Lipoxygenase proteins and nucleic acids

Isolated and purified lipoxygenase proteins and nucleic acids are described. Particularly, a novel human 15(S) lipoxygenase (15-Lox-2) protein and cDNA and a cDNA for mouse 8S-lipoxygenase are described. Recombinant host cells, recombinant nucleic acids and recombinant proteins are also described, along with methods of producing each. Isolated and purified antibodies to 15-Lox-2 and 8-Lox, and methods of producing the same, are also described.

TECHNICAL FIELD
 The present invention relates generally to isolated and purified
 lipoxygenase proteins and nucleic acids. More particularly, the present
 invention relates to an isolated and purified second type of human
 15S-lipoxygenase and an isolated and purified nucleic acid encoding the
 same, and to an isolated and purified nucleic acid encoding a mouse
 8S-lipoxygenase.

Table of Abbreviations
 15-Lox-1 Reticulocyte-type of 15S-lipoxygenase
 15-Lox-2 Second type of human 15S-lipoxygenase
 8-Lox mouse 8S-lipoxygenase
 PMA Phorbol-12-myristate-13-acetate
 H (P) ETE Hydro (pero) xyeicosatetraenoic acid
 HODE Hydroxyoctadecadienoic acid
 HPLC High pressure liquid chromatography
 PCR Polymerase chain reaction
 RACE Rapid amplification of cDNA ends
 BACKGROUND ART
 The lipoxygenases are a structurally related family of non-heme iron
 dioxygenases that function in the production of fatty acid hydroperoxides.
 Three lipoxygenases have been identified and cloned in humans. Funk, C. D.
 (1993) Prog. Nuc. Acid Res. Mol. Biol. 45:67-98; Matsumoto et al. (1988)
 Proc. Natl. Acad. Sci. USA 85: 26-30; Dixon et al. (1988) Proc. Natl.
 Acad. Sci. USA 85: 416-420; Funk et al. (1990) Proc. Natl. Acad. Sci. USA
 87: 5638-5642; Izumi et al. (1990) Proc. Natl. Acad. Sci. USA
 87:7477-7481; Yoshimoto et al. (1990) Biochem. Biophys. Res. Comm.
 172:1230-1235; Sigal et al. (1988) Biochem. Biophys. Res. Comm.
 157:457-464). They oxygenate arachidonic acid in different positions along
 the carbon chain and form the corresponding 5S-, 12S- or
 15S-hydroperoxides (hydroperoxy-eicosatetraenoic acids, HPETEs). The three
 enzymes are known mainly from the blood cell types in which they are
 strongly expressed--the 5S-lipoxygenase of leukocytes, the
 12S-lipoxygenase of platelets, and the 15S-lipoxygenase of reticulocytes,
 eosinophils and macrophages. While these are the most widely recognized
 cellular sources, selective expression is well documented in other
 tissues. For example, both the 12S- and 15S-lipoxygenases are detected in
 skin. Nugteren et al. (1987) Biochim. Biophys. Acta 921:135-141;
 Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297;
 Takahashi et al. (1993) J. Biol. Chem. 268:16443-16448; Hussain et al.
 (1994) Amer. J. Physiol. 266:C243-C253.
 Potentially, the three cloned lipoxygenases could account for all enzymatic
 synthesis of arachidonate hydroperoxides in humans, but there are reasons
 to consider that other lipoxygenases may exist. For example, in the mouse
 there are five known lipoxygenases, three that correspond to the known
 human enzymes, Chen et al. (1994) J. Biol. Chem. 269:13979-13987; Chen et
 al. (1995) J. Biol. Chem. 270:17993-17999 and two others, Furstenberger et
 al. (1991) J. Biol. Chem. 266:15738-15745; Funk et al. (1996) J. Biol.
 Chem. 271:23338-23344.
 Three of the five distinct mouse lipoxygenase enzymes are best known for
 their occurrence in different types of blood cells. In common with other
 mammals, a 5S-lipoxygenase is present in leukocytes and is responsible for
 synthesis of the pro-inflammatory mediators, the leukotrienes. Chen et al.
 (1995) J. Biol. Chem. 270:17993-17999; Chen et al. (1994) Nature
 372:179-182. A 12S-lipoxygenase is found in platelets and several other
 tissues including skin. Nugteren et al. (1987) Biochim. Biophys. Acta
 921:135-141; Chen et al. (1994) J. Biol. Chem. 269:13979-13987; Sun et al.
 (1996) J. Biol. Chem. 271:24055-24062.
 A second type of 12S-lipoxygenase which is closely related in sequence to
 the human and rabbit "reticulocyte-type" of 15S-lipoxygenases occurs in
 certain macrophages. Sun et al. (1996) J. Biol. Chem. 271, 24055-24062.
 The fourth mouse lipoxygenase to be characterized is another enzyme to
 have 12S-lipoxygenase activity; it was cloned recently from mouse skin and
 has been classified as an epidermal 12S-lipoxygenase. van Dijk et al.
 (1995) Biochim. Biophys. Acta 1259:4-8; Funk et al. (1996) J. Biol. Chem.
 271:23338-23344. All four of these murine lipoxygenases enzymes have been
 characterized at the cDNA and genomic levels.
 The fifth known mouse lipoxygenase was described originally in 1986 by
 Furstenberger, Marks and colleagues as an enzyme in skin forming 8-HETE
 and inducible by phorbol ester treatment. Gschwendt et al. (1986)
 Carcinogenesis 7:449-455. It was shown subsequently that this enzyme forms
 the 8S enantiomer (Hughes et al. (1991) Biochim. Biophys. Acta
 1081:347-354) and isolation of the corresponding hydroperoxide confirmed
 identification of the enzyme as a lipoxygenase. Furstenberger et al.
 (1991) J. Biol. Chem. 266:15738-15745. Mouse skin is the only reported
 site of synthesis of 8S-HETE in animal tissues, and there is no indication
 from the literature pointing to a potential homologue of the mouse
 8S-lipoxygenase in other mammals. Additionally, no nucleic acid,
 particularly a cDNA, which encodes this lipoxygenase has been
 characterized.
 Despite the description in the art of the enzymes presented above, along
 with the catalytic activities covered by these enzymes, there remains an
 open question whether a lipoxygenase rather than a cytochrome P450 might
 account for the synthesis of 12R-hydroxy arachidonic acid (12R-HETE),
 Hammarstrom et al. (1975) Proc. Natl. Acad. Sci. USA 72:5130-5134;
 Woollard, P. M. (1986) Biochem. Biophys. Res. Commun. 136(1):169-175; Baer
 et al. (1991) J. Lipid Research 32:341-347; Holtzman et al. (1989) J.
 Clin. Invest. 84:1446-1453; Brash et al. (1996) J. Biol. Chem.
 271:20549-20557, a prominent arachidonate metabolite in the skin disease
 of psoriasis and other proliferative dermatol (Hammarstrom et al. (1975)
 Proc. Natl. Acad. Sci. USA 72:5130-5134; Baer et al. (1991) J. Lipid
 Research 32:341-347; Baer et al. (1995) J. Invest. Dermatol. 104:251-255).
 Therefore, what is needed, then, is further characterization of
 lipoxygenase enzymes in vertebrates, particularly in mammals, and more
 particularly in humans. A novel isolated and purified lipoxygenase and a
 nucleic acid encoding the same would have broad utility to due its role in
 arachidonic acid metabolism, a critical metabolic pathway.
 DISCLOSURE OF THE INVENTION
 A key aspect of this invention pertains to the discovery of a novel
 15S-lipoxygenase (15-Lox-2) protein and nucleic acid encoding the 15-Lox-2
 protein. Preferred nucleic acid and amino acid sequences for 15-Lox-2 are
 described in SEQ ID NO:1 and SEQ ID NO:2.
 It is another aspect of this invention that the novel 15-Lox-2 protein acts
 in the metabolism of arachidonic acid to 15S-Hydro(pero)xyeicosatetraenoic
 acid.
 Another key aspect of this invention is isolation and purification of a
 nucleic acid encoding mouse 8S-lipoxygenase (8-Lox). A preferred
 embodiment of this nucleic acid is described in SEQ ID NO:3.
 Thus, in one aspect, the present invention provides an isolated and
 purified polynucleotide that encodes a lipoxygenase polypeptide wherein
 the lipoxygenase polypeptide includes an iron ligand comprising a serine.
 Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. In
 a preferred embodiment, a polynucleotide of the present invention is a DNA
 molecule from a vertebrate species. A preferred vertebrate is a mammal. A
 preferred mammal is a human. More preferably, a polynucleotide of the
 present invention encodes polypeptides designated 15-Lox-2 and 8-Lox. Even
 more preferred, a polynucleotide of the present invention encodes a
 polypeptide comprising the amino acid residue sequence of SEQ ID NO:2 or
 SEQ ID NO:4. Most preferably, an isolated and purified polynucleotide of
 the invention comprises the nucleotide base sequences of SEQ ID NO:1 or
 SEQ ID NO:3 or their homologues from other vertebrate species.
 Yet another aspect of the present invention contemplates an isolated and
 purified polynucleotide comprising a base sequence that is identical or
 complementary to a segment of at least 10 contiguous bases of SEQ ID NO:1
 wherein the polynucleotide hybridizes to a polynucleotide that encodes a
 lipoxygenase polypeptide wherein the lipoxygenase polypeptide includes an
 iron ligand comprising a serine. Preferably, the lipoxygenase polypeptide
 reacts with arachidonic acid. Preferably, the isolated and purified
 polynucleotide comprises a base sequence that is identical or
 complementary to a segment of at least 25 to 70 contiguous bases of SEQ ID
 NO:1. For example, a polynucleotide of the invention can comprise a
 segment of bases identical or complementary to 40 or 55 contiguous bases
 of the disclosed nucleotide sequences.
 In another embodiment, the present invention contemplates an isolated and
 purified lipoxygenase polypeptide wherein the lipoxygenase polypeptide
 includes an iron ligand comprising a serine. Preferably, the lipoxygenase
 polypeptide reacts with arachidonic acid. More preferably, a polypeptide
 of the invention is a recombinant polypeptide. Even more preferably, a
 polypeptide of the present invention is 15-Lox-2. Even more preferably, a
 polypeptide of the present invention comprises the amino acid residue
 sequence of SEQ ID NO:2.
 In an alternative embodiment, the present invention provides an expression
 vector comprising a polynucleotide that encodes a lipoxygenase polypeptide
 that includes an iron ligand comprising a serine. Preferably, the
 lipoxygenase polypeptide reacts with arachidonic acid. Also preferably, an
 expression vector of the present invention comprises a polynucleotide that
 encodes 15-Lox-2 or 8-Lox. More preferably, an expression vector of the
 present invention comprises a polynucleotide that encodes a polypeptide
 comprising the amino acid residue sequence of SEQ ID NO:2 or SEQ ID NO:4.
 More preferably, an expression vector of the present invention comprises a
 polynucleotide comprising the nucleotide base sequence of SEQ ID NO:1 or
 SEQ ID NO:3. Even more preferably, an expression vector of the invention
 comprises a polynucleotide operatively linked to an enhancer-promoter.
 More preferably still, an expression vector of the invention comprises a
 polynucleotide operatively linked to a prokaryotic promoter.
 Alternatively, an expression vector of the present invention comprises a
 polynucleotide operatively linked to an enhancer-promoter that is a
 eukaryotic promoter, and the expression vector further comprises a
 polyadenylation signal that is positioned 3' of the carboxy-terminal amino
 acid and within a transcriptional unit of the encoded polypeptide.
 In yet another embodiment, the present invention provides a recombinant
 host cell transfected with a polynucleotide that encodes a lipoxygenase
 polypeptide which includes an iron ligand comprising a serine. Preferably,
 the lipoxygenase polypeptide reacts with arachidonic acid. SEQ ID NO:1;
 SEQ ID NO: 2 SEQ ID NO:3; and SEQ ID NO: 4 set forth nucleotide and amino
 acid sequences from the exemplary vertebrates human and mouse. Also
 contemplated by the present invention are homologous or biologically
 equivalent polynucleotides and lipoxygenase polypeptides found in other
 vertebrates. Preferably, a recombinant host cell of the present invention
 is transfected with the polynucleotide that encodes 15-Lox-2 or 8-Lox.
 More preferably, a recombinant host cell of the present invention is
 transfected with the polynucleotide sequence of SEQ ID NO:1 or SEQ ID
 NO:3. Even more preferably, a host cell of the invention is a eukaryotic
 host cell. Still more preferably, a recombinant host cell of the present
 invention is a vertebrate cell. Preferably, a recombinant host cell of the
 invention is a mammalian cell.
 In another aspect, a recombinant host cell of the present invention is a
 prokaryotic host cell. Preferably, a recombinant host cell of the
 invention is a bacterial cell, preferably a strain of Escherichia coli.
 More preferably, a recombinant host cell comprises a polynucleotide under
 the transcriptional control of regulatory signals functional in the
 recombinant host cell, wherein the regulatory signals appropriately
 control expression of a lipoxygenase polypeptide that metabolizes
 arachidonic acid in a manner to enable all necessary transcriptional and
 post-transcriptional modification.
 In yet another embodiment, the present invention contemplates a process of
 preparing a lipoxygenase polypeptide comprising transfecting a cell with
 polynucleotide that encodes a lipoxygenase polypeptide which includes an
 iron ligand comprising a serine, to produce a transformed host cell; and
 maintaining the transformed host cell under biological conditions
 sufficient for expression of the polypeptide. Preferably, the lipoxygenase
 polypeptide that is produced reacts with arachidonic acid. More
 preferably, the transformed host cell is a eukaryotic cell. More
 preferably still, the eukaryotic cell is a vertebrate cell. Alternatively,
 the host cell is a prokaryotic cell. More preferably, the prokaryotic cell
 is a bacterial cell of the DH5.alpha. strain of Escherichia coli. Even
 more preferably, a polynucleotide transfected into the transformed cell
 comprises the nucleotide base sequence of SEQ ID NO:1 or SEQ ID NO:3. SEQ
 ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and SEQ ID NO:4 set forth nucleotide
 and amino acid sequences for the exemplary vertebrates human and mouse.
 Also contemplated by the present invention are homologues or biologically
 equivalent lipoxygenase polynucleotides and polypeptides found in other
 vertebrates.
 In still another embodiment, the present invention provides an antibody
 immunoreactive with a lipoxygenase polypeptide which includes an iron
 ligand comprising a serine. Preferably, the lipoxygenase polypeptide
 reacts with arachidonic acid. SEQ ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and
 SEQ ID NO:4 set forth nucleotide and amino acid sequences from the
 exemplary vertebrates human and mouse. Also contemplated by the present
 invention are antibodies immunoreactive with homologues or biologically
 equivalent lipoxygenase polynucleotides and polypeptides found in other
 vertebrates. Preferably, an antibody of the invention is a monoclonal
 antibody. More preferably, the lipoxygenase polypeptide comprises 15-Lox-2
 or 8-Lox. Even more preferably, a polypeptide comprises the amino acid
 residue sequence of SEQ ID NO:2 or SEQ ID NO:4.
 In another aspect, the present invention contemplates a process of
 producing an antibody immunoreactive with a lipoxygenase polypeptide which
 includes an iron ligand comprising a serine, the process comprising the
 steps of (a) transfecting a recombinant host cell with a polynucleotide
 that encodes a lipoxygenase polypeptide which includes an iron ligand
 comprising a serine; (b) culturing the host cell under conditions
 sufficient for expression of the polypeptide; (c) recovering the
 polypeptide; and (d) preparing the antibody to the polypeptide.
 Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. SEQ
 ID NO:1; SEQ ID NO:2; SEQ ID NO:3; and SEQ ID NO:4 set forth nucleotide
 and amino acid sequences from the exemplary vertebrates mouse and human.
 Preferably, the host cell is transfected with the polynucleotide of SEQ ID
 NO:1 or SEQ ID NO:3. Even more preferably, the present invention provides
 an antibody prepared according to the process described above. Also
 contemplated by the present invention is the use of homologues or
 biologically equivalent polynucleotides and polypeptides found in other
 vertebrates to produce antibodies.
 Alternatively, the present invention provides a process of detecting a
 lipoxygenase polypeptide that metabolizes arachidonic acid, wherein the
 process comprises immunoreacting the polypeptide with an antibody prepared
 according to the process described above to form an antibody-polypeptide
 conjugate, and detecting the conjugate.
 In yet another embodiment, the present invention contemplates a process of
 detecting a messenger RNA transcript that encodes a lipoxygenase
 polypeptide which includes an iron ligand comprising a serine, wherein the
 process comprises hybridizing the messenger RNA transcript with a
 polynucleotide sequence that encodes that polypeptide to form a duplex;
 and detecting the duplex. Alternatively, the present invention provides a
 process of detecting a DNA molecule that encodes a lipoxygenase
 polypeptide, wherein the process comprises hybridizing DNA molecules with
 a polynucleotide that encodes a lipoxygenase polypeptide which includes an
 iron ligand comprising a serine to form a duplex; and detecting the
 duplex. For both such processes, it is preferred that the detected
 lipoxygenase polypeptide is capable of reacting with arachidonic acid.
 In another aspect, the present invention contemplates a diagnostic assay
 kit for detecting the presence of a lipoxygenase polypeptide in a
 biological sample, where the kit comprises a first container containing a
 first antibody capable of immunoreacting with a lipoxygenase polypeptide
 which includes an iron ligand comprising a serine, with the first antibody
 present in an amount sufficient to perform at least one assay. Preferably,
 an assay kit of the invention further comprises a second container
 containing a second antibody that immunoreacts with the first antibody.
 More preferably, the antibodies used in an assay kit of the present
 invention are monoclonal antibodies. Even more preferably, the first
 antibody is affixed to a solid support. More preferably still, the first
 and second antibodies comprise an indicator, and, preferably, the
 indicator is a radioactive label or an enzyme.
 In an alternative aspect, the present invention provides a diagnostic assay
 kit for detecting the presence, in biological samples, of a lipoxygenase
 polypeptide, the kits comprising a first container that contains a second
 polynucleotide identical or complementary to a segment of at least 10
 contiguous nucleotide bases of a polynucleotide that encodes a
 lipoxygenase polypeptide which includes an iron ligand comprising a
 serine. Preferably, the polynucleotide encodes a lipoxygenase polypeptide
 capable of reacting with arachidonic acid. More preferably, the
 polynucleotide encodes 15-Lox-2 or 8-Lox.
 In another embodiment, the present invention contemplates a diagnostic
 assay kit for detecting the presence, in a biological sample, of an
 antibody immunoreactive with a lipoxygenase polypeptide, the kit
 comprising a first container containing a lipoxygenase polypeptide which
 includes an iron ligand comprising a serine that immunoreacts with the
 antibody, with the polypeptide present in an amount sufficient to perform
 at least one assay. Preferably, the lipoxygenase polypeptide is capable of
 reacting with arachidonic acid. More preferably, the polypeptide comprises
 15-Lox-2 or 8-Lox.
 The foregoing aspects and embodiments have broad utility given the
 biological significance of the arachidonic acid pathway, as is known in
 the art. By way of example, the foregoing aspects and embodiments are
 useful in the preparation of screening assays and assay kits that are used
 to identify compounds that affect arachidonic acid metabolism, or that are
 used to detect the presence of the proteins and nucleic acids of this
 invention in biological samples. Additionally, it is well known that
 isolated and purified polypeptides have utility as feed additives for
 livestock and further polynucleotides encoding the polypeptides are thus
 useful in producing the polypeptides.
 Following long-standing patent law convention, the terms "a" and "an" mean
 "one or more" when used in this application, including the claims.
 Some of the aspects and objects of the invention having been stated
 hereinabove, other aspects and objects will become evident as the
 description proceeds, when taken in connection with the accompanying
 drawings as best described hereinbelow.

DETAILED DESCRIPTION OF THE INVENTION
 The lipoxygenase metabolism of arachidonic acid occurs in specific blood
 cell types and epithelial tissues, and is activated in inflammation and
 tissue injury. In the course of studying lipoxygenase expression in human
 skin, a previously unrecognized enzyme was detected and characterized that
 at least partly accounts for the 15S-lipoxygenase metabolism of
 arachidonic acid in certain epithelial tissues. The cDNA was cloned from
 human hair roots, and expression of the mRNA was detected also in
 prostate, lung, and cornea; an additional sixteen human tissues, including
 peripheral blood leukocytes, were negative for the mRNA. The cDNA encodes
 a protein of 676 amino acids with a calculated molecular weight of about
 76 kD. The amino acid sequence has approximately 40% identity to the known
 human 5S-, 12S- and 15S-lipoxygenases.
 When expressed in human embryonic kidney (HEK) 293 cells, the new enzyme
 converts arachidonic acid exclusively to 15S-hydroperoxyeicosatetraenoic
 acid, while linoleic acid is less well metabolized. These features
 contrast with the previously reported 15S-lipoxygenase which oxygenates
 arachidonic acid mainly at C-15, but also partly at C-12, and for which
 linoleic acid is an excellent substrate. The different catalytic
 activities and tissue distribution suggest a distinct function for the new
 enzyme compared to the previously reported human 15S-lipoxygenase.
 It is known that human hair roots metabolize arachidonic acid
 (Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297), and
 that in addition to a relatively prominent synthesis of 12S-HETE and
 15S-HETE, formation of minor amounts of 12R-HETE is detectable. (Baer et
 al. (1993) J. Lipid Research 34:1505-1514). Therefore freshly plucked
 human hair follicles were as a source of RNA for the RT-PCR experiments
 described in Example 1. As described in Example 1, these experiments led
 to the detection of a new lipoxygenase, a 15S-lipoxygenase (referred to
 herein as "15-Lox-2") with a distinctive distribution in tissues.
 Definitions and Techniques Affecting Gene Products and Genes
 The present invention concerns DNA segments, isolatable from vertebrate
 tissue, and preferably mammalian tissue, which are free from genomic DNA
 and which are capable of conferring arachidonic acid metabolism activity
 in a recombinant host cell when incorporated into the recombinant host
 cell. As used herein, the term "mammalian tissue" refers to, among others,
 normal mammalian epithelial tissues, as exemplified by, but not limited
 to, human embryonic kidney (HEK) 293 cell lines. DNA segments capable of
 conferring arachidonic acid metabolism activity may encode complete
 lipoxygenase gene products, cleavage products and biologically actively
 functional domains thereof.
 The terms "lipoxygenase gene product", "lipoxygenase", "Lox", "15-Lox-2
 gene product", "15-Lox2", "8-Lox gene product" and "8-Lox" as used in the
 specification and in the claims refer to proteins having amino acid
 sequences which are substantially identical to the respective native
 lipoxygenase amino acid sequences and which are biologically active in
 that they are capable of reacting with arachidonic acid or are capable of
 cross-reacting with an anti-Lox antibody raised against a lipoxygenase,
 such as 15-Lox-2 or 8-Lox. Such sequences are disclosed herein. The terms
 "lipoxygenase gene product", "lipoxygenase", "Lox", "15-Lox-2 gene
 product", "15-Lox-2", "8-Lox gene product" and "8-Lox" also include
 analogs of lipoxygenase molecules which exhibit at least some biological
 activity in common with native lipoxygenase, 15-Lox-2, or 8-Lox.
 Furthermore, those skilled in the art of mutagenesis will appreciate that
 other analogs, as yet undisclosed or undiscovered, may be used to
 construct lipoxygenase analogs. There is no need for a "lipoxygenase" or
 "Lox", or a "15-Lox-2" or "8-Lox" to comprise all, or substantially all,
 of the amino acid sequence of the native lipoxygenase genes. Shorter or
 longer sequences are anticipated to be of use in the invention.
 The terms "lipoxygenase gene", "15-lox-2 gene" and "8-Lox gene" refer to
 any DNA sequence that is substantially identical to a DNA sequence
 encoding a lipoxygenase, 15-Lox-2 or 8-Lox as defined above. The terms
 also refer to RNA, or antisense sequences, compatible with such DNA
 sequences. A "lipoxygenase gene", a "15-lox-2 gene" or a "8-Lox gene" may
 also comprise any combination of associated control sequences.
 The term "substantially identical", when used to define either a
 lipoxygenase, a 15-lox-2 or a 8-Lox amino acid sequence, or a
 lipoxygenase, a 15-lox-2 or a 8-Lox nucleic acid sequence, means that a
 particular sequence, for example, a mutant sequence, varies from the
 sequence of a natural lipoxygenase, 15-lox-2 or 8-Lox by one or more
 deletions, substitutions, or additions, the net effect of which is to
 retain at least some of biological activity of the lipoxygenase, the
 15-lox-2 or the 8-Lox protein. Alternatively, DNA analog sequences are
 "substantially identical" to specific DNA sequences disclosed herein if:
 (a) the DNA analog sequence is derived from coding regions of the natural
 lipoxygenase, 15-lox-2 or 8-Lox gene; or (b) the DNA analog sequence is
 capable of hybridization of DNA sequences of (a) under moderately
 stringent conditions and which encode biologically active lipoxygenase,
 15-lox-2 or 8-Lox gene; or (c) the DNA sequences are degenerative as a
 result of the genetic code to the DNA analog sequences defined in (a)
 and/or (b). Substantially identical analog proteins will be greater than
 about 60% identical to the corresponding sequence of the native protein.
 Sequences having lesser degrees of similarity but comparable biological
 activity are considered to be equivalents. In determining nucleic acid
 sequences, all subject nucleic acid sequences capable of encoding
 substantially similar amino acid sequences are considered to be
 substantially similar to a reference nucleic acid sequence, regardless of
 differences in codon sequences.
 Percent Similarity
 Percent similarity may be determined, for example, by comparing sequence
 information using the GAP computer program, available from the University
 of Wisconsin Geneticist Computer Group. The GAP program utilizes the
 alignment method of Needleman et al. 1970, as revised by Smith et al.
 1981. Briefly, the GAP program defines similarity as the number of aligned
 symbols (i.e. nucleotides or amino acids) which are similar, divided by
 the total number of symbols in the shorter of the two sequences. The
 preferred default parameters for the GAP program include: (1) a unitary
 comparison matrix (containing a value of 1 for identities and 0 for
 non-identities) of nucleotides and the weighted comparison matrix of
 Gribskov et al., 1986, as described by Schwartz et al., 1979; (2) a
 penalty of 3.0 for each gap and an additional 0.01 penalty for each symbol
 and each gap; and (3) no penalty for end gaps.
 The term "homology" describes a mathematically based comparison of sequence
 similarities which is used to identify genes or proteins with similar
 functions or motifs. Accordingly, the term "homology" is synonymous with
 the term "similarity" and "percent similarity" as defined above. Thus, the
 phrases "substantial homology" or "substantial similarity" have similar
 meanings.
 Nucleic Acid Sequences
 In certain embodiments, the invention concerns the use of lipoxygenase
 genes and gene products, such as the 15-lox-2 and 8-Lox gene products,
 that include within their respective sequences a sequence which is
 essentially that of a lipoxygenase, 15-lox-2 or 8-Lox gene, or the
 corresponding proteins. The term "a sequence essentially as that of
 lipoxygenase, 15-lox-2 or 8-Lox gene or gene product", means that the
 sequence substantially corresponds to a portion of a lipoxygenase,
 15-lox-2 or 8-Lox gene or gene product and has relatively few bases or
 amino acids (whether DNA or protein) which are not identical to those of a
 lipoxygenase, 15-lox-2 or 8-Lox gene or gene product, (or a biologically
 functional equivalent of, when referring to proteins). The term
 "biologically functional equivalent" is well understood in the art and is
 further defined in detail herein. Accordingly, sequences which have
 between about 70% and about 80%; or more preferably, between about 81% and
 about 90%; or even more preferably, between about 91% and about 99%; of
 amino acids which are identical or functionally equivalent to the amino
 acids of a lipoxygenase, 15-lox-2 or 8-Lox gene or gene product, will be
 sequences which are "essentially the same".
 Lipoxygenase, 15-lox-2 and 8-Lox genes which have functionally equivalent
 codons are also covered by the invention. The term "functionally
 equivalent codon" is used herein to refer to codons that encode the same
 amino acid, such as the six codons for arginine or serine, and also to
 refer to codons that encode biologically equivalent amino acids (see Table
 1).
 TABLE 1
 Functionally Equivalent Codons.
 Amino Acids Condons
 Alanine Ala A GCA GCC GCG GCU
 Cysteine Cys C UGC UGU
 Aspartic Acid Asp D GAC GAU
 Glumatic acid Glu E GAA GAG
 Phenylalanine Phe F UUC UUU
 Glycine Gly G GGA GGC GGG GGU
 Histidine His H CAC CAU
 Isoleucine Ile I AUA AUC AUU
 Lysine Lys K AAA AAG
 Leucine Leu L UUA UUG CUA CUC CUG CUU
 Methionine Met M AUG
 Asparagine Asn N AAC AAU
 Proline Pro P CCA CCC CCG CCU
 Glutamine Gln Q CAA CAG
 Arginine Arg R AGA AGG CGA CGC CGG CGU
 Serine Ser S ACG AGU UCA UCC UCG UCU
 Threonine Thr T ACA ACC ACG ACU
 Valine Val V GUA GUC GUG GUU
 Tryptophan Trp W UGG
 Tyrosine Tyr Y UAC UAU
 It will also be understood that amino acid and nucleic acid sequences may
 include additional residues, such as additional N- or C-terminal amino
 acids or 5' or 3' sequences, and yet still be essentially as set forth in
 one of the sequences disclosed herein, so long as the sequence meets the
 criteria set forth above, including the maintenance of biological protein
 activity where protein expression is concerned. The addition of terminal
 sequences particularly applies to nucleic acid sequences which may, for
 example, include various non-coding sequences flanking either of the 5' or
 3' portions of the coding region or may include various internal
 sequences, i.e., introns, which are known to occur within genes.
 The present invention also encompasses the use of DNA segments which are
 complementary, or essentially complementary, to the sequences set forth in
 the specification. Nucleic acid sequences which are "complementary" are
 those which are base-pairing according to the standard Watson-Crick
 complementarity rules. As used herein, the term "complementary sequences"
 means nucleic acid sequences which are substantially complementary, as may
 be assessed by the same nucleotide comparison set forth above, or as
 defined as being capable of hybridizing to the nucleic acid segment in
 question under relatively stringent conditions such as those described
 herein.
 Nucleic acid hybridization will be affected by such conditions as salt
 concentration, temperature, or organic solvents, in addition to the base
 composition, length of the complementary strands, and the number of
 nucleotide base mismatches between the hybridizing nucleic acids, as will
 be readily appreciated by those skilled in the art. Stringent temperature
 conditions will generally include temperatures in excess of 30.degree. C.,
 typically in excess of 37.degree. C., and preferably in excess of
 45.degree. C. Stringent salt conditions will ordinarily be less than 1,000
 mM, typically less than 500 mM, and preferably less than 200 mM. However,
 the combination of parameters is much more important than the measure of
 any single parameter. (See, e.g., Wetmur & Davidson, 1968).
 Probe sequences may also hybridize specifically to duplex DNA under certain
 conditions to form triplex or other higher order DNA complexes. The
 preparation of such probes and suitable hybridization conditions are well
 known in the art.
 As used herein, the term "DNA segment" refers to a DNA molecule which has
 been isolated free of total genomic DNA of a particular species.
 Furthermore, a DNA segment encoding a lipoxygenase, 15-lox-2 or 8-Lox gene
 product refers to a DNA segment which contains lipoxygenase, 15-lox-2 or
 8-Lox coding sequences, yet is isolated away from, or purified free from,
 total genomic DNA of Homo sapiens. Included within the term "DNA segment"
 are DNA segments and smaller fragments of such segments, and also
 recombinant vectors, including, for example, plasmids, cosmids, phages,
 viruses, and the like.
 Similarly, a DNA segment comprising an isolated or purified lipoxygenase,
 15-lox-2 or 8-Lox gene refers to a DNA segment including lipoxygenase,
 15-lox-2 or 8-Lox coding sequences isolated substantially away from other
 naturally occurring genes or protein encoding sequences. In this respect,
 the term "gene" is used for simplicity to refer to a functional protein,
 polypeptide or peptide encoding unit. As will be understood by those in
 the art, this functional term includes both genomic sequences and cDNA
 sequences. "Isolated substantially away from other coding sequences" means
 that the gene of interest, in this case, the lipoxygenase, 15-lox-2 or
 8-Lox gene, forms the significant part of the coding region of the DNA
 segment, and that the DNA segment does not contain large portions of
 naturally-occurring coding DNA, such as large chromosomal fragments or
 other functional genes or cDNA coding regions. Of course, this refers to
 the DNA segment as originally isolated, and does not exclude genes or
 coding regions later added to the segment by the hand of man.
 In particular embodiments, the invention concerns isolated DNA segments and
 recombinant vectors incorporating DNA sequences which encode a 15-Lox-2
 protein that includes within its amino acid sequence the amino acid
 sequence of SEQ ID NO:2. In other particular embodiments, the invention
 concerns isolated DNA segments and recombinant vectors incorporating DNA
 sequences which encode a protein that includes within its amino acid
 sequence the amino acid sequence of the 15-Lox-2 protein corresponding to
 human epithelial tissue.
 In particular embodiments, the invention concerns isolated DNA segments and
 recombinant vectors incorporating DNA sequences which encode a 8-Lox
 protein that includes within its amino acid sequence the amino acid
 sequence of SEQ ID NO:4. In other particular embodiments, the invention
 concerns isolated DNA segments and recombinant vectors incorporating DNA
 sequences which encode a protein that includes within its amino acid
 sequence the amino acid sequence of the 8-Lox protein corresponding to
 mouse epithelial tissue.
 It will also be understood that this invention is not limited to the
 particular nucleic acid and amino acid sequences of SEQ ID NOS:1, 2, 3 and
 4. Recombinant vectors and isolated DNA segments may therefore variously
 include the 15-Lox-2 and 8-Lox encoding regions themselves, include coding
 regions bearing selected alterations or modifications in the basic coding
 region, or include encoded larger polypeptides which nevertheless include
 15-Lox-2 or 8-Lox encoding regions or may encode biologically functional
 equivalent proteins or peptides which have variant amino acid sequences.
 In certain embodiments, the invention concerns isolated DNA segments and
 recombinant vectors which encode a protein or peptide that includes within
 its amino acid sequence an amino acid sequence essentially as set forth in
 SEQ ID NO:2 or SEQ ID NO:4. Naturally, where the DNA segment or vector
 encodes a full length 15-Lox-2 or 8-Lox gene product, the most preferred
 sequences are those which are essentially as set forth in SEQ ID NO:1 and
 SEQ ID NO:3 and which encode a protein that exhibits arachidonic acid
 reactivity in HEK 293 cells, as may be determined by HPLC analysis, as
 disclosed herein.
 The term "a sequence essentially as set forth in SEQ ID NO:2" means that
 the sequence substantially corresponds to a portion of SEQ ID NO:2 and has
 relatively few amino acids which are not identical to, or a biologically
 functional equivalent of, the amino acids of SEQ ID NO:2. The term
 "biologically functional equivalent" is well understood in the art and is
 further defined in detail herein. Accordingly, sequences, which have
 between about 70% and about 80%; or more preferably, between about 81% and
 about 90%; or even more preferably, between about 91% and about 99%; of
 amino acids which are identical or functionally equivalent to the amino
 acids of SEQ ID NO:2, will be sequences which are "essentially as set
 forth in SEQ ID NO:2". The term "a sequence essentially set forth in SEQ
 ID NO:4" has a similar meaning.
 In particular embodiments, the invention concerns gene therapy methods that
 use isolated DNA segments and recombinant vectors incorporating DNA
 sequences which encode a protein that includes within its amino acid
 sequence an amino acid sequence in accordance with SEQ ID NO:2 or in
 accordance with SEQ ID NO:4, SEQ ID NO:2 and SEQ ID NO:4 derived from
 epithelial tissue from Homo sapiens. In other particular embodiments, the
 invention concerns isolated DNA sequences and recombinant DNA vectors
 incorporating DNA sequences which encode a protein that includes within
 its amino acid sequence the amino acid sequence of the 15-Lox-2 protein
 from human epithelial tissue, or which encode a protein that includes
 within its amino acid sequence the amino acid sequence of the 8-Lox
 protein from mouse epithelial tissue.
 In certain other embodiments, the invention concerns isolated DNA segments
 and recombinant vectors that include within their sequence a nucleic acid
 sequence essentially as set forth in SEQ ID NO:1, or a nucleic acid
 sequence essentially as set forth in SEQ ID NO:3. The term "essentially as
 set forth in SEQ ID NO:1" is used in the same sense as described above and
 means that the nucleic acid sequence substantially corresponds to a
 portion of SEQ ID NO:1, respectively, and has relatively few codons which
 are not identical, or functionally equivalent, to the codons of SEQ ID
 NO:1, respectively. Again, DNA segments which encode gene products
 exhibiting arachidonic acid metabolism activity of the 15-Lox-2 and 8-Lox
 gene products will be most preferred. The term "functionally equivalent
 codon" is used herein to refer to codons that encode the same amino acid,
 such as the six codons for arginine or serine, and also to refer to codons
 that encode biologically equivalent amino acids (see Table 1). The term
 "essentially as set forth in SEQ ID NO:3" has a similar meaning.
 The nucleic acid segments of the present invention, regardless of the
 length of the coding sequence itself, may be combined with other DNA
 sequences, such as promoters, enhancers, polyadenylation signals,
 additional restriction enzyme sites, multiple cloning sites, other coding
 segments, and the like, such that their overall length may vary
 considerably. It is therefore contemplated that a nucleic acid fragment of
 almost any length may be employed, with the total length preferably being
 limited by the ease of preparation and use in the intended recombinant DNA
 protocol. For example, nucleic acid fragments may be prepared which
 include a short stretch complementary to SEQ ID NO:1 or SEQ ID NO:3, such
 as about 10 nucleotides, and which are up to 10,000 or 5,000 base pairs in
 length, with segments of 3,000 being preferred in certain cases. DNA
 segments with total lengths of about 1,000, 500, 200, 100 and about 50
 base pairs in length are also contemplated to be useful.
 The DNA segments of the present invention encompass biologically functional
 equivalent 15-Lox-2 and 8-Lox proteins and peptides. Such sequences may
 rise as a consequence of codon redundancy and functional equivalency which
 are known to occur naturally within nucleic acid sequences and the
 proteins thus encoded. Alternatively, functionally equivalent proteins or
 peptides may be created via the application of recombinant DNA technology,
 in which changes in the protein structure may be engineered, based on
 considerations of the properties of the amino acids being exchanged.
 Changes designed by man may be introduced through the application of
 site-directed mutagenesis techniques, e.g., to introduce improvements to
 the antigenicity of the protein or to test 15-Lox-2 and 8-Lox mutants in
 order to examine arachidonic acid reactivity at the molecular level.
 If desired, one may also prepare fusion proteins and peptides, e.g., where
 the 15-Lox-2 or 8-Lox coding regions are aligned within the same
 expression unit with other proteins or peptides having desired functions,
 such as for purification or immunodetection purposes (e.g., proteins which
 may be purified by affinity chromatography and enzyme label coding
 regions, respectively).
 Recombinant vectors form important further aspects of the present
 invention. Particularly useful vectors are contemplated to be those
 vectors in which the coding portion of the DNA segment is positioned under
 the control of a promoter. The promoter may be in the form of the promoter
 which is naturally associated with the 15-Lox-2 or 8-Lox gene(s), e.g., in
 epithelial cells, as may be obtained by isolating the 5' non-coding
 sequences located upstream of the coding segment or exon, for example,
 using recombinant cloning and/or PCR technology, in connection with the
 compositions disclosed herein.
 In other embodiments, it is contemplated that certain advantages will be
 gained by positioning the coding DNA segment under the control of a
 recombinant, or heterologous, promoter. As used herein, a recombinant or
 heterologous promoter is intended to refer to a promoter that is not
 normally associated with a 15-Lox-2 or 8-Lox gene in its natural
 environment. Such promoters may include promoters isolated from bacterial,
 viral, eukaryotic, or mammalian cells. Naturally, it will be important to
 employ a promoter that effectively directs the expression of the DNA
 segment in the cell type chosen for expression. The use of promoter and
 cell type combinations for protein expression is generally known to those
 of skill in the art of molecular biology, for example, see Sambrook et
 al., 1989, specifically incorporated herein by reference. The promoters
 employed may be constitutive, or inducible, and can be used under the
 appropriate conditions to direct high level expression of the introduced
 DNA segment, such as is advantageous in the large-scale production of
 recombinant proteins or peptides. Appropriate promoter systems
 contemplated for use in high-level expression include, but are not limited
 to, the vaccina virus promoter, which is more fully described below.
 As mentioned above, in connection with expression embodiments to prepare
 recombinant 15-Lox-2 and 8-Lox proteins and peptides, it is contemplated
 that longer DNA segments will most often be used, with DNA segments
 encoding the entire 15-Lox-2 or 8-Lox protein, functional domains or
 cleavage products thereof, being most preferred. However, it will be
 appreciated that the use of shorter DNA segments to direct the expression
 of 15-Lox-2 and 8-Lox peptides or epitopic core regions, such as may be
 used to generate anti-15-Lox-2 or anti-8-Lox antibodies, also falls within
 the scope of the invention.
 DNA segments which encode peptide antigens from about 15 to about 50 amino
 acids in length, or more preferably, from about 15 to about 30 amino acids
 in length are contemplated to be particularly useful. DNA segments
 encoding peptides will generally have a minimum coding length in the order
 of about 45 to about 150, or to about 90 nucleotides. DNA segments
 encoding full length proteins may have a minimum coding length on the
 order of about 5,600 nucleotides for a protein in accordance with SEQ ID
 NO:2 or a minimum coding length on the order of about 10,300 nucleotides
 for a protein in accordance with SEQ ID NO:4.
 Naturally, the present invention also encompasses DNA segments which are
 complementary, or essentially complementary, to the sequence set forth in
 SEQ ID NO:1 or the sequence set forth in SEQ ID NO:3. The terms
 "complementary" and "essentially complementary" are defined above.
 Excepting intronic or flanking regions, and allowing for the degeneracy of
 the genetic code, sequences which have between about 70% and about 80%; or
 more preferably, between about 81% and about 90%; or even more preferably,
 between about 91% and about 99%; of nucleotides which are identical or
 functionally equivalent (i.e. encoding the same amino acid) of nucleotides
 of SEQ ID NO:1 or to the nucleotides of SEQ ID NO:3, will be respectively
 sequences which are "essentially as set forth in SEQ ID NO:1" and will be
 sequences which are "essentially as set forth in SEQ ID NO:3". Sequences
 which are essentially the same as those set forth in SEQ ID NO:1 or as
 those set forth in SEQ ID NO:3 may also be functionally defined as
 sequences which are capable of hybridizing to a nucleic acid segment
 containing the complement of SEQ ID NO:1 or to a nucleic acid segment
 containing the complement of SEQ ID NO:3 under relatively stringent
 conditions. Suitable relatively stringent hybridization conditions are
 described herein and will be well known to those of skill in the art.
 Biologically Functional Equivalents
 As mentioned above, modification and changes may be made in the structure
 of the lipoxygenase proteins and peptides, including 15-Lox-2 and 8-Lox,
 described herein and still obtain a molecule having like or otherwise
 desirable characteristics. For example, certain amino acids may be
 substituted for other amino acids in a protein structure without
 appreciable loss of interactive binding capacity with structures such as,
 for example, C-15 carbon or C-8 carbon of arachidonic acid. Since it is
 the interactive capacity and nature of a protein that defines that
 protein's biological functional activity, certain amino acid sequence
 substitutions can be made in a protein sequence (or, of course, its
 underlying DNA coding sequence) and nevertheless obtain a protein with
 like or even countervailing properties (e.g., antagonistic v. agonistic).
 It is thus contemplated by the inventors that various changes may be made
 in the sequence of the lipoxygenase proteins and peptides, including
 15-Lox-2 and 8-Lox, (or underlying DNA) without appreciable loss of their
 biological utility or activity.
 It is also well understood by the skilled artisan that, inherent in the
 definition of a biologically functional equivalent protein or peptide, is
 the concept that there is a limit to the number of changes that may be
 made within a defined portion of the molecule and still result in a
 molecule with an acceptable level of equivalent biological activity.
 Biologically functional equivalent peptides are thus defined herein as
 those peptides in which certain, not most or all, of the amino acids may
 be substituted. Of course, a plurality of distinct proteins/peptides with
 different substitutions may easily be made and used in accordance with the
 invention.
 It is also well understood that where certain residues are shown to be
 particularly important to the biological or structural properties of a
 protein or peptide, e.g., residues in active sites, such residues may not
 generally be exchanged. This is the case in the present invention, where
 it any changes, for example, in an iron ligand moiety of 15-Lox-2 that
 render the peptide incapable of metabolism of arachidonic acid to
 15S-Hydro(pero)xyeicosatetraenoic acid would result in a loss of utility
 of the resulting peptide for the present invention.
 Amino acid substitutions, such as those which might be employed in
 modifying the lipoxygenase proteins and peptides, including 15-Lox-2 and
 8-Lox, described herein, are generally based on the relative similarity of
 the amino acid side-chain substituents, for example, their hydrophobicity,
 hydrophilicity, charge, size, and the like. An analysis of the size, shape
 and type of the amino acid side-chain substituents reveals that arginine,
 lysine and histidine are all positively charged residues; that alanine,
 glycine and serine are all a similar size; and that phenylalanine,
 tryptophan and tyrosine all have a generally similar shape. Therefore,
 based upon these considerations, arginine, lysine and histidine; alanine,
 glycine and serine; and phenylalanine, tryptophan and tyrosine; are
 defined herein as biologically functional equivalents.
 In making such changes, the hydropathic index of amino acids may be
 considered. Each amino acid has been assigned a hydropathic index on the
 basis of their hydrophobicity and charge characteristics, these are:
 isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);
 cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine
 (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine
 (-1.3); praline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine
 (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
 (-4.5).
 The importance of the hydropathic amino acid index in conferring
 interactive biological function on a protein is generally understood in
 the art (Kyte & Doolittle, 1982, incorporated herein by reference). It is
 known that certain amino acids may be substituted for other amino acids
 having a similar hydropathic index or score and still retain a similar
 biological activity. In making changes based upon the hydropathic index,
 the substitution of amino acids whose hydropathic indices are within .+-.2
 is preferred, those which are within .+-.1 are particularly preferred, and
 those within .+-.0.5 are even more particularly preferred.
 It is also understood in the art that the substitution of like amino acids
 can be made effectively on the basis of hydrophilicity. U.S. Pat. No.
 4,554,101, incorporated herein by reference, states that the greatest
 local average hydrophilicity of a protein, as governed by the
 hydrophilicity of its adjacent amino acids, correlates with its
 immunogenicity and antigenicity, i.e. with a biological property of the
 protein. It is understood that an amino acid can be substituted for
 another having a similar hydrophilicity value and still obtain a
 biologically equivalent protein.
 As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicity values
 have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0);
 aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine
 (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline
 (-0.5.+-.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine
 (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
 phenylalanine (-2.5); tryptophan (-3.4).
 In making changes based upon similar hydrophilicity values, the
 substitution of amino acids whose hydrophilicity values are within .+-.2
 is preferred, those which are within .+-.1 are particularly preferred, and
 those within +0.5 are even more particularly preferred.
 While discussion has focused on functionally equivalent polypeptides
 arising from amino acid changes, it will be appreciated that these changes
 may be effected by alteration of the encoding DNA; taking into
 consideration also that the genetic code is degenerate and that two or
 more codons may code for the same amino acid.
 Sequence Modification Techniques
 Modifications to the lipoxygenase proteins and peptides, including 15-Lox-2
 and 8-Lox, described herein may be carried out using techniques such as
 site directed mutagenesis. Site-specific mutagenesis is a technique useful
 in the preparation of individual peptides, or biologically functional
 equivalent proteins or peptides, through specific mutagenesis of the
 underlying DNA. The technique further provides a ready ability to prepare
 and test sequence variants, for example, incorporating one or more of the
 foregoing considerations, by introducing one or more nucleotide sequence
 changes into the DNA. Site-specific mutagenesis allows the production of
 mutants through the use of specific oligonucleotide sequences which encode
 the DNA sequence of the desired mutation, as well as a sufficient number
 of adjacent nucleotides, to provide a primer sequence of sufficient size
 and sequence complexity to form a stable duplex on both sides of the
 deletion junction being traversed. Typically, a primer of about 17 to 25
 nucleotides in length is preferred, with about 5 to 10 residues on both
 sides of the junction of the sequence being altered.
 In general, the technique of site-specific mutagenesis is well known in the
 art as exemplified by publications (e.g., Adelman et al., 1983). As will
 be appreciated, the technique typically employs a phage vector which
 exists in both a single stranded and double stranded form. Typical vectors
 useful in site-directed mutagenesis include vectors such as the M13 phage
 (Messing et al., 1981). These phage are readily commercially available and
 their use is generally well known to those skilled in the art. Double
 stranded plasmids are also routinely employed in site directed mutagenesis
 which eliminates the step of transferring the gene of interest from a
 plasmid to a phage.
 In general, site-directed mutagenesis in accordance herewith is performed
 by first obtaining a single-stranded vector or melting apart the two
 strands of a double stranded vector which includes within its sequence a
 DNA sequence which encodes, for example, the 15-Lox-2 and the 8-Lox gene.
 An oligonucleotide primer bearing the desired mutated sequence is
 prepared, generally synthetically, for example by the method of Crea et
 al. (1978). This primer is then annealed with the single-stranded vector,
 and subjected to DNA polymerizing enzymes such as E. coli polymerase I
 Klenow fragment, in order to complete the synthesis of the
 mutation-bearing strand. Thus, a heteroduplex is formed wherein one strand
 encodes the original non-mutated sequence and the second strand bears the
 desired mutation. This heteroduplex vector is then used to transform
 appropriate cells, such as E. coli cells, and clones are selected which
 include recombinant vectors bearing the mutated sequence arrangement.
 The preparation of sequence variants of the selected gene using
 site-directed mutagenesis is provided as a means of producing potentially
 useful 15-Lox-2, 8-Lox or other arachidonic acid metabolizing species and
 is not meant to be limiting as there are other ways in which sequence
 variants of these peptides may be obtained. For example, recombinant
 vectors encoding the desired genes may be treated with mutagenic agents to
 obtain sequence variants (see, e.g., a method described by Eichenlaub,
 1979) for the mutagenesis of plasmid DNA using hydroxylamine.
 Other Structural Equivalents
 In addition to the lipoxygenase peptidyl compounds described herein, the
 inventors also contemplate that other sterically similar compounds may be
 formulated to mimic the key portions of the peptide structure. Such
 compounds may be used in the same manner as the peptides of the invention
 and hence are also functional equivalents. The generation of a structural
 functional equivalent may be achieved by the techniques of modeling and
 chemical design known to those of skill in the art. It will be understood
 that all such sterically similar constructs fall within the scope of the
 present invention.
 Introduction of Gene Products
 Where the gene itself is employed to introduce the gene products, a
 convenient method of introduction will be through the use of a recombinant
 vector which incorporates the desired gene, together with its associated
 control sequences. The preparation of recombinant vectors is well known to
 those of skill in the art and described in many references, such as, for
 example, Sambrook et al. (1989), specifically incorporated herein by
 reference.
 In vectors, it is understood that the DNA coding sequences to be expressed,
 in this case those encoding the lipoxygenase gene products, are positioned
 adjacent to and under the control of a promoter. It is understood in the
 art that to bring a coding sequence under the control of such a promoter,
 one generally positions the 5' end of the transcription initiation site of
 the transcriptional reading frame of the gene product to be expressed
 between about 1 and about 50 nucleotides "downstream" of (i.e., 3' of) the
 chosen promoter. One may also desire to incorporate into the
 transcriptional unit of the vector an appropriate polyadenylation site
 (e.g., 5'-AATAAA-3'), if one was not contained within the original
 inserted DNA. Typically, these poly A addition sites are placed about 30
 to 2000 nucleotides "downstream" of the coding sequence at a position
 prior to transcription termination.
 While use of the control sequences of the specific gene (i.e., the 15-Lox-2
 promoter for 15-Lox-2) will be preferred, there is no reason why other
 control sequences could not be employed, so long as they are compatible
 with the genotype of the cell being treated. Thus, one may mention other
 useful promoters by way of example, including, e.g., an SV40 early
 promoter, a long terminal repeat promoter from retrovirus, an actin
 promoter, a heat shock promoter, a metallothionein promoter, and the like.
 As is known in the art, a promoter is a region of a DNA molecule typically
 within about 100 nucleotide pairs in front of (upstream of) the point at
 which transcription begins (i.e., a transcription start site). That region
 typically contains several types of DNA sequence elements that are located
 in similar relative positions in different genes. As used herein, the term
 "promoter" includes what is referred to in the art as an upstream promoter
 region, a promoter region or a promoter of a generalized eukaryotic RNA
 Polymerase II transcription unit.
 Another type of discrete transcription regulatory sequence element is an
 enhancer. An enhancer provides specificity of time, location and
 expression level for a particular encoding region (e.g., gene). A major
 function of an enhancer is to increase the level of transcription of a
 coding sequence in a cell that contains one or more transcription factors
 that bind to that enhancer. Unlike a promoter, an enhancer can function
 when located at variable distances from transcription start sites so long
 as a promoter is present.
 As used herein, the phrase "enhancer-promoter" means a composite unit that
 contains both enhancer and promoter elements. An enhancer-promoter is
 operatively linked to a coding sequence that encodes at least one gene
 product. As used herein, the phrase "operatively linked" means that an
 enhancer-promoter is connected to a coding sequence in such a way that the
 transcription of that coding sequence is controlled and regulated by that
 enhancer-promoter. Means for operatively linking an enhancer-promoter to a
 coding sequence are well known in the art. As is also well known in the
 art, the precise orientation and location relative to a coding sequence
 whose transcription is controlled, is dependent inter alia upon the
 specific nature of the enhancer-promoter. Thus, a TATA box minimal
 promoter is typically located from about 25 to about 30 base pairs
 upstream of a transcription initiation site and an upstream promoter
 element is typically located from about 100 to about 200 base pairs
 upstream of a transcription initiation site. In contrast, an enhancer can
 be located downstream from the initiation site and can be at a
 considerable distance from that site.
 An enhancer-promoter used in a vector construct of the present invention
 can be any enhancer-promoter that drives expression in a cell to be
 transfected. By employing an enhancer-promoter with well-known properties,
 the level and pattern of gene product expression can be optimized.
 For introduction of, for example, the 15-Lox-2 or 8-Lox genes, it is
 proposed that one will desire to preferably employ a vector construct that
 will deliver the desired gene to the affected cells. This will, of course,
 generally require that the construct be delivered to the targeted cells,
 for example, epthelial cells. It is proposed that this may be achieved
 most preferably by introduction of the desired gene through the use of a
 viral vector to carry either the 15-Lox-2 sequence or the 8-Lox sequence
 to efficiently infect the cells. These vectors will preferably be an
 adenoviral, a retroviral, a vaccinia viral vector or adeno-associated
 virus. These vectors are preferred because they have been successfully
 used to deliver desired sequences to cells and tend to have a high
 infection efficiency.
 Commonly used viral promoters for expression vectors are derived from
 polyoma, cytomegalovirus, Adenovirus 2, and Simian Virus 40 (SV40). The
 early and late promoters of SV40 virus are particularly useful because
 both are obtained easily from the virus as a fragment which also contains
 the SV40 viral origin of replication. Smaller or larger SV40 fragments may
 also be used, provided there is included the approximately 250 bp sequence
 extending from the Hind III site toward the Bg1 I site located in the
 viral origin of replication. Further, it is also possible, and often
 desirable, to utilize promoter or control sequences normally associated
 with the desired gene sequence, provided such control sequences are
 compatible with the host cell systems.
 The origin of replication may be provided either by construction of the
 vector to include an exogenous origin, such as may be derived from SV40 or
 other viral (e.g., Polyoma, Adeno, VSV, BPV) source, or may be provided by
 the host cell chromosomal replication mechanism. If the vector is
 integrated into the host cell chromosome, the latter is often sufficient.
 Where the 15-Lox-2 or 8-Lox genes themselves are employed it will be most
 convenient to simply use the wild type 15-Lox-2 gene or 8-Lox gene
 directly. However, it is contemplated that certain regions of either the
 15-Lox-2 gene or the 8-Lox gene may be employed exclusively without
 employing the entire wild type 15-Lox-2 or 8-Lox gene. It is proposed that
 it will ultimately be preferable to employ the smallest region needed to
 regulate the metabolism of arachidonic acid to
 15S-hydro(pero)xyeicosatetraenoic acid or to
 8S-hydro(pero)xyeicosatetraenoic acid so that one is not introducing
 unnecessary DNA into cells which receive either a 15-Lox-2 gene construct
 or an 8-Lox gene construct. Techniques well known to those of skill in the
 art, such as the use of restriction enzymes, will allow for the generation
 of small regions of the 15-Lox-2 or 8-Lox genes. The ability of these
 regions to regulate the metabolism of arachidonic acid to
 15S-hydro(pero)xyeicosatetraenoic acid or to
 8S-hydro(pero)xyeicosatetraenoic acid can easily be determined by the
 assays reported in the Examples. In general, techniques for assessing
 metabolism of arachidonic acid to 15S-Hydro(pero)xyeicosatetraenoic acid
 or to 8S-hydro(pero)xyeicosatetraenoic acid are well known in the art.
 Generation of Antibodies
 In still another embodiment, the present invention provides an antibody
 immunoreactive with a polypeptide of the present invention. Preferably, an
 antibody of the invention is a monoclonal antibody. Means for preparing
 and characterizing antibodies are well known in the art (See, e.g.,
 Antibodies A Laboratory Manual, E. Howell and D. Lane, Cold Spring Harbor
 Laboratory, 1988).
 Briefly, a polyclonal antibody is prepared by immunizing an animal with an
 immunogen comprising a polypeptide or polynucleotide of the present
 invention, and collecting antisera from that immunized animal. A wide
 range of animal species can be used for the production of antisera.
 Typically an animal used for production of anti-antisera is a rabbit, a
 mouse, a rat, a hamster or a guinea pig. Because of the relatively large
 blood volume of rabbits, a rabbit is a preferred choice for production of
 polyclonal antibodies.
 As is well known in the art, a given polypeptide or polynucleotide may vary
 in its immunogenicity. It is often necessary therefore to couple the
 immunogen (e.g., a polypeptide or polynucleotide) of the present
 invention) with a carrier. Exemplary and preferred carriers are keyhole
 limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins
 such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be
 used as carriers.
 Means for conjugating a polypeptide or a polynucleotide to a carrier
 protein are well known in the art and include glutaraldehyde,
 m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and
 bis-biazotized benzidine.
 As is also well known in the art, immunogencity to a particular immunogen
 can be enhanced by the use of non-specific stimulators of the immune
 response known as adjuvants. Exemplary and preferred adjuvants include
 complete Freund's adjuvant, incomplete Freund's adjuvants and aluminum
 hydroxide adjuvant.
 The amount of immunogen used of the production of polyclonal antibodies
 varies inter alia, upon the nature of the immunogen as well as the animal
 used for immunization. A variety of routes can be used to administer the
 immunogen (subcutaneous, intramuscular, intradermal, intravenous and
 intraperitoneal. The production of polyclonal antibodies is monitored by
 sampling blood of the immunized animal at various points following
 immunization. When a desired level of immunogenicity is obtained, the
 immunized animal can be bled and the serum isolated and stored.
 In another aspect, the present invention contemplates a process of
 producing an antibody immunoreactive with a lipoxygenase polypeptide, such
 as 15-Lox-2 or 8-Lox, the process comprising the steps of (a) transfecting
 recombinant host cells with a polynucleotide that encodes that
 polypeptide; (b) culturing the host cells under conditions sufficient for
 expression of the polypeptide; (c) recovering the polypeptide; and (d)
 preparing antibodies to the polypeptide. Preferably, the lipoxygenase
 polypeptide is capable of metabolizing arachidonic acid. Even more
 preferably, the present invention provides antibodies prepared according
 to the process described above.
 A monoclonal antibody of the present invention can be readily prepared
 through use of well-known techniques such as those exemplified in U.S.
 Pat. No. 4,196,265, herein incorporated by reference. Typically, a
 technique involves first immunizing a suitable animal with a selected
 antigen (e.g., a polypeptide or polynucleotide of the present invention)
 in a manner sufficient to provide an immune response. Rodents such as mice
 and rats are preferred animals. Spleen cells from the immunized animal are
 then fused with cells of an immortal myeloma cell. Where the immunized
 animal is a mouse, a preferred myeloma cell is a murine NS-1 myeloma cell.
 The fused spleen/myeloma cells are cultured in a selective medium to select
 fused spleen/myeloma cells from the parental cells. Fused cells are
 separated from the mixture of non-fused parental cells, for example, by
 the addition of agents that block the de novo synthesis of nucleotides in
 the tissue culture media. Exemplary and preferred agents are aminopterin,
 methotrexate, and azaserine. Aminopterin and methotrexate block de novo
 synthesis of both purines and pyrimidines, whereas azaserine blocks only
 purine synthesis. Where aminopterin or methotrexate is used, the media is
 supplemented with hypoxanthine and thymidine as a source of nucleotides.
 Where azaserine is used, the media is supplemented with hypoxanthine.
 This culturing provides a population of hybridomas from which specific
 hybridomas are selected. Typically, selection of hybridomas is performed
 by culturing the cells by single-clone dilution in microtiter plates,
 followed by testing the individual clonal supernatants for reactivity with
 an antigen-polypeptides. The selected clones can then be propagated
 indefinitely to provide the monoclonal antibody.
 By way of specific example, to produce an antibody of the present
 invention, mice are injected intraperitoneally with between about 1-200 mg
 of an antigen comprising a polypeptide of the present invention. B
 lymphocyte cells are stimulated to grow by injecting the antigen in
 association with an adjuvant such as complete Freund's adjuvant (a
 non-specific stimulator of the immune response containing killed
 Mycobacterium tuberculosis). At some time (e.g., at least two weeks) after
 the first injection, mice are boosted by injection with a second dose of
 the antigen mixed with incomplete Freund's adjuvant.
 A few weeks after the second injection, mice are tail bled and the sera
 titered by immunoprecipitation against radiolabeled antigen. Preferably,
 the process of boosting and titering is repeated until a suitable titer is
 achieved. The spleen of the mouse with the highest titer is removed and
 the spleen lymphocytes are obtained by homogenizing the spleen with a
 syringe. Typically, a spleen from an immunized mouse contains
 approximately 5.times.10.sup.7 to 2.times.10.sup.8 lymphocytes.
 Mutant lymphocyte cells known as myeloma cells are obtained from laboratory
 animals in which such cells have been induced to grow by a variety of
 well-known methods. Myeloma cells lack the salvage pathway of nucleotide
 biosynthesis. Because myeloma cells are tumor cells, they can be
 propagated indefinitely in tissue culture, and are thus denominated
 immortal. Numerous cultured cell lines of myeloma cells from mice and
 rats, such as murine NS-1 myeloma cells, have been established.
 Myeloma cells are combined under conditions appropriate to foster fusion
 with the normal antibody-producing cells from the spleen of the mouse or
 rat injected with the antigen/polypeptide of the present invention. Fusion
 conditions include, for example, the presence of polyethylene glycol. The
 resulting fused cells are hybridoma cells. Like myeloma cells, hybridoma
 cells grow indefinitely in culture.
 Hybridoma ceils are separated from unfused myeloma cells by culturing in a
 selection medium such as HAT media (hypoxanthine, aminopterin, thymidine).
 Unfused myeloma cells lack the enzymes necessary to synthesize nucleotides
 from the salvage pathway because they are killed in the presence of
 aminopterin, methotrexate, or azaserine. Unfused lymphocytes also do not
 continue to grow in tissue culture. Thus, only cells that have
 successfully fused (hybridoma cells) can grow in the selection media.
 Each of the surviving hybridoma cells produces a single antibody. These
 cells are then screened for the production of the specific antibody
 immunoreactive with an antigen/polypeptide of the present invention.
 Single cell hybridomas are isolated by limiting dilutions of the
 hybridomas. The hybridomas are serially diluted many times and, after the
 dilutions are allowed to grow, the supernatant is tested for the presence
 of the monoclonal antibody. The clones producing that antibody are then
 cultured in large amounts to produce an antibody of the present invention
 in convenient quantity.
 By use of a monoclonal antibody of the present invention, specific
 polypeptides and polynucleotide of the invention can be recognized as
 antigens, and thus identified. Once identified, those polypeptides and
 polynucleotide can be isolated and purified by techniques such as
 antibody-affinity chromatography. In antibody-affinity chromatography, a
 monoclonal antibody is bound to a solid substrate and exposed to a
 solution containing the desired antigen. The antigen is removed from the
 solution through an immunospecific reaction with the bound antibody. The
 polypeptide or polynucleotide is then easily removed from the substrate
 and purified.
 Detecting a Polynucleotide or a Polypeptide of the Present Invention
 Alternatively, the present invention provides a process of detecting a
 polypeptide of the present invention, wherein the process comprises
 immunoreacting the polypeptides with antibodies prepared according to the
 process described above to form antibody-polypeptide conjugates, and
 detecting the conjugates.
 In yet another embodiment, the present invention contemplates a process of
 detecting messenger RNA transcripts that encode a polypeptide of the
 present invention, wherein the process comprises hybridizing the messenger
 RNA transcripts with polynucleotide sequences that encode the polypeptide
 to form duplexes; and detecting the duplex. Alternatively, the present
 invention provides a process of detecting DNA molecules that encode a
 polypeptide of the present invention, wherein the process comprises
 hybridizing DNA molecules with a polynucleotide that encodes that
 polypeptide to form duplexes; and detecting the duplexes.
 Screening Assays
 In yet another aspect, the present invention contemplates a process of
 screening substances for their ability to affect arachidonic acid
 metabolism comprising the steps of providing a cell that contains a
 functional polypeptide of the present invention and testing the ability of
 selected substances to affect arachidonic acid metabolism in that cell.
 Utilizing the methods and compositions of the present invention, screening
 assays for the testing of candidate substances can be derived. A candidate
 substance is a substance which potentially can promote or inhibit
 arachidonic acid metabolism, by binding or other intramolecular
 interaction, with a lipoxygenase polypeptide, such as 15-Lox-2 or 8-Lox,
 that metabolizes arachidonic acid.
 A screening assay of the present invention generally involves determining
 the ability of a candidate substance to affect metabolism of arachidonic
 acid in a target cell, such as the screening of candidate substances to
 identify those that inhibit or promote metabolism of arachidonic acid.
 Target cells can be either naturally occurring cells known to contain a
 polypeptide of the present invention or transformed cell produced in
 accordance with a process of transformation set forth hereinbefore.
 As is well known in the art, a screening assay provides a cell under
 conditions suitable for testing arachidonic acid metabolism. These
 conditions include but are not limited to pH, temperature, tonicity, the
 presence of relevant factors involved in arachidonic acid metabolism
 (e.g., metal ions such as for example Ca.sup.++, growth factor,
 interleukins, or colony stimulating factors), and relevant modifications
 to the polypeptide such as glycosylation or prenylation. It is
 contemplated that a polypeptide of the present invention can be expressed
 and utilized in a prokaryotic or eukaryotic cell. The host cell can also
 be fractionated into sub-cellular fractions where the receptor can be
 found. For example, cells expressing the polypeptide can be fractionated
 into the nuclei, the endoplasmic reticulum, vesicles, or the membrane
 surfaces of the cell.
 pH is preferably from about a value of 6.0 to a value of about 8.0, more
 preferably from about a value of about 6.8 to a value of about 7.8 and,
 most preferably about 7.4. In a preferred embodiment, temperature is from
 about 20.degree. C. to about 50.degree. C., more preferably from about
 30.degree. C. to about 40.degree. C. and, even more preferably about
 37.degree. C. Osmolality is preferably from about 5 milliosmols per liter
 (mosm/L) to about 400 mosm/l and, more preferably from about 200
 milliosmols per liter to about 400 mosm/l and, even more preferably from
 about 290 mosm/L to about 310 mosm/L. The presence of factors can be
 required for the proper testing of arachidonic acid metabolism in specific
 cells. Such factors include, for example, the presence and absence
 (withdrawal) of growth factor, interleukins, or colony stimulating
 factors.
 In one embodiment, a screening assay is designed to be capable of
 discriminating candidate substances having selective ability to interact
 with one or more of the polypeptides of the present invention but which
 polypeptides are without a substantially overlapping activity with another
 of those polypeptides identified herein.
 Screening Assays for a Polypeptide of the Present Invention
 The present invention provides a process of screening a biological sample
 for the presence of a lipoxygenase polypeptide, such as 15-Lox-2 or 8-Lox.
 Preferably, the lipoxygenase polypeptide reacts with arachidonic acid. A
 biological sample to be screened can be a biological fluid such as
 extracellular or intracellular fluid or a cell or tissue extract or
 homogenate. A biological sample can also be an isolated cell (e.g., in
 culture) or a collection of cells such as in a tissue sample or histology
 sample. A tissue sample can be suspended in a liquid medium or fixed onto
 a solid support such as a microscope slide.
 In accordance with a screening assay process, a biological sample is
 exposed to an antibody immunoreactive with the polypeptide whose presence
 is being assayed. Typically, exposure is accomplished by forming an
 admixture in a liquid medium that contains both the antibody and the
 candidate polypeptide. Either the antibody or the sample with the
 polypeptide can be affixed to a solid support (e.g., a column or a
 microtiter plate).
 The biological sample is exposed to the antibody under biological reaction
 conditions and for a period of time sufficient for antibody-polypeptide
 conjugate formation. Biological reaction conditions include ionic
 composition and concentration, temperature, pH and the like.
 Ionic composition and concentration can range from that of distilled water
 to a 2 molal solution of NaCl. Preferably, osmolality is from about 100
 mosmols/l to about 400 mosmols/l and, more preferably from about 200
 mosmols/l to about 300 mosmols/l. Temperature preferably is from about
 4.degree. C. to about 100.degree. C., more preferably from about
 15.degree. C. to about 50.degree. C. and, even more preferably from about
 25.degree. C. to about 40.degree. C. pH is preferably from about a value
 of 4.0 to a value of about 9.0, more preferably from about a value of 6.5
 to a value of about 8.5 and, even more preferably from about a value of
 7.0 to a value of about 7.5. The only limit on biological reaction
 conditions is that the conditions selected allow for antibody-polypeptide
 conjugate formation and that the conditions do not adversely affect either
 the antibody or the polypeptide.
 Exposure time will vary inter alia with the biological conditions used, the
 concentration of antibody and polypeptide and the nature of the sample
 (e.g., fluid or tissue sample). Means for determining exposure time are
 well known to one of ordinary skill in the art. Typically, where the
 sample is fluid and the concentration of polypeptide in that sample is
 about 10.sup.-10 M, exposure time is from about 10 minutes to about 200
 minutes.
 The presence of polypeptide in the sample is detected by detecting the
 formation and presence of antibody-polypeptide conjugates. Means for
 detecting such antibody-antigen (e.g., receptor polypeptide) conjugates or
 complexes are well known in the art and include such procedures as
 centrifugation, affinity chromatography and the like, binding of a
 secondary antibody to the antibody-candidate receptor complex.
 In one embodiment, detection is accomplished by detecting an indicator
 affixed to the antibody. Exemplary and well known such indicators include
 radioactive labels (e.g., .sup.32 P, .sup.125 I, .sup.14 C), a second
 antibody or an enzyme such as horse radish peroxidase. Means for affixing
 indicators to antibodies are well known in the art. Commercial kits are
 available.
 Screening Assay for Anti-Polypeptide Antibody
 In another aspect, the present invention provides a process of screening a
 biological sample for the presence of antibodies immunoreactive with a
 lipoxygenase polypeptide, such as 15-Lox-2 or 8-Lox. Preferably the
 lipoxygenase polypeptide reacts with arachidonic acid. In accordance with
 such a process, a biological sample is exposed to a lipoxygenase
 polypeptide, such as 15-Lox-2 or 8-Lox, under biological conditions and
 for a period of time sufficient for antibody-polypeptide conjugate
 formation and the formed conjugates are detected.
 Screening Assay for Polynucleotide That Encodes a Lipoxygenase Polypeptide,
 such as 15-Lox-2 or 8-Lox
 A DNA molecule and, particularly a probe molecule, can be used for
 hybridizing as an oligonucleotide probe to a DNA source suspected of
 encoding a lipoxygenase polypeptide, such as 15-Lox-2 or 8-Lox. Preferably
 the lipoxygenase polypeptide reacts with arachidonic acid. The probing is
 usually accomplished by hybridizing the oligonucleotide to a DNA source
 suspected of possessing a lipoxygenase gene. In some cases, the probes
 constitute only a single probe, and in others, the probes constitute a
 collection of probes based on a certain amino acid sequence or sequences
 of the polypeptide and account in their diversity for the redundancy
 inherent in the genetic code.
 A suitable source of DNA for probing in this manner is capable of
 expressing a polypeptide of the present invention and can be a genomic
 library of a cell line of interest. Alternatively, a source of DNA can
 include total DNA from the cell line of interest. Once the hybridization
 process of the invention has identified a candidate DNA segment, one
 confirms that a positive clone has been obtained by further hybridization,
 restriction enzyme mapping, sequencing and/or expression and testing.
 Alternatively, such DNA molecules can be used in a number of techniques
 including their use as: (1) diagnostic tools to detect normal and abnormal
 DNA sequences in DNA derived from patient's cells; (2) means for detecting
 and isolating other members of the polypeptide family and related
 polypeptides from a DNA library potentially containing such sequences; (3)
 primers for hybridizing to related sequences for the purpose of amplifying
 those sequences; (4) primers for altering native lipoxygenase DNA
 sequences; as well as other techniques which rely on the similarity of the
 DNA sequences to those of the DNA segments herein disclosed.
 As set forth above, in certain aspects, DNA sequence information provided
 by the invention allows for the preparation of relatively short DNA (or
 RNA) sequences (e.g., probes) that specifically hybridize to encoding
 sequences of a selected lipoxygenase gene. In these aspects, nucleic acid
 probes of an appropriate length are prepared based on a consideration of
 the encoding sequence for a polypeptide of this invention. The ability of
 such nucleic acid probes to specifically hybridize to other encoding
 sequences lend them particular utility in a variety of embodiments. Most
 importantly, the probes can be used in a variety of assays for detecting
 the presence of complementary sequences in a given sample. However, uses
 are envisioned, including the use of the sequence information for the
 preparation of mutant species primers, or primers for use in preparing
 other genetic constructions.
 To provide certain of the advantages in accordance with the invention, a
 preferred nucleic acid sequence employed for hybridization studies or
 assays includes probe sequences that are complementary to at least a 14 to
 40 or so long nucleotide stretch of a nucleic acid sequence of the present
 invention, such as that shown in SEQ ID NO:1. A size of at least 14
 nucleotides in length helps to ensure that the fragment is of sufficient
 length to form a duplex molecule that is both stable and selective.
 Molecules having complementary sequences over stretches greater than 14
 bases in length are generally preferred, though, to increase stability and
 selectivity of the hybrid, and thereby improve the quality and degree of
 specific hybrid molecules obtained. One will generally prefer to design
 nucleic acid molecules having gene-complementary stretches of 14 to 20
 nucleotides, or even longer where desired. Such fragments can be readily
 prepared by, for example, directly synthesizing the fragment by chemical
 means, by application of nucleic acid reproduction technology, such as the
 PCR technology of U.S. Pat. No. 4,683,202, herein incorporated by
 reference, or by introducing selected sequences into recombinant vectors
 for recombinant production.
 Accordingly, a nucleotide sequence of the present invention can be used for
 its ability to selectively form duplex molecules with complementary
 stretches of the gene. Depending on the application envisioned, one
 employs varying conditions of hybridization to achieve varying degrees of
 selectivity of the probe toward the target sequence. For applications
 requiring a high degree of selectivity, one typically employs relatively
 stringent conditions to form the hybrids. For example, one selects
 relatively low salt and/or high temperature conditions, such as provided
 by 0.02M-0.15M NaCl at temperatures of 50.degree. C. to 70.degree. C. Such
 conditions are particularly selective, and tolerate little, if any,
 mismatch between the probe and the template or target strand.
 Of course, for some applications, for example, where one desires to prepare
 mutants employing a mutant primer strand hybridized to an underlying
 template or where one seeks to isolate polypeptide coding sequences from
 related species, functional equivalents, or the like, less stringent
 hybridization conditions are typically needed to allow formation of the
 heteroduplex. Under such circumstances, one employs conditions such as
 0.15M-0.9M salt, at temperatures ranging from 20.degree. C. to 55.degree.
 C. Cross-hybridizing species can thereby be readily identified as
 positively hybridizing signals with respect to control hybridizations. In
 any case, it is generally appreciated that conditions can be rendered more
 stringent by the addition of increasing amounts of formamide, which serves
 to destabilize the hybrid duplex in the same manner as increased
 temperature. Thus, hybridization conditions can be readily manipulated,
 and thus will generally be a method of choice depending on the desired
 results.
 In certain embodiments, it is advantageous to employ a nucleic acid
 sequence of the present invention in combination with an appropriate
 means, such as a label, for determining hybridization. A wide variety of
 appropriate indicator means are known in the art, including radioactive,
 enzymatic or other ligands, such as avidin/biotin, which are capable of
 giving a detectable signal. In preferred embodiments, one likely employs
 an enzyme tag such a urease, alkaline phosphatase or peroxidase, instead
 of radioactive or other environmentally undesirable reagents. In the case
 of enzyme tags, calorimetric indicator substrates are known which can be
 employed to provide a means visible to the human eye or
 spectrophotometrically, to identify specific hybridization with
 complementary nucleic acid-containing samples.
 In general, it is envisioned that the hybridization probes described herein
 are useful both as reagents in solution hybridization as well as in
 embodiments employing a solid phase. In embodiments involving a solid
 phase, the sample containing test DNA (or RNA) is adsorbed or otherwise
 affixed to a selected matrix or surface. This fixed, single-stranded
 nucleic acid is then subjected to specific hybridization with selected
 probes under desired conditions. The selected conditions depend inter alia
 on the particular circumstances based on the particular criteria required
 (depending, for example, on the G+ C contents, type of target nucleic
 acid, source of nucleic acid, size of hybridization probe, etc.).
 Following washing of the hybridized surface so as to remove
 nonspecifically bound probe molecules, specific hybridization is detected,
 or even quantified, by means of the label.
 Assay Kits
 In another aspect, the present invention contemplates diagnostic assay kits
 for detecting the presence of a polypeptide of the present invention in
 biological samples, where the kits comprise a first container containing a
 first antibody capable of immunoreacting with the polypeptide, with the
 first antibody present in an amount sufficient to perform at least one
 assay. Preferably, the assay kits of the invention further comprise a
 second container containing a second antibody that immunoreacts with the
 first antibody. More preferably, the antibodies used in the assay kits of
 the present invention are monoclonal antibodies. Even more preferably, the
 first antibody is affixed to a solid support. More preferably still, the
 first and second antibodies comprise an indicator, and, preferably, the
 indicator is a radioactive label or an enzyme.
 The present invention also contemplates a diagnostic kit for screening
 agents. Such a kit can contain a polypeptide of the present invention. The
 kit can contain reagents for detecting an interaction between an agent and
 a receptor of the present invention. The provided reagent can be
 radiolabelled. The kit can contain a known radiolabelled agent capable of
 binding or interacting with a receptor of the present invention.
 In an alternative aspect, the present invention provides diagnostic assay
 kits for detecting the presence, in biological samples, of a
 polynucleotide that encodes a polypeptide of the present invention, the
 kits comprising a first container that contains a second polynucleotide
 identical or complementary to a segment of at least 10 contiguous
 nucleotide bases of, as a preferred example, SEQ ID NO:1 or SEQ ID NO:3.
 In another embodiment, the present invention contemplates diagnostic assay
 kits for detecting the presence, in a biological sample, of antibodies
 immunoreactive with a polypeptide of the present invention, the kits
 comprising a first container containing a lipoxygenase polypeptide, such
 as 15-Lox-2 or 8-Lox, that immunoreacts with the antibodies, with the
 polypeptide present in an amount sufficient to perform at least one assay.
 Preferably, the lipoxygenase polypeptide metabolizes arachidonic acid. The
 reagents of the kit can be provided as a liquid solution, attached to a
 solid support or as a dried powder. Preferably, when the reagent is
 provided in a liquid solution, the liquid solution is an aqueous solution.
 Preferably, when the reagent provided is attached to a solid support, the
 solid support can be chromatograph media or a microscope slide. When the
 reagent provided is a dry powder, the powder can be reconstituted by the
 addition of a suitable solvent. The solvent can be provided.
 The following examples have been included to illustrate preferred modes of
 the invention. Certain aspects of the following examples are described in
 terms of techniques and procedures found or contemplated by the present
 inventors to work well in the practice of the invention. These examples
 are exemplified through the use of standard laboratory practices of the
 inventors. In light of the present disclosure and the general level of
 skill in the art, those of skill will appreciate that the following
 examples are intended to be exemplary only and that numerous changes,
 modifications and alterations can be employed without departing from the
 spirit and scope of the invention.
 EXAMPLE 1
 ISOLATION OF A SECOND 15S-LIPOXYGENASE (15-LOX-2)
 Preparation of total RNA, and cDNA synthesis--For each RNA preparation,
 about 50 human scalp hairs were plucked individually from a volunteer.
 About 30 hair roots, mainly from anagen follicles (Baden et al. (1979) J.
 Amer. Acad. Dermatol. 1:121-122), were cut off and dropped into 1 ml of
 guanidinium thiocyanate solution, the lysis buffer from the RNeasy RNA
 extraction kit (Qiagen). After a brief sonication using an ultrasonic
 probe (2 sec, twice), total RNA was extracted according to the
 manufacturer's instructions. Approximately 5-10 .mu.g of total RNA was
 recovered in 50 .mu.l of water. In some experiments, RNA was prepared from
 psoriatic scales using essentially the same procedure. Thirty microliter
 aliquots of RNA were used in 50 .mu.l reactions for first strand cDNA
 synthesis using either an oligo-dT-adaptor primer, random hexamer primers,
 or the Marathon RACE procedure (Clontech) as described previously (Brash
 et al. (1996) J. Biol. Chem. 271, 20549-20557). One microliter aliquots of
 cDNA were used directly in PCR reactions.
 PCR experiments--The primers encoded conserved sequences in animal and
 plant lipoxygenases. Two upstream primers encoded the sequence WLLAK (SEQ
 ID NO:5) from the middle of the lipoxygenase primary structure. This
 sequence forms the beginning of a long helix that crosses the center of
 the protein and includes two of the histidine iron ligands. The two
 upstream primers differed only in using alternative codons for the 3'
 lysine, AAA or AAG, and were designated as WLLAK-(AAA) and WLLAK-(AAG):
 5'-GAC-GTC-TGG-YTi-YTi-GCi-AAA, (SEQ ID NO:6) or -AAG-3' (SEQ ID NO:7)
 (where i encodes inosine). The human 5S-lipoxygenase and the blood cell
 15S-lipoxygenase are encoded as WLLAK-(AAA) (SEQ ID NO:6) (Matsumoto et
 al. (1988) Proc. Natl. Acad. Sci. USA 85; 26-30; Dixon et al. (1988) Proc.
 Natl. Acad. Sci. USA 85, 416-420; Sigal et al. (1988) Biochem. Biophys.
 Res. Comm. 157, 457-464), whereas the platelet 12-lipoxygenase uses
 WLLAK-(AAG) (SEQ ID NO:7) (Table 2) (Funk et al. (1990) Proc. Natl. Acad.
 Sci. USA 87, 5638-5642; Izumi et al. (1990) Proc. Natl. Acad. Sci. USA 87,
 7477-7481). (One of the three papers on the human platelet 12-lipoxygenase
 reports a different sequence around this lysine, Yoshimoto et al. (1990)
 Biochem. Biophys. Res. Comm. 172, 1230-1235.)
 For the first round PCR, each upstream primer was used in separate
 reactions against a set of downstream primers encoding an amino acid
 sequence that occurs seven amino acids downstream of the most 3' histidine
 ligand to the lipoxygenase iron on a second long helix. The sequence GQLDW
 (SEQ ID NO:8) occurs in the human 12S- and 15S-lipoxygenases beginning at
 amino acid position 546 and was encoded (with an additional three amino
 acids of consensus sequence on the 5' end) as
 5'-CCA-AGT-GTA-CCA-RTC-NAG-YTG-NCC-3' (SEQ ID NO:9). The sequence GQYDW
 (SEQ ID NO:35) occurs in the equivalent position in the human
 5S-lipoxygenase and this primer differed only in changing one amino acid
 code from leucine to tyrosine (5'-CCA-AGT-GTA-CCA-RTC-RTA-YTG-NCC-3') (SEQ
 ID NO:10).
 The first round PCR reaction was primed with human hair follicle cDNA and
 in some experiments with cDNA prepared from psoriatic scales (1 .mu.l from
 a 50 .mu.l reaction using 5 .mu.g total RNA) per 50 .mu.l PCR reaction,
 and using 10 mM Tris, pH 8.3, 50 mM KCl, 3 mM MgCl.sub.2 with 0.2 mM of
 each dNTP and 0.25 .mu.l (1.25 units) AmpliTaq DNA polymerase (Perkin
 Elmer) in a Perkin Elmer 480 thermocycler. After addition of cDNA at
 80.degree. (hot start), the PCR was programmed as follows: 94.degree. for
 2 min, 1 cycle; 50.degree. for 1 min, 72.degree. for 1 min, 94.degree. for
 1 min, 30 cycles; 72.degree. for 10 min, 1 cycle, and then the block
 temperature was held at 4.degree. C.
 For second round PCR, the upstream primer was either retained as before
 (WLLAK-(AAA) (SEQ ID NO:6) or WLLAK-(AAG) (SEQ ID NO:7)), or changed to a
 nested upstream primer modified very slightly from that used by Funk and
 colleagues (Funk et al. (1990) Proc. Natl. Acad. Sci. USA 87:5638-5642)
 for cloning of the human 12S-lipoxygenase and encoding the sequence
 XVDWLLAKXWVR (SEQ ID NO:36):
 5'-TA-GTC-GAC-TGG-CTT-YTG-GCC-AAA-iiC-TGG-GTS-CG-3' (where S ("strong")
 encodes C or G) (SEQ ID NO:11). The downstream primer for all second round
 reactions (nested PCR) encoded the sequence ELQXWWR (SEQ ID NO:26) and
 included a BamHI restriction site at the 5' end:
 5'-G-CGG-ATC-CCT-CCA-CCA-GGN-YTG-SAG-YTC-3' (SEQ ID NO:12). The second
 round PCR reactions used 1 .mu.l of 10-times dilute first round PCR
 products as cDNA and otherwise the conditions differed only in using
 either 55.degree. or 58.degree. as annealing temperature.
 3' RACE and 5' RACE--The 3' sequence was obtained using established
 upstream sequence for the new human lipoxygenase (first round:
 5'-GGT-ATC-TAC-TAC-CCA-AGT-GAT-GAG-3' (SEQ ID NO:13); second round:
 5'-TAC-CCA-AGT-GAT-GAG-TCT-GTC-3' (SEQ ID NO:14)) against a downstream
 primer based on the adaptor-linked oligo-dT primer used for cDNA
 synthesis, as described previously (Brash et al. (1996) J. Biol. Chem.
 271:20549-20557). The 5' RACE was accomplished using the Marathon cDNA
 Amplication Kit (Clontech) (Brash et al. (1996) J. Biol. Chem.
 271:20549-20557) using 4 .mu.g of total RNA from beard hair follicles. The
 gene-specific downstream primers were 5'-GAA-GAC-CTC-AGG-CAG-CAG-ATG-TG-3'
 (SEQ ID NO:15) and 5'-TC-ATG-GAA-GGA-GAA-CTC-GGC-AT-3' (SEQ ID NO:16). A
 full length clone was obtained by PCR using primers purified by HPLC
 (Brash et al. (1996) J. Biol. Chem. 271:20549-20557) and using a
 proof-reading mixture of Taq/Pwo DNA polymerases (Expand High Fidelity,
 Boehringer-Mannheim) as described (Brash et al. (1996) J. Biol. Chem.
 271:20549-20557). The upstream primer encoded the N-terminus with a BamHI
 site added at the 5' end to facilitate subcloning: 5'
 AC-GGA-TCC-AGC-ATG-GCC-GAG-TTC-AGG-GTC-AG 3' (SEQ ID NO:17), and the
 downstream primer encoded the C-terminus of the protein with an added
 5'EcoRI site to facilitate subcloning: 5'
 CGG-AAT-TCA-TGT-CAT-CTG-GGC-CTG-TGT-TCC 3' (SEQ ID NO:18). After a hot
 start at 80.degree. C., the reaction conditions were 94.degree., 2 min, 1
 cycle; 58.degree. for 30 sec, 72.degree. for 1 min 30 sec, 96.degree. 15
 sec, 3 cycles; 68.degree. for 2 min, 96.degree. 15 sec, 30 cycles;
 72.degree. 10 min, 1 cycle; hold at 4.degree. C.
 Northern analysis--Two nylon membranes containing mRNA from human tissues
 (Clontech, Palo Alto, Calif.) were probed using a .sup.32 P-labeled 1059
 bp fragment of the new human lipoxygenase prepared from the plasmid by PCR
 (with primers 5'-TG-CCT-CTC-GCC-ATC-CAG-CT-3' (SEQ ID NO:19) and 5'
 TG-TTC-CCC-TGG-GAT-TTA-GAT-GGA-3') (SEQ ID NO:20) and labeled by Rediprime
 random priming (Amersham). After hybridization in ExpressHyb solution
 (Clontech) at 68.degree. C. for 1 hr, the membranes were washed finally in
 0.1.times.SSC/0.1% SDS at 50.degree. C. for 40 min and exposed to film.
 Detection of the cDNA in human cornea--RNA was prepared using Tri Reagent
 (Molecular Research Center, Inc., Cincinnati, Ohio) from corneal
 epithelial cells scraped from eye bank corneas unsuitable for
 transplantation. The RNA samples were treated with DNAse 1, then reverse
 transcribed to cDNA. PCR reactions were run with human cornea cDNA as
 template, and also with rabbit cornea cDNA and buffer alone as negative
 controls. Additional negative controls using RNA without the reverse
 transcriptase step confirmed the absence of DNA contaminants in the
 samples. Two pairs of primers were used: GGT-ATC-TAC-TAC-CCA-AGT-GAT-GAG
 (SEQ ID NO:21) with 5'-TGGGATGTCATCTGGGCCTGT-3' (SEQ ID NO:22) giving a
 589 bp product (#1), and from the 3' untranslated region (UTR), #2:
 5'-AACTCACCCCCACCACCATACACA-3' (SEQ ID NO:23) with
 5'-TTCCCGCCTCCATCTCCCAAAGT-3' (SEQ ID NO:24) giving a 351 bp product (#2).
 Both reactions were run using an annealing temperature of 65.degree. in
 the PCR. Northern analysis of eye tissues used approximately 1 .mu.g of
 poly A-selected RNA and the same hybridization protocol as given above.
 DNA sequencing--PCR products were subcloned into the pCR2.1 vector
 (Invitrogen) and sequenced using the Oncor Fidelity manual dideoxy chain
 termination method or by automated sequencing on a ABI Prism 310 Genetic
 analyzer and fluorescence-tagged dye terminator cycle sequencing (Perkin
 Elmer).
 Expression of cDNA, HPLC analysis of lipoxygenase metabolism--The PCR
 products corresponding to the open reading frame of the cDNA were
 subcloned into the pCDNA3 vector (Invitrogen), or in some experiments
 ligated directly into pCR3 (Invitrogen), and expressed by transient
 transfection in human embryonic kidney (HEK) 293 cells as described (Funk
 et al. (1996) J. Biol. Chem. 271, 23338-23344). Following incubation with
 substrate (100 .mu.M [1-.sup.14 C]arachidonic acid or [1-.sup.14
 C]linoleic acid) for 30 min at 37.degree. C., products were extracted
 using the Bligh and Dyer procedure (Bligh et al. (1959) Can. J. Biochem.
 Physiol. 37:911-917) and the extracts were analyzed by reversed-phase
 HPLC, straight phase HPLC and chiral column analysis (Brash et al. (1990)
 Method. Enzymol. 187:187-192).
 Results of PCR experiments--As described more fully in Experimental
 Procedures and summarized in Table 2, a PCR strategy was developed using
 sets of degenerate upstream and downstream primers that would resolve the
 known 5S-, 12S-, and 15S-lipoxygenases into separate tubes. The reactions
 were run under non-stringent conditions to permit detection of related
 sequences. After two rounds of reactions (nested PCR, see Experimental
 Procedures), successful amplification was expected to give a PCR product
 of approximately 500 bp.
 When the reactions were carried out using different human hair root cDNAs
 as template, bands of .apprxeq.500 bp were evident in tubes corresponding
 to several of the original combinations of primer. Many of the bands were
 found to represent the known 12S- and 15S-lipoxygenase sequences. These
 two cDNAs were successfully resolved into separate PCR reactions by making
 use of their different codon usages for lysine 344 at the 3' end of the
 upstream primer (Table 2, and Experimental Procedures). Over 60 clones
 from the first two primer combinations in Table 2 were categorized as 12-
 or 15-lipoxygenase by sequencing and/or restriction enzyme digest with
 ApaI and HindIII.
 Particular attention was paid to the 500 bp product obtained from the
 fourth primer set in Table 2, as this combination of sequences is not
 found in the three previously cloned human lipoxygenases. Of 41 clones
 with the correct sized insert, 39 cut with ApaI as expected of the human
 12S-lipoxygenase. These clones appeared to correspond to 12S-lipoxygenase
 cDNA that had annealed to the slightly mismatched primers under the
 non-stringent conditions of PCR; a limited number were sequenced and all
 were identical to the human 12S-lipoxygenase. Two of the 41 positive
 clones did not cut with ApaI or HindIII, and sequencing indicated these
 clones represented a new lipoxygenase cDNA. The complete cDNA sequence of
 this new lipoxygenase was extended by 3' RACE and 5' RACE, and full length
 clones corresponding to the open reading frame were obtained by PCR. Two
 of the active clones (see below) were fully sequenced. The percent
 identity to the reported amino acid sequences of the 5S-, 12S- and
 15S-lipoxygenases are approximately 44% to the 5-lipoxygenase, and 38-39%
 to the 12- and 15-lipoxygenases. FIG. 1 shows the deduced amino acid
 sequence (SEQ ID NO:1) in alignment with the 15S-lipoxygenase of human
 blood cells (SEQ ID NO:25) (Sigal et al. (1988) Biochem. Biophys. Res.
 Comm. 157, 457-464).
 Results of Expression studies--Initially, five full length clones were
 expressed in HEK 293 cells and the lipoxygenase activity evaluated by
 incubation with [.sup.14 C]arachidonic acid followed by HPLC analysis.
 Three of the PCR clones expressed with equivalent activity. The active
 clones made a single product, identified as 15-HETE (after reduction of
 the HPETE) on the basis of its retention time on reversed-phase HPLC (FIG.
 2A) and SP-HPLC, and its characteristic uv spectrum (Ingram et al. (1988)
 Lipids 23:340-344); it was exclusively the 15S enantiomer as determined by
 chiral column analysis (FIG. 2B). The same product was formed following
 expression in Hela cells and Cos cells, and in these experiments another
 twenty clones, eight active, were evaluated. Addition of calcium (2 mM) or
 ATP (2 mM) to the incubation media had no significant effect on enzymatic
 activity.
 Differences from the 15S-lipoxygenase of blood cells--Applicants looked
 carefully for any 12-HETE or other HETE by-products of the new
 15S-lipoxygenase and unexpectedly, found none. This is in sharp contrast
 to the 15S-lipoxygenase of human blood cells that was analyzed in the same
 experiments; as reported before, the blood cell 15S-lipoxygenase forms
 10-20% 12S-HETE in addition to 15S-HETE (Bryant et al. (1982) J. Biol.
 Chem. 257:6050-6055).
 A comparison of the metabolism of arachidonic acid and linoleic acid
 revealed a second significant difference between the two enzymes. Linoleic
 acid is an excellent substrate for the blood cell 15S-lipoxygenase
 (Soberman et al. (1985) J. Biol. Chem. 260:4508-4515); in applicants'
 experiments it was metabolized more extensively than arachidonic acid.
 Although the new 15-lipoxygenase did metabolize linoleic acid, it was not
 as good a substrate. In two experiments, linoleic acid was 11% and 37%
 metabolized by the new enzyme, while the respective values for arachidonic
 acid were 30% and 83% conversion.
 Expression in other tissues--Multiple tissue Northern blots showed fourteen
 tissues negative for the new 15S-lipoxygenase mRNA (heart, brain,
 placenta, liver, skeletal muscle, kidney, pancreas, spleen, thymus,
 testis, ovary, small intestine, colon, and peripheral blood leukocytes)
 and two distinctly positive (FIG. 3). The positive tissues, lung and
 prostate, showed a transcript estimated as 2.5-3 kb, compatible with the
 established size of the cDNA (2.7 kb). Applicants also checked for the
 presence in cornea (originally because of a suspected connection to
 12R-HETE synthesis). As determined by RT-PCR, human cornea is positive for
 the new lipoxygenase mRNA (FIG. 4a), and Northern analysis confirmed the
 presence of the new lipoxygenase transcript (FIG. 4b).
 The human lipoxygenases can be distinguished by their positional
 specificity, by other distinctive features of their catalytic activities
 such as their ability to metabolize C18 fatty acid substrates, by their
 cellular distribution, and functionally, in their physiological roles.
 Funk, C. D. (1993) Prog. Nuc. Acid Res. Mol. Biol. 45:67-98. The new
 15S-lipoxygenase characterized herein has a distinctive substrate
 specificity, a unique tissue distribution, and a different physiological
 role from the previously known human 15S-lipoxygenase.
 The primary structure of the new enzyme has the features typical of a
 lipoxygenase. It has about 40% amino acid sequence identity to the blood
 cell 15S-lipoxygenase and other reported mammalian lipoxygenases.
 The sequence contains the absolutely conserved iron-binding histidines and
 the carboxy terminal isoleucine that also functions as an iron ligand
 (Boyington et al. (1993) Science 260:1482-1486; Minor et al. (1996)
 Biochemistry 35:10687-10701). One difference from other members of the
 lipoxygenase gene family is a change in the putative fifth iron ligand,
 normally a histidine or asparagine (N693 in the soybean L1 enzyme
 (Boyington et al. (1993) Science 260:1482-1486; Minor et al. (1996)
 Biochemistry 35:10687-10701), H544 in the human blood cell
 15S-lipoxygenase (Sigal et al. (1988) Biochem. Biophys. Res. Comm.
 157:457-464). In the new human lipoxygenase the equivalent residue is
 changed to a serine (S558).
 Catalytically the enzyme differs from the blood cell 15S-lipoxygenase in
 two important respects: it oxygenates more exclusively at the 15 carbon,
 and linoleic acid is a relatively poor substrate. These two features of
 the new 15S-lipoxygenase, the high positional specificity and the
 preference for arachidonic acid, have a parallel among 12-lipoxygenases in
 the properties of the 12S-lipoxygenase of platelets (Nugteren, D. H.
 (1975) Biochim. Bikophys. Acta 380:299-307; Hada et al. (1991) Biochim.
 Biophys. Acta 1083:89-93). Given this analogy in catalytic activities, it
 is likely that the new enzyme will be a comparatively poor metabolizer of
 esterified fatty acids, in contrast to the blood cell 15S-lipoxygenase
 (Schewe et al. (1975) FEBS Lett. 60:149-153; Murray et al. (1988) Arch.
 Biochem. Biophys. 265:514-523).
 The four tissues in which the new enzyme is located, skin, lung, prostate
 and cornea, are all reported sites of 15-HETE synthesis. In human skin,
 applicants have now established the occurrence of both types of
 15S-lipoxygenase. In lung, the 15-HETE synthesis has been ascribed to the
 blood cell type of 15-lipoxygenase (Sigal et al. (1992) Am. J. Physiol.
 262:L392-398) and this enzyme has been detected by immunohistochemistry
 (Nadel et al. (1991) J. Clin. Invest. 87:1139-1145; Shannon et al. (1991)
 Am. J. Physiol. 261:L399-405; Shannon et al. (1993) Am. Rev. Respir. Dis.
 147:1024-1028). But, clearly the possibility that the new 15-lipoxygenase
 contributes to the synthesis in certain cell types should be re-examined.
 Applicants' finding of the mRNA in prostate is compatible with the reports
 by Oliw and colleagues of the occurrence of 15-lipoxygenase in
 prostasomes, components of semen secreted by the prostate gland. Oliw et
 al. (1989) Biochim. Biophys. Acta 1002:283-291; Oliw et al. (1993) J.
 Reprod. Fertil. 99, 195-199. Similarly, our detection of the cDNA in
 cornea is in accord with metabolism studies by Oliw and coworkers; they
 established that human cornea synthesizes 15S-HETE from [.sup.14
 C]arachidonic acid. Liminga et al. (1994) Biochim. Biophys. Acta
 1210:288-296. Additional studies using immunohistochemistry indicated
 expression of the blood cell type of 15S-lipoxygenase in human cornea.
 Liminga et al. (1994) Exp. Eye Res. 59, 313-321. In cornea, as in skin, it
 is likely that both types of 15S-lipoxygenase are expressed.
 It appears that this enzyme is a lipoxygenase specific for certain
 epithelial tissues. Based on the Northern result on colon and small
 intestine, the enzyme is not expressed in all epithelia, but the tissues
 in which it is identified so far are epithelial or have a significant
 epithelial component. As described in Example 2, the new human enzyme is
 related in primary structure to the phorbol ester inducible
 8S-lipoxygenase of mouse skin. Thus, regulation of the expression of the
 new human enzyme is a significant feature of its involvement in the
 pathophysiology of skin and other tissues.
 TABLE 2
 Primers (first round PCR) to resolve human
 lipoxygenases
 Reference for
 Upstream Downstream Match to known known
 Primer Primer.sup.a lipoxygenase lipoxygenase
 WLLAK-(AAA) GQLDW 15S-lipoxygenase Sigal et al.
 (SEQ ID NO:6) (SEQ ID NO:8) (1988)
 WLLAK-(AAG) GQLDW 12S-lipoxygenase Funk et al.
 (SEQ ID NO:7) (SEQ ID NO:8) (1990); Izumi
 et al. (1990)
 WLLAK-(AAA) GQYDW 5S-lipoxygenase Matsumoto et
 (SEQ ID NO:6) (SEQ ID NO:35) al. (1988);
 Dixon et al.
 (1988)
 WLLAK- (AAG) GQYDW NONE NONE
 (SEQ ID NO:7) (SEQ ID NO:35)
 .sup.a All second round PCR reactions used the nested primer ELQXWWR (SEQ
 ID NO:26 and SEQ ID NO:12) described in Experimental Procedures.
 EXAMPLE 2
 MOLECULAR CLONING AND FUNCTIONAL EXPRESSION OF A PHORBOL ESTER-INDUCIBLE
 8S-LIPOXYGENASE (8-LOX) FROM MOUSE SKIN
 As described above, in the course of studies on HETE synthesis in skin, a
 second type of 15S-lipoxygenase from human skin was cloned. This enzyme is
 different from the well known reticulocyte-type of 15S-lipoxygenase in
 that it oxygenates arachidonic acid purely at C-15 and linoleic acid is a
 relatively poor substrate. Continuing with the abbreviations adopted
 above, the reticulocyte-type of 15S-lipoxygenase is referred to as
 15-Lox-1 and the enzyme that is an aspect of this instant invention is
 referred to as 15-Lox-2 in this example.
 It was not clear a priori what is the animal homologue of the new human
 lipoxygenase, 15-Lox-2. In searching for a potential murine homologue a
 series of PCR reactions using mouse skin were performed. This led to the
 detection of a new mouse cDNA that is characterized in this example.
 One of the effects of topical application of phorbol ester to mouse skin is
 the induction of an 8S-lipoxygenase in association with the inflammatory
 response. This example describes the molecular cloning and
 characterization of this enzyme. The cDNA was isolated by PCR from mouse
 epidermis and subsequently from a mouse epidermal cDNA library. The cDNA
 encodes a protein of 677 amino acids with a calculated molecular weight of
 76 kDa. The amino acid sequence has 78% identity to 15-Lox-2, and
 approximately 40% to other mammalian lipoxygenases. When expressed in
 vaccinia virus-infected Hela cells, the mouse enzyme converts arachidonic
 acid exclusively to 8S-hydroperoxyeicosatetraenoic acid, while linoleic
 acid is converted to 9S-hydroperoxy-linoleic acid in lower efficiency.
 Phorbol ester treatment of mouse skin is associated with strong induction
 of 8S-lipoxygenase mRNA and protein. By Northern analysis, expression of
 8S-lipoxygenase mRNA was also detected in brain. Immunohistochemical
 analysis of phorbol ester-treated mouse skin showed the strongest reaction
 to 8S-lipoxygenase in the differentiated epidermal layer, the stratum
 granulosum. The inducibility of this enzyme is likely a characteristic
 feature of the mouse 8S-lipoxygenase and its human 15S-lipoxygenase
 homologue.
 Preparation of mouse epidermal total RNA, and cDNA synthesis--Phorbol ester
 (PMA, 10 nmol) dissolved in 50 ml of acetone was applied topically onto
 dorsal skin of 6-7-day-old mice. At 21-24 h after PMA-treatment, the mice
 were euthanized, and epidermis was prepared from the frozen dorsal skin as
 previously described (Hughes et al. (1991) Biochim. Biophys. Acta
 1081:347-354). The frozen epidermis was dropped into guanidinium
 thiocyanate solution, the lysis buffer from the RNeasy RNA extraction kit
 (QIAGEN). After a brief sonication using an ultrasonic probe (2 sec,
 twice), total RNA was extracted according to the manufacturer's
 instructions. Approximately 50 mg of total RNA was recovered in 50 ml of
 water. Twenty microliter aliquots were used in 50 ml reactions for first
 strand cDNA synthesis using an oligo dT-adaptor primer (Brash et al.
 (1996) J. Biol. Chem. 271:20949-20957). One microliter aliquots of cDNA
 were used directly in PCR reactions.
 PCR cloning of epidermal lipoxygenase cDNA--initial PCR clone--Two upstream
 degenerate primers encoded the sequence DVWLLAK (SEQ ID NO:27). The two
 primers differed only in using alternative codons for the 3' lysine, AAA
 or AAG, and they are referred to as WLLAK-(AAA) (SEQ ID NO:6) and
 WLLAK-(AAG) (SEQ ID NO:7). For the first round PCR reaction, each upstream
 primer was used in separate reactions against a set of downstream primers
 encoding three amino acid sequences beginning GQ that occur seven amino
 acids downstream of the most 3' histidine ligand to the iron: the sequence
 GQLDW (SEQ ID NO:8) occurs in mammalian 12S- and 15S-lipoxygenases, GQYDW
 (SEQ ID NO:35) occurs in 5S-lipoxygenases, and GQFDS (SEQ ID NO:28) occurs
 in the new human 15S-lipoxygenase, 15-Lox-2. The primer sequences are the
 same as those described in Example 1 above, except for the new degenerate
 downstream primer encoding GQFDS (SEQ ID NO:28):
 5'-CCA-AGC-GCA-SSA-RTC-RAA-YTG-NCC (where S, "strong", encodes C or G)
 (SEQ ID NO:29). For the second round nested PCR reaction, the upstream
 primer was retained as before [WLLAK-(AAA) (SEQ ID NO:6) or WLLAK-(AAG)
 (SEQ ID NO:7)], while the downstream primer was changed in all reactions
 to encode the sequence ELQXWWR (SEQ ID NO:26). After the second round PCR,
 only the reaction that originally used the WLLAK-(AAG) (SEQ ID NO:7) and
 GQFDS primers (SEQ ID NO:29) yielded a visible PCR product. This product
 was 500 bp in size. The first round PCR reaction was primed with cDNA from
 phorbol ester-treated mouse epidermis, 1 ml per 50 ml PCR reaction (from a
 50 ml cDNA synthesis using 20 mg total RNA), and using 10 mM Tris, pH 8.3,
 50 mM KCl, 3 mM MgCl.sub.2 with 0.2 mM of each dNTP and 0.25 ml (1.25
 units) of AmpliTaq DNA polymerase (Perkin Elmer) in a Perkin Elmer 480
 thermocycler. After the addition of the cDNA at 80.degree. C. (hot start),
 the PCR was programmed as follows: 94.degree. C. for 2 min, 1 cycle;
 50.degree. C. for 1 min, 72.degree. C. for 1 min, 94.degree. C. for 1 min,
 30 cycles; 72.degree. C. for 10 min, 1 cycle, and then the block
 temperature was held at 4.degree. C. The second round reaction was primed
 with the equivalent of 0.1 ml of the first round reaction products (added
 as a 10-fold dilution). The protocol was 94.degree. C. for 2 min, 1 cycle;
 58.degree. C. for 1 min, 72.degree. C. for 1 min, 94.degree. C. for 1 min
 for 30 cycles; the protocol was completed with one cycle at 72.degree. C.
 for 10 min, and then the block temperature was held at 4.degree. C.
 3'-RACE and 5'-RACE--The 3' sequence was obtained using established
 upstream sequence 5' G-AGC-TTT-GTC-TCT-GAA-ATA-GTC-AG 3' (SEQ ID NO:30)
 against a downstream primer based on the adaptor-linked oligo-dT primer
 used for cDNA synthesis (Brash et al. (1996) J. Biol. Chem.
 271:20949-20957). The 5' RACE was accomplished using a kit from GIBCO BRL
 according to the manufacturer's instructions. The gene-specific downstream
 primers were 5' GTG-AGG-AAT-CAA-TAG-CTT-GAA-GAG 3' (SEQ ID NO:31), and 5'
 G-ATG-TGT-GAC-AGC-CTC-ATG-GAT-G 3' (SEQ ID NO:32).
 Full-length clones obtained by PCR--The upstream primer encoded the
 N-terminus with a HindIII site added at the 5' end to facilitate
 subcloning: 5' C-AAG-CTT-AGG-AGG-ATG-GCG-AAA-TGC-AGG 3' (SEQ ID NO:33),
 and the downstream primer encoded the C-terminus of the protein with an
 added 5'EcoRI site: 5' G-GAA-TTC-ATG-TTA-GAT-GGA-GAC-ACT-GTT 3' (SEQ ID
 NO:34). These two primers were purified by HPLC with the DMT protecting
 groups on (Brash et al. (1996) J. Biol. Chem. 271:20949-20957). After
 deprotection they were used in PCR reactions with a proof-reading mixture
 of Taq/Pwo DNA polymerase (Expand High Fidelity, Boehringer-Mannheim)
 according to the manufacturer's instructions. The reaction conditions were
 94.degree. C., 2 min, 1 cycle; 58.degree. C. for 30 sec, 72.degree. C. for
 1 min 30 sec, 96.degree. C. for 15 sec, 3 cycles; 68.degree. C. for 2 min,
 96.degree. C. 15 sec, 30 cycles; 72.degree. C. for 10 min, 1 cycle; hold
 at 4.degree. C.
 DNA Sequencing--cDNAs were sequenced using the Oncor Fidelity manual
 dideoxy chain termination method or by automated sequencing on a ABI Prism
 310 Genetic analyzer and fluorescence-tagged dye terminator cycle
 sequencing (Perkin Elmer).
 HPLC Analysis of Lipoxygenase Metabolism--The lipoxygenase metabolism of
 [1-.sup.14 C]arachidonic acid or [1-.sup.14 C]linoleic acid was evaluated
 essentially as described previously (Hughes et al. (1991) Biochim.
 Biophys. Acta 1081:347-354). Following incubation with 100 mM of
 substrate, products were extracted using the Bligh and Dyer procedure
 (Bligh et al. (1959) Can. J. Biochem. Physiol. 37:911-917), and the
 extracts were analyzed by RP-HPLC, SP-HPLC and chiral column analysis
 (Brash et al. (1990) Methods Enzymol. 187:187-192). The hydroperoxide
 products were reduced with triphenylphosphine, methylated with
 diazomethane, purified by SP-HPLC, and then the stereochemistry was
 analyzed using a Chiralcel OD column.
 Expression of Mouse 8s-lipoxygenase Clones--The PCR products corresponding
 to the open reading frame of the cDNA were ligated directly into pCR3.1
 (Invitrogen), and expressed by transient transfection in HeLa cells using
 VTF-7, a recombinant vaccinia virus containing the T7 RNA polymerase gene
 (Blakely et al. (1991) Anal. Biochem. 194:302-308), or in human embryonic
 kidney (HEK) 293 cells using the adenovirus VA RNA gene. Funk et al.
 (1996) J. Biol. Chem. 271:23338-23344. In the former system, cells plated
 at 1.times.10.sup.6 cells/35 mm well 48 h earlier were transfected with 1
 mg of plasmid DNA and 3 mg of lipofectin, and harvested after 12 hours. In
 the HEK system, cells plated at 1.times.10.sup.6 cells/10 cm dish 24 h
 earlier were transfected with 10 mg of plasmid DNA by the calcium
 phosphate method, and harvested after two to three (2-3) days. Funk et al.
 (1996) J. Biol. Chem. 271:23338-23344. The harvested cells were sonicated
 on ice, and the resulting homogenates were incubated with 100 mM
 [1-.sup.14 C]arachidonic acid or [1-.sup.14 C]linoleic acid for 45 min at
 room temperature. The metabolites were extracted and analyzed as described
 above.
 Screening of cDNA Library--The library was a commercial 1 Unizap.XR skin
 cDNA library prepared using poly(A)+ RNA isolated from whole skin of
 C57/Black female mice (Stratagene). It was screened with a 347 bp BsaMI
 fragment of the mouse 8S-lipoxygenase cDNA (PCR clone) as probe.
 Northern Analysis--Poly(A)+ RNA was prepared from PMA- or acetone-treated
 frozen dorsal skin using TRI REAGENT.RTM. (Molecular Research Center,
 Inc.) and Oligotex.TM. (Qiagen) according to the manufacturers'
 instructions. The poly(A).sup.+ RNA was electrophoresed in 1%
 agarose/formaldehyde gel and blotted to a Hybond-H.sup.+ nylon membrane
 (Amersham). The membrane was hybridized with .sup.32 P-labeled DNA probe
 (complementary with a 0.6-kb EcoRV/BamHI fragment of mouse epidermal
 8S-lipoxygenase) prepared using the Multiprime DNA labeling kit and
 Rapid-hybridization buffer (Amersham), and then washed according to the
 manufacturer's specifications. Blots were exposed to Fuji x-ray film at
 -80.degree. C. Mouse cyclophilin cDNA was used as a house-keeping gene to
 access loading of RNA.
 Western Analysis--After quantitation by Bradford assay (Bio-Rad), protein
 was separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and
 then transferred electrophoretically to Hybond ECL nitrocellulose
 membranes (Amersham). These were probed using a rabbit polyclonal antibody
 raised against the human 15-Lox-2. This antibody recognises 15-Lox-2 and
 the mouse 8S-lipoxygenase, but not the human reticulocyte type of
 15S-lipoxygenase, as more fully described below. Donkey anti-rabbit Ig
 linked with horseradish peroxidase (Amersham) was the secondary antibody.
 Specifically bound protein was detected by chemiluminescence using the ECL
 Western blotting detection reagents (Amersham).
 Immunohistochemical Analysis--The dorsal and tail skin of six to seven
 (6-7)-day-old mice were treated with acetone or PMA (10 nmol for dorsal
 skin, 2 nmol for tail skin). After 24 h, the animals were euthanized and
 the dorsal and tail skin were washed with soap and then rinsed thoroughly
 with water. Whole dorsal and tail skin was immersion-fixed for 24 h in 4%
 paraformaldehyde in phosphate-buffered saline (pH 7.4), dehydrated in
 ethanolic solutions and xylenes, and embedded in paraffin. Skin sections
 were deparaffinized, rehydrated in graded alcohols. Endogenous peroxidase
 activity was blocked in 3% hydrogen peroxide/methanol for 20 min followed
 by incubation in 10% goat serum for 20 min. Sections were incubated at
 room temperature for 1 h in a 1/2500 dilution of either primary rabbit
 antisera that was used for Western analyses of 8S-lipoxygenase or
 pre-immune sera. After rinsing in PBS, sections were incubated with the
 biotinylated secondary antisera and peroxidase-labeled tertiary antisera
 supplied with an ABC Elite kit (Vector Corp, Guyrlingame, Calif.) followed
 by visualization of immunoprecipitate with DAB chromagen (Biogenix, Ban
 Ramon, Calif.).
 Results of molecular cloning by RT-PCR--A series of PCR reactions were
 carried out with cDNA prepared from phorbol ester-treated mouse skin as
 template, and using degenerate primers based on well conserved sequences
 in mammalian lipoxygenases. The primers were identical to those used in
 the cloning of a novel 15S-lipoxygenase (15-Lox-2) from human skin as
 described in Example 1, with the addition of an extra downstream primer
 that better represented the sequence of the new human enzyme. After
 running the protocol of nested PCR reactions, a strong band of the
 expected size of 500 bp was obtained in one of the reactions that used the
 new downstream primer (see above for details). The sequence of this PCR
 product showed a striking homology to the human lipoxygenase sequence. The
 remainder of the mouse cDNA was cloned by conventional 3'- and 5'-RACE.
 cDNA corresponding to the open reading frame was prepared by PCR using a
 proof-reading mixture of Taq/Pwo as DNA polymerase. Eight of these clones
 were selected for expression studies. Seven clones were fully sequenced.
 Subsequently, a partial cDNA sequence was used to screen a mouse skin cDNA
 library and two full length cDNAs of 3.2 kb were isolated and sequenced.
 These were identical to each other and also matched exactly one of the PCR
 products in the open reading frame (FIGS. 5A-5C). FIG. 6 shows an
 alignment with the human 15-Lox-2. The sequences are 78% identical at both
 the DNA and protein levels.
 Transient expression in HEK and Hela cells--The library clone was obtained
 relatively late in this study, and therefore much of the expression work
 described here was carried out using several of the PCR products. It
 became apparent very early on that there is some problem in expression of
 this mouse lipoxygenase. Using a standard transient expression system in
 HEK 293 cells, it was only very occasionally that applicants detected
 enzymatic activity in the expressed mouse lipoxygenase. Positive controls
 using the human reticulocyte-type of 15S-lipoxygenase (15-Lox-1) or the
 second type of human 15S-lipoxygenase (15-Lox-2) were run in every
 experiment, and these cDNAs always expressed with readily detectible
 activity. In the few instances when active mouse lipoxygenase was obtained
 in HEK cell expression, the enzyme converted arachidonic acid to
 8S-hydroperoxyeicosatetraenoic acid (8S-HPETE).
 Consistent expression of the mouse skin lipoxygenase was obtained using
 HeLa cells infected with vaccinia virus encoding the T7 RNA polymerase.
 Blakely et al. (1991) Anal. Biochem. 194:302-308. In this system, using
 sonicated cells from a 35 mm well, typically 30-40% of added arachidonic
 acid (100 .mu..E-backward.M) was converted to 8S-HPETE as the sole
 enzymatic product (FIG. 7). The percentage conversion of arachidonic acid
 in this system was always similar to that obtained using the human
 15-Lox-2 as a positive control. Active enzyme was obtained using several
 of the PCR clones (that encode one, two, or three different amino acids
 from the library clone, FIGS. 5A-5C description) and the library clone
 itself.
 Using the vaccinia expression system, linoleic acid was found to be a
 substrate for the mouse 8S-lipoxygenase although the conversion was two to
 three (2-3)-fold lower compared to arachidonic acid. The enzyme converted
 linoleic acid exclusively to 9S-HODE (FIG. 8).
 Effect of phorbol ester on expression of 8S-lipoxygenase in mouse skin--The
 expression level of 8S-lipoxygenase in mouse skin is known to be strongly
 strain-dependent. Fischer et al. (1987) Cancer Res. 47:3174-3179; Fischer
 et al. (1987) Carcinogenesis 8:421-424; Fischer et al. (1988) Cancer Res.
 48:658-664. Also, the highest activity is inducible in six to ten
 (6-10)-day-old animals. Gschwendt et al. (1986) Carcinogenesis 7:449-455;
 Furstenberger et al. (1991) J. Biol. Chem. 266:15738-15745. Applicants
 examined several strains of mice and observed major differences in the
 level of constitutive expression (with no phorbol ester) and in the level
 after phorbol ester treatment. For example, using the Sencar strain
 applicants observed high constitutive 8S-lipoxygenase activity in six to
 ten (6-10)-day-old pups, with little extra induction by phorbol ester. The
 results shown here were obtained using a mixed breed of black Swiss
 animals that have low constitutive activity of 8S-lipoxygenase and exhibit
 strong induction with phorbol ester. Using six to ten (6-10)-day-old pups,
 the inducing effect of phorbol ester clearly is related to induction of
 both mRNA and protein (FIG. 9).
 Cellular localization of 8S-lipoxygenase in mouse skin--The localization of
 mouse 8S-lipoxygenase protein in dorsal skin and tail skin was observed
 and characterized by immunohistochemical analysis. A thickened
 hyperproliferative epidermis after PMA treatment was observed. An increase
 in 8S-lipoxygenase is due to an expansion in cellularity in the stratum
 granulosum compartment of the epidermis. Baseline staining of the stratum
 granulosum for 8S-lipoxygenase in skin receiving vehicle alone(acetone)
 was performed. The absence of immunoreactivity after incubation of PMA
 treated skin with pre-immune antisera was also observed.
 Thus, expression of the 8S-lipoxygenase protein was examined in normal
 mouse skin following treatment with phorbol ester in acetone (PMA) or
 acetone alone using the strain of black Swiss animals responsive to PMA.
 The histological analysis of skin from two differing body locations (thin
 dorsal skin and thick tail skin) revealed a marked hyperproliferative
 response to PMA and a diminished response to the acetone vehicle alone.
 Most notable was an increase in the number of differentiated cells within
 the outer epidermal compartment, the stratum granulosum. The net result
 was more 8S-lipoxygenase positive cells in the the PMA treated samples as
 compared to the samples receiving acetone alone. No immunoreactivity was
 detected in any samples reacted with pre-immune serum. Hair follicles
 positioned within the underlying dermis also showed positive staining for
 8S-lipoxygenase in differentiated cell layers. Staining in these locations
 did not show a modulation in response to topical treatment with phorbol
 ester.
 Tissue distribution of 8S-lipoxygenase--As the related human
 15S-lipoxygenase, 15-Lox-2, is expressed in prostate, an activity assay
 was used (HPLC analysis of products formed from [1-.sup.14 C]arachidonic
 acid) to examine for 8S-lipoxygenase activity in mouse prostate. Using
 young adult males of 8 weeks of age, high levels of cyclooxygenase and
 12S-lipoxygenase activities were found in the prostate, but no
 8S-lipoxygenase products were detected. Occurrence of the 8S-lipoxygenase
 transcript was examined in several different tissues by Northern analysis.
 This revealed expression of 8S-lipoxygenase transcript in mouse brain,
 with no detectible expression in heart, spleen, lung, liver, skeletal
 muscle, kidney and testis (FIG. 10).
 Mouse 8S-lipoxygenase cDNA was cloned by PCR using primers related to the
 characterized human 15S-lipoxygenase (15-Lox-2) also described herein.
 These two lipoxygenases have 78% amino acid identity, and the differences
 are mainly conservative substitutions. The two enzymes have 30-45%
 identity to other mammalian lipoxygenases. The primary structure of the
 mouse 8S-lipoxygenase contains the absolutely conserved iron-binding
 histidines of lipoxygenases and the C-terminal isoleucine that is also an
 iron ligand. Boyington et al. (1993) Science 260:1482-1486; Minor et al.
 (1996) Biochemistry 35:10687-10701. A notable feature of the mouse
 8S-lipoxygenase primary structure is the presence of a serine at amino
 acid position 558 as the putative 5th iron ligand. Minor et al. (1996)
 Biochemistry 35:10687-10701. The equivalent residue in all other
 lipoxygenases is either a histidine or asparagine, with the exception of
 the human 15-Lox-2 in which a serine is also present. Based on the
 sequence similarity, the 8S-lipoxygenase is the mouse homologue of the
 human 15-Lox-2.
 Initially, there was difficulty in studying the mouse enzyme as reliable
 expression of active lipoxygenase could not be obtained using a
 conventional HEK cell system. Chen et al. (1995) J. Biol. Chem.
 270:17993-17999; Chen et al. (1994) J. Biol. Chem. 269:13979-13987. The
 problem was solved by use of the recombinant vaccinia virus in a
 co-transfection system in Hela cells. In this procedure, the cells are
 co-transfected with the plasmid cDNA and vaccinia virus encoding the T7
 RNA polymerase. The virus protein induces high level expression via the T7
 promoter upstream of the lipoxygenase cDNA. The cells are harvested after
 12 hours. In this system, the mouse enzyme expressed with equivalent
 activity to either the 15-Lox-1 or 15-Lox-2 positive controls. Each of
 these lipoxygenases was expressed at a much higher level in the viral
 infected Hela cells than in the other procedure using the HEK cells.
 The Western results show clearly that HEK cells produce the 8S-lipoxygenase
 protein, although at lower levels than the positive controls (FIG. 7C).
 Whether this is a transfection problem, or related to translation or
 protein stability is not resolved. A similar poor expression of activity
 of the mouse epidermal-type of 12S-lipoxygenase was found in HEK cells.
 Funk et al. (1996) J. Biol. Chem. 271:23338-23344. The lipoxygenase
 proteins expressed in HEK and HeLa cells were indistinguishable in size by
 Western analysis. The very same preparations of 8S-lipoxygenase plasmid
 cDNAs that failed to express active 8S-lipoxygenase in HEK cells were
 expressed with good activity in the vaccinia system. Changing the vector
 from pCR3 to pCDNA3 had no effect on HEK cell expression. Extracts of Hela
 cells (+/-viral infection or vector alone) did not restore catalytic
 activity to HEK cells transfected with 8S-lipoxygenase. Furthermore,
 changing the vaccinia virus system over to HEK cells failed to induce
 expression of 8S-lipoxygenase, whereas a 15-Lox-2 positive control gave
 easily measureable 15S-lipoxygenase activity. HEK cells may lack some
 factor that helps the effective expression of certain lipoxygenase
 enzymes.
 Linoleic acid was converted with about 3-fold lower efficiency than
 arachidonic acid by the 8S-lipoxygenase. It was, however, specifically
 oxygenated to 9S-HPODE and likely participates in the biosynthesis of this
 product in vivo. Linoleic acid is an abundant polyunsaturated fatty acid
 in mouse skin and thus, potentially, this substrate is available. Ziboh et
 al. (1988) Prog. Lipid Res. 27:81-105. Lehmann and colleagues noted that
 the levels of 8-HETE and 9-HODE in mouse skin tend to change in parallel.
 Both are strikingly elevated in mouse skin papillomas are lower than
 normal in skin carcinomas. Lehmann et al. (1992) Anal. Biochem.
 204:158-170. The 9S chirality of the product from linoleic acid is one
 criterion that could be used to assess the contribution of the
 8S-lipoxygenase to formation of 9-HODE in mouse skin. The main
 cyclooxygenase product from linoleic acid is the enantiomeric 9R-HODE
 (Hamberg et al. (1980) Biochim. Biophys. Acta 617:545-547), while
 non-enzymatic reactions would give racemic product.
 Northern and Western analyses, as well as the activity assay, showed that
 PMA treatment strongly induced de novo synthesis of mouse 8S-lipoxygenase
 in the dorsal skin of the outbred mice used in this experiment. The
 histochemical analyses further defined the effect of PMA. Immunoreactive
 8S-lipoxygenase protein was most prominent in a layer of differentiated
 epidermis, the stratum granulosum. The thickness of this cell layer
 increased markedly following 24 hours of treatment with PMA. An increase
 in the number of cells that produce 8S-lipoxygenase, therefore, is one of
 the causes of the increased 8S-lipoxygenase activity induced by PMA.
 In the Northern analysis using a multiple tissue blot, 8S-lipoxygenase mRNA
 was detected clearly in brain, but not in the other seven tissues
 examined. Both the stratum granulosum of the epidermis and the neuronal
 tissues of the central nervous system were originally derived from the
 same ectodermal layer in early embryonic development, and both represent
 highly differentiated cell types. Occurrence of the 8S-lipoxygenase
 transcript in brain was unexpected as lipoxygenase-catalyzed formation of
 8-HETE has not been reported in neuronal tissues. The negative reaction in
 liver is of interest in relation to the reported activity of 8S-HETE as a
 strong activator of the peroxisome proliferator-activated receptor,
 P-a. Yu et al. (1995) J. Biol. Chem. 270:23975-23983. In liver, there
 is the possibility of synthesis of 8-HETE via the microsomal cytochrome
 P-450 system, although in vitro this gives a nearly racemic 8-HETE
 product. Capdevila et al. (1986) Biochem. Biophys. Res. Commun.
 141:1007-1011.
 The absence of 8S-lipoxygenase signal in the Northern analysis of liver,
 could, however, be related to the lack of induction in normal tissue. The
 same issue applies to the absence of detectible 8S-lipoxygenase activity
 in normal mouse prostate from young adult males. Although the human
 homologue of the mouse 8S-lipoxygenase, 15-Lox2, was readily detectible in
 human prostate, as described in Example 1, the pooled human sample would
 include tissue from older subjects, the majority of which are expected to
 exhibit benign prostatic hyperplasia. Oesterling, J. E. (1996) Prostate
 6:67-73. The induction of 8S-lipoxygenase in mouse skin by phorbol ester
 certainly is thus a striking feature of this enzyme.
 REFERENCES
 The references listed below as well as all references cited in the
 specification are incorporated herein by reference to the extent that they
 supplement, explain, provide a background for or teach methodology,
 techniques and/or compositions employed herein.
 Adelman et al. (1983) DNA 2:183.
 Ausubel, F. M., et al. Current Protocols in Molecular Biology, (J. Wylie &
 Sons, N.Y.), 1992.
 Baden et al. (1979) J. Amer. Acad. Dermatol. 1:121-122.
 Baer et al. (1991) J. Lipid Research 32:341-347.
 Baer et al. (1993) J. Lipid Research 34:1505-1514.
 Baer et al. (1995) J. Invest. Dermatol. 104:251-255.
 Blakely et al. (1991) Anal. Biochem. 194:302-308.
 Bligh et al. (1959) Can. J. Biochem. Physiol. 37:911-917.
 Boyington et al. (1993) Science 260:1482-1486.
 Brash et al. (1990) Method. Enzymol. 187:187-192.
 Brash et al. (1996) J. Biol.Chem. 271:20549-20557.
 Brash et al. (1996) J. Biol. Chem. 271:20949-20957.
 Bryant et al. (1982) J. Biol. Chem. 257:6050-6055.
 Capdevila et al. (1986) Biochem. Biophys. Res. Commun. 141:1007-1011.
 Chen et al. (1994) J. Biol. Chem. 269:13979-13987.
 Chen et al. (1994) Nature 372:179-182.
 Chen et al. (1995) J. Biol. Chem. 270:17993-17999.
 Crea et al., (1978) Proc. Natl. Acad. Sci. U.S.A, 75:5765.
 Dixon et al. (1988) Proc. Natl. Acad. Sci. USA 85:416-420.
 Eichenlaub, R. J. Bacteriol 138:559-566, 1979.
 Fischer et al. (1987) Carcinogenesis 8:421-424.
 Fischer et al. (1987) Cancer Res. 47:3174-3179.
 Fischer et al. (1988) Cancer Res. 48:658-664.
 Funk et al. (1990) Proc. Natl. Acad. Sci. USA 87:5638-5642.
 Funk et al. (1993) Prog. Nuc. Acid Res. Mol. Biol. 45:67-98.
 Funk et al. (1996) J. Biol. Chem. 271:23338-23344.
 Furstenberger et al. (1991) J. Biol. Chem. 266:15738-15745.
 Gschwendt et al. (1986) Carcinogenesis 7:449-455.
 Hada et al. (1991) Biochim. Biophys. Acta 1083:89-93.
 Hamberg et al. (1980) Biochim. Biophys. Acta 617:545-547.
 Hammarstrom et al. (1975) Proc. Natl. Acad. Sci.USA 72:5130-5134.
 Henneicke-von Zepelin et al. (1991) J. Invest. Dermatol. 97:291-297.
 Holtzman et al. (1989) J. Clin. Invest. 84:1446-1453.
 Hopp, U.S. Pat. No. 4,554,101.
 Howell et al. Antibodies A Laboratory Manual, Cold Spring Harbor
 Laboratory, 1988.
 Hughes et al. (1991) Biochim. Biophys. Acta 1081:347-354.
 Hussain et al. (1994) Amer. J. Physiol. 266:C243-C253.
 Ingram et al. (1988) Lipids 23:340-344.
 Izumi et al. (1990) Proc. Natl. Acad. Sci. USA 87:7477-7481.
 Kyte, J., and R. F. Doolittle (1982) J. Mol. Biol. 157: 105.
 Lehmann et al. (1992) Anal. Biochem. 204:158-170
 Liminga et al. (1994) Exp. Eye Res. 59:313-321.
 Liminga et al. (1994) Biochim. Biophys. Acta 1210:288-296.
 Matsumoto et al. (1988) Proc. Natl. Acad. Sci. USA 85:26-30.
 Messing et al., Third Cleveland Symposium on Macromolecules and Recombinant
 DNA, Editor A. Walton, Elsevier, Amsterdam (1981).
 Minor et al. (1996) Biochemistry 35:10687-10701.
 Murray et al. (1988) Arch. Biochem. Biophys. 265:514-523.
 Nadel et al. (1991) J. Clin. Invest. 87:1139-1145.
 Nugteren et al. (1975) Biochim. Bikophys. Acta 380:299-307.
 Nugteren et al. (1987) Biochim. Biophys. Acta 921:135-141
 Oesterling et al. (1996) Prostate 6:67-73.
 Oliw et al. (1989) Biochim. Biophys. Acta 1002:283-291.
 Oliw et al. (1993) J. Reprod. Fertil. 99:195-199.
 Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual (Cold
 Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
 Schewe et al. (1975) FEBS Lett. 60:149-153.
 Shannon et al. (1991) Am. J. Physiol. 261:L399-405.
 Shannon et al. (1993) Am. Rev. Respir. Dis. 147:1024-1028.
 Sigal et al. (1988) Biochem. Biophys. Res. Comm. 157: 457-464.
 Sigal et al. (1992) Am. J. Physiol. 262:L392-398.
 Soberman et al. (1985) J. Biol. Chem. 260:4508-4515.
 Sun et al. (1996) J. Biol. Chem. 271, 24055-24062.
 Takahashi et al. (1993) J. Biol. Chem. 268:16443-16448.
 U.S. Pat. No. 4,196,265
 U.S. Pat. No. 4,683,202
 van Dijk et al. (1995) Biochim. Biophys. Acta 1259:4-8.
 Wetmur & Davidson, J. Mol. Biol. 31:349-370, 1968.
 Woollard et al. (1986) Biochem. Biophys. Res. Commun. 136(1):169-175.
 Yoshimoto et al. (1990) Biochem. Biophys. Res. Comm. 172:1230-1235.
 Yu et al. (1995) J. Biol. Chem. 270:23975-23983.
 Ziboh et al. (1988) Prog. Lipid Res. 27:81-105.
 It will be understood that various details of the invention may be changed
 without departing from the scope of the invention. Furthermore, the
 foregoing description is for the purpose of illustration only, and not for
 the purpose of limitation--the invention being defined by the claims.