Cloning, Sequencing and Tissue Distribution of Rat Flavin-Containing Monooxygenase 4: Two Different Forms Are Produced by Tissue-Specific Alternative Splicing
- Unité de Toxicologie et de Métabolisme Comparés des Xénobiotiques, Unité Mixte Recherche, Institut National de la Recherche Agronomique, et Direction Générale de l'Enseignement et de la Recherche, Ecole Nationale Vétérinaire de Lyon, Marcy l'étoile, France
- Etienne Benoit, Unité de Toxicologie et de Métabolisme Comparés des Xénobiotiques, UMR INRA et DGER, Ecole Nationale Vétérinaire de Lyon, BP 83, 69280 Marcy l'étoile, France. E-mail: e.benoit{at}vet-lyon.fr
Abstract
The nucleotide sequence of rat flavin-containing monooxygenase 4 (FMO4) mRNA was obtained by reverse transcription-polymerase chain reaction (RT-PCR) and 5′/3′ terminal extension. Complete cDNA was amplified, cloned, and sequenced from the mRNA obtained from rat kidney and brain. Two different transcripts (short and long) stemming from the splicing of an internal region of 189 bases pair, corresponding to exon 4 were identified. This alternative splicing seems to be specific of the brain. The long cDNA encodes a protein of 560 amino acids with a predicted molecular mass of 63,395 Da. The short cDNA encodes a protein of 497 amino acids with a predicted molecular mass of 55,871 Da. Both of these encoded sequences contain the NADPH- and FAD-binding sites and a hydrophilic carboxyl terminus. These sequences are 80 and 79% identical to the sequences of human and rabbit FMO4. By Northern blotting and/or RT-PCR, the long-form FMO4 mRNA was detected in the rat kidney, intestine, and liver and the short form particularly in the brain. For the first time, the expression of FMO4 protein was demonstrated. By Western blotting using the two different forms of FMO4 antibodies, a long FMO4 protein was detected in the rat kidney, whereas in the rat brain, only the short form of FMO4 was observed.
Footnotes
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This study was funded in part by a grant from DGER “Formation par la Recherche”, Ministère de l'Agriculture, France.
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The nucleotide sequences reported in this article have been submitted to GenBank with accession numbers AF458416 and AF458417 for long and short FMO4, respectively.
- Abbreviations:
- FMO
- flavin-containing monooxygenase
- FAD
- flavin adenine dinucleotide
- RACE
- rapid amplification of cDNA ends
- RT-PCR
- reverse transcription polymerase chain reaction
- PAGE
- polyacrylamide gel electrophoresis
- MMLV-RT
- Moloney murine leukemia virus reverse-transcriptase
- bp
- base pair(s)
- SSC
- standard saline citrate
- KLH
- keyhole limpet hemocyanin
- ORF
- open reading frame
- GST
- glutathione S-transferase
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- Received May 17, 2002.
- Accepted September 30, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
