Isolation of cytochrome P-450 cDNA clones from the higher plant Catharanthus roseus by a PCR strategy.
Cytochrome P-450 monooxygenases are membrane-bound enzymes involved in a wide range of biosynthetic pathways in plants. An efficient PCR strategy for isolating cytochrome P-450 cDNA clones from plant cDNA libraries is described. A set of degenerate primers for PCR amplification was designed to recognize nucleotide sequences specifying the highly conserved haembinding region of cytochrome P-450 proteins. Using this primer set and a non-specific primer, complementary to either the poly(A) tail of the cDNA clones or a phage vector sequence, we isolated 16 different cytochrome P-450 cDNA sequences from a cDNA library of Catharanthus roseus.
Meijer
A H
AH
Centre for Bio-Pharmaceutical Sciences, Leiden University, Gorlaeus Laboratories, Netherlands.
Souer
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E
Verpoorte
R
R
Hoge
J H
JH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000235:genetics; D008969:Molecular Sequence Data; D010944:Plants / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid
D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C081675:geraniol 10-hydroxylase