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• W2055666951 abstract "Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoenzymes that catalyze the oxidation of heteroatom centers in numerous drugs and xenobiotics. FMO2, or “pulmonary” FMO, one of five forms of the enzyme identified in mammals, is expressed predominantly in lung and differs from other FMOs in that it can catalyze the N-oxidation of certain primary alkylamines. We describe here the isolation and characterization of cDNAs for human FMO2. Analysis of the sequence of the cDNAs and of a section of the corresponding gene revealed that the major FMO2 allele of humans encodes a polypeptide that, compared with the orthologous protein of other mammals, lacks 64 amino acid residues from its C terminus. Heterologous expression of the cDNA revealed that the truncated polypeptide was catalytically inactive. The nonsense mutation that gave rise to the truncated polypeptide, a C → T transition in codon 472, is not present in theFMO2 gene of closely related primates, including gorilla and chimpanzee, and must therefore have arisen in the human lineage after the divergence of the Homo and Pan clades. Possible mechanisms for the fixation of the mutation in the human population and the potential significance of the loss of functional FMO2 in humans are discussed. Flavin-containing monooxygenases (FMOs) are NADPH-dependent flavoenzymes that catalyze the oxidation of heteroatom centers in numerous drugs and xenobiotics. FMO2, or “pulmonary” FMO, one of five forms of the enzyme identified in mammals, is expressed predominantly in lung and differs from other FMOs in that it can catalyze the N-oxidation of certain primary alkylamines. We describe here the isolation and characterization of cDNAs for human FMO2. Analysis of the sequence of the cDNAs and of a section of the corresponding gene revealed that the major FMO2 allele of humans encodes a polypeptide that, compared with the orthologous protein of other mammals, lacks 64 amino acid residues from its C terminus. Heterologous expression of the cDNA revealed that the truncated polypeptide was catalytically inactive. The nonsense mutation that gave rise to the truncated polypeptide, a C → T transition in codon 472, is not present in theFMO2 gene of closely related primates, including gorilla and chimpanzee, and must therefore have arisen in the human lineage after the divergence of the Homo and Pan clades. Possible mechanisms for the fixation of the mutation in the human population and the potential significance of the loss of functional FMO2 in humans are discussed. flavin-containing monooxygenase open reading frame pBluescript polymerase chain reaction rapid amplification of cDNA ends base pair(s) kilobase pair(s). The flavin-containing monooxygenases (FMOs)1 (EC 1.14.13.8) are NADPH-dependent flavoenzymes that catalyze oxidation of soft nucleophilic heteroatom centers in a range of structurally diverse compounds, including drugs, pesticides, and other xenobiotics (1Ziegler D.M. Trends Pharmacol. Sci. 1990; 11: 321-324Abstract Full Text PDF PubMed Scopus (136) Google Scholar, 2Ziegler D.M. Annu. Rev. Pharmacol. Toxicol. 1993; 33: 179-199Crossref PubMed Scopus (244) Google Scholar). Functionally, FMOs differ from other monooxygenases in that the active oxygenating species, the C(4a)hydroperoxide derivative of the flavin cofactor, exists stably within the protein in the absence of substrate, thereby enabling the enzyme to oxidize any soft nucleophile able to gain access to the active site. FMO was originally characterized by using a homogeneous enzyme preparation isolated from pig liver (3Ziegler D.M. Mitchell C.H. Arch. Biochem. Biophys. 1972; 150: 116-125Crossref PubMed Scopus (228) Google Scholar). Proteins with equivalent catalytic properties and substrate profiles were later isolated from the livers of several other species (4Hlavica P. Hulsmann S. Biochem. J. 1979; 182: 109-116Crossref PubMed Scopus (39) Google Scholar, 5Kimura T. Kodama M. Nagata C. Biochem. Biophys. Res. Commun. 1983; 110: 640-645Crossref PubMed Scopus (45) Google Scholar, 6Sabourin P.J. Smyser B.P. Hodgson E. Int. J. Biochem. 1984; 16: 713-720Crossref PubMed Scopus (77) Google Scholar). However, contemporaneous studies indicated that rabbit lung contained a form of FMO which, although clearly related to the liver enzyme, possessed distinct physicochemical properties (7Devereux T.R. Fouts J.R. Chem. Biol. Interact. 1974; 8: 91-105Crossref PubMed Scopus (41) Google Scholar) and substrate preferences (8Ohmiya Y. Mehendale H.M. Pharmacology. 1981; 22: 172-182Crossref PubMed Scopus (13) Google Scholar, 9Ohmiya Y. Mehendale H.M. Biochem. Pharmacol. 1982; 31: 157-162Crossref PubMed Scopus (25) Google Scholar). The purification of this “lung” or “pulmonary” FMO (10Williams D.E. Ziegler D.M. Nordin D.J. Hale S.E. Masters B.S.S. Biochem. Biophys. Res. Commun. 1984; 125: 116-122Crossref PubMed Scopus (99) Google Scholar, 11Williams D.E. Hale S.E. Meurhoff A.S. Masters B.S.S. Mol. Pharmacol. 1984; 28: 381-390Google Scholar, 12Tynes R.E. Sabourin P.J. Hodgson E. Biochem. Biophys. Res. Commun. 1985; 126: 1069-1075Crossref PubMed Scopus (101) Google Scholar) confirmed the existence of two distinct FMOs, distinguishable both immunochemically and by substrate preference (10Williams D.E. Ziegler D.M. Nordin D.J. Hale S.E. Masters B.S.S. Biochem. Biophys. Res. Commun. 1984; 125: 116-122Crossref PubMed Scopus (99) Google Scholar, 11Williams D.E. Hale S.E. Meurhoff A.S. Masters B.S.S. Mol. Pharmacol. 1984; 28: 381-390Google Scholar, 12Tynes R.E. Sabourin P.J. Hodgson E. Biochem. Biophys. Res. Commun. 1985; 126: 1069-1075Crossref PubMed Scopus (101) Google Scholar), and the subsequent isolation and characterization of the corresponding cDNA clones (13Lawton M.P. Gasser R. Tynes R.E. Hodgson E. Philpot R.M. J. Biol. Chem. 1990; 265: 5855-5861Abstract Full Text PDF PubMed Google Scholar) demonstrated that each was the product of a separate gene. Following the identification of “liver” and “lung” FMO as discrete enzymes, evidence accumulated indicating the presence in these tissues of additional forms of the enzyme. The existence of multiple mammalian FMOs was subsequently confirmed when new forms were identified, either by direct sequencing of purified proteins (14Ozols J. Arch. Biochem. Biophys. 1991; 290: 103-115Crossref PubMed Scopus (41) Google Scholar, 15Ozols J. Biochemistry. 1994; 33: 3751-3755Crossref PubMed Scopus (17) Google Scholar), or via the isolation and characterization of cDNA clones (16Dolphin C.T. Shephard E.A. Povey S. Smith R.L. Phillips I.R. Biochem. J. 1992; 287: 261-267Crossref PubMed Scopus (47) Google Scholar, 17Lomri N. Gu Q. Cashman J.R. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1685-1689Crossref PubMed Scopus (97) Google Scholar, 18Atta-Asafo-Adjei E. Lawton M.P. Philpot R.M. J. Biol. Chem. 1993; 268: 9681-9689Abstract Full Text PDF PubMed Google Scholar). To date, five distinct forms of FMO, designated FMOs 1–5, each of which is encoded by its own gene and which exhibits approximately 50–60% pairwise amino acid sequence similarity, have been identified in mammals (19Lawton M.P. Cashman J.R. Cresteil T. Dolphin C.T. Elfarra A.A. Hines R.N. Hodgson E. Kimura T. Ozols J. Phillips I.R. Philpot R.M. Poulsen L.L. Rettie A.E. Shephard E.A. Williams D.E. Ziegler D.M. Arch. Biochem. Biophys. 1994; 308: 254-257Crossref PubMed Scopus (193) Google Scholar). According to the present nomenclature (19Lawton M.P. Cashman J.R. Cresteil T. Dolphin C.T. Elfarra A.A. Hines R.N. Hodgson E. Kimura T. Ozols J. Phillips I.R. Philpot R.M. Poulsen L.L. Rettie A.E. Shephard E.A. Williams D.E. Ziegler D.M. Arch. Biochem. Biophys. 1994; 308: 254-257Crossref PubMed Scopus (193) Google Scholar) “liver” and “lung” FMO are designated FMO1 and FMO2, respectively, whereas the forms identified subsequently have been designated FMOs 3, 4, and 5. Although detected at other sites, such as the kidney (13Lawton M.P. Gasser R. Tynes R.E. Hodgson E. Philpot R.M. J. Biol. Chem. 1990; 265: 5855-5861Abstract Full Text PDF PubMed Google Scholar, 20Tynes R.E. Philpot R.M. Mol. Pharmacol. 1987; 31: 569-574PubMed Google Scholar, 21Shehin-Johnson S.E. Williams D.E. Larsen-Su S. Stresser D.M. Hines R.N. J. Pharmacol. Exp. Ther. 1995; 272: 1293-1299PubMed Google Scholar), FMO2 is expressed predominantly in pulmonary tissue (10Williams D.E. Ziegler D.M. Nordin D.J. Hale S.E. Masters B.S.S. Biochem. Biophys. Res. Commun. 1984; 125: 116-122Crossref PubMed Scopus (99) Google Scholar, 11Williams D.E. Hale S.E. Meurhoff A.S. Masters B.S.S. Mol. Pharmacol. 1984; 28: 381-390Google Scholar, 13Lawton M.P. Gasser R. Tynes R.E. Hodgson E. Philpot R.M. J. Biol. Chem. 1990; 265: 5855-5861Abstract Full Text PDF PubMed Google Scholar, 20Tynes R.E. Philpot R.M. Mol. Pharmacol. 1987; 31: 569-574PubMed Google Scholar, 21Shehin-Johnson S.E. Williams D.E. Larsen-Su S. Stresser D.M. Hines R.N. J. Pharmacol. Exp. Ther. 1995; 272: 1293-1299PubMed Google Scholar, 22Tynes R.E. Hodgson E. Arch. Biochem. Biophys. 1985; 240: 77-93Crossref PubMed Scopus (94) Google Scholar, 23Yueh M-H Krueger S.K. Williams D.E. Biochim. Biophys. Acta. 1997; 1350: 267-271Crossref PubMed Scopus (29) Google Scholar) where, in the rabbit, it has been localized to the nonciliated bronchiolar epithelial (clara) cells and endothelial type I and II cells (24Overby L. Nishio S.J. Lawton M.P. Plopper C.G. Philpot R.M. Exp. Lung Res. 1992; 18: 131-144Crossref PubMed Scopus (25) Google Scholar). FMO2 gene expression has been demonstrated to be regulated by sex hormones in experimental animals (25Lee M-Y. Clark J.E. Williams D.E. Arch. Biochem. Biophys. 1993; 302: 332-336Crossref PubMed Scopus (44) Google Scholar, 26Lee M-Y. Smiley S. Kadkhodayan S. Hines R.N. Williams D.E. Chem. Biol. Interact. 1995; 96: 75-85Crossref PubMed Scopus (25) Google Scholar) and putative glucocorticoid responsive elements have been identified in the 5′-flanking region of the rabbit FMO2 gene (27Wyatt M.K. Philpot R.M. Carver G. Lawton M.P. Nikbakht K.N. Drug Metab. Dispos. 1996; 24: 1320-1327PubMed Google Scholar). FMO2 displays catalytic characteristics that distinguish it from other forms of FMO. For instance, although able to oxidize many typical FMO substrates, it is inactive toward certain tertiary amines, such as imipramine and chlorpromazine (9Ohmiya Y. Mehendale H.M. Biochem. Pharmacol. 1982; 31: 157-162Crossref PubMed Scopus (25) Google Scholar, 10Williams D.E. Ziegler D.M. Nordin D.J. Hale S.E. Masters B.S.S. Biochem. Biophys. Res. Commun. 1984; 125: 116-122Crossref PubMed Scopus (99) Google Scholar), that are good substrates for other forms of the enzyme. Furthermore, in contrast to other FMOs, FMO2 is capable of mediating the N-oxidation of some primary alkylamines to their oximes, via anN-hydroxylamine intermediate (28Tynes R.E. Sabourin P.J. Hodgson E. Philpot R.M. Arch. Biochem. Biophys. 1986; 251: 654-664Crossref PubMed Scopus (47) Google Scholar, 29Poulsen L.L. Taylor K. Williams D.E. Masters B.S.S. Ziegler D.M. Mol. Pharmacol. 1986; 30: 680-685PubMed Google Scholar). We have previously reported the isolation of cDNAs encoding human FMOs 1 (30Dolphin C. Shephard E.A. Povey S. Palmer C.N.A. Ziegler D.M. Ayesh R. Smith R.L. Phillips I.R. J. Biol. Chem. 1991; 266: 12379-12385Abstract Full Text PDF PubMed Google Scholar), 4 (16Dolphin C.T. Shephard E.A. Povey S. Smith R.L. Phillips I.R. Biochem. J. 1992; 287: 261-267Crossref PubMed Scopus (47) Google Scholar) (previously designated FMO2), and 3 (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar) and have determined that the corresponding genes, plus the genes encoding human FMOs 2 and 5, are all located on the long arm of chromosome 1 (16Dolphin C.T. Shephard E.A. Povey S. Smith R.L. Phillips I.R. Biochem. J. 1992; 287: 261-267Crossref PubMed Scopus (47) Google Scholar, 30Dolphin C. Shephard E.A. Povey S. Palmer C.N.A. Ziegler D.M. Ayesh R. Smith R.L. Phillips I.R. J. Biol. Chem. 1991; 266: 12379-12385Abstract Full Text PDF PubMed Google Scholar,32Shephard E.A. Dolphin C.T. Fox M.F. Povey S. Smith R. Phillips I.R. Genomics. 1993; 16: 85-89Crossref PubMed Scopus (36) Google Scholar, 33McCombie R.R. Dolphin C.T. Povey S. Shephard E.A. Phillips I.R. Genomics. 1996; 34: 426-429Crossref PubMed Scopus (32) Google Scholar). In this report we describe the isolation and characterization of cDNA clones encoding human FMO2 and demonstrate that, in common with human FMOs 1 and 3 (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar), expression of the corresponding gene is subject to both ontogenic and tissue-specific regulation. Furthermore, we report that, in contrast to apparently all other mammalian species, including closely related primates, in humans the major FMO2allele encodes a truncated polypeptide which is catalytically inactive. Oligonucleotides were synthesized on a PCR-MATE DNA synthesizer (model 391, Applied Biosystems, Warrington, UK). Human adult and fetal tissue samples were obtained as described previously (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar) except for single adult lung and kidney samples, which were obtained from the International Institute for the Advancement of Medicine (Exton, PA). Total RNA was prepared by the guanidinium thiocyanate/LiCl method (34Cathala G. Savouret J-F. Mendez B. West B.L. Karin M. Martial J.A. Baxter J.D. DNA. 1983; 2: 329-335Crossref PubMed Scopus (1229) Google Scholar), resuspended in diethylpyrocarbonate-treated water and stored in aliquots at −80 °C. RNA concentration was determined from absorbance at 260 nm. Human genomic DNA was isolated from whole blood by the method of Lahiri and Nurnberger (35Lahiri D.K. Nurnberger J.I. Nucleic Acids Res. 1991; 19: 5444Crossref PubMed Scopus (1982) Google Scholar) and from solid tissue by use of a commercial DNA isolation kit (Nucleon Biosciences, Coatbridge, Scotland). Gorilla and chimpanzee genomic DNA was isolated by the method of Blin and Stafford (36Blin N. Stafford D.W. Nucleic Acids Res. 1976; 3: 2303-2308Crossref PubMed Scopus (2335) Google Scholar) from post-mortem tissue samples. Reverse-transcription of rabbit (New Zealand White) lung total RNA (20 μg) was primed with 200 pg of random hexamer oligonucleotides (Pharmacia Biotech, St. Albans, UK) and catalyzed by 200 units of Moloney murine leukemia virus reverse-transcriptase (Life Technologies, Paisley, Scotland) in a total volume of 20 μl according to the supplier's recommendations. The reaction mix was then incubated for 5 min at 75 °C and the volume increased to 100 μl with water. Oligonucleotides 102 (5′-GACGCAGTCATGGTCTGCAGTGGC-3′) and 180 (5′-GATGTAATTGGTCTGCAGTTTCTG-3′), designed from the rabbit FMO2 cDNA sequence (13Lawton M.P. Gasser R. Tynes R.E. Hodgson E. Philpot R.M. J. Biol. Chem. 1990; 265: 5855-5861Abstract Full Text PDF PubMed Google Scholar) and which incorporated PstI restriction endonuclease sites present within the rabbit sequence, were used to prime the amplification, by PCR, of 879 bp of coding region. The PCR was performed in a volume of 40 μl containing 15 pmol of each primer, 66.7 mm Tris-HCl (pH 8.4), 1.25 mmMgCl2, 16.7 mm(NH4)2SO4, 0.1% (v/v) Tween 20, 50 μm of each dNTP, and a 5-μl aliquot of the reverse-transcription product. After an initial denaturation at 94 °C for 1 min, 0.5 μl (2.5 units) of Taq DNA polymerase (BioLine, London, UK) was added, and the reaction mix was incubated (TR1, Hybaid, Ashford, UK) for 32 cycles at 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 1 min, followed by an additional 5 min at 72 °C. The PCR product was incubated withPstI, gel-purified, and ligated intoPstI-digested pBS (Stratagene, Cambridge, UK) to give the plasmid pRABLUNG. The identity of the cDNA insert was confirmed by restriction mapping and partial DNA sequencing. The insert from pRABLUNG was excised by incubation with PstI, gel-purified, radiolabeled by the oligonucleotide random primer method (37Feinberg A.P. Vogelstein B. Anal. Biochem. 1983; 132: 6-13Crossref PubMed Scopus (16651) Google Scholar) to a specific radioactivity of approximately 109 cpm/μg with [α–32P]dCTP (800 Ci/mmol, Amersham International, Amersham, UK), and used to screen an adult human lung cDNA library constructed in λgt11 (gift of Dr. K. Reid) plated at a density of 1.5 × 105 plaque-forming units per 20 × 20-cm plate (Nalge Nunc, Hereford, UK). Duplicate filters were prehybridized, hybridized, washed, and subjected to autoradiography as described previously (30Dolphin C. Shephard E.A. Povey S. Palmer C.N.A. Ziegler D.M. Ayesh R. Smith R.L. Phillips I.R. J. Biol. Chem. 1991; 266: 12379-12385Abstract Full Text PDF PubMed Google Scholar). Because of loss of the EcoRI cloning sites during library construction, insert DNAs could not be excised from positive phage clones by restriction digestion and were therefore recovered by PCR as described previously (30Dolphin C. Shephard E.A. Povey S. Palmer C.N.A. Ziegler D.M. Ayesh R. Smith R.L. Phillips I.R. J. Biol. Chem. 1991; 266: 12379-12385Abstract Full Text PDF PubMed Google Scholar). Reverse-transcription of human or cynomolgus macaque (Macaca fascicularis) lung total RNA (10 μg), and first-round and seminested 5′-RACE-PCRs, using the human FMO2 cDNA-specific primers 498 (5′-CAGGAAGTTTGGAAAATCTTCAGGC-3′) and 423 (5′-CTCAAGTCCCTCATCCACACAGC-3′), respectively, and the common adapter primer 365 (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar), were performed as described previously (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar). The products of the seminested 5′-RACE-PCRs were blunt-ended with T4 DNA polymerase, phosphorylated with T4 polynucleotide kinase (New England Biolabs, Hitchin, UK), then purifed (SpinBind, FMC Bioproducts, Rockland, ME) and inserted into EcoRV-digested pBS. 3′-RACE-PCR was performed essentially as described by Frohman et al. (38Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Crossref PubMed Scopus (4341) Google Scholar). Reverse-transcription of human lung total RNA (10 μg) was performed as described previously (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar), except that reactions were primed with 100 ng each of the anchored oligo-d(T) primers 366, 367, and 368 (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar). First-round and seminested 3′-RACE-PCRs were performed as described above for 5′-RACE-PCR, except that they were primed with 15 pmol each of the universal primer 365 (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar) and the FMO2 cDNA-specific primers 359 (5′-ATGATGTCCCAAGTCGTCTACT-3′) and 356 (5′-TACTCTGTGGAGCCATCAAG-3′), respectively. Seminested 3′-RACE-PCR products were cloned as descibed above for 5′-RACE-PCR products. Reverse-transcription of human or macaque lung total RNA (10 μg) and the subsequent removal of RNA with RNases A and H were performed as described previously (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar), except that reverse-transcription was catalyzed by 200 units of SuperScript II reverse-transcriptase (Life Technologies) in a volume of 20 μl according to the supplier's recommendations. Oligonucleotides 392 (5′-CTAGAATTCTAGAATGGCAAAGAAGGTAGCTGTGATTG-3′) and 394 (5′-TACTGGATCCTGACAAGATAATAAAGCCCAAAG-3′), containing uniqueXbaI and BamHI restriction enzyme sites, respectively, were designed to prime the amplification of the entire protein-coding region of the human FMO2 cDNA plus a small stretch of the 3′-untranslated region. PCR was performed in a volume of 40 μl containing 15 pmol of each primer, 10 mm KCl, 10 mm (NH4)2SO4, 20 mm Tris-HCl (pH 8.8), 2 mm MgSO4, 0.1% (v/v) Triton X-100, bovine serum albumin (100 μg/ml), 200 mm of each dNTP, and a 2-μl aliquot of the reverse-transcription product. After an initial denaturation step at 94 °C for 1 min, 1 μl (2.5 units) of Pfu DNA polymerase (Stratagene) was added and the reaction incubated for 36 cycles at 94 °C for 75 s, 52 °C for 75 s, and 72 °C for 4 min, and then for an additional 10 min at 72 °C. The resulting PCR product was incubated with XbaI and BamHI, gel-purified, and ligated into pBS, previously digested withXbaI and BamHI, to give the plasmid pBSFMO2/2. Amplification of the entire protein-coding region of the cynomolgus macaque FMO2 cDNA, plus small stretches of both the 5′- and 3′-untranslated regions, was achieved with the forward primer 618 (5′-CGTTGAATTCCAAGGGAGAAAACTATTCTGTC-3′), designed from the sequence of the 5′-RACE-PCR products (data not shown), and the human FMO2 cDNA-specific reverse primer 394. PCR conditions were as described above for the amplification of the partial-length rabbit FMO2 cDNA, except that a 5-μl aliquot of the reverse-transcription product was used as template and the reaction mix was incubated for 35 cycles at 94 °C for 30 s, 56 °C for 1 min, and 72 °C for 2 min, followed by an additional 5 min at 72 °C. The PCR product was purified from the reaction mix (SpinBind, FMC) and sequenced directly. An equivalent 235-bp region of the human, gorilla (Gorilla gorilla), and chimpanzee (Pan troglodytes) FMO2 gene was amplified by PCR using the primers 422 (5′-TTCAAAGATCCTAAACTGGCTGTGAG-3′) and 394. Reaction mixtures containing approximately 200 ng of genomic DNA as template were incubated for 38 cycles at 94 °C for 45 s, 56 °C for 45 s, and 72 °C for 45 s, followed by an additional 5 min at 72 °C. Reaction conditions were as described above for the amplification of the partial-length rabbit FMO2 cDNA. PCR products were purified from the reaction mix (SpinBind, FMC) and sequenced directly. Plasmid DNA and purified PCR products were sequenced by the dideoxy chain-termination method (39Sanger F. Nicklen S. Coulson A.R. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 5463-5467Crossref PubMed Scopus (52771) Google Scholar) using DNA sequencing kits (Sequenase II, Amersham International; Cyclist, Stratagene) and either vector- or insert-specific primers. The insert of pBSFMO2/2 and the subcloned products of the human FMO2 3′-RACE-PCRs and of the human and cynomolgus macaque FMO2 5′-RACE-PCRs were sequenced completely on both strands (Fig. 1), whereas the full-length cDNA encoding cynomolgus macaque FMO2 was only partially sequenced. Sequence data were analyzed with MacVector (Oxford Molecular, Oxford, UK) or Genetics Computer Group (Madison, WI) sequence analysis software. The human FMO2 RNase protection plasmid, pBSFMO2/2/15, was constructed by excising a 985-bp section from within the insert of pBSFMO2/2 (by digestion withHindIII and StyI), followed by blunt-ending of the DNA with T4 DNA polymerase and self-ligation of the larger vector fragment. pBSFMO2/2/15 (10 μg) was linearized by digestion withMscI, after which the reaction mix was treated with proteinase K and SDS (40Shephard E.A. Palmer C.N.A. Segall H.J. Phillips I.R. Arch. Biochem. Biophys. 1992; 294: 168-172Crossref PubMed Scopus (48) Google Scholar), and extracted with phenol-chloroform (1:1, v/v). The linearized plasmid was ethanol-precipitated and resuspended in diethylpyrocarbonate-treated water. In vitro synthesis of radiolabeled antisense RNA, probe purification, and RNase protection assays were performed as described previously (31Dolphin C.T. Cullingford T.E. Shephard E.A. Smith R.L. Phillips I.R. Eur. J. Biochem. 1996; 235: 683-689Crossref PubMed Scopus (129) Google Scholar, 40Shephard E.A. Palmer C.N.A. Segall H.J. Phillips I.R. Arch. Biochem. Biophys. 1992; 294: 168-172Crossref PubMed Scopus (48) Google Scholar, 41Akrawi M. Rogiers V. Vandenberghe Y. Palmer C.N.A. Vercruysse A. Shephard E.A. Phillips I.R. Biochem. Pharmacol. 1993; 45: 1583-1591Crossref PubMed Scopus (53) Google Scholar). Comparison of the autoradiographic signal derived from the protected species with a standard curve of undigested probe permitted quantification of FMO2 mRNA in terms of molecules/μg of total RNA. This was converted to molecules/cell by using the average RNA content of a mammalian cell (5 pg) (42Little P.F.R. Jackson I.J. Glover D.M. DNA Cloning; Volume III. A Practical Approach. IRL Press, Oxford1987: 1-18Google Scholar). Human genomic DNA (20 μg) was incubated overnight at 37 °C with 300 units of EcoRI orHindIII (New England Biolabs), extracted with phenol-chloroform (1:1, v/v), ethanol-precipitated, electrophoresed through a 0.8% agarose gel, and transferred to a nylon membrane (Hybond, Amersham International). The membrane was prehybridized at 42 °C for 3 h in 50% deionized formamide, 5× SSPE (1× SSPE, 0.18 m NaCl, 10 mm sodium phosphate (pH 7.4), 1 mm EDTA), 5× Denhardt's reagent, 2% (w/v) SDS, and denatured salmon sperm DNA (100 μg/ml), then hybridized overnight at 42 °C in 50% deionized formamide, 5× SSPE, 2% (w/v) SDS and 10% (w/v) dextran sulfate, containing radiolabeled probe (synthesized as described above for cDNA library screening) at a final concentration of 2–5 ng/ml. After hybridization the membrane was washed once in 2× SSPE/1% (w/v) SDS, (15 min at room temperature), twice in 1× SSPE/1% SDS (15 min each at room temperature), and twice in 0.1× SSPE/1% SDS (15 min each, once at room temperature then at 55 °C), then autoradiographed for 72 h at −80 °C with an intensifying screen. RNA samples (15 μg) were denatured in formaldehyde and electrophoresed through a 1% agarose gel (43Fourney R. Miyakoshi J. Day R.S. Patterson M.C. Focus (BRL). 1988; 10: 5-7Google Scholar). After transfer to a nylon membrane (BDH, Poole, UK) RNA was immobilized by baking the membrane at 80 °C, followed by UV cross-linking (Stratalinker 1800, Stratagene). The membrane was prehybridized, hybridized, washed, and subjected to autoradiography as described above. To construct FMO2X472 recombinant baculovirus, pBSFMO2/2 was incubated withXbaI and PstI, and the smaller of the resulting restriction fragments, comprising the 1413-bp ORF, 192 bp of associated 3′-untranslated region, and approximately 50 bp of pBS, was ligated toXbaI/PstI-digested pFastBac (Life Technologies) to give pFastFMO2/2/8. To construct recombinant virus containing FMO2Q472, two oligonucleotides, 576 (5′-CGAGCTCGCCTTAGAGATAGGTGCG-3′) and 394, were used to prime the amplification of a 330-bp contiguous region of the FMO2 gene that encompassed codon 472 and the second in-frame stop signal located 192 bp downstream (see Fig. 2). PCR conditions were as described above for FMO2gene amplification, except that genomic DNA (100 ng) isolated from an individual previously identified as being heterozygous for TAG and CAG triplets at codon 472 was used as a template. The PCR product was ligated directly into EcoRV-linearized pBS, and, after propagation in Escherichia coli, a plasmid clone containing CAG at codon 472 in the amplified region of theFMO2 gene was identified by DNA sequencing. The insert of this plasmid was excised by incubation with SacI and BamHI, gel-purified, and ligated, in a three-fragment reaction, with a SacI/XbaI restriction fragment of pBSFMO2/2 that contained the remainder of the FMO2 coding region, and XbaI/BamHI-digested pBS, to give pBSFMO2/2/16. The extended FMO2 ORF was excised from pBSFMO2/2/16, by incubation with XbaI and PstI, then gel-purified and ligated into pFastBac to give pFastFMO2/2/16. In order to bring the translational initiation codon of each of the inserted cDNAs closer to the position of that of the original polyhedrin gene, approximately 50 bp were excised from the multiple cloning sites of both pFastFMO2/2/8 and pFastFMO2/2/16 by digestion with EcoRI and XbaI. After religation of the blunt-ended vector portions to give pFastFMO2X472 and pFastFMO2Q472, the respective recombinant baculoviruses, AcFMO2X472 and AcFMO2Q472, were generated by transfection of Spodoptera frugiperda(Sf) 9 cells with the corresponding recombinant bacmid DNA, obtained via site-specific transposition using the Bac-to-Bac system (Life Technologies) according to the supplier's recommendations. Sf9 and Tricoplusia ni insect cells were grown and passaged in shaker cultures using Sf-900II (Life Technologies) and Excell 405 (JRH Biosciences-Europe, Marlow, UK) serum-free media, respectively, containing amphotericin B (5 μg/ml), penicillin G (100 units/ml), and streptomycin sulfate (50 μg/ml). For amplification of virus, Sf9 cells were infected at a multiplicity of infecton of ≤0.1. For expression, 100 ml of T. ni cells, grown to a density of 1 × 106 cells/ml in a 500-ml Erlenmeyer flask, were infected with virus at a multiplicity of infecton of 7 and incubated by shaking at 130 rpm in an orbital shaker (New Brunswick Scientific (UK) Ltd., Hatfield, Herts) at 28 °C for 60 h. Cells were pelleted, resuspended in 30 ml 0.154 m KCl, 50 mmTris-HCl (pH 7.4), 0.2 mm phenylmethylsulfonyl fluoride, and subjected to two 30-s bursts of sonication (Dynatech, model 150) on ice. The cell lysate was centrifuged at 10,000 × g for 15 min at 4 °C. The microsomal fraction was obtained by centrifugation of the resulting supernatant at 100,000 ×g for 1 h at 4 °C. Microsomal pellets were resuspended in 5 ml of 0.154 m KCl, 10 mm HEPES (pH 7.5), 1 mm EDTA, 20% (v/v) glycerol, and stored in aliquots (150 μl) at −80 °C until use. Pr" @default.
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• W2055666951 date "1998-11-01" @default.
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• W2055666951 title "The Flavin-containing Monooxygenase 2 Gene (FMO2) of Humans, but Not of Other Primates, Encodes a Truncated, Nonfunctional Protein" @default.
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