome P-450 reductases, and 30% identity with that of Bacillus megaterium. 4. Comparison of the N-terminal FMN-binding domain with flavodoxin, and the C-terminal FAD- and NADP-binding domain with ferredoxin-NADP reductase, indicates the presence of several functionally conserved regions. 5. The Sc. pombe cytochrome P-450 reductase gene was shown to contain no introns.
Miles
J S
JS
Department of Biochemistry, University of Glasgow, U.K.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000235:genetics*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D012568:Schizosaccharomyces / Q000235:genetics*; D016415:Sequence Alignment
D005656:Fungal Proteins; D009838:Oligodeoxyribonucleotides; D004247:DNA; D009251:NADPH-Ferrihemoprotein Reductase