cDNA Cloning, Expression, Mutagenesis, Intracellular Localization, and Gene Chromosomal Assignment of Mouse 5-Lipoxygenase

Abstract

5-Lipoxygenase of mouse macrophages and bone marrow-derived mast cells (BMMC) was investigated. Indirect immunocytofluorescence combined with confocal microscopy provided evidence for distinct intracellular expression patterns and trafficking of 5-lipoxygenase upon cellular activation. In resting BMMC, 5-lipoxygenase was found within the nucleus co-localizing with the nuclear stain Yo-Pro-1. When BMMC were IgE/antigen-activated the 5-lipoxygenase immunofluorescence pattern was changed from nuclear to perinuclear. The absence of divalent cations in the incubation medium, or calcium ionophore A23187 challenge, altered the predominantly nuclear expression pattern to new sites both cytosolic and intranuclear. The cDNA for murine macrophage 5-lipoxygenase was cloned by the polymerase chain reaction and would predict a 674 amino acid protein. Using control cells obtained from 5-lipoxygenase-deficient mice it was determined that a single isoform accounts for both soluble and membrane-bound and nuclear and cytosolic-localized enzyme in macrophages and BMMC. A mutation at amino acid 672 (Val ⟶ Met) introduced serendipitously during the cloning process was found to completely abolish 5-lipoxygenase enzyme activity when the enzyme was expressed in human embryonic kidney 293 cells. This subtle change is proposed to affect the ability of the COOH-terminal isoleucine to coordinate the essential non-heme iron atom. In macrophages and BMMC obtained from 5-lipoxygenase-deficient mice, compensatory changes in expression of genes involved in the biosynthesis of leukotriene B4 were investigated. 5-Lipoxygenase-activating protein expression was reduced by 50%, while leukotriene A4 hydrolase expression was unaltered. The 5-lipoxygenase gene was mapped to the central region of mouse chromosome 6 in a region that shares homology with human chromosome 10 by interspecific backcross analysis. These studies provide a global picture of the murine 5-lipoxygenase system and raise questions about the role of 5-lipoxygenase and leukotrienes within the nucleus. 5-Lipoxygenase of mouse macrophages and bone marrow-derived mast cells (BMMC) was investigated. Indirect immunocytofluorescence combined with confocal microscopy provided evidence for distinct intracellular expression patterns and trafficking of 5-lipoxygenase upon cellular activation. In resting BMMC, 5-lipoxygenase was found within the nucleus co-localizing with the nuclear stain Yo-Pro-1. When BMMC were IgE/antigen-activated the 5-lipoxygenase immunofluorescence pattern was changed from nuclear to perinuclear. The absence of divalent cations in the incubation medium, or calcium ionophore A23187 challenge, altered the predominantly nuclear expression pattern to new sites both cytosolic and intranuclear. The cDNA for murine macrophage 5-lipoxygenase was cloned by the polymerase chain reaction and would predict a 674 amino acid protein. Using control cells obtained from 5-lipoxygenase-deficient mice it was determined that a single isoform accounts for both soluble and membrane-bound and nuclear and cytosolic-localized enzyme in macrophages and BMMC. A mutation at amino acid 672 (Val ⟶ Met) introduced serendipitously during the cloning process was found to completely abolish 5-lipoxygenase enzyme activity when the enzyme was expressed in human embryonic kidney 293 cells. This subtle change is proposed to affect the ability of the COOH-terminal isoleucine to coordinate the essential non-heme iron atom. In macrophages and BMMC obtained from 5-lipoxygenase-deficient mice, compensatory changes in expression of genes involved in the biosynthesis of leukotriene B4 were investigated. 5-Lipoxygenase-activating protein expression was reduced by 50%, while leukotriene A4 hydrolase expression was unaltered. The 5-lipoxygenase gene was mapped to the central region of mouse chromosome 6 in a region that shares homology with human chromosome 10 by interspecific backcross analysis. These studies provide a global picture of the murine 5-lipoxygenase system and raise questions about the role of 5-lipoxygenase and leukotrienes within the nucleus. The enzyme 5-lipoxygenase (arachidonate:oxygen 5-oxidoreductase, EC 1.13.11.34) catalyzes the formation of 5-hydroperoxy-6,8,11,14-eicosatetraenoic acid (5-HPETE)1 1The abbreviations used are: 5-H(P)ETE5-hydro(pero)xy-eicosatetraenoic acidLTleukotrieneFLAP5-lipoxygenase-activating proteinBMMCbone marrow-derived mast cellPCRpolymerase chain reactionHEKhuman embryonic kidneyRACErapid amplification of cDNA endsDNP-BSAdinitrophenyl bovine serum albumin13-H(P)ODE13-hydro(pero)xy-octadecadienoic acidRP-HPLCreversed phase-high performance liquid chromatographykbkilobase(s). and its subsequent conversion to leukotriene (LT)A4 (5,6-oxido-7,9,11,14-eicosatetraenoic acid). LTA4 is a pivotal intermediate in the biosynthesis of inflammatory and anaphylactic mediators which include leukotriene B4 (5S,12R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid and the peptidyl leukotrienes (LTC4, LTD4, and LTE4; see Refs. 1, 2 for reviews). In human neutrophils, 5-lipoxygenase undergoes a Ca2+-dependent translocation from the cytosol to a membrane site which appears to be the nuclear envelope(3Rouzer C.A. Kargman S. J. Biol. Chem. 1988; 263: 10980-10988Google Scholar, 4Woods J.W. Evans J.F. Ethier D. Scott S. Vickers P.J. Hearn L. Heilbein J. Charleson S. Singer I.I. J. Exp. Med. 1993; 178: 1935-1946Google Scholar). 5-Lipoxygenase activating protein (FLAP), an 18-kDa membrane protein found in the nuclear envelope, acts apparently as an arachidonate-binding protein to facilitate the concerted formation of LTA4(5Dixon R.A.F. Diehl R.E. Opas E. Rands E. Vickers P.J. Evans J.F. Gillard J.W. Miller D.K. Nature. 1990; 343: 282-284Google Scholar, 6Abramovitz M. Wong E. Cox M.E. Richardson C.D. Li C. Vickers P.J. Eur. J. Biochem. 1993; 215: 105-111Google Scholar). 5-hydro(pero)xy-eicosatetraenoic acid leukotriene 5-lipoxygenase-activating protein bone marrow-derived mast cell polymerase chain reaction human embryonic kidney rapid amplification of cDNA ends dinitrophenyl bovine serum albumin 13-hydro(pero)xy-octadecadienoic acid reversed phase-high performance liquid chromatography kilobase(s). In alveolar macrophages there is evidence for the existence of two 5-lipoxygenase “pools,” cytosolic and membrane-bound forms(7Coffey M. Peters-Golden M. Fantone J.C. Sporn P.H.S. J. Biol. Chem. 1992; 267: 570-576Google Scholar). Recent data by the same investigators has established nuclear soluble and nuclear bound 5-lipoxygenase expression patterns in rat basophilic leukemia cells(8Brock T.G. Paine R. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Google Scholar). Lepley and Fitzpatrick (9Lepley R.A. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 24163-24168Google Scholar) have obtained in vitro data that 5-lipoxygenase can bind to cytoskeletal proteins and the signaling protein Grb2 via an SH3-binding domain interaction. Thus, in addition to the carrier-mediated export of leukotrienes(10Lam B.K. Owen Jr., W.F. Austen K.F. Soberman R.J. J. Biol. Chem. 1989; 264: 12885-12889Google Scholar, 11Lam B.K. Gagnon L. Austen K.F. Soberman R.J. J. Biol. Chem. 1990; 265: 13438-13441Google Scholar), the concept is emerging that 5-lipoxygenase may have novel intracellular functions, possibly independent of leukotriene biosynthesis, and its intracellular location may be dictated by specific protein-protein interactions. cDNAs encoding the human (12Matsumoto T. Funk C.D. Rdmark O. Hg J.-O. Jrnvall H. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85 (and correction p. 3406): 26-30Google Scholar, 13Dixon R.A.F. Jones R.E. Diehl R.E. Bennett C.D. Kargman S. Rouzer C.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 416-420Google Scholar) and rat (14Balcarek J.M. Theisen T.W. Cook M.N. Varrichio A. Hwang S.-M. Strohsacker M.W. Crooke S.T. J. Biol. Chem. 1988; 263: 13937-13941Google Scholar) 5-lipoxygenases have been isolated, and the human 5-lipoxygenase genomic structure (15Funk C.D. Hoshiko S. Matsumoto T. Rdmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2587-2591Google Scholar) has been elucidated. Recently, in our efforts to better understand the biology of 5-lipoxygenase and leukotrienes we created 5-lipoxygenase-deficient mice by gene targeting(16Funk C.D. Kurre U. Griffis G. Ann. N. Y. Acad. Sci. 1994; 714: 253-258Google Scholar, 17Chen X.-S. Sheller J.R. Johnson E. Funk C.D. Nature. 1994; 372: 179-181Google Scholar). We realized the importance of ascertaining more clearly the existence, or not, of 5-lipoxygenase isoforms and their relationship to distinct intracellular pools of 5-lipoxygenase in resting and activated cells. Our studies have focused primarily on bone marrow-derived mast cells (BMMC) and macrophages. Here we show evidence for different 5-lipoxygenase intracellular locations depending on the state of cell activation in BMMC. Moreover, studies with 5-lipoxygenase-deficient mice show that the different 5-lipoxygenase pools in alveolar macrophages derive from the same gene product. Additionally, we demonstrate the importance of the amino acid residue two positions upstream of the carboxyl terminus for 5-lipoxygenase activity, the chromosomal location of the murine 5-lipoxygenase gene, and studies with macrophages and BMMC of 5-lipoxygenase-deficient mice designed to examine compensatory expression of other proteins key to the formation of leukotrienes (FLAP and LTA4 hydrolase). C57BL/6 × 129 mixed genetic background mice were maintained in the animal barrier facility of Vanderbilt University on a 12 h light/12 h dark cycle with water and food provided ad libitum. The generation of 5-lipoxygenase-deficient mice has been described(17Chen X.-S. Sheller J.R. Johnson E. Funk C.D. Nature. 1994; 372: 179-181Google Scholar). BMMC were prepared from cells flushed from femurs and tibiae of wild-type and 5-lipoxygenase-deficient mice and were cultured in the presence of 50% WEHI-3b conditioned medium, 50% RPMI 1640 (complete medium) for 3-6 weeks(18Razin E. Mencia-Huerta J.-M. Stevens R.L. Lewis R.A. Liu F.-T. Corey E.J. Austen K.F. J. Exp. Med. 1983; 157: 189-201Google Scholar). Cell purity estimated by cell morphology and staining with toluidine blue was 90-95%. Peritoneal macrophages (19Cohn Z.A. Benson B. J. Exp. Med. 1965; 121: 153-170Google Scholar) were obtained by lavage from the peritoneal cavity with 3 ml of Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 5 units/ml heparin. Cells were plated in a humidified 95% air, 5% CO2 atmosphere at 37°C in tissue culture dishes. After adherence for 1 h, the cells were washed three times and used for experiments. Cell purity was estimated to be >97% based on cell morphology and staining with nonspecific esterase. Pulmonary alveolar macrophages were isolated by a published procedure(20Rouzer C.A. Scott W.A. Hamill A.L. Cohn Z.A. J. Exp. Med. 1982; 155: 720-733Google Scholar). Cultured BMMC obtained from wild-type and 5-lipoxygenase-deficient mice were washed three times with modified Tyrode's buffer (contains 0.32 mM Ca2+; (18Razin E. Mencia-Huerta J.-M. Stevens R.L. Lewis R.A. Liu F.-T. Corey E.J. Austen K.F. J. Exp. Med. 1983; 157: 189-201Google Scholar)) at 4°C. One group of cells was sensitized with monoclonal IgE directed against DNP-human serum albumin (Sigma; 100 μg/ml) for 1 h at 37°C, followed by three washes, and subsequent incubation with DNP-BSA (50 ng/ml) for 30 min. Other groups of cells were incubated with calcium ionophore A23187 (0.5 μM), EDTA (2 mM) or no additions for 30 min in modified Tyrode's. The cells were quickly washed two times with complete medium, with or without EDTA, at 4°C. Cells were placed on glass microscope slides using a Shandon cytocentrifuge (550 revolutions/min for 5 min). Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline for 15 min and permeabilized with 0.2% Triton X-100 for 10 min. Cells were incubated with 3% bovine serum albumin for 30 min followed by 5% donkey serum for 30 min to block nonspecific binding. The cells were incubated with a rabbit polyclonal anti-5-lipoxygenase antiserum (1:2500 dilution; see below) for 5 h at room temperature or overnight at 4°C. The slides were washed three times with phosphate-buffered saline and incubated with Cy3-labeled donkey anti-rabbit antibody (Jackson ImmunoResearch Laboratories; 1:4000 dilution). The slides were washed three times with phosphate-buffered saline and incubated with the nuclear stain Yo-Pro-1 (Molecular Probes; 1:5000 dilution) for 15 min. The slides were air-dried and mounted with Aqua-Poly/Mount (Polysciences Inc.). Slides were examined under oil with a confocal microscope using or were for and for Yo-Pro-1 data were using and Cells were for two of 15 on and at × at 4°C. and were was by and prepared for was as in the 5 using polyclonal and leukotriene A4 hydrolase and human 5-lipoxygenase as of J. was by combined with using from were estimated by of using BMMC in 1 ml of modified Tyrode's buffer were incubated in the presence and absence of or for or 30 min at 37°C as acid was were with of with and were by as described(17Chen X.-S. Sheller J.R. Johnson E. Funk C.D. Nature. 1994; 372: 179-181Google Scholar). the complete region for the murine 5-lipoxygenase cDNA based on the human and rat (12Matsumoto T. Funk C.D. Rdmark O. Hg J.-O. Jrnvall H. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85 (and correction p. 3406): 26-30Google Scholar, 13Dixon R.A.F. Jones R.E. Diehl R.E. Bennett C.D. Kargman S. Rouzer C.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 416-420Google Scholar, J.M. Theisen T.W. Cook M.N. Varrichio A. Hwang S.-M. Strohsacker M.W. Crooke S.T. J. Biol. Chem. 1988; 263: 13937-13941Google Scholar) were designed 1, and and were prepared based on on the we obtained from a genomic containing of the murine gene and was prepared by the of and N. Biochem. Scholar). obtained from peritoneal macrophages of C57BL/6 × 129 mice was used as the for by C.D. J. Biol. Chem. Scholar). for 3 using of the cDNA 1 min 30 for A was by and glass and an was for an A was using using the same without subsequent were cloned the and the were by the chain expression was prepared in the by of the two 5-lipoxygenase cDNA the of the 5-lipoxygenase cDNA was the as an After of the the was as an by was human embryonic kidney 293 cells as by the calcium X.-S. Funk C.D. Eur. J. Biochem. 1993; Scholar, G. 1990; Scholar). h enzyme activity was C.D. H. H. T. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: Scholar). A was prepared for expression The for in the was changed to in a reaction using 5 and 6 and altered is and site for cloning is The change was by and a was to of the was by site and a using an and with cDNA was to the at backcross was as Scholar) by using from × and A of mice were used to the for enzyme and were as B.K. J. 1982; Scholar). The was a cDNA by with with using a was to a of × at A of was in and a of was in M. The presence or absence of the M. was followed in backcross A of the and for the to the and has been C.A. A. E. H. 1993; Scholar, A. S. B. Scholar). were as and in University Scholar) using the was determined by the of to the we isolated a mouse genomic 5-lipoxygenase that for a region of C.D. Kurre U. Griffis G. Ann. N. Y. Acad. Sci. 1994; 714: 253-258Google Scholar). examine 5-lipoxygenase we to the complete murine 5-lipoxygenase cDNA by from macrophage were obtained The cDNA a protein of 674 amino the with a of no evidence for by of and subsequent analysis. Moreover, of mouse genomic with 5-lipoxygenase cDNA under reduced or patterns These with our data that there is a single 5-lipoxygenase C.D. Kurre U. Griffis G. Ann. N. Y. Acad. Sci. 1994; 714: 253-258Google Scholar). expression was prepared by the two at an site of expression human embryonic kidney 293 cells to expression of 5-lipoxygenase protein of the of enzyme activity of the mouse with human and rat and of the 2 a in the for the amino acid two from the terminus acid a residue that is in would be at of to and of the expression was In to expression in 293 5-lipoxygenase enzyme activity as with protein A and 5-lipoxygenase protein and enzyme activity were in cells. A with mouse macrophage cDNA and an upstream the presence of a at The intracellular expression pattern of 5-lipoxygenase in BMMC was by immunocytofluorescence and confocal In BMMC from wild-type mice 5-lipoxygenase was primarily within the nucleus This was by the with the nuclear stain Yo-Pro-1 and at followed by specific immunofluorescence was in BMMC from 5-lipoxygenase-deficient mice or when the antiserum was with serum A and the divalent EDTA was to the modified Tyrode's buffer for the incubation there was a in the pattern of 5-lipoxygenase enzyme within the nucleus there was evidence for 5-lipoxygenase the the cells or divalent cations to the cell which min pattern When BMMC were activated with the 5-lipoxygenase to a location the nuclear to be on the In the cells were with ionophore A23187 the immunofluorescence pattern was was as a possibly with both nuclear and was with and resting and BMMC obtained from wild-type mice using BMMC obtained from 5-lipoxygenase-deficient mice to leukotriene of by IgE and calcium ionophore A23187 BMMC cells. cells obtained from wild-type cells obtained from 5-lipoxygenase-deficient leukotrienes were by cells resting or cells The at and min with and Recent data on the of 5-lipoxygenase in alveolar macrophages the presence of enzyme in both membrane and soluble nuclear in rat basophilic leukemia M. Peters-Golden M. Fantone J.C. Sporn P.H.S. J. Biol. Chem. 1992; 267: 570-576Google Scholar, T.G. Paine R. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Google Scholar, M. Biochem. 1993; Scholar). This the of the existence of distinct 5-lipoxygenase a single 5-lipoxygenase in alveolar macrophages from wild-type mice no in 5-lipoxygenase-deficient mice by gene Moreover, a with leukotriene was by alveolar macrophages from wild-type mice 5-lipoxygenase-deficient mice We examined the expression of genes involved in the and of leukotriene formation (FLAP and at the protein to see there was a compensatory or in expression in the absence of of BMMC and macrophages from three mice using of protein that leukotriene A4 hydrolase protein were 50% in 5-lipoxygenase-deficient mice as by analysis. The 5-lipoxygenase chromosomal location was determined by interspecific backcross using from of × M. This interspecific backcross has been for that the as as the Scholar). and M. were with and by for using a mouse cDNA The M. was used to the of the in backcross The that is in the central region of mouse chromosome 6 to and mice were for and in the to mice were for of was in for using the The of the of mice to the of mice for of and the gene are: The as genetic in the were and in in that the two within of other 95% 5-Lipoxygenase as a single isoform in mice and to intracellular sites in activated BMMC. Using immunocytofluorescence combined with confocal 5-lipoxygenase was found within the nucleus of resting BMMC. A expression pattern was with the rat basophilic cell These basophilic cells to mast cells mast cell T.G. Paine R. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Google Scholar, A. Exp. Cell Scholar). when resting BMMC were incubated in the absence of divalent cations (2 mM for 30 min there was or of 5-lipoxygenase from the nucleus the within the nucleus. The EDTA a of intracellular divalent in addition to by of and of a or divalent to nuclear 5-lipoxygenase intracellular change with ionophore in a of nuclear 5-lipoxygenase to a pattern the nuclear the data that 5-lipoxygenase can bind cytoskeletal proteins by domain (9Lepley R.A. Fitzpatrick F.A. J. Biol. Chem. 1994; 269: 24163-24168Google Scholar) and data that 5-lipoxygenase undergoes a Ca2+-dependent translocation to membrane sites that C.A. Kargman S. J. Biol. Chem. 1988; 263: 10980-10988Google Scholar, A. Cook M.N. Hwang Scholar) it is that 5-lipoxygenase is with nuclear proteins or other cytoskeletal proteins that to the nuclear protein-protein interactions. data be with In in human alveolar macrophages using 5-lipoxygenase with the in resting cells. A23187 in translocation to the nuclear J.W. T.G. Singer I.I. Peters-Golden M. J. Scholar). of BMMC by a to of intracellular in and rat basophilic leukemia A. Cook M.N. Hwang Crooke S.T. 1992; Scholar), translocation of The enzyme predominantly to a with in the R. R. J. Biol. Chem. 1993; Scholar) a translocation of 5-lipoxygenase in mast cells upon from a to by analysis. The of membrane bound 5-lipoxygenase was upon of the R. R. J. Biol. Chem. 1993; Scholar). their with is to the different of analysis. questions to be nuclear control trafficking of protein-protein and or other divalent 5-lipoxygenase and in vitro data obtained on cells with in cellular it is that the generation of leukotrienes and their subsequent have to be modified with novel of 5-lipoxygenase within the nucleus. The murine 5-lipoxygenase cDNA was cloned by based on homology with the human (12Matsumoto T. Funk C.D. Rdmark O. Hg J.-O. Jrnvall H. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1988; 85 (and correction p. 3406): 26-30Google Scholar, 13Dixon R.A.F. Jones R.E. Diehl R.E. Bennett C.D. Kargman S. Rouzer C.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 416-420Google Scholar) and rat (14Balcarek J.M. Theisen T.W. Cook M.N. Varrichio A. Hwang S.-M. Strohsacker M.W. Crooke S.T. J. Biol. Chem. 1988; 263: 13937-13941Google Scholar) the same a at the terminus of the rat J.M. Theisen T.W. Cook M.N. Varrichio A. Hwang S.-M. Strohsacker M.W. Crooke S.T. J. Biol. Chem. 1988; 263: 13937-13941Google Scholar, J. B. 1993; Scholar). The mouse is to the rat and to the human shares the and COOH-terminal isoleucine found in on the structure of the as for the non-heme iron J. B. 1993; Scholar, J.C. 1993; Scholar). a cloning the of the amino acid 2 upstream of the COOH-terminal isoleucine during expression in 293 cells. A to 5-lipoxygenase activity at This would in a chain in the of isoleucine the ability of residue to coordinate the iron atom. we of the COOH-terminal isoleucine and to different using mouse and the essential importance of residue for enzyme X.-S. Kurre U. Funk C.D. J. Biol. Chem. 1994; 269: Scholar). be for isoleucine with of Moreover, Y. Rdmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1992; Scholar) found that of 6 amino from the terminus of human 5-lipoxygenase enzyme the importance of the of the 3 COOH-terminal amino of which to the ability of the chain to and with the essential iron atom. 5-Lipoxygenase as a single in the mouse which has two distinct isoforms by X.-S. Kurre U. Funk C.D. J. Biol. Chem. 1994; 269: Scholar). we were to cDNA of of the 5-lipoxygenase gene 5-lipoxygenase protein and enzyme activity in alveolar macrophages to both soluble and membrane-bound and in IgE/antigen-activated BMMC nuclear and expression patterns were The polyclonal antibody used in studies with M. Peters-Golden M. Fantone J.C. Sporn P.H.S. J. Biol. Chem. 1992; 267: 570-576Google Scholar, T.G. Paine R. Peters-Golden M. J. Biol. Chem. 1994; 269: 22059-22066Google Scholar), and mouse 5-lipoxygenases and would by different genes 5-lipoxygenases homology our data using with genomic has a single gene with no at in mice and C.D. Hoshiko S. Matsumoto T. Rdmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2587-2591Google Scholar, C.D. Kurre U. Griffis G. Ann. N. Y. Acad. Sci. 1994; 714: 253-258Google Scholar). A single human 5-lipoxygenase R.J. Proc. Natl. Acad. Sci. U. S. A. 1992; Scholar) there is in the or or that were of the 5-lipoxygenase gene in mice expression of leukotriene A4 an enzyme in the of leukotriene B4 in macrophages and BMMC. expression was reduced about may as an acid M. Wong E. Cox M.E. Richardson C.D. Li C. Vickers P.J. Eur. J. Biochem. 1993; 215: 105-111Google Scholar). The or for the reduced expression is The human and 5-lipoxygenase genes on different and have different C.D. Hoshiko S. Matsumoto T. Rdmark O. Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2587-2591Google Scholar, Diehl R.E. Y. M. R.A.F. J. Biol. Chem. Scholar, C.D. Funk Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1992; Scholar). intracellular leukotrienes can by a to gene or and is in 5-lipoxygenase-deficient The 5-lipoxygenase gene to mouse chromosome 6 by interspecific backcross analysis. We have the interspecific of chromosome 6 with a mouse that the location of mouse by M. T. T. H. A. L. and D. and provided from a data maintained at The mapped in a region of the that mouse with a that be for an in with was the of an in 5-lipoxygenase-deficient mice under X.-S. Sheller J.R. Johnson E. Funk C.D. Nature. 1994; 372: 179-181Google Scholar, C.D. X.-S. Kurre U. Griffis G. Scholar). mice inflammatory in of The central region of mouse chromosome 6 shares of homology with human 3 and 10 in The human of has been to human chromosome C.D. Funk Samuelsson B. Proc. Natl. Acad. Sci. U. S. A. 1992; Scholar). The of the mouse gene in region of mouse chromosome 6 and region of homology mouse and human In the murine 5-lipoxygenase has been at A single 5-lipoxygenase is within the nucleus in mast cells and apparently to different sites upon cellular activation. The of 5-lipoxygenase-deficient mice in the of nuclear We Griffis and for and and for We to and the Vanderbilt for with the immunocytofluorescence and confocal Evans is for