DNA encoding human leukotriene C4 synthase, polypeptides and uses therefor

An isolated nucleotide sequence encoding human leukotriene C.sub.4 synthase or variants of human leukotriene C.sub.4 synthase polypeptide, is provided. One embodiment is an isolated DNA sequence (SEQ ID NO.:1) encoding a human leukotriene C.sub.4 synthase polypeptide, that has three hydrophobic transmembrane domains. Also described are recombinant cells and plasmids containing the foregoing isolated DNA, preferably linked to a promoter. Isolated leukotriene C.sub.4 synthase is provided, having at least three hydrophobic transmembrane domains (SEQ ID NO.:2). Portions of the foregoing isolated human leukotriene C.sub.4 synthase polypeptide are also described. Antibodies with selective binding specificity for the polypeptides of the invention also are provided as well as a sensitive method for assay of human leukotriene C.sub.4. Methods for producing leukotriene C4 synthase as well as methods for testing for modulators of leukotriene C4 synthase activity are also described.

BACKGROUND OF THE INVENTION
 Leukotrienes are lipid-derived cell mediators that are released in response
 to a variety of immunologic and inflammatory stimuli. They are products of
 arachidonic acid metabolism derived through the 5-lipoxygenase pathway.
 Briefly, the initial step in leukotriene production involves oxygenation
 of arachidonic acid to produce 5S-hydroperoxy-6,8-trans-11,14
 cis-eicosatetraenoic acid (5-HPETE), a subsequent dehydrase step producing
 the epoxide intermediate,
 5,6-trans-oxido-7,9-trans-11,14-cis-eicosatetraenoic acid (LTA.sub.4). Two
 routes of metabolism from LTA.sub.4 lead to the production of biologically
 active products. One of these pathways involves conjugation of LTA.sub.4
 with glutathione (GSH) via LTC.sub.4 synthase to produce the
 sulfur-containing leukotriene 5S-hydroxy-6R-S-glutathionyl-7,9-trans-11,14
 cis-eicosatetraenoic acid (LTC.sub.4). It is generally believed that
 LTC.sub.4 synthase is a member of the glutathione-S transferase enzyme
 family.
 LTC.sub.4 has been implicated in a wide variety of diseases and pathologic
 conditions. LTC.sub.4 has been identified in fluids from psoriatic lesions
 and bronchial secretions associated with adult respiratory distress
 syndrome and neonatal pulmonary hypertension. For review, see Lewis et
 al., New England J. Med., 323: 645 (1990), incorporated herein by
 reference.
 Although the other enzymatic members of the 5-lipoxygenase pathway have
 been cloned, the cloning of LTC.sub.4 synthase has been problematic. This
 is partly because the synthase is very labile in partially purified form
 and because the endogenous production of LTC.sub.4 synthase in normal
 human cells is extremely small. LTC.sub.4 synthase is present only in
 limited types of normal human cells, namely granulocytes derived from bone
 marrow. Moreover, oligonucleotides developed from the N-terminal region of
 the LTC.sub.4 synthase polypeptide have not been specific enough to
 develop an effective screen because the N-terminal region is highly
 degenerate. In addition, an effective immunoassay for LTC.sub.4 which
 relies on incubation of substrate has also been problematic since
 breakdown products of the substrate have been shown to cross-react with
 antibodies used in the assay.
 It has already been established that inhibitors of 5-lipoxygenase and of
 the cell receptors for leukotrienes are of substantial efficacy in the
 management of patients with bronchial asthma. Given that that are only
 three points at which the leukotriene metabolic system can be disrupted:
 the activation and function of 5-lipoxygenase; the receptor for the
 leukotriene; or the function of LTC.sub.4 synthase; characterization of
 LTC.sub.4 synthase would be important, notwithstanding the problems
 associated with its cloning.
 SUMMARY OF THE INVENTION
 Human leukotriene C.sub.4 synthase (also referred to herein as "LTC.sub.4
 synthase") has been cloned in an expression cloning system using a highly
 sensitive assay for LTC.sub.4, the product of the reaction catalyzed by
 LTC.sub.4 synthase. According to one aspect of the invention, an isolated
 nucleotide sequence encoding a human leukotriene C.sub.4 synthase
 polypeptide or unique fragments of human leukotriene C.sub.4 synthase
 polypeptide, is provided. One embodiment is an isolated DNA sequence
 encoding a human leukotriene C.sub.4 synthase polypeptide that has three
 hydrophobic transmembrane domains. Additionally, the invention relates to
 mammalian leukotriene C.sub.4 synthase nucleotide sequences isolated from
 murine, porcine, ovine, bovine, feline, equine, or canine, as well as
 primate (e.g. simian) sources.
 Also provided are recombinant cells and plasmids containing the foregoing
 isolated DNA, preferably linked to a promoter. Portions of the foregoing
 nucleotide sequences are also included in the invention. One such portion
 is contained in a vector within a host cell.
 According to another aspect of the invention, isolated human leukotriene
 C.sub.4 synthase polypetide is provided, having three hydrophobic
 transmembrane domains. Portions of the foregoing isolated human
 leukotriene C.sub.4 synthase polypeptides are also included in the
 invention. Antibodies with selective binding specificity for the
 polypeptides of the invention also are provided.
 Another aspect of the invention is a method for producing human leukotriene
 C.sub.4 synthase polypeptide. The method includes providing an expression
 vector to a host, the vector containing a DNA sequence of the invention
 encoding for human leukotriene C.sub.4 synthase polypeptide, allowing the
 host to express the human leukotriene C.sub.4 synthase polypeptide, and
 isolating the expressed polypeptide.
 A further aspect of the invention is an isolated nucleotide sequence
 capable of hybridizing to a target nucleotide sequence encoding human
 leukotriene C.sub.4 synthase polypeptide. The target includes a nucleotide
 sequence encoding a human leukotriene C.sub.4 synthase polypetide with
 three transmembrane domains. The nucleotide sequence also can encode a
 human leukotriene C.sub.4 synthase polypeptide having amino acid sequences
 unique to the polypeptide.
 The novel molecules of the invention can be employed in experimental or
 therapeutic protocols. For example, a method for interfering with the
 activity of a human leukotriene C.sub.4 synthase gene is provided, in
 which a construct is arranged to include a human leukotriene C.sub.4
 synthase nucleotide sequence that, when inserted into the genome of a
 cell, either inactivates transcription of messenger RNA for human
 leukotriene C.sub.4 synthase polypeptide and/or inactivates translation of
 messenger RNA into human leukotriene C.sub.4 synthase polypeptide in that
 cell. This construct further has a promotor operatively liked to the
 leukotriene C.sub.4 sequence. Next, the construct is introduced into a
 cell, and the construct is allowed to recombine with complementary
 sequences of the cell genome. Finally, cells lacking the ability to
 express human leukotriene C.sub.4 synthase polypeptide are selected.
 A further aspect of the invention is an assay method for identifying a
 modulator of a human leukotriene C.sub.4 synthase polypeptide. The method
 includes providing a target cell containing an isolated nucleotide
 sequence which encodes for a human leukotriene C.sub.4 synthase
 polypeptide. The target cell is maintained under conditions and for a time
 sufficient for the synthase to be expressed in the target cell. The target
 cell is then exposed to a compound suspected of modulating human
 leukotriene C.sub.4 synthase polypeptide activity and a property of the
 target cell is measured in the presence of the modulator. This property is
 also measured in an identical target cell in the absence of the modulator.
 An altered property of the target cell exposed to the modulator is
 indicative of a modulating effect of the compound.
 A highly sensitive assay for LTC.sub.4, the product of the reaction
 catalyzed by LTC.sub.4 synthase, is also described which includes the
 steps of contacting a carrier having bound to it an amount of an LTC.sub.4
 analogue (e.g., LTC.sub.2) and incubating the carrier in the presence of a
 solution containing an unknown amount of leukotriene C.sub.4 synthase.
 Next, the carrier and solution are contacted with an amount of
 anti-leukotriene C.sub.4 antibody under conditions and for a time
 sufficient for the anti-leukotriene C.sub.4 antibody to bind with
 leukotriene C.sub.4 in solution and with analogue (LTC.sub.2) on the
 carrier. Unbound anti-leukotriene C.sub.4 antibody is separated from the
 carrier and then the carrier is contacted with a second antibody linked to
 a fluorescent label under conditions and for a time sufficient for the
 second antibody to bind with anti-leukotriene C.sub.4 antibody associated
 with the carrier. The unbound second antibody is separated from the
 carrier and cell surface fluorescence of the carrier is analyzed by flow
 cytometry.

BRIEF DESCRIPTION OF THE SEQUENCES
 SEQ ID NO.: 1 is the isolated cDNA sequence of human leukotriene C.sub.4
 synthase;
 SEQ ID NO.: 2 is the deduced amino acid sequence of human leukotriene
 C.sub.4 synthase based upon SEQ ID NO.: 1;
 SEQ ID NO.: 3 (VSPPLTTGPPEFER) is a 14 amino acid sequence that is an
 internal tryptic fragment of native leukotriene C.sub.4 synthase;
 SEQ ID NO.: 4 (AGCGTTCCCCAGCTCGCCTTC) and SEQ ID NO.: 5
 (CGGTCACTAGAACTTTAATGATAGAG) are a pair of oligonucleotide primers for PCR
 amplification of the human leukotriene C.sub.4 synthase gene;
 SEQ ID NOS. 6,7 and 8 are the three extramembrane ("loop") amino acid
 sequences of SEQ ID NO.: 2;
 SEQ IN NO.: 9 (MKDEVALLAAVTLLGVLLQAYF) is the N-terminal 22 amino acid
 sequence of leukotriene C.sub.4 synthase purified from native KG-1 cells.
 DETAILED DESCRIPTION OF THE INVENTION
 The novel polypetide of the present invention, hereinafter called, "human
 leukotriene C.sub.4 synthase polypeptide" is a 150-amino acid residue
 integral membrane protein with three hydrophobic transmembrane domains.
 The nucleotide sequence of the leukotriene C.sub.4 synthase of the
 invention is a 694 base pair complementary DNA sequence which encodes for
 the functional LTC.sub.4 synthase enzyme.
 The nucleotide and amino acid sequences of the full length enzyme, the
 configuration and number of transmembrane hydrophobic domains, and the
 lack of homology to known sequences, define a unique nucleotide and
 polypeptide structure. In this regard, the term "homology or homologous"
 is necessarily defined relative to a comparsion between two sequences.
 Given the known pattern of codon degeneracy, any identity between two
 nucleotide sequences above the codon degeneracy "noise", is considered to
 be a "signal" of homology. Preferably, at least 50% identity of nucleotide
 sequence is indicative of a "homologous" sequence.
 One embodiment of a human leukotriene C.sub.4 synthase molecule, according
 to the invention, is the isolated nucleotide sequence shown in SEQ ID NO.:
 1. "Isolated", when applied to the nucleotide sequences encoding the
 polypeptides of the present invention means an RNA or DNA polymer, portion
 of genomic nucleic acid, cDNA, or synthetic nucleic acid which, by virtue
 of its origin or manipulation: (i) is not associated with all of a nucleic
 acid with which it is associated in nature (e.g., is present in a host
 cell as a portion of an expression vector); or (ii) is linked to a nucleic
 acid or other chemical moiety other than that to which it is linked in
 nature; or (iii) does not occur in nature.
 By "isolated" it is further meant a nucleic acid sequence: (i) amplified in
 vitro by, for example, polymerase chain reaction (PCR); (ii) synthesized
 by, for example, chemical synthesis; (iii) recombinantly produced by
 cloning; or (iv) purified, as by cleavage and gel separation.
 SEQ ID NO.: 1 (see also FIG. 1) is a 694 base pair complementary DNA
 sequence encoding for leukotriene C.sub.4 synthase that has been isolated
 from human myelocytic cells. An open reading frame of 450 base pairs is
 identified from nucleotides 55 to 505 (TGA stop codon) of SEQ ID NO.: 1
 (FIG. 1) and predicts an amino acid sequence of 150 amino acids (SEQ ID
 NO.: 2-FIG. 2). Numbering of nucleotides follows the convention of
 starting with the first base pair (A) of SEQ ID NO.: 1 as base number 1;
 amino acid residues begin with the start codon (ATG-bases 55-57) as
 residue number 1.
 The cDNA contains a 54 nucleotide, 5' non-translated region; 450
 nucleotides of translated sequence; an 193 nucleotide, 3' untranslated
 region that includes an ATTAAA polyadenylation signal (in bold lettering
 in FIG. 1), and a poly+ A tail (72 base pairs long--not shown in FIG. 1)
 indicating its full length. The cDNA sequence of SEQ ID NO.: 1 shows no
 significant homology with nucleotide sequences in GENBANK of EMBL
 databanks (using Molecular Biology Computer Research Resources (MBCR)
 software) for known cytosolic or microsomal GSH-S transferases. SEQ NO.: 1
 encodes for a 150 amino acid residue protein with a calculated molecular
 weight and isoelectric point (pI) of 16,567 and 11.05, respectively. SEQ
 NO.: 1 is identical to two other clones having LTC.sub.4 synthase activity
 (see Example 1). SEQ ID NO.: 1 was deposited with GENBANK and has been
 given GENBANK Accession Number U09353.
 Another embodiment of the human LTC.sub.4 synthase of the invention is the
 deduced, isolated amino acid sequence encoded by SEQ ID NO.: 1. The
 isolated polypeptide is given in SEQ ID NO. 2. The term "isolated", when
 applied to the polypeptides of the present invention means polypeptides:
 (i) encoded by nucleic acids using recombinant DNA methods; or (ii);
 synthesized by, for example, chemical synthetic methods; or (iii)
 separated from naturally-occurring biological materials, and then purified
 using protein analytical procedures; or (iv) associated with chemical
 moieties (e.g. polypeptides; carbohydrates, fatty acids, and the like)
 other than those associated with the polypeptide in its
 naturally-occurring state; or (v) that do not occur in nature. SEQ ID NO.:
 2 contains 2 cysteine residues (residues 56 and 82), and two putative
 protein kinase phosphorylation sites (residues 28-30 and 111-113).
 A search of protein sequence databases (SWISSPROT and PIR using BLAST.TM.
 and MBCRR software), relative to the deduced amino acid sequence of SEQ ID
 NO.: 2 reveals that SEQ ID NO.: 2 shares 31 percent overall homology with
 the 5-lipoxygenase activating protein (FLAP- see Dixon et al., Nature 343:
 282-284 (1990)). This homology increases to 44 percent between the
 N-terminal two thirds of SEQ ID NO.: 2 (residues 4-97) and the N-terminal
 end of FLAP (amino acids 9-101). Within this N-terminal region, there are
 portions of near identity at residues 7-13 of LTC.sub.4 (6 of 8 residues
 identical) and residues 46-52 of LTC.sub.4 (6 of 7 residues identical).
 Alignment of the coding nucleotide sequence according to regions of amino
 acid homology reveals 52 percent homology between FLAP and LTC.sub.4 in
 this N-terminal region. There is no nucleotide homology at the 3' end of
 the transcript for LTC.sub.4 and the corresponding region of FLAP. FLAP
 then extends for an additional 300 base pairs.
 A hydropathy analysis of the leukotriene C.sub.4 synthase (Kyte, J. and R.
 Doolittle, J. Molec. Biol., 157: 105-132, 1982) of SEQ ID NO.: 2 was
 performed using a window of 6 amino acids. Briefly, a hydropathy analysis
 progressively evaluates the hydrophilic and hydrophobic properties of a
 protein as a scan along its amino acid sequence. There is a singular
 correspondence between interior portions of soluble, globular proteins and
 hydrophobicity, and a correspondence between exterior portions and
 hydrophilicity.
 The hydropathy analysis reveals three potential transmembrane domains.
 Potential membrane spanning (hydrophobic) regions extend between amino
 acid residues 5-24, 59-89, and 114-135. The terms "hydrophilic and
 hydrophobic" in this context are primarily a function of the size of the
 amino acid "window" used in the hydropathy analysis. For the present
 purposes, "hydrophilic" refers to a stretch of amino acid sequences at
 least 20 residues long and scoring less than 0 on a Kyte-Doolittle plot;
 the scores are derived using a window of preferably at least 6 amino
 acids.
 Native LTC.sub.4 synthase protein from myelocytic KG-1 cells was
 solubilized, purified and sequenced. (See Example 1). In addition,
 recombinant LTC.sub.4 protein derived from transfected COS-7 cells was
 purified and analysed using SDS-PAGE electrophoresis. (See Example 1). The
 predicted molecular weight of 16,567 for SEQ ID NO. 2 is in agreement with
 the observed mobility (18 kDa) of a native integral membrane protein.
 Furthermore, recombinant LTC.sub.4 synthase purified from transfected
 cells also shows a molecular weight of approximately 18 kDa on SDS-PAGE.
 Furthermore, SEQ ID NO.: 2 matched the N-terminal 22 amino acids of the
 LTC.sub.4 protein isolated and sequenced from KG-1 cells (SEQ ID NO.: 9)
 and 14 of 14 internal amino acids (SEQ ID NO.: 3) from tryptic fragments
 of the native protein. These 14 amino acids were identical to amino acid
 residues 35-48 of SEQ ID NO.: 2. SEQ ID NO.: 2 also matched 34 of 35
 N-terminal amino acids purified from human leukemic THP-1 cell line. See
 Nicholson et al., Proc. Nat. Acad. Sci. USA, 90: 2015-2019 (1993).
 Using the nucleotide sequence information provide in SEQ ID NO. 1, cell
 lines expressing the polypeptide of SEQ ID NO.: 2 can be established
 (Example 3). Likewise, homologues to SEQ ID NO.: 1 from other mammalian
 species can be identified using conventional techniques, described in
 greater detail below. Such genetic engineering techniques are well within
 the scope of those of ordinary skill in the art.
 Northern blot analysis was employed to study steady state transcription of
 LTC.sub.4 synthase and its distribution in human eosinophils and the KG-1
 cell line. A 0.7 kb mRNA transcript was observed in these cells, both of
 which are known to contain LTC.sub.4 synthase. The size of the mRNA (0.7
 kb) is similar to that of SEQ ID NO.1, consistent with SEQ ID NO.: 1 being
 full length. (see Example 1).
 A nucleotide sequence encoding leukotriene C.sub.4 synthase has been
 cloned, isolated and expressed. A general protocol is presented below.
 This protocol is intended to obtain a cDNA having a complete reading frame
 for the polypeptide.
 A. Cloning Human Leukotriene C.sub.4 Synthase Polypeptide
 A cDNA encoding leukotriene C.sub.4 synthase is cloned by expressing a
 leukotriene C.sub.4 synthase from a cDNA expression library in mammalian
 COS-7 cells. (See Example 1). Briefly, mRNA is isolated from cells
 containing LTC.sub.4 synthase. Next, mRNA is used to prepare a cDNA
 library using an in vitro expression vector system. cDNA is synthesized,
 separated by gel electrophoresis and ligated into an expression vector.
 This synthetic cDNA library is used to transform a bacterial host and
 bacteria are then subjected to extraction of their plasmid cDNA. Plasmids
 are used to transfect mammalian cells. The cDNA library was screened for
 expression of LTC.sub.4 synthase in transfectants by a highly sensitive,
 fluorescence-linked competitive immunoassay (See Example 1).
 For sequencing, plasmid cDNA from the clones is extracted, then cloned into
 a vector for DNA sequencing, using standard methods. See for example,
 Sambrook, J. et al., Molecular Cloning, Cold spring Harbor Press, N.Y. See
 also, Example 1.
 B. Cloning Other Homologues of Human Leukotriene C.sub.4 Synthase
 Polypeptide
 Now that the cDNA sequence of human leukotriene C.sub.4 synthase has been
 characterized, one can use a variety of approaches for cloning other human
 homologues. One approach used to screen a DNA library for the presence of
 a human leukotriene C.sub.4 synthase nucleotide coding sequence
 corresponding to a human homologue includes generating preferred probes
 using the polymerase chain reaction. The probes are produced by using, for
 example, a human granulocyte or myelocytic cell line (i.e., KG-1, THP-1)
 cDNA library as a template for polymerase chain reaction (PCR). Based on
 the degree of codon degeneracy of the predicted amino acid sequence, PCR
 primers are derived from the human leukotriene C.sub.4 synthase nucleotide
 sequence of SEQ ID NO.: 1. Examples of suitable PCR primer pairs include
 SEQ ID NO.: 4 (AGCGTTCCCCAGCTCGCCTTC) and SEQ ID NO.: 5
 (CGGTCACTAGAACTTTAATGATAGAG). See Example 2.
 The product of the PCR reaction is cloned and the human cDNA library is
 rescreened using the PCR product as the probe(s). This preferred method,
 however, requires identifying tissue that expresses leukotriene C.sub.4
 synthase as a source of RNA.
 Other tissues suspected of expressing the human homologue can, however, be
 identified by RNA analysis, i.e., Northern blot analysis under low
 stringency conditions. Confirmation of a human tissue as an RNA source and
 identification of additional sources of tissue can be accomplished by
 preparing RNA from the selected tissue and performing Northern blot
 analysis under low stringency conditions using PCR product as the
 probe(s). A suitable range of such stringency conditions is described in
 Krause, M. H., and Aaronson, S. A., 1991, Methods in Enzymology 200:
 546-556. Additionally, genomic libraries can be screened for the presence
 of the human homolog coding sequence using a PCR generated probe(s).
 C. Testing and Cloning Related Molecules
 The invention also pertains to a more general protocol for isolating the
 gene for leukotriene C.sub.4 synthase. In this approach, total mRNA can be
 isolated from mammalian tissues or from cell lines likely to express
 leukotriene C.sub.4 synthase polypeptide. In general, total RNA from the
 selected tissue or cell culture is isolated using conventional methods.
 Subsequent isolation of mRNA is typically accomplished by oligo (dT)
 chromatography. Messenger RNA for Northern analysis is size-fractionated
 by electrophoresis and the RNA transcripts are transferred to
 nitrocellulose according to conventional protocols (Sambrook, J. et al.,
 Molecular Cloning, Cold spring Harbor Press, N.Y.).
 A labelled PCR-generated probe capable of hybridizing with the human
 leukotriene C.sub.4 synthase nucleotide (SEQ ID NO.: 1) can serve to
 identify RNA transcripts complementary to at least a portion of the human
 leukotriene C.sub.4 synthase gene. For example, if Northern analysis
 indicates that RNA isolated from murine lung tissue hybridizes with the
 labelled probe, then a murine lung cDNA library is a likely candidate for
 screening and identification of a clone containing the coding sequence for
 a murine homolog of human leukotriene C.sub.4 synthase polypeptide.
 Northern analysis is used to confirm the presence of mRNA fragments which
 hybridize to a probe corresponding to all or part of the human leukotriene
 C.sub.4 synthase polypeptide. Northern analysis indicates the presence and
 size of the transcript. This allows one to determine whether a given cDNA
 clone is long enough to encompass the entire transcript or whether it is
 necessary to obtain further cDNA clones, i.e., if the length of the cDNA
 clone is less than the length of RNA transcripts as seen by Northern
 analysis. If the cDNA is not long enough, it is necessary to perform
 several steps such as: (i) rescreen the same library with the longest
 probes available to identify a longer cDNA; (ii) screen a different cDNA
 library with the longest probe; and (iii) prepare a primer-extended cDNA
 library using a specific nucleotide primer corresponding to a region close
 to, but not at, the most 5' available region. This nucleotide sequence is
 used to prime reverse transcription. The primer extended library is then
 screened with the probe corresponding to available sequences located at 5'
 to the primer. See for example, Rupp et al., Neuron, 6: 811-823 (1991).
 The preferred clone of leukotriene C.sub.4 synthase has a complete coding
 sequence, i.e., one that begins with methionine, ends with a stop codon,
 and preferably has another in-frame stop codon 5' to the first methionine.
 It is also desirable to have a cDNA that is "full length", i.e. includes
 all of the 5' and 3' untranslated sequences. To assemble a long clone from
 short fragments, the full-length sequence is determined by aligning the
 fragments based upon overlapping sequences. Thereafter, the full-length
 clone is prepared by ligating the fragments together using the appropriate
 restriction enzymes.
 As discussed above, PCR-generated probes may be used in the protocol for
 isolating mammalian homologues to human leukotriene C.sub.4 synthase
 polypeptide. Moreover, probes to be used in the general method for
 isolating mammalian leukotriene C.sub.4 synthase can now include
 oligonucleotides, all of which encode at least part of the sequence shown
 in SEQ ID NO.: 1. Unlike the PCR approach to generating a probe, the
 above-identified probes do not require prior isolation of RNA from a
 tissue expressing the vertebrate homolog.
 An oligodeoxyribonucleotide probe typically has a sequence somewhat longer
 than that used for the PCR primers. A longer sequence is preferable for
 the probe, and it is important that codon degeneracy be minimized. A
 representative protocol for the preparation of an oligonucleotide probe
 for screening a cDNA library is described in Sambrook, J. et al.,
 Molecular Cloning, Cold Spring Harbor Press, New York, 1989. In general,
 the probe is labelled, e.g., with .sup.32 P, and used to screen clones of
 a cDNA or genomic library.
 Alternately, an expression library can be screened using conventional
 immunization techniques, such as those descried in Harlowe and Lane, D.
 (1988), Antibodies, Cold Spring Harbor Press, New York. Antibodies
 prepared using purified leukotriene C.sub.4 synthase as an immunogen are
 preferably first tested for cross reactivity with the homolog of
 leukotriene C.sub.4 synthase from other species. Other approaches to
 preparing antibodies for use in screening DNA libraries, as well as for
 use in diagnostic and research applications, are described below. See
 Example 3.
 D. Nucleic Acid and Protein Sequences
 The nucleic acid sequence of the human leukotriene C.sub.4 synthase is
 depicted in SEQ ID NO.: 1. This sequence, its functional equivalents, or
 fragments of this sequence may be used in accordance with the invention.
 The term "fragments" refers to portions of the human leukotriene C.sub.4
 synthase nucleic acid sequence that may find no counterpart in the known
 sequences of other polypeptides. Subsequences comprising hybridizable
 portions of the LTC.sub.4 synthase sequence have use, e.g., in nucleic
 acid hybridization assays, Southern and Northern blot analyses, etc.
 Exemplary nucleotide subsequences of SEQ ID NO.: 1 are those encoding
 extramembrane, "loop" portions of LTC.sub.4 synthase, such as nucleotide
 sequences or portions of nucleotide sequences encoding (with reference to
 the residues of SEQ ID NO.: 2) amino acid residues 25 to 58 (SEQ ID NO.:
 6); amino acid residues 90 to 113 (SEQ ID NO.: 7); and amino acid residues
 136 to 150 (SEQ ID NO.: 8).
 Moreover, the nucleic acid sequence depicted in SEQ ID NO: 1 can be altered
 by mutations such a substitutions, additions or deletions that provide for
 functionally equivalent nucleic acid sequences. According to the present
 invention, a nucleic acid sequence is "functionally equivalent" compared
 with the nucleic acid sequence depicted in SEQ ID NO.: 1, if it satisfies
 at least one of the following conditions: (i) the nucleic acid sequence
 has the ability to hybridize to a human leukotriene C.sub.4 synthase
 nucleotide sequence, but it does not necessarily hybridize to that
 sequence with an affinity that is the same as that of the naturally
 occurring human leukotriene C.sub.4 synthase nucleic acid sequence; and/or
 (ii) the nucleic acid can serve as a probe to distinguish between the
 present human leukotriene C.sub.4 synthase sequences and other nucleotide
 sequences.
 The term "probe", therefore, refers to a ligand of known qualities that can
 bind selectively to a target. As applied to the nucleic acid sequences of
 the invention, the term "probe" refers to a strand of nucleic acid having
 a base sequence complementary to a target sequence. Preferred nucleotide
 sequences may hybridize if they contain sequences that have at least 50%
 identity to a target sequence. A preferred probe that can distinguish
 between a human leukotriene C.sub.4 synthase sequence and other sequences
 refers to a probe that includes SEQ ID NO.: 1, functional variants, or
 fragments thereof.
 Because the nucleic acid sequence of leukotriene C.sub.4 synthase is now
 known, those of ordinary skill in the art can readily determine nucleic
 acid sequences of the human leukotriene C.sub.4 synthase that are not
 homologous to any other nucleic acid sequence, including other human
 leukotriene C.sub.4 synthase sequences. These non-homologous sequences,
 and peptides encoded by them, are referred to as "unique" fragments and
 are meant to be included within the scope of the present invention.
 Moreover, due to the degeneracy of nucleotide coding sequences, other
 nucleic acid sequences may be used in the practice of the present
 invention. These include, but are not limed to, sequences comprising all
 or portions of the human leukotriene C.sub.4 synthase sequences depicted
 in SEQ ID NO.: 1 which are altered by the substitution of different codons
 that encode the same amino acid residue within the sequence, thus
 producing a silent change. Such altered sequences are regarded as
 equivalents of the specifically claimed sequences.
 Human leukotriene C.sub.4 synthase polypeptide or fragments or other
 derivatives thereof include, but are not limited to, those containing as a
 primary amino acid sequence all, or unique parts of the amino acid
 residues substantially as depicted in SEQ ID NO.: 2, including altered
 sequences in which functionally equivalent amino acid residues are
 substituted for residues within the sequence, resulting in a silent
 change. According to the invention, an amino acid sequence is
 "functionally equivalent" compared with a sequence depicted in SEQ ID NO.:
 2 if the amino acid sequence contains one or more amino acid residues
 within the sequence which can be substituted by another amino acid of a
 similar polarity which acts as a conservative substitution (i.e., a
 functional equivalent). For example, at least one of the tyrosine residues
 at positions 50, 93 and 109 of SEQ ID NO. 2 may be substituted by a
 phenylalanine, yielding a total of 2.sup.3 or 8 separate functional
 equivalents of SEQ ID NO.: 2.
 In addition, substitutes for an amino acid within the sequence may also be
 selected from other members of the class to which the amino acid belongs.
 The non-polar (hydrophobic) amino acids include alanine, leucine,
 isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The
 polar neutral amino acids include glycine, serine, threonine, cysteine,
 tyrosine, asparagine, and glutamine. The positively charged (basic) amino
 acids include arginine, lysine and histidine. The negatively charged
 (acidic) amino acids include aspartic acid and glutamic acid.
 Substantial changes in functional or, for example, immunological properties
 may be made by selecting substitutes that differ from the original amino
 acid residue. More significantly, the substitutions are chosen for their
 effect on: (i) maintaining the structure of the peptide backbone in the
 area of the substitution, for example, as a sheet or helical conformation;
 (ii) maintaining the charge or hydrophobicity of the molecule at the
 target side; or (iii) maintaining the bulk of the side chain.
 The substitutions that in general are expected to induce greater changes in
 the functional properties of LTC.sub.4 synthase are those in which: (a)
 glycine and/or proline is substituted by another amino acid or is deleted
 or inserted; (b) a hydrophilic residue, e.g., seryl or threonyl, is
 substituted for (or by) a hydrophobic residue, e.g., leucyl, isoleucyl,
 phenylalanyl, or alanyl; (c) a cysteine residue is substituted for (or by)
 any other residue; (d) a residue having an electropositive side chain,
 e.g., lysyl, arginyl, or histidyl, is substituted for (or by) a residue
 having an electronegative charge, e.g., glutamyl or aspartyl, or (e) a
 residue having a bulky side chain, e.g., phenylalanine, is substituted for
 one (or by) one not having such a side chain, e.g., glycine.
 In particular, any change in the number of arginine residues will have an
 effect on the isoelectric point (pI) of the LTC.sub.4 synthase. Arginine
 has the highest pI (10.76) of all the standard amino acids. Fewer arginine
 residues would be expected to result in an LTC.sub.4 synthase with a lower
 pI and may be expected to result in an enzyme with reduced activity.
 Substitution of an arginine residue of SEQ ID NO.: 2 with an aspartic acid
 (pI=2.77) is a particularly effective substitution for lowering the
 LTC.sub.4 synthase isoelectic point. For example, SEQ ID NO.: 2 has 13
 arginine residues at positions 30, 31, 34, 48, 51, 90, 92, 99, 104, 113,
 136, 142, and 144. Substitution of at least one aspartic acid for at least
 one of these arginines would yield a total of 2.sup.13 or 8192 separate
 modifications of SEQ ID NO.: 2.
 In addition, substitution of at least one arginine residue with at least
 one glutamic acid (pI=3.22) is another substitution likely to lower the
 LTC.sub.4 synthase isoelectric point. Substitution of at least one
 glutamic acid for at least one of these arginines would also yield a total
 of 2.sup.13 or 8192 separate modifications of SEQ ID NO.: 2. Synthesis of
 all of these sequences is well within the present level of skill in the
 art.
 Most deletions and insertions in the human leukotriene C.sub.4 synthase
 polypeptide, and substitutions in particular, are not expected to produce
 radical changes in the characteristics of the polypeptide. Nevertheless,
 when it is difficult to predict the exact effect of the substitution,
 deletion, or insertion in advance of doing so, one skilled in the art will
 appreciate that the effect will be evaluated using routine screening
 assays as described below. For example, a change in the immunological
 character of the human leukotriene C.sub.4 synthase polypeptide, such as
 binding to a given antibody, is measured by an immunoassay such as a
 competitive type immunoassay.
 Also included within the scope of the invention are human leukotriene
 C.sub.4 synthase polypeptides or unique fragments or derivatives thereof
 which are differentially modified during or after translation, e.g., by
 phosphorylation, glycosylation, crosslinking, acylation, proteolytic
 cleavage, linkage to an antibody molecule, membrane molecule or other
 ligand, (Ferguson, et al., 1988, Ann. Rev. Biochem. 57:285-320).
 In addition, the recombinant human leukotriene C.sub.4 synthase
 polypeptide-encoding nucleic acid sequences of the invention may be
 engineered so as to modify processing or expression of the human
 leukotriene C.sub.4 synthase polypeptide. For example, and not by way of
 limitation, the human leukotriene C.sub.4 synthase nucleotide sequence(s)
 may be combined with a promoter sequence and/or a ribosome binding site
 using well characterized methods, and thereby facilitate harvesting or
 bioavailability.
 Now that the cDNA of human LTC.sub.4 synthase has been identified, it will
 be readily appreciated that the human leukotriene C.sub.4 synthase
 nucleotide sequence can be mutated in vitro or in vivo, to create
 variations in coding regions and/or form new restriction endonuclease
 sites or destroy preexisting ones, to facilitate further in vitro
 modification. Any technique for mutagenesis known in the art can be used
 including, but not limited to, in vitro site-directed mutagenesis
 (Hutchinson, et al., 1978, J. Biol. Chem. 253:6551), use of TAB.RTM.
 linkers (Pharmacia), PCR-directed mutagenesis, and the like.
 In addition to generating fragments of leukotriene C.sub.4 synthase from
 expression of cloned partial sequences of human leukotriene C.sub.4
 synthase polypeptide DNA, fragments of human leukotriene C.sub.4 synthase
 polypeptide can be generated directly from the intact polypeptide.
 Proteins are specifically cleaved by proteolytic enzymes, including, but
 not limited to, trypsin, chymotrypsin or pepsin. Each of these enzymes is
 specific for the type of peptide bond it attacks. Trypsin catalyzes the
 hydrolysis of peptide bonds whose carbonyl group is from a basic amino
 acid, usually arginine or lysine. Pepsin and chymotrypsin catalyze the
 hydrolysis of peptide bonds from aromatic amino acids, particularly
 tryptophan, tyrosine and phenylalanine. Alternate sets of cleaved
 polypeptide fragments are generated by preventing cleavage at a site which
 is susceptible to a proteolytic enzyme. For example, reaction of the
 .epsilon.-amino groups of lysine with ethyltrifluorothioacetate in mildly
 basic solution yields a blocked amino acid residue whose adjacent peptide
 bond is no longer susceptible to hydrolysis by trypsin (Goldberger et al.
 Biochem., 1:401 (1962)). Treatment of such a polypeptide with trypsin thus
 cleaves only at the arginyl residues.
 Polypeptides also can be modified to create peptide linkages that are
 susceptible to proteolytic enzyme catalyzed hydrolysis. For example,
 alkylation of cysteine residues with .beta.-halo ethylamines yields
 peptide linkages that are hydrolyzed by trypsin (Lindley, Nature, 178: 647
 (1956)). In addition, chemical reagents that cleave polypeptide chains at
 specific residues can be used (Withcop, Adv. Protein Chem. 16: 221
 (1961)). For example, cyanogen bromide cleaves polypeptides at methionine
 residues (Gross & Witkip, J. Am Chem Soc., 83: 1510 (1961)). Thus, by
 treating leukotriene C.sub.4 synthase or fragments thereof with various
 combinations of modifiers, proteolytic enzymes and/or chemical reagents,
 numerous discrete overlapping peptides of varying sizes are generated.
 These peptide fragments can be isolated and purified from such digests by
 chromatographic methods.
 Alternatively, human leukotriene C.sub.4 synthase polypeptides can be
 synthesized using an appropriate solid state synthetic procedure (Steward
 and Young, Solid Phase Peptide Synthesis, Freemantle, San Francisco,
 Calif. (1968)). A preferred method is the Merrifield process (Merrifield,
 Recent Progress in Hormone Res., 23: 451 (1967)). The activity of these
 peptide fragments may conveniently be tested using, for example, a COS-7
 expression assay as described herein.
 The human leukotriene C.sub.4 synthase sequences of the invention also
 include non-human homologues of the amino acid sequence of SEQ ID NO.: 2.
 The non-human leukotriene C.sub.4 synthases of the invention may be
 prepared by recombinant nucleic acid expression techniques or by chemical
 synthesis using standard peptide synthesis techniques.
 Also within the scope of the invention are nucleic acid sequences or
 proteins encoded by nucleic acid sequences derived from the same gene but
 lacking one or more structural features as a result of alternative
 splicing of transcripts from a gene that also encodes the complete human
 leukotriene C.sub.4 synthase polypeptide gene, as defined previously.
 Nucleic acid sequences complementary to DNA or RNA sequences encoding
 leukotriene C.sub.4 synthase or a functionally active portion(s) thereof
 are also provided. In animals, particularly transgenic animals, RNA
 transcripts of a desired gene or genes may be translated into polypeptide
 products having a host of phenotypic actions.
 In a particular aspect of the invention, antisense oligonucleotides and
 oligonucleotide analogs can be synthesized. Antisense oligonucleotides or
 analogs specifically bind to the complementary sequence of either pre-mRNA
 or mature mRNA, as defined by Watson-Crick base pairing, inhibiting the
 flow of genetic information from DNA to protein. These oligonucleotides
 may have activity in their own right, such as antisense reagents which
 block translation or inhibit RNA function. Where leukotriene C.sub.4
 synthase is to be produced utilizing the nucleotide sequences of this
 invention, the DNA sequence may also be in an inverted orientation which
 gives rise to a negative sense RNA on transcription. This RNA is not
 capable of being translated to the desired human leukotriene C.sub.4
 synthase polypeptide product, as it is in the wrong orientation and would
 give a nonsensical product if translated, thus modulating production of
 LTC.sub.4 synthase. In this regard, "oligonucleotide" refers to a
 polynucleotide formed from naturally occurring bases and cyclofuranasyl
 groups joined by phosphodiester bonds. "Oligonucleotide analog", refers to
 moieties which function similarly to anti-sense oligonucleotides but which
 have non-naturally-occurring portions which are not closely homologous.
 Thus, oligonucleotides may have altered sugar moities or inter-sugar
 linkages. Exemplary are phosphorothioate and other sulfur linkages species
 known in the art. Such analogs are functional equivalents of the
 anti-sense oligonucleotides of the invention. The most direct effect which
 antisense oligonucleotides have on intact cells that can be easily
 quantified is specific inhibition of LTC.sub.4 synthase activity (See
 Example 1).
 E. Expression of Polypeptide
 The present invention also permits the expression, isolation, and
 purification of the human leukotriene C.sub.4 synthase polypeptide. A
 human leukotriene C.sub.4 synthase nucleotide sequence may be cloned or
 subcloned using any method known in the art. It will be appreciated that
 some post-transitional events such as glycosylation, phosphorylation,
 and/or subunit assembly may not be carried out in the same manner in all
 eukaryotic cells. The preferred expression systems utilize mammalian cells
 and cell lines. A large number of vector-mammalian host systems known in
 the art may be used. Possible vectors include, but are not limited to,
 cosmids, plasmids or modified viruses, but the vector system must be
 compatible with the host cell used. Viral vectors include, but are not
 limited to, vaccinia virus, or lambda derivatives. Plasmids include, but
 are not limited to, pBR322, pUC, or Bluescript.RTM. (Stratagene) plasmid
 derivatives. Recombinant human leukotriene C.sub.4 synthase polypeptide
 molecules can be introduced into host cells via transformation,
 transfection, infection, electroporation, etc. Generally, introduction of
 human leukotriene C.sub.4 synthase polypeptide molecules into a host is
 accomplished using a vector containing human leukotriene C.sub.4 synthase
 polypeptide DNA under control by regulatory regions of the DNA that
 function in the host cell.
 In one method of expressing human leukotriene C.sub.4 synthase polypeptide,
 the cDNA that corresponds to the entire coding region (SEQ ID NO.: 1) is
 moved by way of a eukaryotic expression vector into cells derived from the
 simian kidney (e.g., COS-7 cells). Expression is monitored after
 transfection by measuring the production of LTC.sub.4. See Examples 1 and
 3. The details of this experimental approach for transfection, selection
 and characterization of the leukotriene C.sub.4 synthase are similar to
 those that have been used previously for other polypeptides (see, for
 example, Birnir, B. et al., Biochim. Biophys. Acta, 1048: 100-104 (1990),
 the entire contents of which are incorporated herein by reference.
 Once the polypeptide is expressed, it may be isolated and purified by
 standard methods including chromatography (e.g., ion exchange, affinity,
 and sizing column chromatography), centrifugation, differential
 solubility, or by any other standard technique for the purification of
 proteins. In particular, leukotriene C.sub.4 synthase may be isolated by
 binding to an affinity column comprising antibodies to leukotriene C.sub.4
 synthase bound to a stationary support.
 F. Preparation of Antibodies to the Polypeptide
 The term "antibodies" is meant to include monoclonal antibodies, polyclonal
 antibodies and antibodies prepared by recombinant nucleic acid techniques
 that are selectively reactive with human leukotriene C.sub.4 synthase
 polypeptide. The term "selectively reactive" refers to those antibodies
 that react with one or more antigenic determinants of human leukotriene
 C.sub.4 synthase polypeptide, and do not react with other transporter
 polypeptides. Determinants usually consist of chemically active surface
 groupings of molecules such as amino acids or sugar side chains and have
 specific three dimensional structural characteristics as well as specific
 charge characteristics. Antibodies include those raised against the human
 polypeptide of SEQ ID NO.: 2 and intended to cross-react with other human
 homologs but not with non-human LTC.sub.4 synthase. These antibodies may
 be useful for diagnostic applications. Other antibodies include those
 raised against non-human (i.e., mouse or goat) leukotriene C.sub.4
 synthase, which antibodies may be generally used for research purposes.
 These antibodies include those raised against short, synthetic peptides of
 the non-human sequence.
 Antibodies may be raised against human LTC.sub.4 synthase and isolated by
 standard protein purification methods. Generally, a peptide immunogen is
 first attached to a carrier to enhance the immunogenic response. Although
 the peptide immunogen can correspond to any portion of the amino acid
 sequence of the human leukotriene C.sub.4 synthase or to variants of the
 sequence, such as the amino acid sequences corresponding to the primers
 and probes described, certain peptides are more likely than others to
 provoke an immediate response. For example, a peptide including the
 C-terminal amino acid is more likely to generate an antibody response.
 Other alternatives to preparing antibodies reactive with human LTC.sub.4
 synthase include: immunizing an animal with a protein expressed by a
 procaryotic (e.g., bacterial) or eucaryotic cell, which cell includes the
 coding sequence for: (i) all or part of human LTC.sub.4 synthase; or (ii)
 the coding sequence for all or part of a non-human (i.e., mouse)
 leukotriene C.sub.4 synthase. Antibodies can also be prepared by
 immunizing an animal with whole cells that are expressing all or a part of
 a cDNA encoding the human leukotriene C.sub.4 synthase polypeptide. For
 example, cDNA encoding the leukotriene C.sub.4 synthase of the present
 invention (e.g., SEQ ID NO.: 1) may be expressed in a host using standard
 techniques (see Sambrook et al., Molecular Cloning; A Laboratory Manual,
 Cold Spring Harbor Press, Cole Spring Harbor, N.Y. (1989) or methods
 described herein such that 5-20% of the total of protein recovered is
 human leukotriene C.sub.4 synthase polypeptide. Proteins are
 electrophoresed using PAGE, the appropriate band cut, the protein eluted,
 and prepared for immunization. Mice are immunized twice intraperitoneally
 with 50 micrograms protein immunogen per mouse. Their sera is tested for
 antibody activity by immunohistology or immunocytology on any leukotriene
 C.sub.4 synthase expressing cell system (e.g., transfected COS-7 cells)
 and/or by immunoassay with the expressed human leukotriene C.sub.4
 synthase polypeptide. For immunohistology, a biotin-conjugated anti-mouse
 immunoglobulin may be used followed by avidin-peroxidase, and a
 chromogenic peroxidase substrate. Such preparations are commercially
 available; for example, from Zymad Corp., San Francisco, Calif. Animals
 with serum antibodies are sacrificed three days later and their spleens
 taken for fusion and hybridoma production, as above. Positive supernatants
 are tested as above and by, for example, Western blot analysis.
 To further improve the likelihood of producing an anti-human leukotriene
 C.sub.4 synthase immune response, the amino acid sequence of the
 leukotriene C.sub.4 synthase may be analyzed in order to identify portions
 of the molecule which may be associated with increased immunogenicity. For
 example, the amino acid sequence may be subjected to computer analysis to
 identify surface epitopes which present computer-generated plots of
 antigenic index, an amphophilic helix, amphophilic sheet, hydrophilicity,
 and the like. Alternatively, the deduced amino acid sequences of
 leukotriene C.sub.4 synthase from different species could be compared, and
 relatively non-homologous regions identified. These non-homologous regions
 would be more likely to be immunogenic across various species.
 For preparation of monoclonal antibodies directed toward human leukotriene
 C.sub.4 synthase polypeptide, any technique which provides for the
 production of antibody molecules by continuous cell lines and culture may
 be used. For example, the hybridoma technique originally developed by
 Kohler and Milstein (Nature, 256: 495-497, 1973), as well as the trioma
 technique, the human B-cell hybridoma technique (Kozbor et al., Immunology
 Today, 4:72), and the EBV-hybridoma technique to produce human monoclonal
 antibodies, and the like, are within the scope of the present invention.
 See, generally Larrick et al., U.S. Pat. No. 5,001,065 and references
 cited therein. Further, single-chain antibody (SCA) methods are also
 available to form anti-human leukotriene C.sub.4 synthase antibodies
 (Ladner et al. U.S. Pat. Nos. 4,946,778 and 5,260,203). Recent
 developments in production of human monoclonal antibodies, involving
 insertion of human heavy and light-chain genetic loci into mice in which
 endogenous production of heavy and light chains is disrupted, has lead to
 mice that can synthesize human antibodies specific for human antigens, and
 can be employed to produce hybridomas making human antibodies. See,
 Lonberg, et al., Nature 368: 856-859 (1994) and Green et al., Nature
 Genet., 7: 13-21 (1994), incorporated herein by reference.
 The monoclonal antibodies may be human monoclonal antibodies or chimeric
 human-mouse (or other species) monoclonal antibodies. The present
 invention provides for antibody molecules as well as fragments of such
 antibody molecules.
 Those of ordinary skill in the art will recognize that a large variety of
 possible moieties can be coupled to anti-human leukotriene C.sub.4
 synthase polypeptide monoclonal antibodies or other molecules of the
 invention. See, for example, "Conjugate Vaccines", Contributions to
 Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger
 Press, New York, (1989), the entire contents of which are incorporated
 herein by reference.
 Coupling may be accomplished by any chemical reaction that will bind the
 two molecules so long as the antibody and the other moiety retains their
 respective activities. This linkage can include many chemical mechanisms,
 for instance covalent binding, affinity binding, intercalation, coordinate
 binding and complexation. The preferred binding is, however, covalent
 binding. The covalent binding can be achieved either by direct
 condensation of existing side chains or by the incorporation of external
 bridging molecules. Many bivalent or polyvalent linking agents are useful
 in coupling protein molecules, such as an anti-human leukotriene C.sub.4
 synthase monoclonal antibody, to other molecules. For example,
 representative coupling agents can include organic compounds such as
 thioesters, carbodiimides, succinimide ester, diisocyanates,
 gluteraldehydes, diazobenzenes and hexamethylene diamines. This listing is
 not intended to be exhaustive of the various classes of coupling agents
 known in the art but, rather, is exemplary of the more common coupling
 agents. (See Killen and Lindstrom 1984, "Specific killing of lymphocytes
 that cause experimental Autoimmune Myesthenia Gravis by
 toxin-acetylcholine receptor conjugates." Jour. Immun. 133:1335-2549;
 Jansen, F. K., H. E. Blythman, D. Carriere, P. Casella, O. Gros, P. Gros,
 J. C. Invent, F. Paolucci, B. Pau, P. Poncelet, G. Richer, H. Vidal, and
 G. A. Voisin. 1982. "Immunotoxins: Hybrid molecules combining high
 specificity and potent cytotoxicity". Immunological Reviews 62:185-216;
 and Vitetta et al., supra).
 Preferred linkers are described in the literature. See, for example,
 Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) describing use of
 MBS (M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, Umemoto et
 al. U.S. Pat. No. 5,030,719, describing use of halogenated acetyl
 hydrazide derivative coupled to an antibody by way of an oligopeptide
 linker. Particularly preferred linkers include: (i) EDC
 (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide hydrochloride; (ii) SMPT
 (4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)-toluene
 (Pierce Chem. Co., Cat. #21558G); (iii) SPDP (succinimidyl-6
 [3-(2-pyridyldithio) propionamido] hexanoate (Pierce Chem. Co., Cat
 #21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6
 [3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.
 #2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem. Co.,
 Cat. #24510) conjugated to EDC.
 The linkers described above contain components that have different
 attributes, thus leading to conjugates with differing physio-chemical
 properties. For example, sulfo-NHS esters of alkyl carboxylates are more
 stable than sulfo-NHS esters of aromatic carboxylates. NHS-ester
 containing linkers are less soluble than sulfo-NHS esters. Further, the
 linker SMPT contains a sterically hindered disulfide bond, and can form
 conjugates with increased stability. Disulfide linkages, are in general,
 less stable than other linkages because the disulfide linkage is cleaved
 in vitro, resulting in less conjugate available. Sulfo-NHS, in particular,
 can enhance the stability of carbodimide couplings. Carbodimide couplings
 (such as EDC) when used in conjunction with sulfo-NHS, forms esters that
 are more resistant to hydrolysis than the carbodimide coupling reaction
 alone.
 In other embodiments, compositions of the invention can be used as reagents
 in immunoassays to detect antibodies against human leukotriene C.sub.4
 synthase. Immunoassays can be any of the conventional assay types. For
 example, a sandwich assay can be performed in which the leukotriene
 C.sub.4 synthase of the invention is affixed to a solid phase. A liquid
 sample such as bronchial fluid containing, or suspected of containing,
 antibodies directed against leukotriene C.sub.4 synthase is incubated with
 the solid phase. Incubation is maintained for a sufficient period of time
 to allow the antibody in the sample to bind to the immobilized polypeptide
 on the solid phase. After this first incubation, the solid phase is
 separated from the sample. The solid phase is washed to remove unbound
 materials and interfering substances such as non-specific proteins which
 may also be present in the sample. The solid phase containing the antibody
 of interest bound to the immobilized polypeptide of the present invention
 is subsequently incubated with labeled antibody or antibody bound to a
 coupling agent such as biotin or avidin. Labels for antibodies are
 well-known in the art and include radionuclides, enzymes (e.g. maleate
 dehydrogenase, horseradish peroxidase, glucose oxidase, catalase), fluors
 (fluorescein isothiocyanate, rhodamine, phycocyanin, fluorescamine),
 biotin, and the like. The labeled antibodies are incubated with the solid
 phase and the label bound to the solid phase is measured, the amount of
 the label detected serving as a measure of the amount of anti-human
 leukotriene C.sub.4 synthase antibody present in the sample. These and
 other immunoassays can be easily performed by those of ordinary skill in
 the art using the present compositions as reagents. Such fragments are
 typically produced by proteolytic cleavage using enzymes such a papain or
 pepsin, using methods well known in the art.
 Radioactive isotopes can be detected by such means as the use of a gamma
 counter or assimilation counter or by autoradiography. For example,
 reference by Work, T. S. et al., laboratory techniques and biochemistry
 and molecular biology, North Holland Publishing Company, New York 1978.
 G. Assays/Utilities
 The present invention provides for assay systems in which activity or
 activities resulting from exposure to a peptide or non-peptide compound
 may be detected by measuring a physiological response to the compound in a
 cell or cell line which expresses the molecules of the invention. A
 "physiological response" may comprise any biological response, including
 but not limited to transcriptional activation of certain nucleic acid
 sequences (e.g.. promoter/enhancer elements as well as structural genes),
 translation, or phosphorylation, or the induction of human leukotriene
 C.sub.4 synthesis.
 The present invention thus provides for the development of novel assay
 systems which may be utilized in the screening of compounds directed
 against human leukotriene C.sub.4 synthase. Target cells expressing human
 leukotriene C.sub.4 synthase polypeptide, which are modulated (i.e.,
 activated and/or inhibited) by the compounds, may be produced by
 transfection with human leukotriene C.sub.4 synthase polypeptide-encoding
 nucleic acid.
 A convenient assay method for identifying a modulator of a human
 leukotriene C.sub.4 synthase polypeptide includes providing a human
 leukotriene C.sub.4 synthase messenger RNA to a target cell such as an
 mammalian COS-7 cell; incubating the cell in the presence of the
 modulating compound; and measuring synthesis of the product of the
 LTC.sub.4 synthase reaction (LTC.sub.4). Alternately, one could measure
 expression of the messenger RNA into the human leukotriene C.sub.4
 synthase polypeptide using the sensitive LTC.sub.4 synthase assay
 described herein. In particular, one can screen many compounds of interest
 in a short period of time using this sensitive LTC.sub.4 synthase assay.
 An exemplary assay method for identifying a modulator of a human
 leukotriene C.sub.4 synthase polypeptide may include providing a target
 cell containing an isolated nucleotide sequence which encodes for a human
 leukotriene C.sub.4 synthase polypeptide; maintaining the target cell
 under conditions and for a time sufficient for the leukotriene C.sub.4
 synthase to be expressed in the target cell; exposing the target cell to a
 compound suspected of modulating leukotriene C.sub.4 synthase activity;
 measuring a property of the target cell in the presence of the modulator;
 and comparing this property to that of a target cell in the absence of the
 modulator but containing the isolated nucleotide sequence. An altered
 property of the target cell exposed to the modulator is indicative of a
 modulating effect of the compound. Transfection of mammalian cell lines
 with eukaryotic DNA is well known and the techniques have been described
 extensively in the literature. See, for example Sambrook, J. et al.,
 Molecular Cloning, Cold Spring Harbor Press, New York, 1989, the entire
 contents of which are incorporated herein by reference.
 Once target cell lines are produced or identified, it may be desirable to
 select for cells which are exceptionally sensitive to a particular
 compound. Such target cells may express large amounts of human leukotriene
 C.sub.4 synthase polypeptide. Target cells expressing a relative abundance
 of the polypeptide could be identified by selecting target cells which,
 when incubated with a compound/tag, produce a relatively higher degree of
 human leukotriene C.sub.4 synthesis. Alternatively, cell lines which are
 exceptionally sensitive to a compound may exhibit a relatively strong
 biological response, such as a sharp increase in immediate early gene
 products such as c-fos or c-jun, in response to leukotriene C.sub.4
 synthase expression. By developing assay systems using target cells which
 are extremely sensitive to a compound, the present invention provides for
 methods of screening for low levels of human leukotriene C.sub.4 synthase
 activity.
 In particular, using recombinant DNA techniques, the present invention
 provides for human leukotriene C.sub.4 synthase target cells which are
 engineered to be highly sensitive to modulating compounds. For example,
 the human leukotriene C.sub.4 synthase gene, cloned according to the
 methods set forth above, may be inserted into cells which naturally
 express leukotriene C.sub.4 synthase such that the recombinant human
 leukotriene C.sub.4 synthase gene is expressed at high levels.
 The present invention also provides for experimental model systems for
 studying the physiological role of the native human leukotriene C.sub.4
 synthase polypeptide. In these model systems, human leukotriene C.sub.4
 synthase polypeptide, peptide fragment, or a derivation thereof, may be
 either supplied to the system or produced within the system. Such model
 systems could be used to study the effects of human leukotriene C.sub.4
 synthase excess or depletion. The experimental model systems may be used
 to study the effects of increased or decreased response to ligand in cell
 or tissue cultures, in whole animals, or in particular cells or tissues
 within whole animals or tissue culture systems, or over specified time
 intervals (including during embryogenesis).
 In additional embodiments of the invention, a human leukotriene C.sub.4
 synthase sequence may be used to inactivate the endogenous gene by
 homologous recombination, and thereby create a human leukotriene C.sub.4
 synthase-deficient cell, tissue, or animal. For example, and not by way of
 limitation, a recombinant human leukotriene C.sub.4 synthase nucleotide
 sequence may be engineered to contain an insertional mutation (e.g., the
 neo gene) which, when inserted, inactivates transcription of human
 leukotriene C.sub.4 synthase polypeptide. Such a construct, under the
 control of a suitable promoter operatively linked to the human leukotriene
 C.sub.4 synthase nucleotide sequence, may be introduced into a cell by a
 technique such as transfection, transduction, injection, etc. In
 particular, stem cells lacking an intact human leukotriene C.sub.4
 synthase gene may generate transgenic animals deficient in human
 leukotriene C.sub.4 synthase polypeptide. A "transgenic animal" is an
 animal having cells that contain DNA which has been artificially inserted
 into a cell, which DNA becomes part of the genome of the animal which
 develops from that cell. The preferred DNA encodes for leukotriene C.sub.4
 synthase and may be entirely foreign to the transgenic animal or may be
 homologous to the natural leukotriene C.sub.4 synthase of the transgenic
 animal, but which is inserted into the animal's genome at a location which
 differs from that of the natural homolog.
 In a specific embodiment of the invention (See Example 5), the endogenous
 human leukotriene C.sub.4 synthase gene of a cell may be inactivated by
 homologous recombination with a mutant human leukotriene C.sub.4 synthase
 gene to form a transgenic animal lacking the ability to express human
 leukotriene C.sub.4 synthase polypeptide. In another embodiment, a
 construct can be provided that, upon transcription, produces an
 "anti-sense" nucleic acid sequence which, upon translation, will not
 produce the required human leukotriene C.sub.4 synthase polypetide.
 In a further embodiment of the invention, leukotriene C.sub.4 synthase
 expression may be reduced by providing human leukotriene C.sub.4 synthase
 polypeptide-expressing cells, preferably in a transgenic animal, with an
 amount of human leukotriene C.sub.4 synthase polypeptide anti-sense RNA or
 DNA effective to reduce expression of human leukotriene C.sub.4 synthase
 polypeptide.
 A transgenic animal (preferably a non-human mammal) can also be provided
 with a human leukotriene C.sub.4 synthase DNA sequence that also encodes a
 repressor protein that can bind to a specific DNA sequence of human
 leukotriene C.sub.4 synthase polypeptide, thereby reducing ("repressing")
 the level of transcription of human leukotriene C.sub.4 synthase DNA.
 Transgenic animals of the invention which have attenuated levels of
 leukotriene C.sub.4 synthase expression have general applicability to the
 field of transgenic animal generation, as they permit control of the level
 of expression of genes.
 According to the present invention, human leukotriene C.sub.4 synthase
 probes may be used to identify cells and tissues of transgenic animals
 which lack the ability to transcribe human leukotriene C.sub.4 synthase
 nucleotide sequences. Leukotriene C.sub.4 synthase expression may be
 evidenced by transcription of human leukotriene C.sub.4 synthase mRNA or
 production of human leukotriene C.sub.4 synthase polypeptide, determined
 using probes as described above. One variety of probe which may be used to
 detect human leukotriene C.sub.4 synthase expression is a nucleic acid
 probe, containing at least a portion of SEQ ID NO.: 1. Detection of human
 leukotriene C.sub.4 synthase-encoding mRNA may be easily accomplished by
 any method known in the art, including, but not limited to, in situ
 hybridization, Northern blot analysis, or PCR related techniques. Probes
 of SEQ ID NO.: 1 or derived therefrom, may be used to screen tissues of
 patients with, for example, asthma in order to detect the presence of
 aberrant leukotriene C.sub.4 synthase encoding mRNA. Another variety of
 probe which may be used is an anti-human LTC.sub.4 synthase antibody to
 screen patients for abnormal levels of the enzyme.
 The above-mentioned probes may be used experimentally to identify cells or
 tissues which hitherto had not been shown to express human leukotriene
 C.sub.4 synthase polypeptide. Furthermore, these methods may be used to
 identify the expression of LTC.sub.4 synthase by aberrant tissues, such as
 malignancies.
 Pharmaceutical Compositions
 Those of ordinary skill in the art will recognize that modulators of human
 LTC.sub.4 synthase may have potential therapeutic applications. The
 compositions and assays described herein, by modulating synthesis of
 LTC.sub.4, can provide clinicians with strategies for the treatment of
 patients with inflammatory conditions such as cardiac ischemia,
 anaphylactic shock, cold-induced asthma, exercise-induced asthma,
 aspirin-induced asthma, and allergic rhinitis. Therefore, the development
 of drugs which selectively inhibit human leukotriene C.sub.4 synthesis is
 expected to provide an important advantage. Full-length LTC.sub.4
 synthase, modulators of the synthase, functional equivalents,
 modifications, and/or nucleic acids capable of encoding them may be used
 in pharmaceutical compositions. An exemplary pharmaceutical composition
 comprises a therapeutically effective amount of active ingredient(s) (e.g.
 LTC.sub.4 synthase, or modification thereof and/or a nucleic acid capable
 of encoding them) and optionally includes a pharmaceutically-acceptable
 and compatible carrier(s).
 It is contemplated that pharmaceutical compositions comprising nucleic
 acids that are capable of encoding full-length LTC.sub.4 synthase or
 modification thereof could be used in gene therapy procedures.
 The term "pharmaceutically-acceptable and compatible carrier" as used
 herein, and described more fully below, refers to (i) one or more
 compatible solid or liquid filler diluents or encapsulating substances
 that are suitable for administration to a human or other animal, and/or
 (ii) a system, such as a retroviral vector, capable of delivering a
 nucleic acid to a target cell.
 In the present invention, the term "carrier" thus denotes an organic or
 inorganic ingredient, natural or synthetic, with which the active
 ingredient is combined to facilitate application. The term
 "therapeutically-effective amount" is that amount of the present
 pharmaceutical composition that produces a desired result or exerts a
 desired influence. In particular, a "therapeutically-effective" amount of
 the pharmaceutical compositions of the present invention is an amount that
 detectably inhibits or enhances activity of LTC.sub.4 synthase in vivo or
 in vitro. Those of ordinary skill in the art will recognize that
 modulators of human LTC.sub.4 synthase may have potential therapeutic
 applications. Any LTC.sub.4 synthase assay may be used to detect
 inhibition or enhancement of LTC.sub.4 synthase, including those LTC.sub.4
 assays described herein (see Example 1).
 The term "compatible", as used herein, means that the components of the
 pharmaceutical compositions are capable of being commingled with the
 nucleic acid and/or polypeptides of the present invention, and with each
 other, in a manner such that there is no interaction that would
 substantially impair the desired pharmaceutical efficacy.
 The moleclules of the invention may be administered per se (neat) or in the
 form of a pharmaceutically acceptable salt. When used in medicine, the
 salts should be pharmaceutically acceptable, but non-pharmaceutically
 acceptable salts may conveniently be used to prepare pharmaceutically
 acceptable salts thereof and are not excluded from the scope of this
 invention. Such pharmaceutically acceptable salts include, but are not
 limited to, those prepared from the following acids: hydrochloric,
 hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic,
 p-toluene-sulfonic, tartaric, citric, methanesulphonic, formic, malonic,
 succinic, naphthalene-2-sulfonic, and benzenesulphonic. Also,
 pharmaceutically acceptable salts can be prepared as alkaline metal or
 alkaline earth salts, such as sodium, potassium or calcium salts of the
 carboxylic acid group.
 The pharmaceutical compositions of the invention include those suitable for
 oral, rectal, topical, nasal, ophthalmic or parenteral administration, all
 of which may be used as routes of administration using the materials of
 the present invention. Other suitable routes of admiration include
 intrathecal administration directly into spinal fluid (CSF), direct
 injection onto an arterial surface and intraparenchymal injection directly
 into targeted areas of an organ. The term "parenteral" includes
 subcutaneous injections, intravenous, intramuscular, intrasternal
 injection or infusion techniques.
 Pharmaceutical compositions of the present invention may be in a form
 suitable for oral administration. For example, pharmaceutical compositions
 of the invention may be presented as capsules, cachets, tablets, troches,
 lozenges, aqueous or oily suspensions, dispersible powders or granules,
 emulsions, hard or soft capsules, or syrups or elixirs. Capsules, cachets,
 tablets, and lozenges may contain the active ingredient in liposomes or as
 a suspension in an aqueous liquor or non-aqueous liquid such as a syrup,
 an elixir, or an emulsion.
 Aqueous suspensions may contain the active materials in admixture with
 excipients suitable for the manufacture of aqueous suspensions. Such
 excipients may be (1) suspending agents such as sodium
 carboxymethoylcellulose, methylcellulose, hydroxypropylmethylcellulose,
 sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; (2)
 dispersing or wetting agents which may be (a) a naturally-occurring
 phosphatide such as lecithin, (b) a condensation product of an alykylene
 oxide with a fatty acid, for example, polyoxyethylene stearate, (c) a
 condensation product of ethylene oxide with a long chain aliphatic
 alcohol, for example, heptadecaethyleneoxycetanol, (d) a condensation
 product of ethylene oxide with a partial ester derived from a fatty acid
 and a hexitol such as polyoxyethylene sorbitol monooleate, or (e) a
 condensation product of ethylene oxide with a partial ester derived from a
 fatty acid and a hexitol anhydride, for example polyoxyethylene sorbitan
 monooleate.
 The aqueous suspensions may also contain one or more preservatives, for
 example, ethyl or n-propyl p-hydroxybenzoate; one or more coloring agents;
 one or more flavoring agents; and one or more sweetening agents such as
 sucrose or saccharin.
 Oily suspensions may be formulated by suspending the active ingredient in a
 vegetable oil, for example arachis oil, olive oil, sesame oil or coconut
 oil, or in a mineral oil such as liquid paraffin. The oily suspensions may
 contain a thickening agent, for example beeswax, hard paraffin or cetyl
 alcohol. Sweetening agents and flavoring agents may be added to provide a
 palatable oral preparation. These compositions may be preserved by the
 addition of an antioxidant such as ascorbic acid.
 The pharmaceutical compositions of the invention may also be in the form of
 oil-in-water emulsions. The oily phase may be a vegetable oil such as
 olive oil or arachis oils, or mineral oil such as liquid paraffin or
 mixture thereof.
 Suitable emulsifying agents may be (1) naturally-occurring gums such as gum
 acacia and gum tragacanth, (2) naturally-occurring phosphatides such as
 soy bean and lechithin, (3) esters or partial esters derived from fatty
 acids and hexitol anhydrides, for example, sorbitan monooleate, (4)
 condensation products of said partial esters with ethylene oxide, for
 example, polyoxyethylene sorbitan monooleate. The emulsions may also
 contain sweetening and flavoring agents.
 Syrups and elixirs may be formulated with sweetening agents, for example,
 glycerol, propylene glycol, sorbitol or sucrose. Such formulations may
 also contain a demulcent, a preservative and flavoring and coloring
 agents.
 The pharmaceutical compositions may be in the form of a sterile injectable
 aqueous or oleagenous suspension. This suspension may be formulated
 according to known methods using those suitable dispersing or wetting
 agents and suspending agents which have been mentioned above. The sterile
 injectable preparation may also be a sterile injectable solution or
 suspension in a non-toxic parenterally-acceptable diluent or solvent, for
 example as a solution in 1,3-butane diol. Among the acceptable vehicles
 and solvents that may be employed are water, Ringer's solution and
 isotonic sodium chloride solution. In addition, sterile, fixed oils are
 conventionally employed as a solvent or suspending medium. For this
 purpose any bland fixed oil may be employed including synthetic monoor
 diglycerides. In addition, fatty acids such as oleic acid find use in the
 preparation of injectibles.
 The pharmaceutical compositions of the invention may include a sustained
 release delivery system. Preferred sustained release delivery systems are
 those that can provide for release of the active compounds in sustained
 release pellets or capsules. Many types of sustained release delivery
 systems are available (see, for example, U.S. Pat. No. 5,252,318). These
 include, but are not limited to, (i) erosional systems in which the active
 compounds are contained within a matrix (see, for example, U.S. Pat. Nos.
 4,452,775; 4,667,014; 4,748,024; and 5,239,660; and (ii) diffusional
 systems in which the active compounds permeate at a controlled rate
 through a polymer (see, for example, U.S. Pat. Nos. 3,832,252; and
 3,854,480).
 Alternatively, the pharmaceutical compositions of the invention may include
 a delivery system such as, for example, a liposome delivery system.
 Liposomes are single- or multi-compartmented bodies obtained when lipids
 are dispersed in aqueous dispension. The walls of the compartments are
 membranes that are composed of a continuous lipid bi-layer that encloses
 an inner space. Liposomes can be used, for example, to encapsulate and
 deliver pharmaceutical agents.
 The invention will be further illustrated by the following, non-limiting
 examples:
 EXAMPLE 1
 Cloning and Sequencing the Human LTC.sub.4 Synthase Gene
 Materials and Methods
 LTC.sub.2 (see Nicholson et al., Eur. J. Biochem., 209: 725-FIG. 6B) and
 LTA.sub.4 -methyl ester were synthesized by Dr. Bernd Spur while he was at
 Harvard University. MK-886, mouse monoclonal LTC.sub.4 antibodies and a
 FLAP cDNA probe (A. Ford-Hutchinson, Merck Frosst) and A79175 (G. Carter,
 Abbott Laboratories) were generous gifts. Total RNA from human eosinophils
 developed in vitro from human umbilical cord blood mononuclear leukocytes
 cultured for 28 days with human recombinant interleukin 3 and interleukin
 5 was provided by Joshua Boyce (Harvard Medical School). LTA.sub.4 was
 hydrolyzed as described by Maycock et al., J. Biol. Chem. 257: 13911-13914
 (1982). LTC.sub.2 -LC-Biotin was synthesized by coupling LTC.sub.2 with
 NHS-LC-Biotin (1:10 ratio) at pH 7.4 in Tris-HCl buffer at room
 temperature for 120 min with a conjugation rate of over 95%. The mixture
 was purified by high performance liquid chromatography (HPLC) with an
 isocratic solvent system containing 24.9% H.sub.2 O, 1% acetic acid, 75%
 methanol, pH 5.0.
 KG-1 cells and Raji cells (American Type Culture Collection) were cultured
 in RPMI-1640 supplemented with 10% fetal calf serum and 15 .mu.g/ml
 gentamicin under a humidified atmosphere of 95% O.sub.2 /5% CO.sub.2 at
 37.degree. C. COS-7 cells were cultured in RPMI-1640 supplemented with 10%
 heat-inactivated fetal calf serum and 15 .mu.g/ml gentamycin under
 identical culture conditions. Human PMN and eosinophils were obtained from
 normal volunteers and isolated as described by Weller et al., Proc. Nat.
 Acad. Sci. USA, 80: 7626-7630 (1983).
 mRNA Isolation and pcDNA3 Library Construction
 Messenger RNA (mRNA) was prepared from 2.times.10.sup.8 KG-1 cells with a
 FastTrack mRNA (InVitrogen, San Diego, Calif.) isolation kit according to
 the manufacturer's instructions. A plasmid cDNA library (pcDNA) was
 constructed from the mRNA by InVitrogen briefly as follows: The first
 strand cDNA was synthesized from mRNA using Not oligo T priming. cDNAs
 were sized by agarose gel electrophoresis, and those greater than 500 base
 pairs (bp) were ligated with a EcoRI/BstXI adaptor and cloned into the
 pcDNA3 mammalian expression vector (obtained from InVitrogen, San Diego,
 Calif.). The pcDNA3 library was used to transform E. coli strain Top
 10F.sup.1 with a complexity of 1.76 million original clones.
 Development of cDNA Library
 Exactly 96 pools of 2500 E. coli strain Top 10F.sup.1 colonies each were
 aliquoted from the KG-1 pcDNA3 library and grown in 100 .mu.l SB medium
 (containing 5 g/l NaCl, 32 g/l tryptone, 20 g/l yeast extract, and 50 mg/l
 ampicillin) in a 96-well flat-bottom microtiter plate at 37.degree. C.
 overnight to amplify each pool. Then, 20-.mu.l samples of bacterial
 culture from each well were transferred separately into a 10-ml conical
 tube containing 3 ml of SB medium, and the remaining 80 .mu.l were frozen
 at -20.degree. C. in 15% glycerol for later rounds of screening. Bacterial
 cultures in the conical tubes were grown at 37.degree. C. for 18 h, and a
 1.5-ml sample of each was subjected to alkaline hydrolysis to develop
 miniature preparations of plasmids. See, Freeman et al., J. Immunol., 143:
 2714-2722 (1989), incorporated herein by reference. Plasmids were
 dissolved in 25 .mu.l of Tris-EDTA buffer and a 5-.mu.l sample of each was
 used for COS cell transfection.
 Transfection and Screening
 COS-7 cells were transfected with plasmid DNA from the KG-1 pcDNA3 library
 using a DEAE-dextran technique as described by Seed and Aruffo, Proc. Nat.
 Acad. Sci. USA, 84: 3365-3369 (1987). Briefly, COS-7 cells at about 50%
 confluence are transfected in 1.5 ml of Dulbecco's or Iscove's modified
 Eagle's medium (DMEM or IMDM) with 10% NuSerum (Collaborative Research,
 Waltham, Mass.); 400 micrograms DEAE-dextran per ml; 100 micromolar
 chloroquine diphosphate; and miniprep plasmid DNA.
 Seventy-two hours after transfection, the COS cells were harvested by
 trypsinization. Twenty thousand cells from each plate were resuspended in
 500 .mu.l of culture medium and incubated with 25 .mu.M LTA.sub.4
 substrate for 30 min on ice to allow the synthesis of intracellular
 leukotriene product (LTC.sub.4). Cells were washed and resuspended in
 37.degree. C. medium containing 20 mM serine-borate for 10 min to release
 intracellular LTC.sub.4 into the medium. Cells were pelleted, and the
 LTC.sub.4 in the supernatants was measured by immunoassay.
 A positive pool was defined as having produced an amount of LTC.sub.4 that
 was 2 standard deviations above the mean value for all plates in that
 particular transfection experiment. Any pools that met the definition for
 positive were simultaneously rescreened in duplicate, and the amounts of
 LTC.sub.4 produced were compared to the mean of 10 controls that were
 transfected with irrelevant plasmid DNA (CD40 plasmid DNA). A positive
 pool at this stage was defined as producing an amount of LTC.sub.4 that
 was 5 standard deviations above the mean of the control transfectants.
 Once a positive pool was identified, its frozen stock from the 96-well
 microtiter plate was thawed, titered, and sub-divided into smaller pools
 of about 50 colonies each; these pools were distributed to SB-agar plates
 containing 50 .mu.g/ml ampicillin. After overnight growth, each plate was
 replicated by overlaying a nitrocellulose membrane and transferring the
 membrane to an empty plate. Each membrane was then submerged in 10 ml of
 SB medium and subjected to intermittent agitation for 10 min by pipeting
 SB medium to elute the bacterial colonies from the nitrocellulose
 membranes. The replicate eluted colonies were grown at 37.degree. C.
 overnight for plasmid preparation. The original plates were cultured at
 37.degree. C. for an additional 6 h to replenish the bacterial colonies
 and were held at 4.degree. C. until a final screening by plasmid
 preparations of individual colonies of a positive plate.
 For these later rounds of screening, samples of transfected COS cells
 (5.times.10.sup.5 cells each) were incubated with 20 .mu.M LTA.sub.4
 -methyl ester (ME) substrate for 10 min at 37.degree. C. in the presence
 of 20 mM serine-borate. Reactions were terminated by the addition of 3
 volumes of methanol containing prostaglandin B.sub.2 (PGB.sub.2). Total
 LTC.sub.4 -ME was quantitated by reverse phase-high performance liquid
 chromatography (RP-HPLC) to confirm the identity of the product by
 retention time and UV spectra. LTA.sub.4 -ME was used as substrate instead
 of the free acid because it provides a high V.sub.max (Yoshimoto et al.,
 J. Clin. Invest., 81: 866-871 (1988) and because we had devised an
 automated HPLC system to detect and quantitate LTC.sub.4 -ME every 18 min.
 This assay had the additional advantage of confirming that the product
 released by transfected COS cells after incubation with LTA.sub.4 -ME was
 LTC.sub.4 -ME based upon its retention time and the on-line UV spectra.
 DNA Sequencing
 Plasmids were prepared with a Nucleobond isolation kit (Nest) and were
 sequenced as described by Sanger et al., Proc. Nat. Acad. Sci. USA, 74:
 5463-5467 (1977) using dye-labeled dideoxy nucleotides as terminators.
 Samples were analyzed on an Applied Biosystems model 373A automated DNA
 sequencer at the Molecular Biology Core Facility, Dana Farber Cancer
 Institute. See also, Smith et al., Nature 321: 674-679 (1986). Double
 strand sequencing was performed on one clone producing LTC.sub.4 activity
 (clone 56-12-8), whereas the other two clones producing LT C.sub.4
 activity (clones 56-13-25 and 56-16-3) were sequenced on the sense strand
 only.
 Reverse Phase-HPLC
 HPLC was carried out with a model 126 dual pump system and model 167
 scanning UV detector (Beckman Instruments) controlled by an IBM PS2/50
 computer using Beckman System Gold software. Samples were applied to a
 5-.mu.m 4.6.times.250-mm C18 Ultrasphere reverse phase column (Beckman
 Instruments) equilibrated with a solvent of
 methanol/acetonitrile/water/acetic acid (10:15:100:0.2, v/v), pH 6.0
 (solvent A). After injection of the sample, the column was eluted at a
 flow rate of 1 ml/min with a programmed concave gradient (System Gold
 curve 6) to 30% solvent A and 70% pure methanol (solvent B) over 0.2 min.
 After 2.8 min more, solvent B was increased linearly to 90% over 2 min and
 was maintained at this level for an additional 10 min. UV absorbance at
 280 nm and the UV spectra were recorded simultaneously. The retention
 times for PGB.sub.2 and LTC.sub.4 -ME were 8.5 and 10.1 min, respectively.
 LTC.sub.4 -ME was quantitated by calculating the ratio of the peak area to
 the area of the internal standard PGB.sub.2.
 Native and Recombinant Protein Purification and Sequencing
 Subcellular localization of LTC.sub.4 synthase followed the procedure of
 Penrose et al., Proc. Nat. Acad. Sci., USA, 89: 11603-11606 (1992),
 incorporated herein by reference. Briefly, native KG-1 or COS-7 cells were
 harvested by centrifugation at 1000.times.g for 10 min at 4.degree. C. and
 washed in a small amount of buffer A (50 mM HEPES/5 mM 2-mercaptoethanol/1
 mM EDTA (ph 7.6)). The cells were suspended in buffer containing 50 mM
 HEPES, 0.25 M sucrose, 5 mM 2-mercaptoethanol, 1 mM EDTA and 10% glycerol
 (pH 7.6) and sonicated on ice. The sonicate was centrifuged and
 1000.times.g for 10 min to sediment cell debris. The supernatant was
 centrifuged at 10,000.times.g for 10 min, decanted to new centrifuge
 tubes, and spun at 100,000.times.g to obtain pellets containing microsomes
 and supernatants containing cytosol. Microsomal and cytosolic fractions
 were then assayed for LTC.sub.4 synthase activity (see immunoassay
 described herein) to determine subcellular localization of LTC.sub.4
 synthase (i.e., microsome vs. cytosol).
 Detergent-solubilize LTC.sub.4 synthase from 6.times.10.sup.10 native KG-1
 cells (using 0.4% deoxycholate, 0.4% Triton X-102, 10% glycerol) was
 purified by S-hexylglutathione-agarose chromatography using procedures
 adapted from Penrose et al., Proc. Nat. Acad. Sci., USA, 89:, above,
 followed by LTC.sub.2 affinity chromatography. Active fractions purified
 from the S-hexylglutathione-agarose column were combined, concentrated
 (Amicon, Danvers, Mass.) and then loaded onto an LTC.sub.2 affinity column
 equilibrated with buffer A and 0.1% Triton X-102. The column was washed
 with the same buffer and the enzyme was eluted with buffer A, containing
 0.1% Triton X-102, 0.5 M NaCl and 5 mM reduced glutathione.
 The LTC.sub.4 synthase activity contained a single 18-kDa protein by sodium
 dodecyl sulfate-polyacrylamide gel electrophoresis (NaDodSO.sub.4 -PAGE)
 with silver staining. This fraction did not conjugate GSH to
 1-chloro-2,4-dintrobenzene. A sample of the purified protein was
 concentrated and added to an equal volume of reducing buffer, and the
 mixture was boiled for 5 min. The reduced protein in this solution was
 bound to a polyvinylidene membrane by incubation overnight at 4.degree. C.
 The bound protein was analyzed for N-terminal amino acid sequence by
 automated Edman degradation or was digested in situ with trypsin.
 Fernandez et al., Anal. Biochem., 201: 255-264 (1992). The resulting
 peptide mixture was separated by narrow-bore HPLC, and a prominent peptide
 was sequenced by automated Edman degradation on an Applied Biosystems 477A
 protein sequencer at the Harvard Microchemistry Department. See also, Lane
 et al., J. Prot. Chem., 10: 151-160 (1991).
 LTC.sub.4 synthase was purified from COS-7 cell transfectants by the
 identical sequential S-hexylglutathione and LTC.sub.2 affinity
 chromatography procedures described above.
 Northern Blot Analysis
 Total RNA (10 micrograms) from human eosinophils were developed in vitro
 were electrophoresed in formaldehyde/agarose gels, transblotted onto
 Zetabind and probed under conditions of high stringency (wash temperature
 65.degree. C.) with an LTC.sub.4 synthase cDNA probe (nucleotides 1-520 of
 SEQ ID NO.: 1). The probe was produced by digesting 10 micrograms of SEQ
 ED NO.: 1 with 10 units each of EcoRI and SMAI at 37.degree. C. overnight
 and separated on 1 percent agarose gel. The RNA blot was then striped and
 probed with cDNA derived from FLAP. Autoradiography exposure was 24 hours
 at -80.degree. C. with two enhancing screens.
 Functioning of LTC.sub.4 Synthase in COS Cells: Fluorescence-linked
 Competitive Immunoassay for LTC.sub.4
 This is a novel assay based, in part, on the discovery of a distinct
 LTC.sub.4 cell transport step following biosynthesis. Release of LTC.sub.4
 from human eosinophils is time dependent at 37.degree. C. but release of
 LTC.sub.4 formed at this temperature, or even at 0.degree. C., is fully
 inhibited at zero degrees, resulting in the intracellular retention of
 accumulated LTC.sub.4. Thus, cells can be preloaded with LTC.sub.4 at low
 temperatures. See Lam et al., J. Biol. Chem., 264: 12885-12889 (1989),
 incorporated herein by reference.
 COS cells possess a low basal capability to conjugate glutathione to
 LTA.sub.4, perhaps due to a cytosolic glutathione S-transferase.
 Therefore, screening for enzymatic activity in transfected cells requires
 an assay that is sensitive enough to distinguish the incremental
 production of LTC.sub.4 by a single clone within a pool. We developed a
 competitive fluorescence-linked immunoassay for LTC.sub.4, with exquisite
 sensitivity and the high volume efficiency necessary for expression
 cloning of the LTC.sub.4 synthase. In addition, the signal to background
 ratio for the production and release of LTC.sub.4 from transfected cells
 was optimized by providing substrate LTA.sub.4 at 4.degree. C. This step
 allowed LTA.sub.4 uptake and LTC.sub.4 biosynthesis without release until
 the cells had been washed and warmed. The transfected COS-7 cells
 exhibited the same temperature-dependent LTC.sub.4 export step(s)
 previously observed. See Lam et al., J. Biol. Chem., id. Because of the
 wash step and the assay sensitivity, a signal to noise ratio was achieved
 that proved suitable for assaying all pools at the same cell number to
 minimize the plate-to-plate variation in cell numbers of transfected COS
 cells per plate.
 Leukotriene C.sub.4 synthase was generated from COS-7 cells transfected
 with cDNA from a KG-1 pcDNA mammalian KG-1 expression library, as
 described above. Cells were held at 4.degree. C. to ensure intracellular
 retention of LTC.sub.4 generated during the incubation with substrate. The
 temperature was then raised to 37.degree. C. to allow for the export of
 LTC.sub.4 into the incubation medium.
 KG-1 cells were harvested by centrifugation and resuspended in Hanks'
 balanced salt solution (HBSS) at 10.sup.7 cells/ml. After warming to
 37.degree. C., NHS-LC-Biotin was added to achieve a final concentration of
 350 .mu.g/ml, and the mixture was incubated for 30 min to biotinylate the
 KG-1 cell membranes. The biotinylated cells were washed twice with HBSS
 containing 2 mg/ml bovine serum albumin (HBSA) to remove excess and
 un-reacted biotin and were incubated with 2 mg/ml avidin in HBSA. After 30
 min, the biotin-avidin-coupled KG-1 cells were washed twice to remove
 excess avidin and were incubated with 600 ng/ml LTC.sub.2 -LC-Biotin for
 30 min at 37.degree. C. to link the LTC.sub.2 -biotin complex onto the
 cell surface via the previously bound avidin.
 After washing the KG-1 cells, samples of 10.sup.5 cells each were then
 incubated either with (i) 0-200 pg of synthetic LTC.sub.4 or (ii) with
 5-10 .mu.l portions of the warmed incubation medium from transfected COS
 cells. The incubation medium contained unknown amounts of released
 LTC.sub.4. After incubation for 3 min, mouse monoclonal anti-LTC.sub.4
 antibodies were added (final dilution 1:10,000), and the KG-1 cells were
 incubated for 30 min at room temperature, washed, and incubated with
 fluorescein isothiocyanate-conjugated goat anti-mouse Fab.sup.1 antiserum
 (1:30 dilution) for 40 min on ice in the dark. The cells were washed,
 resuspended in 1 ml of HBSA containing 1 mM EDTA, and analyzed by flow
 cytometry for cell surface fluorescence intensities.
 This immunoassay measures the competition between released LTC.sub.4 in
 solution and membrane bound LTC.sub.2 for anti-LTC.sub.4 as analysed by
 decrements in binding of fluorescence-linked secondary antibody. Thus, the
 more LTC.sub.4 that is in solution, the less antibody that will be
 available for binding to the LTC.sub.2 on the cell surface. The
 immunoassay is capable of detecting as little as 2.5 pg of LTC.sub.4 with
 a linear dose response between 10 and 100 pg of LTC.sub.4.
 It will be appreciated by those having ordinary skill in the art that inert
 materials may be substituted for KG-1 cells in this assay. That is, an
 inert carrier such as, for example, agarose beads may be coupled to avidin
 and used in the method, provided that the beads are of sufficient size to
 be assayed with flow cytometry. The assay described herein may be used to
 detect and quantify any product that can be biotinylated and linked to a
 carrier.
 Dose-Response Assay using MK886
 Cell lysates of the COS-7 cell transfectant containing SEQ ID NO.: 1 were
 incubated with 20 micromolar LTA.sub.4 -ME, 20 mM GSH, 5 mM MgCl.sub.2 in
 HEPES buffer (pH 7.6), in the presence of 0-10 micromolar MK-886 for 10
 minutes at room temperature. Reactions were stopped by addition of 2
 volumes of methanol containing 200 ng PGB2. Samples were analyzed by
 RP-HPLC.
 RESULTS
 Expression Cloning
 Of the plasmids from the 96 pools (2500 colonies each) of the KG-1 pcDNA3
 library transfected into COS-7 cells, only a single pool produced an
 LTC.sub.4 value (207 pg/10.sup.4 cells) that was 5 standard deviations
 above the mean (64.2.+-.8 pg/10.sup.4 cells).
 This pool was divided into smaller pools of about 50 colonies each,
 colonies plated on SB-agar, and subjected to a second round of screening
 with LTA.sub.4 -ME as substrate. Three highly positive plates, numbers
 56-12 (4,988 pg/10.sup.4 cells), 56-13 (3,419 pb/10.sup.4 cells), and
 56-16 (3,850 pg/10.sup.4 cells), were identified. COS-7 cells transfected
 with plasmid from other plates did not make detectable amounts of
 LTC.sub.4 -ME.
 When individual colonies from each plate were grown and their plasmids
 prepared and transfected into COS-7 cells, only one colony producing
 LTC.sub.4 synthase activity was identified from each plate, namely, clones
 56-12-8 (7,485 pg/10.sup.4 cells), 56-13-25 (6,391 pg/10.sup.4 cells), and
 56-16-3 (7,583 pg/10.sup.4 cells).
 Nucleotide Sequence of cDNA
 Clone 56-12-8 contained a 694-bp insert with an open reading frame of 450
 bp terminated by a TGA stop codon (SEQ ID NO.: 1). The nucleotide sequence
 was identical for the two other clones, 56-13-25 and 56-16-3, producing
 LTC.sub.4 synthase activity at high levels.
 Consensus Amino Acid Sequence of LTC.sub.4 Synthase and RNA Blot Analysis
 The LTC.sub.4 synthase protein obtained by sequential S-hexyl glutathione
 and LTC.sub.2 affinity chromatography of solubilized LTC.sub.4 from native
 KG-1 cells provided a 22 amino acid N-terminal sequence of
 MKDEVALLAAVTLLGVLLQAYF (SEQ ID NO. 9) that corresponds exactly to the
 N-terminal amino acid sequence (SEQ ID NO.: 2) deduced from the cDNA of
 SEQ ID NO.: 1. An internal peptide fragment of the protein provided a
 sequence of VSPPLTTGPPEFER (SEQ ID NO. 3) in which all 14 amino acid
 residues are identical to the deduced amino acid sequence amino acid
 residues 35-48 of SEQ ID NO.: 2.
 RNA isolated from human eosinophils developed in vitro demonstrated a
 0.7-kilobase pair mRNA transcript (data not shown). The FLAP transcript in
 these same cells was approximately 1.0 kilobase. The 0.7-kilobase
 LTC.sub.4 synthase mRNA transcript was also detected in total RNA from the
 KG-1 cells and less abundantly in peripheral blood eosinophils. No
 transcript was detected in human PMNs and Burkitt's lymphoma Raji cell
 line, which lack LTC.sub.4 synthase function. When compared with FLAP mRNA
 transcript by autoradiography, LTC.sub.4 synthase transcript is less
 intense than those of FLAP. The FLAP transcript in these same cells was
 approximately 1.0 kilobase. The 0.7-kilobase LTC.sub.4 synthase mRNA
 transcript was also detected in total RNA from the KG-1 cells and less
 abundantly in peripheral blood eosinophils. No transcript was detected in
 human PMNs and Burkitt's lymphoma Raji cell line, which lack LTC.sub.4
 synthase function. When compared with FLAP mRNA transcript by
 autoradiography, LTC.sub.4 synthase transcript is less intense than those
 of FLAP.
 Microsomal Localization, Size and Inhibition of Function of the Protein
 Expressed in COS Cells.
 When examined for subcellular localization, 87-89% (n=3) of the activity of
 the recombinant protein was in the microsomal fraction.
 LTC.sub.4 synthase purified from COS cell transfectants by sequential
 S-hexyl-GSH and LTC.sub.2 affinity chromatography migrated as an 18-kDa
 protein by NaDodSO.sub.4 -PAGE, identical in size to the native enzyme
 purified from KG-1 cells (data not shown).
 Lysates from transfected COS cells were analysed for LTC.sub.4, the product
 of conjugating reduced glutathione with
 5,6-oxido-7,9-E-11,14-Z-eicosatetraenoic acid. MK-886, a FLAP inhibitor
 (see Dixon et al., Nature 343: 6255 (1990) and Gillard et al., Can. J.
 Physil ol. Pharmac., 67: 456 (1989), both of which incorporated herein by
 reference), dose-dependently inhibited the conversion of 20 uM LTA.sub.4
 -ME to LTC.sub.4 -ME by COS cell lysates with an IC.sub.50 of less than 3
 .mu.M (data not shown). At 10 .mu.M, MK-886 inhibits more than 90% of the
 enzyme activity. In contrast, a 5-lipoxygenase inhibitor, A79175, did not
 affect LTC.sub.4 synthase activity in COS cell lysates at a concentration
 of 10 .mu.M.
 EXAMPLE 2
 Isolating a Homolog of LTC.sub.4 Synthase
 A portion of the human leukotriene C.sub.4 synthase gene is amplified from
 human eosinophil DNA using the polymerase chain reaction technique (Saiki,
 R. K., et al., 1985, Science 230 1350-1354) using Notl-Sall sites in the
 PCR primers. The 100 .mu.l reaction contains 10 mM Tris-HCl pH 8.3, 50 mM
 KCl, 0.001% (w/v) Gelatin, 2 mM MgCl2, 200 .mu.M dNTPs, 1.5 .mu.M SEQ ID
 NO.: 1, 1.5 .mu.M primer sequence (e.g., SEQ ID NOS. 4 and 5), 2.5 units
 Taq Polymerase (Perkin Elmer Cetus), and 1.0 .mu.g of human eosinophil
 DNA. The DNA Thermal-cycler (Perkin Elmer Cetus, Model N801) is programmed
 for the following incubations:
 1. 94.degree. C., 2 min. (initial denaturation)
 2. 94.degree. C., 1 min. (denaturation)
 3. 50.degree. C., 1 min. (annealing)
 4. 72.degree. C., 3 min. (elongation)
 5. Steps 2-4 cycle 50 times (amplification)
 6. 4.degree. C., Soak (storage)
 The DNA amplified in this reaction is electrophoresed on 5% polyacrylamide
 gels to verify band length. If the size is determined to be correct, the
 DNA is purified by phenol extraction, then digested with Notl and Sall to
 remove the termini. The DNA is then ligated into the Notl/Sall site of
 vector pUC19 (New England Biolabs). The DNA is transformed into E. coli
 strain DH5-alpha made competent by the CaCl.sub.2 procedure (Hanahan, D.,
 1983, J. Mol. Biol. 155:557). The human leukotriene C.sub.4 synthase is
 then sequenced by the chain-termination method (Sanger, F. et al., 1977,
 Proc. Natl. Acad. Sci. USA 74:5463).
 An alternate cloning procedure for genomic DNA or cDNA encoding human
 leukotriene C.sub.4 synthase includes generating oligonucleotides from the
 polymerase chain reactions described above and radioactively labeling them
 according to the procedure described in Sambrook et al. (1989). These
 oligonucleotides are used to screen a .lambda.gt11 genomic library from a
 human cell line. Alternatively, a .lambda.gt11 cDNA library prepared from
 mRNA from the same human cell line is used. Construction of these
 libraries follows the procedure of Sambrook, J. et al., Molecular Cloning,
 (1989). Alternatively, a commercially available library, available from
 Clontech (Palo Alto, Calif.), is used.
 Hybridization conditions are as described by Cate et al., Cell, 45:165
 (1986), except that the final wash in tetramethyl ammonium chloride is
 omitted. DNA inserts from positive plaques are subcloned directly into the
 plasmid vector pBluescript SKM13+ (Stratagene, Inc. San Diego, Calif.).
 Positive plasmid subclones are identified by colony hybridization, with
 the use of the same oligonucleotide hybridization probe. Minipreparations
 of plasmid DNA are prepared from positive colonies.
 The nucleotide sequence immediately upstream from the oligonucleotide
 binding site is determined by double stand sequencing (Chen and Seeburg,
 DNA, 4:165 1985), using .sup.32 P end-labeled oligonucleotide as
 sequencing primer and non-radioactive nucleotides in the extension
 reactions. Subclones whose codon order upstream from the priming site
 match the known human amino acid sequence (SEQ. ID. NO. 2) are sequenced
 in their entirety by the diideoxy chain termination method, with either
 the Klenow fragment of Escherichia coli DNA polymerase I or modified
 bacteriophage T7 DNA polymerase (Sequenase; United States Biochemicals) in
 the extension reactions. Subclones are sequenced from their termini, from
 both directions from a set of restriction sites. Clones are obtained whose
 codon order is at least partially similar to the amino acid sequence of
 human leukotriene C.sub.4 synthase polypeptide. A full-length genomic or
 cDNA sequence for human leukotriene C.sub.4 synthase polypeptide is
 assembled from overlapping partial clones.
 EXAMPLE 3
 Expression of Polypeptide
 The following method for transient expression of leukotriene C.sub.4
 synthase cDNA in cultured cells is adapted from Birnir et al., supra.
 COS-7 cells, or other cultured cells are used. Tissue culture medium,
 serum, and antibiotics are obtained from GIBCO (Gaithersburg, Md.).
 The eukaryotic expression vector pEUK-C1 is obtained from Clontech (Palo
 Alto, Calif.). Plasmid pEUK-UT2 is constructed by inserting SEQ ID NO.: 1
 cDNA (blunt-ended with T4 DNA polymerase) into the SmaI side of plasmid
 pEUK-C1. The orientation and correct insertion at the 5' end is confirmed
 by DNA sequencing. pEUK-UT2 (15 .mu.g) is transfected into COS-7 cells
 using lipofectin. Briefly, COS-7 cells are seeded onto 35 mm tissue
 culture plates (Falcon, N.J.) in Dulbecco's modified Eagle's medium (DMEM)
 with 10% fetal bovine serum and 1% antimycotic (containing Fungizon-GIBCO)
 and transfected at a confluency of 80-95%. Immediately before
 transfection, cell monolayers are washed twice with OPTI-MEM I medium
 (GIBCO). For each 35 mm plate, 15 .mu.g of plasmid and 15 .mu.g of
 Lipofectin are mixed for 30 min in 0.5 ml of OPTI-MEM I medium and then
 added to the plate.
 After incubation for 24 h at 37.degree. C. in a humidified atmosphere
 containing 5% CO.sub.2, 1 ml of DMEM with 10% serum is added. LTC.sub.4
 synthase is measured 48 to 72 h post-transfection.
 In control experiments, pEUK-C1 plasmid DNA without SEQ ID NO.: 1 is
 transfected. The transfection efficiency is monitored after
 co-transfection with plasmid pCH110 (Clontech), containing a functional
 Lac Z gene and a SV 40 origin of replication. COS-7 cells produce the SV
 40 large tumor antigen which allows replication of plasmids (such as
 pCH110 and pEUK-C1) containing a SV 40 origin. The product of the Lac Z
 gene, beta-galactosidase, is measured using X-Gal. Generally, between
 15-25% of cells are transfected.
 EXAMPLE 4
 Preparation of Constructions for Transfections and Microinjections
 Methods for purification of DNA for microinjection are well known to those
 of ordinary skill in the art. See, for example, Hogan et al., Manipulating
 the Mouse Embryo, Cold spring Harbor Laboratory, Cold Spring Harbor, N.Y.
 (1986); and Palmer et al., Nature, 300: 611 (1982).
 Construction of Transgenic Animals
 A variety of methods are available for the production of transgenic animals
 associated with this invention. DNA can be injected into the pronucleus of
 a fertilized egg before fusion of the male and female pronuclei, or
 injected into the nucleus of an embryonic cell (e.g., the nucleus of a
 two-cell embryo) following the initiation of cell division (Brinster et
 al., Proc. Nat. Acad. Sci. USA, 82: 4438-4442 (1985)). Embryos can be
 infected with viruses, especially retroviruses, modified to bear human
 leukotriene C.sub.4 synthase genes of the invention.
 Pluripotent stem cells derived from the inner cell mass of the embryo and
 stabilized in culture can be manipulated in culture to incorporate human
 leukotriene C.sub.4 synthase genes of the invention. A transgenic animal
 can be produced from such cells through implantation into a blastocyst
 that is implanted into a foster mother and allowed to come to term.
 Animals suitable for transgenic experiments can be obtained from standard
 commercial sources such as Charles River (Wilmington, Mass.), Taconic
 (Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.), etc. Swiss
 Webster female mice are preferred for embryo retrieval and transfer.
 B6D2F.sub.1 males can be used for mating and vasectomized Swiss Webster
 studs can be used to stimulate pseudopregnancy. Vasectomized mice and rats
 can be obtained from the supplier.
 Microinjecion Procedures
 The procedures for manipulation of the rodent embryo and for microinjection
 of DNA into the pronucleus of the zygote are well known to those of
 ordinary skill in the art (Hogan et al., supra). Microinjection procedures
 for fish, amphibian eggs and birds are detailed in Houdebine and
 Chourrout, Experientia, 47: 897-905 (1991). Other procedures for
 introduction of DNA into tissues of animals are described in U.S. Pat. No.
 4,945,050 (Sanford et al., Jul. 30, 1990).
 Transgenic Mice
 Female mice six weeks of age are induced to superovulate with a 5 IU
 injection (0.1 cc, ip) of pregnant mare serum gonadotropin (PMSG; Sigma)
 followed 48 hours later by a 5 IU injection (0.1 cc, ip) of human
 chorionic gonadotropin (hCG; Sigma). Females are placed with males
 immediately after hCG injection. Twenty-one hours after hCG, the mated
 females are sacrificed by CO.sub.2 asphyxiation or cervical dislocation
 and embryos are recovered from excised oviducts and placed in Dulbecco's
 phosphate buffered saline (DPSS) with 0.5% bovine serum albumin (BSA;
 Sigma). Surrounding cumulus cells are removed with hyaluronidase (1
 mg/ml). Pronuclear embryos are then washed and placed in Earle's balanced
 salt solution containing 0.5% BSA (EBSS) in a 37.5.degree. C. incubator
 with a humidified atmosphere at 5% CO.sub.2, 95% air until the time of
 injection.
 Randomly cycling adult female mice are paired with vasectomized males.
 Swiss Webster or other comparable strains can be used for this purpose.
 Recipient females are mated at the same time as donor females. At the time
 of embryo transfer, the recipient females are anesthetized with an
 intraperitoneal injection of 0.015 ml of 2.5% avertin per gram of body
 weight. The oviducts are exposed by a single midline dorsal incision. An
 incision is then made through the body wall directly over the oviduct. The
 ovarian bursa is then torn with watchmakers forceps. Embryos to be
 transferred are placed in DPBS and in the tip of a transfer pipet (about
 10-12 embryos). The pipet tip is inserted into the infundibulum and the
 embryos transferred. After the transfer, the incision is closed by two
 sutures.
 Transgenic Rats
 The procedure for generating transgenic rats is similar to that of mice See
 Hammer et al., Cell, 63:1099-1112 (1990). Thirty day-old female rats are
 given a subcutaneous injection of 20 IU of PMSG (0.1 cc) and 48 hours
 later each female placed with a proven male. At the same time, 40-80 day
 old females are placed in cages with vasectomized males. These will
 provide the foster mothers for embryo transfer. The next morning females
 are checked for vaginal plugs. Females who have mated with vasectomized
 males are held aside until the time of transfer. Donor females that have
 mated are sacrificed (CO.sub.2 asphyxiation) and their oviducts removed,
 placed in DPSS with 0.5% BSA and the embryos collected. Cumulus cells
 surrounding the embryos are removed with hyaluronidase (1 mg/ml). The
 embryos are then washed and placed in EBSS (Earle's balanced salt
 solution) containing 0.5% BSA in a 37.5.degree. C. incubator until the
 time of microinjection.
 Once the embryos are injected, the live embryos are moved to DPBS for
 transfer into foster mothers. The foster mothers are anesthetized with
 ketamine (40 mg/kg, ip) and xylazine (5 mg/kg, ip). A dorsal midline
 incision is made through the skin and the ovary and oviduct are exposed by
 an incision through the muscle layer directly over the ovary. The ovarian
 bursa is torn, the embryos are picked up into the transfer pipet, and the
 tip of the transfer pipet is inserted into the infundibulum. Approximately
 10-12 embryos are transferred into each rat oviduct through the
 infundibulum. The incision is then closed with sutures, and the foster
 mothers are housed singly.
 Embryonic Stem (ES) Cell Methods
 Introduction of DNA into ES cells:
 Methods for the culturing of ES cells and the subsequent production of
 transgenic animals by the introduction of DNA into ES cells using methods
 such as electroporation, calcium phosphate/DNA precipitation; and direct
 injection are well known to those of ordinary skill in the art. See, for
 example, Teratocarcinomas and Embryonic Stem Cells, A Practical Approach,
 E. J. Robertson, ed., IRL Press (1987). Selection of the desired clone of
 thrombospondin-4-containing ES cells is accomplished through one of
 several means. Although embryonic stem cells are currently available for
 mice only, it is expected that similar methods and procedures as described
 and cited here will be effective for embryonic stem cells from different
 species as they become available.
 In cases involving random gene integration, a clone containing the human
 leukotriene C.sub.4 synthase gene of the invention is co-transfected with
 a gene encoding neomycin resistance. Alternatively, the gene encoding
 neomycin resistance is physically linked to the human leukotriene C.sub.4
 synthase gene. Transfection is carried out by any one of several methods
 well known to those of ordinary skill in the art (E. J. Robertson, supra).
 Calcium phosphate/DNA precipitation, direct injection, and electroporation
 are the preferred methods. Following DNA introduction, cells are fed with
 selection medium containing 10% fetal bovine serum in DMEM supplemented
 with G418 (between 200 and 500 .mu.g/ml biological weight). Colonies of
 cells resistant to G418 are isolated using cloning rings and expanded. DNA
 is extracted from drug resistant clones and Southern blotting experiments
 using a transgene-specific DNA probe are used to identify those clones
 carrying the human leukotriene C.sub.4 synthase polypeptide sequences. In
 some experiments, PCR methods are used to identify the clones of interest.
 DNA molecules introduced into ES cells can also be integrated into the
 chromosome through the process of homologous recombination. Copecchi,
 Science, 244: 1288-1292 (1989). Direct injection results in a high
 efficiency of integration. Desired clones are identified through PCR of
 DNA prepared from pools of injected ES cells. Positive cells within the
 pools are identified by PCR subsequent to cell cloning. DNA introduction
 by electroporation is less efficient and requires a selection step.
 Methods for positive selection of the recombination event (i.e., neo
 resistance) and dual positive-negative selection (i.e., neo resistance and
 gancyclovir resistance) and the subsequent identification of the desired
 clones by PCR have been described by Copecchi, supra and Joyner et al.,
 Nature 338: 153-156 (1989), the disclosures of which are incorporated
 herein.
 Embryo Recovery and ES Cell Injection:
 Naturally cycling or superovulated female mice mated with males are used to
 harvest embryos for the implantation of ES cells. It is desirable to use
 the C57BL165 stain for this purpose when using mice. Embryos of the
 appropriate age are recovered approximately 3.5 days after successful
 mating. Mated females are sacrificed by CO.sub.2 asphyxiation or cervical
 dislocation and embryos are flushed from excised uterine horns and placed
 in Dulbecco's modified essential medium plus 10% calf serum for injection
 with ES cells. Approximately 10-20 ES cells are injected into blastocysts
 using a glass microneedle with an internal diameter of approximately 20
 .mu.m.
 Transfer of Embryos to Receptive Females:
 Randomly cycling adult female mice are paired with vasectomized males.
 Mouse strains such as Swiss Webster, ICR or others can be used for this
 purpose. Recipient females are mated such that they will be at 2.5 to 3.5
 days post-mating when required for implantation with blastocysts
 containing ES cells. At the time of embryo transfer, the recipient females
 are anesthetized with an intraperitoneal injection of 0.015 ml of 2.5%
 avertin per gram of body weight. The ovaries are exposed by making an
 incision in the body wall directly over the oviduct and the ovary and
 uterus are externalized. A hole is made in the uterine horn with a 25
 gauge needle through which the blastocysts are transferred. After the
 transfer, the ovary and uterus are pushed back into the body and the
 incision is closed by two sutures. This procedure is repeated on the
 opposite side if additional transfers are to be made.
 Identification of Transgenic Mice and Rats
 Tail samples (1-2 cm) are removed from three week old animals. DNA is
 prepared and analyzed by Southern blot or PCR to detect transgenic founder
 (F.sub.0) animals and their progeny (F.sub.1 and F.sub.2). In this way,
 animals that have become transgenic for the desired human leukotriene
 C.sub.4 synthase genes are identified. Because not every transgenic animal
 expresses the polypeptide, and not all of those that do will have the
 expression pattern anticipated by the experimenter, it is necessary to
 characterize each line of transgenic animals with regard to expression of
 the leukotriene C.sub.4 synthase in different tissues.
 Production of Non-Rodent Transgenic Animals
 Procedures for the production of non-rodent mammals and other animals have
 been discussed by others. See Houdebine and Chourrout, supra; Pursel et
 al., Science 244: 1281-1288 (1989); and Simms et al., Bio/Technology, 6:
 179-183 (1988).
 Identification of Other Transgenic Organisms
 An organism is identified as a potential transgenic by taking a sample of
 the organism for DNA extraction and hybridization analysis with a probe
 complementary to the human leukotriene C.sub.4 synthase gene of interest.
 Alternatively, DNA extracted from the organism can be subjected to PCR
 analysis using PCR primers complementary to the human leukotriene C.sub.4
 synthase gene of interest.
 EXAMPLE 5
 Protocol for Inactivating the Human Leukotriene C.sub.4 Synthase Gene
 Mouse genomic clones are isolated by screening a genomic library from the
 D3 strain of mouse with a human leukotriene C.sub.4 synthase probe.
 Duplicate lifts are hybridized with a radiolabeled probe by established
 protocols (Sambrook, J. et al., The Cloning Manual, Cold Spring Harbor
 Press, N.Y.). Plaques that correspond to positive signal on both lifts are
 isolated and purified by successive screening rounds at decreasing plaque
 density. The validity of the isolated clones is confirmed by nucleotide
 sequencing.
 The genomic clones are used to prepare a gene targeting vector for the
 deletion of human leukotriene C.sub.4 synthase polypeptide in embryonic
 stem cells by homologous recombination. A neomycin resistance gene (neo)
 with its transcriptional and translational signals, is cloned into
 convenient sites that are near the 5' end of the gene. This will disrupt
 the coding sequence of human leukotriene C.sub.4 synthase polypeptide and
 allow for selection by the drug Geneticin (G418) by embryonic stem (ES)
 cells transfected with the vector. The Herpes simplex virus thymidine
 kinase (HSV-tk) gene is placed at the other end of the genomic DNA as a
 second selectable marker. Only stem cells with the neo gene will grow in
 the presence of this drug.
 Random integration of this construct into the ES genome will occur via
 sequences at the ends of the construct. In these cell lines, the HSV-tk
 gene will be functional and the drug gancyclovir will therefore be
 cytotoxic to cells having an integrated sequence of the mutated human
 leukotriene C.sub.4 synthase coding sequence.
 Homologous recombination will also take place between homologous DNA
 sequences of the ES human leukotriene C.sub.4 synthase genome and the
 targeting vector. This usually results in the excision of the HSV-tk gene
 because it is not homologous with the human leukotriene C.sub.4 synthase
 gene.
 Thus, by growing the transfected ES cells in G418 and gancyclovir, the cell
 lines in which homologous recombination has occurred win be highly
 enriched. These cells will contain a disrupted coding sequence of human
 leukotriene C.sub.4 synthase. Individual clones are isolated and grown up
 to produce enough cells for frozen stocks and for preparation of DNA.
 Clones in which the human leukotriene C.sub.4 synthase gene has been
 successfully targeted are identified by Southern blot analysis. The final
 phase of the procedure is to inject targeted ES cells into blastocysts and
 to transfer the blastocysts into pseudopregnant females. The resulting
 chimeric animals are bred and the offspring are analyzed by Southern
 blotting to identify individuals that carry the mutated form of the gene
 in the germ line. These animals will be mated to determine the effect of
 human leukotriene C.sub.4 synthase polypeptide deficiency on murine
 development and physiology.
 DISCUSSION
 LTC.sub.4 synthase, an integral membrane protein that conjugates reduced
 glutathione (GSH) to LTA.sub.4 but not to xenobiotics, provides the parent
 LTC.sub.4 for the cysteinyl leukotriene family, and is the only
 biosynthetic moiety in the 5-lipoxygenase pathway that has not yet been
 defined by its cDNA, protein structure, or gene family. Recently, a
 35-amino-acid N-terminal sequence was obtained for LTC.sub.4 synthase
 extracted and purified from the THP-1 cell line. Nicholson et al., Proc.
 Nat. Acad. Sci., USA 90: 2015-2019 (1993). The amino acid N-terminal
 sequence obtained for the KG-1 cell line described herein (SEQ ID NO.: 9)
 differed from that of the THP-1 cell line only at position 21; an internal
 14 residue peptide (SEQ ID NO.: 3) provided an additional amino acid
 sequence that corresponded to residues 35 to 48 of LT C.sub.4 from KG-1
 cells.
 Nonetheless, the degeneracy of the nucleotides coding for the observed
 THP-1 sequence data is extremely high and did not provide oligonucleotide
 probes capable of hybridizing to clones carrying the sequence of interest
 using a .lambda.gt11 KG-1 cDNA library (unpublished data). We thus
 proceeded to expression cloning with the intent of using the available
 THP-1 protein sequence at the N-terminal amino acid residues for
 reference, but depending on enzymatic function for detection and
 definition of the cDNA and its amino acid sequence.
 We have also demonstrated that FLAP inhibitor, MK-886, inhibits LTC.sub.4
 synthase activity from transfected COS cell lysates in a dose-related
 fashion with an ID.sub.50 of about 3 .mu.M. Since FLAP binds arachidonic
 acid for presentation to 5-lipoxygenase that is engaged in
 calcium-dependent membrane association (Mancini et al., FEBS Lett., 318:
 277-281 (1993) for synthesis of LTA.sub.4, it seems plausible that the
 transmembrane domains of LTC.sub.4 synthase homologous to FLAP will accept
 LTA.sub.4 for conjugation with glutathione at/or in the membrane.
 LTA.sub.4 and reduced glutathione conjugate spontaneously in a basic
 microenvironment. See Radmark et al., J. Biol. Chem., 12339-12345 (1984).
 It may be that the putative binding of LTA.sub.4 to LTC.sub.4 synthase, a
 protein with a pI of 11.05, allows a favorable environment for the
 conjugation with bound or unbound glutathioine with only a modest
 catalytic boost. Thus, LTC.sub.4 synthase may represent a member of the
 lipid-binding family rather than the classical glutathione S-transferase
 family. Irrespective of the catalytic mechanisms yet to be elucidated, it
 is likely that LTC.sub.4 synthase represents a member of a novel gene
 family in which FLAP is also a member.
 EQUIVALENTS
 It should be understood that the preceding is merely a detailed description
 of certain preferred embodiments. It therefore should be apparent to those
 skilled in the art that various modifications and equivalents can be made
 without departing from the spirit or scope of the invention.