Search

The flavin-containing monooxygenase (FMO) gene family is conserved and ancient with representatives present in almost all phyla so far examined. The genes encode FAD-, NADP- and O(2)-dependent enzymes that catalyse oxygenation of soft-nucleophilic heteroatom centres in a range of substrates. Although usually classified as xenobiotic-metabolising enzymes, examples of FMOs exist that have evolved to metabolise specific endogenous substrates as part of a discrete physiological process. The genome of Caenorhabditis elegans contains five predicted genes encoding putative homologs of mammalian FMOs, K08C7.2, K08C7.5, Y39A1A.19, F53F4.5 and H24K24.5, which we have named fmo and numbered fmo-1 to fmo-5, respectively. As a first step towards determining their functional role(s), we have experimentally characterised these C. elegans fmo genes including analysing reporter gene expression patterns and RNAi phenotypes. Two major gene expression patterns were observed, either intestinal or hypodermal, but no gross RNAi phenotypes were found possibly due to functional redundancy. The internal structures of fmo-2, fmo-3 and fmo-4 have been compared with orthologs identified in the related nematode C. briggsae. For each orthologous pair, a global comparison of the paired upstream intergenic regions was performed and a number of conserved noncoding sequences, which may represent potential cis-regulatory elements, identified. Phylogenetic analysis reveals that several of the fmo homologs are the result of gene duplication along the lineage leading to the nematodes.

We report the isolation, by RT-PCR, of partial cDNAs encoding the rat peroxisome proliferator-activated receptor (PPAR) isoforms PPAR alpha, PPAR beta, and PPAR gamma and the rat retinoid X receptor (RXR) isoforms RXR alpha, RXR beta, and RXR gamma. These cDNAs were used to generate antisense RNA probes to permit analysis, by the highly sensitive and discriminatory RNase protection assay, of the corresponding mRNAs in rat brain regions during development. PPAR alpha, PPAR beta, RXR alpha, and RXR beta mRNAs are ubiquitously present in different brain regions during development, PPAR gamma mRNA is essentially undetectable, and RXR gamma mRNA is principally localised to cortex. We demonstrate, for the first time, the presence of PPAR and RXR mRNAs in primary cultures of neonatal meningeal fibroblasts, cerebellar granule neurons (CGNs), and cortical and cerebellar astrocytes and in primary cultures of adult cortical astrocytes. PPAR alpha, PPAR beta, RXR alpha, and RXR beta mRNAs are present in all cell types, albeit that PPAR alpha and RXR alpha mRNAs are at levels near the limit of detection in CGNs. PPAR gamma mRNA is expressed at low levels in most cell types but is present at levels similar to those of PPAR alpha mRNA in adult astrocytes. RXR gamma mRNA is present either at low levels, or below the level of detection of the assay, for all cell types studied.

The expression, in adult human skin, of genes encoding flavin-containing monooxygenases (FMOs) 1, 3, 4, and 5 and cytochromes P450 (CYPs) 2A6, 2B6, and 3A4 was determined by RNase protection. Each FMO and CYP exhibits inter-individual variation in expression in this organ. Of the individuals analysed, all contained CYP2B6 mRNA in their skin, 90% contained FMO5 mRNA and about half contained mRNAs encoding FMOs 1, 3, and 4, and CYPs 2A6 and 3A4. The amount of each of the FMO and CYP mRNAs in skin is much lower than in the organ in which it is most highly expressed, namely the kidney (for FMO1) and the liver (for the others). In contrast to the latter organs, in the skin FMO mRNAs are present in amounts similar to, or greater than, CYP mRNAs. Only the mRNA encoding CYP2B6 decreased in abundance in skin with increasing age of the individual. All of the mRNAs were substantially less abundant in cultures of keratinocytes than in samples of skin from which the cells were derived. In contrast, an immortalized human keratinocyte cell line, HaCaT, expressed FMO3, FMO5, and CYP2B6 mRNAs in amounts that fall within the range detected in the whole skin samples analysed. FMO1, CYP2A6, and CYP3A4 mRNAs were not detected in HaCaT cells, whereas FMO4 expression was markedly increased in this cell line compared to whole skin. In situ hybridization showed that the expression of each of the FMOs and CYPs analysed was localized to the epidermis, sebaceous glands and hair follicles.

Fish-odour syndrome is a highly unpleasant disorder of hepatic trimethylamine (TMA) metabolism characterized by a body odour reminiscent of rotting fish, due to excessive excretion of the malodorous free amine. Although fish-odour syndrome may exhibit as sequelae with other conditions (e.g. liver dy

The flavin-containing monooxygenases (FMOs) are a family of xenobiotic-metabolizing enzymes that are expressed in a species- and tissue-specific manner. FMO2 expression has been observed in pulmonary tissue from several species, but not human. Two human FMO2 point mutations have been reported: a cytosine to thymidine transition at position 1414 resulting in a premature stop codon and a thymidine insertion at position 1589 resulting in a frameshift. To define the frequency of these sequence variations and explore their significance, unrelated African-American, Caucasian, and Korean individuals were genotyped. In the African-American population tested (n = 180), the 1414C allele occurred at a 13% frequency; however, all of the tested Caucasians (n = 52) and Koreans (n = 100) were homozygous for the 1414T allele. The T1589 allele occurred at frequencies of 6.9 and 13.0% in African-Americans (n = 175) and Caucasians (n = 23), respectively, and appears to segregate with the 1414T allele. Thus, it would have no further impact on FMO2 activity. Western blot analysis of pulmonary microsomes failed to detect immunoreactive protein in 1414T homozygotes. A heterozygotic individual did exhibit a single band of the expected size, but no detectable FMO activity in the corresponding lung microsomes. Sequence analysis, however, was consistent with the 1414C allele encoding an active FMO2 enzyme. FMO2 mRNA expression was observed in most individuals, but failed to correlate with genotype or protein expression. In summary, functional FMO2 is expressed in only a small percentage of the overall population. However, in certain ethnic groups, active pulmonary FMO2 enzyme will be present in a significant number of individuals.

We have previously shown that primary trimethylaminuria, or fish-odour syndrome, is caused by an inherited defect in the flavin-containing monooxygenase 3 (FMO3) catalysed N-oxidation of the dietary-derived malodorous amine, trimethylamine (TMA). We now report a novel causative mutation for the disorder identified in a young girl diagnosed by proton nuclear magnetic resonance (NMR) spectroscopy of her urine. Sequence analysis of genomic DNA amplified from the patient revealed that she was homozygous for a T to C missense mutation in exon 3 of the FMO3 gene. The mutation changes an ATG triplet, encoding methionine, at codon 82 to an ACG triplet, encoding threonine. A polymerase chain reaction/restriction enzyme-based assay was devised to genotype individuals for the FMO3Thr82 allele. Wild-type and mutant FMO3, heterologously expressed in a baculovirus-insect cell system, were assayed by ultraviolet spectrophotometry and NMR spectroscopy for their ability to catalyse the N-oxidation of TMA. The latter technique has the advantage of enabling the simultaneous, direct and semi-continuous measurement of both of the products, TMA N-oxide and NADP, and of one of the reactants, NADPH. Results obtained from both techniques demonstrate that the Met82Thr mutation abolishes the catalytic activity of the enzyme and thus represents the genetic basis of the disorder in this individual. The combination of NMR spectroscopy with gene sequence and expression technology provides a powerful means of determining genotype-phenotype relationships in trimethylaminuria.

The human flavin-containing monooxygenase (FMO) gene family comprises at least five distinct members (FMO1 to FMO5) that code for enzymes responsible for the oxidation of a wide variety of soft nucleophilic substrates, including drugs and environmental pollutants. Three of these genes (FMO1, FMO3, and FMO4) have previously been localized to human chromosome 1q, raising the possibility that the entire gene family is clustered in this chromosomal region. Analysis by polymerase chain reaction of DNA isolated from a panel of human-rodent somatic cell hybrids demonstrates that the two remaining identified members of the FMO gene family, FMO2 and FMO5, also are located on chromosome 1q.

We have previously described the isolation and sequencing of cDNA clones encoding flavin-containing monooxygenases (FMOs) 1 and 4 of man [Dolphin, C., Shephard, E. A., Povey, S., Palmer, C. N. A., Ziegler, D. M., Ayesh, R., Smith, R. L. & Phillips, I. R. (1991) J. Biol. Chem. 266, 12379-12385; Dolphin, C., Shephard E. A., Povey, S., Smith, R. L. & Phillips, I. R. (1992) Biochem. J. 287, 261-267]. We present here the isolation of a cDNA for FM03 of man. The sequence of this CDNA and the amino acid sequence deduced from it differ substantially from those previously reported for this member of the FMO family of man. In addition, we have investigated, by quantitative RNase protection assays, the expression in several foetal and adult human tissues of genes encoding FMO1, FMO3 and FMO4, Our results demonstrate that, in the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus it is expressed in both organs. The lack of expression of FMO1 in adult human liver is in marked contrast to the situation in other mammals, such as pig and rabbit, in which FMO1 constitutes a major form of the enzyme in the liver of the adult animal. The mRNA encoding FMO3 is abundant in adult liver and is also present, in low abundance, in some foetal tissues. Thus, FMO1 and FMO3 are both subject to developmental and tissue-specific regulation, with a developmental switch in the expression of the genes taking place in the liver. FMO4 mRNA is present in low abundance in several foetal and adult tissues and thus the corresponding gene appears to be expressed constitutively.

We have used the polymerase chain reaction to map the gene encoding human flavin-containing monooxygenase (FMO) form II (N. Lomri, Q. Gu, and J. R. Cashman, 1992, Proc. Natl. Acad. Sci. USA 89: 1685-1689) to chromosome 1. We propose the designation FMO3 for this gene as it is the third FMO gene to be mapped. The two other human FMO genes identified to date, FMO1 and FMO2, are also located on chromosome 1 (C. Dolphin, E. A. Shephard, S. Povey, C. N. A. Palmer, D. M. Ziegler, R. Ayesh, R. L. Smith, and I. R. Phillips, 1991, J. Biol. Chem. 266: 12379-12385; C. Dolphin, E. A. Shephard, S. F. Povey, R. L. Smith, and I. R. Phillips, 1992, Biochem. J. 286: 261-267). The localization of FMO1, FMO2, and FMO3 has been refined to the long arm of chromosome 1. Analysis of human metaphase chromosomes by in situ hybridization confirmed the mapping of FMO1 and localized this gene more precisely to 1q23-q25.

Background Escherichia coli strain EL350 contains chromosomally integrated phage lambda Red recombinase genes enabling this strain to be used for modifying the sequence of resident clones via recombineering. BAC and fosmid clones are highly suitable for modification by recombineering but, because they are present at low (1-2) copies per cell, the DNA is difficult to isolate in high yield and purity. To overcome this limitation vectors, e.g. pCC1FOS, have been constructed that contain the additional replication origin, oriV, which permits copy-number to be induced transiently when propagated in a suitable host strain, e.g. EPI300, that supplies the cognate trans-replication protein TrfA. Previously, we used EL350 and EPI300 sequentially to recombineer oriV-equipped fosmid genomic clones and, subsequently, to induce copy-number of the resulting recombinant clone. To eliminate these intervening DNA isolation and transformation steps we retrofitted EL350 with a P BAD-driven trfA gene generating strain MW005 that supports, independently, both recombineering and copy-number induction. Results The P BAD-driven copy of cre in EL350 was replaced seamlessly with a copy of trfA, PCR-amplified from EPI300 chromosomal DNA, to generate MW005. This new strain has been used to both generate, via recombineering, a number of reporter gene fusions directly from pCC1FOS-based Caenorhabditis elegans genomic clones and to transiently induce copy-number of fosmid and BAC clones prior to DNA preparation. Conclusions By retrofitting EL350, an established 'recombineering' E. coli strain, with a tightly regulated copy of trfA we have produced a new strain, MW005, which combines recombineering capacity with the useful ability to transiently induce copy-number of oriV-equipped clones. By coupling these two steps in a single strain, use of MW005 will enable the more rapid recombineering-mediated production of recombinant clones in the yield and quality necessary for many downstream purposes.

cDNA clones that code for a pig and human flavin-containing monooxygenase (FMO) have been isolated. The full-length sequence of the human cDNAs revealed that they encode a polypeptide of 532 amino acid residues containing putative FAD- and NADP-binding sites. The deduced amino acid sequence has 88 and 86% identity, respectively, with the pig and rabbit "hepatic" forms of FMO, but is only 58% similar to the rabbit "pulmonary" FMO, and thus represents the human ortholog of the "hepatic" form of FMO. However, as this FMO is present in low abundance in human adult liver, the general term "hepatic" for this form of the enzyme is misleading, and thus we propose the name FMO1 to describe this human FMO and its mammalian orthologs. Northern blot analysis demonstrated that human FMO1 mRNA is more abundant in fetal than in adult liver, indicating that in man the enzyme is subject to developmental regulation. Southern blot hybridization of human genomic DNA suggests that the protein is encoded by a single gene, which has been designated FMO1 and mapped to chromosome 1.

The success of RNA interference (RNAi) in mammalian cells, mediated by siRNAs or shRNA-generating plasmids, is dependent, to an extent, upon transfection efficiency. This is a particular problem with primary cells, which are often difficult to transfect using cationic lipid vehicles. Effective RNAi in primary cells is thus best achieved with viral vectors, and retro-, adeno-, and lentivirus RNAi systems have been described. However, the use of such human viral vectors is inherently problematic, e.g., Class 2 status and requirement of secondary helper functions. Although insect cells are their natural host, baculoviruses also transduce a range of vertebrate cell lines and primary cells with high efficiency. The inability of baculoviral vectors to replicate in mammalian cells, their Class 1 status, and the simplicity of their construction make baculovirus an attractive alternative gene delivery vector. We have developed a baculoviral-based RNAi system designed to express shRNAs and GFP from U6 and CMV promoters, respectively. Transduction of Saos2, HepG2, Huh7, and primary human hepatic stellate cells with a baculoviral construct expressing shRNAs targeting lamin A/C resulted in effective knockdown of the corresponding mRNA and protein. Development of this baculoviral-based system provides an additional shRNA delivery option for RNAi-based investigations in mammalian cells.

Mammalian flavin-containing monooxygenase (FMO) exists as six gene families and metabolizes a plethora of drugs and xenobiotics. The major FMO in adult human liver, FMO3, is responsible for trimethylamine (TMA) N-oxygenation. A number of FMO3 mutant alleles have been described and associated with a disease termed trimethylaminuria (TMAU). The TMAU patient excretes large amounts of TMA in urine and sweat. A more recent ethnically related polymorphism in expression of the major FMO in lung, FMO2, has been described. All Caucasians and Asians genotyped to date are homozygous for a CAG --> TAG amber mutation resulting in a premature stop codon and a nonfunctional protein truncated at AA 472 (wildtype FMO2 is 535 AA). This allele has been designated hFMO2*2A. Twenty-six percent of individuals of African descent and 5% of Hispanics genotyped to date carry at least one allele coding for full-length FMO2 (hFMO2*1 allele). Preliminary evidence indicates that FMO2.1 is very active toward the S-oxygenation of low MW thioureas, including the lung toxicant ethylene thiourea. Polymorphic expression of functional FMO2 in the individuals of African and Hispanic descent may markedly influence drug metabolism and/or xenobiotic toxicity in the lung.

Activated peroxisome proliferator activated receptor alpha (PPAR alpha) protects against the cellular inflammatory response, and is central to fatty acid-mediated upregulation of the gene encoding the key ketogenic enzyme mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mHS). We have previously demonstrated both PPAR alpha and mHS expression in brain, implying that brain-targeted PPAR alpha activators may likewise up-regulate mHS expression in brain. Thus, to attempt pharmacological activation of brain PPAR alpha in vivo, we have administered to rats two drugs with previously defined actions in rat brain, namely the PPAR alpha-selective activator ciprofibrate and the pan-PPAR activator valproate. Using the sensitive and discriminatory RNase protection co-assay, we demonstrate that both ciprofibrate and valproate induce mHS expression in liver, the archetypal PPAR alpha-expressing organ. Furthermore, ciprofibrate potently increases mHS mRNA abundance in rat brain, together with lesser increases in two other PPAR alpha-regulated mRNAs. Thus we demonstrate, for the first time, up-regulation of expression of PPAR alpha-dependent genes including mHS in brain, with implications in the increased elimination of neuro-inflammatory lipids and concomitant increased production of neuro-protective ketone bodies.

Metabolic fuels may be defined as complex carbon molecules that can be exploited for the energy released upon breakage of their carbon-carbon bonds. Fatty acids and hexoses, principally glucose, represent the two major classes of such complex carbon molecules that are used for fuel by eukaryotes. Typically, glucose is first metabolized anaerobically (glycolysis) to three-carbon molecules (pyruvate, phosphoenolpyruvate) which then provide the precursors of the tricarboxylic acid or Krebs cycle, ie: oxaloacetate and acetyl-CoA. The cycling of such intermediates via the Krebs cycle then effects the con

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.

The inherited metabolic disorder trimethylaminuria (fish-odor syndrome) is associated with defective hepatic N-oxidation of dietary-derived trimethylamine catalyzed by flavin-containing monooxygenase (FMO). As FMO3 encodes the major form of FMO expressed in adult human liver, it represents the best candidate gene for the disorder. The structural organization of FMO3 was determined by sequencing the products of exon-to-exon and vectorette PCR, the latter through the use of vectorette libraries constructed directly from genomic DNA. The gene contains one noncoding and eight coding exons. Knowledge of the exon/intron organization of the human FMO3 gene enabled each of the coding exons of the gene, together with their associated flanking intron sequences, to be amplified from genomic DNA and will thus facilitate the identification of mutations in FMO3 in families affected with fish-odor syndrome.

Individuals with primary trimethylaminuria exhibit a body odour reminiscent of rotting fish, due to excessive excretion of trimethylamine (TMA; refs 1-3). The disorder, colloquially known as fish-odour syndrome, is inherited recessively as a defect in hepatic N-oxidation of dietary-derived TMA and cannot be considered benign, as sufferers may display a variety of psychosocial reactions, ranging from social isolation of clinical depression and attempted suicide. TMA oxidation is catalyzed by flavin-containing mono-oxygenase (FMO; refs 7,8), and tissue localization and functional studies have established FMO3 as the form most likely to be defective in fish-odour syndrome. Direct sequencing of the coding exons of FMO3 amplified from a patient with fish-odour syndrome identified two missense mutations. Although one of these represented a common polymorphism, the other, a C-->T transition in exon 4, was found only in an affected pedigree, in which it segregated with the disorder. The latter mutation predicts a proline-->leucine substitution at residue 153 and abolishes FMO3 catalytic activity. Our results indicate that defects in FMO3 underlie fish-odour syndrome and that the Pro 153-->Leu 153 mutation described here is a cause of this distressing condition.

Background Recombineering is a genetic engineering tool that enables facile modification of large episomal clones, e.g. BACs, fosmids. We have previously adapted this technology to generate, directly from fosmid-based genomic clones, fusion gene reporter constructs designed to investigate gene expression patterns in C. elegans. In our adaptation a rpsL-tet(A) positive/negative-selection cassette (RT-cassette) is first inserted and then, under negative selection, seamlessly replaced with the desired sequence. We report here on the generation and application of a resource comprising two sets of constructs designed to facilitate this particular recombineering approach. Results Two complementary sets of constructs were generated. The first contains different fluorescent protein reporter coding sequences and derivatives while the second set of constructs, based in the copy-number inducible vector pCC1Fos, provide a resource designed to simplify RT-cassette-based recombineering. These latter constructs are used in pairs the first member of which provides a template for PCR-amplification of an RT-cassette while the second provides, as an excised restriction fragment, the desired fluorescent protein reporter sequence. As the RT-cassette is flanked by approximately 200 bp from the ends of the reporter sequence the subsequent negative selection replacement step is highly efficient. Furthermore, use of a restriction fragment minimizes artefacts negating the need for final clone sequencing. Utilizing this resource we generated single-, double- and triple-tagged fosmid-based reporters to investigate expression patterns of three C. elegans genes located on a single genomic clone. Conclusions We describe the generation and application of a resource designed to facilitate counter-selection recombineering of fosmid-based C. elegans genomic clones. By choosing the appropriate pair of ‘insertion’ and ‘replacement’ constructs recombineered products, devoid of artefacts, are generated at high efficiency. Gene expression patterns for three genes located on the same genomic clone were investigated via a set of fosmid-based reporter constructs generated with the modified protocol.

cDNA clones encoding five distinct members of the FMO family of man (FMOs 1, 2, 3, 4 and 5) were isolated by a combination of library screening and reverse transcription-polymerase chain reaction techniques. The deduced amino acid sequences of the human FMOs have 82-87% identity with their known orthologues in other mammal but only 51-57% similarity to each other. The hydropathy profiles of the proteins are very similar. From the calculated rate of evolution of FMOs (a 1% change in sequence per 6 million years) it would appear that individual members of the FMO gene family arose by duplication of a common ancestral gene some 250-300 million years ago. Each of the FMO genes was mapped by the polymerase chain reaction to the long arm of human chromosome 1. The localization of the FMO1 gene was further refined to 1q23-q25 by in situ hybridization of human metaphase chromosomes. RNase protection assays demonstrated that in man each FMO gene displays a distinct developmental and tissue-specific pattern of expression. In the adult, FMO1 is expressed in kidney but not in liver, whereas in the foetus its mRNA is abundant in both organs. FMO3 expression is essentially restricted to the liver in the adult and the mRNA is either absent, or present in low amounts, in foetal tissues. FMO4 is expressed more constitutively. Human FMO1 and FMO3 cDNAs were functionally expressed in prokaryotic and eukaryotic cells. FMO1 and FMO3, expressed in either system, displayed product stereoselectivity in their catalysis of the N-oxidation of the pro-chiral tertiary amines, N-ethyl-N-methylaniline (EMA) and pargyline. Both enzymes were stereoselective with respect to the production of the (-)-S-enantiomer of EMA N-oxide. But in the case of pargyline, the enzymes displayed opposite stereoselectivity, FMO1 producing solely the (+)-enantiomer and FMO3 predominantly the (-)-enantiomer of the N-oxide.

A nomenclature based on comparisons of amino acid sequences is proposed for the members of the mammalian flavin-containing monooxygenase (FMO) gene family. This nomenclature is based on evidence of a single gene family composed of five genes. The percentage identities of the amino acid sequences of the five known forms of mammalian FMO are between 52 and 57% in rabbit and between 50 and 58% across species lines. The identities of all orthologs are greater than 82%. There is no evidence for multiple, highly related forms of the enzyme or for more than one mammalian FMO gene family. In the proposed system, the mammalian flavin-containing monooxygenase gene family is designated as "FMO" and the individual genes are distinguished by an Arabic numeral. The FMOs known as the "liver" and "lung" enzymes become FMO1 and FMO2, and the more recently described forms of the enzymes become FMO3, FMO4, and FMO5. Human FMO gene designations, FMO1 and FMO3, remain unchanged, but the gene designated FMO2 becomes FMO4. Following convention, the genes and cDNA designations will be italicized and the mRNA and protein designations will be nonitalicized. The purpose of the proposed nomenclature is to provide for the unambiguous identification of orthologous forms of mammalian FMOs, regardless of the species or tissue in question. The proposed classification considers only members of the mammalian flavin-containing monooxygenase gene family and has no bearing on the generally accepted definition of a multisubstrate flavin-containing monooxygenase.

Recombineering is employed to modify large DNA clones such as fosmids, BACs and PACs. Subtle and seamless modifications can be achieved using counter-selection strategies in which a donor cassette carrying both positive and negative markers inserted in the target clone is replaced by the desired sequence change. We are applying counter-selection recombineering to modify bacmid bMON14272, a recombinant baculoviral genome, as we wish to engineer the virus into a therapeutically useful gene delivery vector with cell targeting characteristics. Initial attempts to replace gp64 with Fusion (F) genes from other baculoviruses resulted in many rearranged clones in which the counter-selection cassette had been deleted. Bacmid bMON14272 contains nine highly homologous regions (hrs) and deletions were mapped to recombination between hr pairs. Recombineering modifications were attempted to decrease intramolecular recombination and/or increase recombineering efficiency. Of these only the use of longer homology arms on the donor molecule proved effective permitting seamless modification. bMON14272, because of the presence of the hr sequences, can be considered equivalent to a highly repetitive BAC and, as such, the optimized method detailed here should prove useful to others applying counter-selection recombineering to modify BACs or PACs containing similar regions of significant repeating homologies.

We have previously reported the cloning of cDNAs for a flavin-containing mono-oxygenase (FMO) of man, designated FMO1 [Dolphin, Shephard, Povey, Palmer, Ziegler, Ayesh, Smith & Phillips (1991) J. Biol. Chem. 266, 12379-12385], that is the orthologue of pig and rabbit hepatic FMOs. We now describe the isolation and characterization of cDNA clones for a second human FMO, which we have designated FMO2. The polypeptide encoded by the cDNAs is 558 amino acid residues long, has a calculated M(r) of 63337, and contains putative FAD- and NADP-binding sites that align exactly with those described in other mammalian FMOs. Human FMO2 has 51-53% primary sequence identity with human FMO1, rabbit pulmonary FMO and rabbit liver FMO form 2, and thus represents a fourth, distinct, member of the mammalian FMO family. The corresponding mRNA is present in low abundance in adult human liver. Southern blot hybridization with single-exon probes demonstrated that human FMO2 and FMO1 are the products of single genes. The gene encoding FMO2 (designated FMO2) was mapped, by the polymerase chain reaction, to human chromosome 1, the same chromosome on which FMO1 is located.

The contribution of the gastrointestinal microflora to the formation of methylthio adducts from paracetamol has been studied by comparing the fate of this drug in conventional mice with that in germ-free and neomycin-treated animals. In both germ-free and neomycin-treated mice there was a highly significant reduction in the urinary excretion of 3-methylthioparacetamol, its glucuronic acid and sulfate conjugates and its sulfoxide, with no other systemic alteration to the overall fate of the drug. These data are consistent with the gut flora playing a major role in the C-S cleavage of paracetamol-3-cysteine, thereby reducing the excretion of the array of methylthio adducts subsequently formed by tissue enzymes from 3-thioparacetamol, the putative product of the C-S cleavage.

The effect of stimulation of interferon (IFN) synthesis with the immunomodulators, polyriboinosinic polyribocytidylic acid (poly rI:rC) and Newcastle Disease Virus (NDV) upon the in vivo metabolism of [carboxyl-14C]-aspirin was investigated in male mice of three different strains. Following poly rI:rC administration to DBA/2 mice, the metabolic conjugation of salicylic acid with glycine was significantly increased, glucuronidation was little changed and there was an approximately 10-fold reduction in the excretion of the oxidation product, gentisic acid. Prolongation of hexobarbitone-induced sleeping time confirmed the ability of this agent to depress oxidative metabolism in vivo. Poly rI:rC administration to C57BL/6By and BALB/cBy mice resulted in similar changes in aspirin metabolism in vivo. Treatment of C57BL/6By mice with NDV produced high levels of circulating IFN and produced alterations in aspirin metabolism similar to those seen after poly rI:rC treatment. Comparable studies in BALB/cBy mice, which did not produce detectable levels of IFN in response to NDV challenge, revealed no change in the fate of aspirin metabolism following treatment with this agent. These data indicate that the in vivo oxidation of salicylate, but not its conjugation, can be depressed, at least indirectly, by an interferon-associated mechanism.

Interferon synthesis was induced in inbred strains of mice with either polyriboinosinic-polyribocytidylic acid or Newcastle disease virus. The strains, BALB/cBy and C57BL/6By, respond to Newcastle disease virus by producing ‘low’ and ‘high’ levels of serum interferon, respectively. 24 h after interferon induction mice received [carboxyl-14C]-aspirin orally. The metabolic conjugation of aspirin was little changed after either treatment while the urinary excretion of gentisic acid, a minor product of metabolic oxidation, was significantly depressed after polyriboinosinic-polyribocytidylic acid in both strains and in C57BL/6By mice after Newcastle disease virus. The results suggest that such a metabolic impairment in aspirin metabolism would not significantly affect salicylate clearance and hence are unlikely to be directly related to the aetiology of Reye's syndrome which has been associated with both a prodromal viral infection and aspirin therapy.