cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of beta-naphthoflavone-induced rabbit liver P-450 LM4 and LM6.
A cDNA library was constructed from liver mRNA of a beta-naphthoflavone-induced rabbit. Two clones pLM4-1 and pLM6-1 containing 2.2-kbp inserts that hybridized at low stringincy with a mouse P1 P-450 probe were selected. The clone pLM4-1 was fully sequenced and found to contain a full-length cDNA coding for cytochrome P-450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6-1 as coding for the major part of cytochrome P-450 LM6. Cloned LM4-1 cDNA was reformed by deletion of the 5' and 3' non-coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6-1 cDNA after replacement of the missing N-terminus-coding sequences by homologous sequences form the pLM4-1 clone resulting in a chimeric cytochrome P-450 coding sequence. Expression of cloned rabbit cytochrome P-450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P-450 production. Yeast synthesized cytochromes P-450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P-450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P-450 that contains the 143 N-terminal amino acids of cytochrome P-450 LM4 and the remaining 375 amino acids of cytochrome P-450 LM6 was found to exhibit most of the authentic cytochrome P-450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are discussed.
Pompon
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Centre de Génétique Moléculaire du CNRS, Laboratoire Propre Associé à l'Université Pierre et Marie Curie, Gif-sur-Yvette, France.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001571:Benzoflavones / Q000494:pharmacology*; D015153:Blotting, Western; D002384:Catalysis; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D005419:Flavonoids / Q000494:pharmacology*; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013053:Spectrophotometry; D014170:Transformation, Genetic; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D005419:Flavonoids; D012333:RNA, Messenger; D019324:beta-Naphthoflavone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D004262:DNA Restriction Enzymes