Novel Human β ₂ Integrin Alpha Subunit

* J_

This application is a continuation-in-part of U.S. Application Serial No. 08/286,889, filed August 5, 1994, which is pending, which in turn is a continuation-in-part of U.S. Application Serial No. 08/173,497, filed December 5 23, 1993, which is pending.

Field of the Invention

The present invention relates to the cloning and expression of polynucleotides encoding a novel human β ₂ integrin subunit, designated _d , which is structurally related to the known human β ₂ integrin subunits, CDlla,

10 CD1 lb and CD1 lc. The present invention also relates to polynucleotides isolated from other species which show homology to human α. _d encoding sequences.

Background of the Invention

The integrins are a class of membrane-associated molecules which actively participate in cellular adhesion. Integrins are transmembrane

15 heterodimers comprising an subunit in noncovalent association with a β subunit.

To date, at least fourteen subunits and eight β subunits have been identified [reviewed in Springer, Nature 346:425-434 (1990)]. The β subunits are generally capable of association with more than one a subunit and the heterodimers sharing a common β subunit have been classified as subfamilies within the integrin

20 population.

One class of human integrins, restricted to expression in white blood cells, is characterized by a common β ₂ subunit. As a result of this cell-

< specific expression, these integrins are commonly referred to as the leukocyte integrins, Leu-CAMs or leukointegrins. Because of the common β ₂ subunit, an

25 alternative designation of this class is the β ₂ integrins. The β ₂ subunit (CD 18) has previously been isolated in association with one of three distinct subunits,

CDlla, CDllb or CDllc. The isolation of a cDNA encoding human CD18 is described in Kishimoto, et al , Cell 48:681-690 (1987). In official WHO nomenclature, the heterodimeric proteins are referred to as CDlla/CD18, CDllb/CD18, and CDllc/CD18; in common nomenclature they are referred to as LFA-1, Mac-1 or Mol and pl50,95 or LeuM5, respectively [Cobbold, et ah, in Leukocyte Typing III, McMichael (ed), Oxford Press, p.788 (1987)]. The human β ₂ integrin α subunits CDlla, CDllb and CDllc have been demonstrated to migrate under reducing condition in electrophoresis with apparent molecular weights of approximately 180 kD, 155 kD and 150 kD, respectively, and DNAs encoding these subunits have been cloned [CDlla, Larson, et al., J. Cell Biol.

108:703-712 (1989); CDllb, Corbi, etal., J.Biol. Chem. 265:12403-12411 (1988) and CDllc, Corbi, et al. EMBO J. 6:4023-4028 (1987)]. Putative homologs of the human β ₂ integrin a and β chains, defined by approximate similarity in molecular weight, have been variously identified in other species including monkeys and other primates [Letvin, et al., Blood 67:408-410 (1983)], mice

[Sanchez-Madrid, et al., J.Exp.Med. 154:1517 (1981)], and dogs [Moore, et al. , Tissue Antigens 56:211-220 (1990)].

The absolute molecular weights of presumed homologs from other species have been shown to vary significantly [see, e.g., Danilenko et al., Tissue Antigens 40: 13-21 (1992)], and in the absence of sequence information, a definitive correlation between human integrin subunits and those identified in other species has not been possible. Moreover, variation in the number of members in a protein family has been observed between different species. Consider, for example, that more IgA isotypes have been isolated in rabbits than in humans [Burnett, et al., EMBO J. 8:4041-4047 (1989) and Schneiderman, et al , Proc.Natl.Acad.Sci. fUSA) 86:7561-7565 (1989)]. Similarly, in humans, at least six variants of the metallothionine protein have been previously identified [Karin and Richards, Nature 299: 797-802 (1982) and Varshney, et al , Mol Cell.Biol. 6:26-37, (1986)], whereas in the mouse, only two such variants are

in evidence [Searle, et al, Mol.Cell.Biol 4:1221-1230 (1984)]. Therefore, existence of multiple members of a protein family in one species does not necessarily imply that corresponding family members exist in another species. In the specific context of β ₂ integrins, in dogs it has been observed that the presumed canine β ₂ counterpart to the human CD 18 is capable of dimer formation with as many as four potentially distinct subunits [Danilenko, et al. , supra]. Antibodies generated by immunizing mice with canine splenocytes resulted in monoclonal antibodies which immunoprecipitated proteins tentatively designated as canine homologs to human CD18, CD1 la, CD1 lb and CD1 lc based mainly on similar, but not identical, molecular weights. Another anti-canine splenocyte antibody, Call.8H2, recognized and immunoprecipitated a fourth - like canine subunit also capable of association with the β ₂ subunit, but having a unique molecular weight and restricted in expression to a subset of differentiated tissue macrophages. Antibodies generated by immunization of hamsters with murine dendritic cells resulted in two anti-integrin antibodies [Metlay, et al,

J.Exp.Med. 171: 1753-1771 (1990)]. One antibody, 2E6, immunoprecipitated a predominant heterodimer with subunits having approximate molecular weights of 180 kD and 90 kD in addition to minor bands in the molecular weight range of 150-160 kD. The second antibody, N418, precipitated another apparent heterodimer with subunits having approximate molecular weights of 150 kD and

90 Kd. Based on cellular adhesion blocking studies, it was hypothesized that antibody 2E6 recognized a murine counterpart to human CD18. While the molecular weight of the N418 antigen suggested recognition of a murine homolog to human CDllc/CD18, further analysis indicated that the murine antigen exhibited a tissue distribution pattern which was inconsistent with that observed for human CDllc/CD18.

The antigens recognized by the canine Call.8H2 antibody and the murine N418 antibody could represent a variant species {e.g. , a glycosylation or splice variant) of a previously identified canine or murine a subunit.

Alternatively, these antigens may represent unique canine and murine integrin a subunits. In the absence of specific information regarding primary structure, these alternatives cannot be distinguished.

In humans, CDl la/CD 18 is expressed on all leukocytes. CDllb/CD18 and CDllc/CD18 are essentially restricted to expression on monocytes, granulocytes, macrophages and natural killer (NK) cells, but CDllc/CD18 is also detected on some B-cell types. In general, CDlla/CD18 predominates on lymphocytes, CDl lb/CD 18 on granulocytes and CDllc/CD18 on macrophages [see review, Arnaout, Blood 75:1037-1050 (1990)]. Expression of the a chains, however, is variable with regard to the state of activation and differentiation of the individual cell types [See review, Larson and Springer, Immunol.Rev. 114: 181-217 (1990).]

The involvement of the β ₂ integrins in human immune and inflammatory responses has been demonstrated using monoclonal antibodies which are capable of blocking β ₂ integrin-associated cell adhesion. For example,

CDlla/CD18, CDl lb/CD 18 and CDllc/CD18 actively participate in natural killer (NK) cell binding to lymphoma and adenocarcinoma cells [Patarroyo, et al. , Immunol.Rev. 114:67-108 (1990)], granulocyte accumulation [Nourshargh, et a , J. Immunol. 742:3193-3198 (1989)], granulocyte-independent plasma leakage [Arfors, et al, Blood 69:338-340 (1987)], chemotactic response of stimulated leukocytes [Arfors, et al. , supra] and leukocyte adhesion to vascular endothelium [Price, et al , J.Immunol. 139:4174-4177 (1987) and Smith, et al , J. Clin. Invest. 85:2008-2017 (1989)]. The fundamental role of β ₂ integrins in immune and inflammatory responses is made apparent in the clinical syndrome referred to as leukocyte adhesion deficiency (LAD), wherein clinical manifestations include recurrent and often life threatening bacterial infections. LAD results from heterogeneous mutations in the β ₂ subunit [Kishimoto, et al , Cell 50:193-202 (1987)] and the severity of the disease state is proportional to the degree of the

deficiency in β ₂ subunit expression. Formation of the complete integrin heterodimer is impaired by the β ₂ mutation [Kishimoto, et al, supra].

Interestingly, at least one antibody specific for CD 18 has been shown to inhibit human immunodeficiency virus type-1 (HTV-1) syncytia formation in vitro, albeit the exact mechanism of this inhibition is unclear

[Hildreth and Orentas, Science 244:1075-1078 (1989)]. This observation is consistent with the discovery that a principal counterreceptor of CDlla CD18, ICAM-1, is also a surface receptor for the major group of rhinovirus serotypes [Greve, et al , Cell 56:839 (1989)]. The significance of β ₂ integrin binding activity in human immune and inflammatory responses underscores the necessity to develop a more complete understanding of this class of surface proteins. Identification of yet unknown members of this subfamily, as well as their counterreceptors, and the generation of monoclonal antibodies or other soluble factors which can alter biological activity of the β ₂ integrins will provide practical means for therapeutic intervention in β ₂ integrin-related immune and inflammatory responses.

Brief Description of the Invention

In one aspect, the present invention provides novel purified and isolated polynucleotides (e.g., DNA and RNA transcripts, both sense and anti- sense strands) encoding a novel human β ₂ integrin α subunit, α _d , and variants thereof (i.e. , deletion, addition or substitution analogs) which possess binding and/or immunological properties inherent to α _d . Preferred DNA molecules of the invention include cDNA, genomic DNA and wholly or partially chemically synthesized DNA molecules. A presently preferred polynucleotide is the DNA as set forth in SEQ ID NO: 1, encoding the polypeptide of SEQ ID NO: 2. Also provided are recombinant plasmid and viral DNA constructions (expression constructs) which include α _d encoding sequences, wherein the α _d encoding

sequence is operatively linked to a homologous or heterologous transcriptional regulatory element or elements.

Also provided by the present invention are isolated and purified mouse and rat polynucleotides which exhibit homology to polynucleotides encoding human α _d . A preferred mouse polynucleotide is set forth in SEQ ID

NO: 52; a preferred rat polynucleotide is set forth in SEQ ID NO: 54.

As another aspect of the invention, prokaryotic or eukaryotic host cells transformed or transfected with DNA sequences of the invention are provided which express α _d polypeptide or variants thereof. Host cells of the invention are particularly useful for large scale production of α. _d polypeptide, which can be isolated from either the host cell itself or from the medium in which the host cell is grown. Host cells which express α. polypeptide on their extracellular membrane surface are also useful as immunogens in the production of α _d -specifιc antibodies. Preferably, host cells transfected with α _d will be co- transfected to express a β ₂ integrin subunit in order to allow surface expression of the heterodimer.

Also provided by the present invention are purified and isolated α _d polypeptides, fragments and variants thereof. Preferred α _d polypeptides are as set forth in SEQ ID NO: 2. Novel α _d products of the invention may be obtained as isolates from natural sources, but, along with a _d variant products, are preferably produced by recombinant procedures involving host cells of the invention. Completely glycosylated, partially glycosylated and wholly de- glycosylated forms of the α _d polypeptide may be generated by varying the host cell selected for recombinant production and/or post-isolation processing. Variant α. _d polypeptides of the invention may comprise water soluble and insoluble α _d polypeptides including analogs wherein one or more of the amino acids are deleted or replaced: (1) without loss, and preferably with enhancement, of one or more biological activities or immunological characteristics specific for α _d ; or (2) with specific disablement of a particular ligand/receptor binding or signalling

function. Fusion polypeptides are also provided, wherein α _d amino acid sequences are expressed contiguously with amino acid sequences from other polypeptides. Such fusion polypeptides may possess modified biological, biochemical, and/or immunological properties in comparison to wild-type α _d . Analog polypeptides including additional amino acid (e.g., lysine or cysteine) residues that facilitate multimer formation are contemplated.

Also comprehended by the present invention are polypeptides and other non-peptide molecules which specifically bind to α _d . Preferred binding molecules include antibodies (e.g., monoclonal and polyclonal antibodies), counterreceptors (e.g. , membrane-associated and soluble forms) and other ligands

(e.g., naturally occurring or synthetic molecules), including those which competitively bind α _d in the presence of α _d monoclonal antibodies and/or specific counterreceptors. Binding molecules are useful for purification of α _d polypeptides and identifying cell types which express o; _d . Binding molecules are also useful for modulating (i.e., inhibiting, blocking or stimulating) of in vivo binding and/or signal transduction activities of o. .

Assays to identify α _d binding molecules are also provided, including immobilized ligand binding assays, solution binding assays, scintillation proximity assays, di-hybrid screening assays, and the like. In vitro assays for identifying antibodies or other compounds that modulate the activity of α. _d may involve, for example, immobilizing α _d or a natural ligand to which α _d binds, detectably labelling the nonimmobilized binding partner, incubating the binding partners together and determining the effect of a test compound on the amount of label bound wherein a reduction in the label bound in the presence of the test compound compared to the amount of label bound in the absence of the test compound indicates that the test agent is an inhibitor of α _d binding.

Another type of assay for identifying compounds that modulate the interaction between α _d and a ligand involves immobilizing α _d or a fragment

thereof on a solid support coated (or impregnated with) a fluorescent agent, labelling the ligand with a compound capable of exciting the fluorescent agent, contacting the immobilized ot with the labelled ligand in the presence and absence of a putative modulator compound, detecting light emission by the fluorescent agent, and identifying modulating compounds as those compounds that affect the emission of light by the fluorescent agent in comparison to the emission of light by the fluorescent agent in the absence of a modulating compound. Alternatively, the α _d ligand may be immobilized and α _d may be labelled in the assay.

Yet another method contemplated by the invention for identifying compounds that modulate the interaction between α _d and a ligand involves transforming or transfecting appropriate host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA-binding domain and an activating domain, expressing in the host cells a first hybrid DNA sequence encoding a first fusion of part or all of α _d and either the DNA binding domain or the activating domain of the transcription factor, expressing in the host cells a second hybrid DNA sequence encoding part or all of the ligand and the DNA binding domain or activating domain of the transcription factor which is not incorporated in the first fusion, evaluating the effect of a putative modulating compound on the interaction between α _d and the ligand by detecting binding of the ligand to α _d in a particular host cell by measuring the production of reporter gene product in the host cell in the presence or absence of the putative modulator, and identifying modulating compounds as those compounds altering production of the reported gene product in comparison to production of the reporter gene product in the absence of the modulating compound. Presently preferred for use in the assay are the lexA promoter, the lexA DNA binding domain, the GAL4 transactivation domain, the lacZ reporter gene, and a yeast host cell.

A modified version of the foregoing assay may be used in isolating a polynucleotide encoding a protein that binds to a _d by transforming or

transfecting appropriate host cells with a DNA construct comprising a reporter gene under the control of a promoter regulated by a transcription factor having a DNA-binding domain and an activating domain, expressing in the host cells a first hybrid DNA sequence encoding a first fusion of part or all of α. _d and either the DNA binding domain or the activating domain of the transcription factor, expressing in the host cells a library of second hybrid DNA sequences encoding second fusions of part or all of putative α _d binding proteins and the DNA binding domain or activating domain of the transcription factor which is not incorporated in the first fusion, detecting binding of an α _d binding protein to α _d in a particular host cell by detecting the production of reporter gene product in the host cell, and isolating second hybrid DNA sequences encoding α _d binding protein from the particular host cell.

Hybridoma cell lines which produce antibodies specific for α. _d are also comprehended by the invention. Techniques for producing hybridomas which secrete monoclonal antibodies are well known in the art. Hybridoma cell lines may be generated after immunizing an animal with purified α _d , variants of α or cells which express α _d or a variant thereof on the extracellular membrane surface. Immunogen cell types include cells which express α _d in vivo, or transfected prokaryotic or eukaryotic cell lines which normally do not normally express α. _d in vivo.

The value of the information contributed through the disclosure of the DNA and amino acid sequences of α _d is manifest. In one series of examples, the disclosed α _d CDNA sequence makes possible the isolation of the human o. _d genomic DNA sequence, including transcriptional control elements for the genomic sequence. Identification of α _d allelic variants and heterologous species

(e.g., rat or mouse) DNAs is also comprehended. Isolation of the human α. _d genomic DNA and heterologous species DNAs can be accomplished by standard DNA/DNA hybridization techniques, under appropriately stringent conditions, using all or part of the α _d cDNA sequence as a probe to screen an appropriate

library. Alternatively, polymerase chain reaction (PCR) using oligonucleotide primers that are designed based on the known cDNA sequence can be used to amplify and identify genomic α _d DNA sequences. Synthetic DNAs encoding the α _d polypeptide, including fragments and other variants thereof, may be produced by conventional synthesis methods.

DNA sequence information of the invention also makes possible the development, by homologous recombination or "knockout" strategies [see, e.g.,

Kapecchi, Science 244:1288-1292 (1989)], to produce rodents that fail to express a functional o: _d polypeptide or that express a variant α _d polypeptide. Such rodents are useful as models for studying the activities of α _d and α _d modulators in vivo.

DNA and amino acid sequences of the invention also make possible the analysis of _d epitopes which actively participate in counterreceptor binding as well as epitopes which may regulate, rather than actively participate in, binding. Identification of epitopes which may participate in transmembrane signal transduction is also comprehended by the invention.

DNA of the invention is also useful for the detection of cell types which express α _d polypeptide. Standard DNA/RNA hybridization techniques which utilize α DNA to detect α _d RNA may be used to determine the constitutive level of α. _d transcription within a cell, as well as changes in the level of transcription in response to internal or external agents. Identification of agents which modify transcription and/or translation of a _d can, in turn, be assessed for potential therapeutic or prophylactic value. DNA of the invention also makes possible in situ hybridization of a _d DNA to cellular RNA to determine the cellular localization of α. _d specific messages within complex cell populations and tissues. DNA of the invention is also useful for identification of non-human polynucleotide sequences which display homology to human α _d sequences. Possession of non-human α. _d DNA sequences permits development of animal models (including, for example, transgenic models) of the human system.

As another aspect of the invention, monoclonal or polyclonal antibodies specific for α _d may be employed in immunohistochemical analysis to localize α _d to subcellular compartments or individual cells within tissues. Immunohistochemical analyses of this type are particularly useful when used in combination with in situ hybridization to localize both α _d mRNA and polypeptide products of the α _d gene.

Identification of cell types which express a _d may have significant ramifications for development of therapeutic and prophylactic agents. It is anticipated that the products of the invention related to _d can be employed in the treatment of diseases wherein macrophages are an essential element of the disease process. Animal models for many pathological conditions associated with macrophage activity have been described in the art. For example, in mice, macrophage recruitment to sites of both chronic and acute inflammation is reported by Jutila, et al, J.Leukocyte Biol. 54:30-39 (1993). In rats, Adams, et al. , [Transplantation 53: 1115-1119(1992) and Transplantation 56:794-799 (1993)] describe a model for graft arteriosclerosis following heterotropic abdominal cardiac allograft transplantation. Rosenfeld, etal , [Arteriosclerosis 7:9-23 (1987) and Arteriosclerosis 7:24-34 (1987)] describe induced atherosclerosis in rabbits fed a cholesterol supplemented diet. Hanenberg, et al. , [Diabetologia 32: 126-134 (1989)] report the spontaneous development of insulin-dependent diabetes in BB rats. Yamada et al , [Gastroenterolgy 104:759-771 (1993)] describe an induced inflammatory bowel disease, chronic granulomatous colitis, in rats following injections of strep tococcal peptidogly can-poly saccharide polymers. Cromartie, et al , [J.Exp.Med. 746: 1585-1602 (1977)] and Schwab, et al , [Infection and Immunity 5 :4436-4442 (1991)] report that injection of streptococcal cell wall protein into rats results in an arthritic condition characterized by inflammation of peripheral joints and subsequent joint destruction. Finally, Huitinga, et al , [Eur.J. Immunol 25:709-715 (1993) describe experimental allergic encephalomyelitis, a model for multiple sclerosis, in Lewis rats. In each of these

models, antibodies, other α _d binding proteins, or soluble forms of α _d are utilized to attenuate the disease state, presumably through inactivation of macrophage activity.

Pharmaceutical compositions for treatment of these and other disease states are provided by the invention. Pharmaceutical compositions are designed for the purpose of inhibiting interaction between α _d and its ligand(s) and include various soluble and membrane-associated forms of a _d (comprising the entire a _d polypeptide, or fragments thereof which actively participate in _d binding), soluble and membrane-associated forms of α _d binding proteins (including antibodies, ligands, and the like), intracellular or extracellular modulators of a _d binding activity, and/or modulators of a _d and/or α. _d -ligand polypeptide expression, including modulators of transcription, translation, post- translational processing and/or intracellular transport. The invention also comprehends methods for treatment of disease states in which _d binding is implicated, wherein a patient suffering from said disease state is provided an amount of a pharmaceutical composition of the invention sufficient to modulate levels of α _d binding. The method of treatment of the invention is applicable to disease states such as, but not limited to, Type I diabetes, atherosclerosis, multiple sclerosis, asthma, psoriasis, and rheumatoid arthritis.

Brief Description of the Drawing

Numerous other aspects and advantages of the present invention will be apparent upon consideration of the following description thereof, reference being made to the drawing wherein:

Figure 1 A through ID comprises an alignment of the human amino acid sequences of CDl lb (SEQ ID NO: 3), CDl lc (SEQ ID NO: 4) and _d (SEQ

ID NO: 2).

Detailed Description of the Invention

The present invention is illustrated by the following examples relating to the isolation of a cDNA clone encoding a _d from a human spleen cDNA library. More particularly, Example 1 illustrates the use of anti-canine «TMI antibody in an attempt to detect a homologous human protein. Example 2 details purification of canine α Mi ^d N-terminal sequencing of the polypeptide to design oligonucleotide primers for PCR amplification of the canine a-^^ gene. Example 3 addresses large scale purification of canine α^Mi ° ^r nternal sequencing in order to design additional PCR primers. Example 4 describes use of the PCR and internal sequence primers to amplify a fragment of the canine ^α TM _l S ^ene - Example 5 addresses cloning of the human α _d -encoding cDNA sequence. Example 6 describes Northern blot hybridization analysis of human tissues and cells for expression of _d mRNA. Example 7 details the construction of human α _d expression plasmids and transfection of COS cells with the resulting plasmids. Example 8 addresses ELISA analysis of α. _d expression in transfected

COS cells. Example 9 describes FACS analysis of COS cells transfected with human _d expression plasmids. Example 10 addresses immunoprecipitation of CD 18 in association with _d in co-transfected COS cells. Example 11 relates to stable transfection of o. _d expression constructs in Chinese hamster ovary cells. Example 12 addresses CD18-dependent binding of α _d to the intercellular adhesion molecule, ICAM-R. Example 13 describes scintillation proximity screening assays to identify inhibitors of _d ligand/anti-ligand binding interactions. Example 14 addresses construction of expression plasmids which encode soluble forms of α _d . Example 15 relates to production of α _d -specific monoclonal antibodies. Example 16 describes analysis of α _d tissue distribution using polyclonal antiserum. Example 17 describes isolation of rat cDNA sequences which show homology to human a _d gene sequences. Example 18 relates to construction of rat α _d I domain expression plasmids, including I domain/IgG fusion proteins, and production of monoclonal antibodies to I domain fusion

proteins. Example 19 addresses isolation of mouse cDNA sequences which show homology to human a _d gene sequences. Example 20 describes isolation of additional mouse α _d cDNA clones used for conformational sequence analysis. Example 21 relates to in situ hybridization analysis of various mouse tissues to determine tissue and cell specific expression of the putative mouse homolog to human α _d . Example 22 describes generation of expression constructs which encode the putative mouse homolog of human a _d . Example 23 addresses design of a "knock-out" mouse wherein the gene encoding the putative mouse homolog of human a _d is disrupted. Example 24 describes isolation of rabbit cDNA clones which show homology to human a _d encoding sequences. Example 25 describes animal models which resemble human disease states wherein modulation of _d is assayed for therapeutic capabilities.

Example 1

Attempt to Detect a Human Homolog of Canine Q-TMI The monoclonal antibody Cal 1.8H2 [Moore, et al. , supra] specific for canine α- _j - _j ^ was tested for cross-reactivity on human peripheral blood leukocytes in an attempt to identify a human homolog of canine α. _TM1 . Cell preparations (typically 1 x 10 ⁶ cells) were incubated with undiluted hybridoma supernatant or a purified mouse IgG-negative control antibody (10 μg/ml) on ice in the presence of 0.1 % sodium azide. Monoclonal antibody binding was detected by subsequent incubation with FITC-conjugated horse anti-mouse IgG (Vector Laboratories, Burlingame, CA) at 6 μg/ml. Stained cells were fixed with 2% w/v paraformaldehyde in phosphate buffered saline (PBS) and were analyzed with a Facstar Plus fluorescence-activated cell sorter (Becton Dickinson, Mountain View, CA). Typically, 10,000 cells were analyzed using logarithmic amplification for fluorescence intensity.

The results indicated that Cal 1.8H2 did not cross-react with surface proteins expressed on human peripheral blood leukocytes, while the control cells,

neoplastic canine peripheral blood lymphocytes, were essentially all positive for ^α TMl-

Because the monoclonal antibody Call.8H2 specific for the canine a subunit did not cross react with a human homolog, isolation of canine α _{j l} DNA was deemed a necessary prerequisite to isolate a counterpart human gene if one existed.

Example 2

Affinity Purification Of Canine Q! _Tjlϊl For N-Terminal Sequencing

Canine α- _j ^i was affinity purified in order to determine N-terminal amino acid sequences for oligonucleotide probe/primer design. Briefly, anti-o. _TM1 monoclonal antibody Call.8H2 was coupled to Affigel 10 chromatographic resin (BioRad, Hercules, CA) and protein was isolated by specific antibody-protein interaction. Antibody was conjugated to the resin, according to the BioRad suggested protocol, at a concentration of approximately 5 mg antibody per ml of resin. Following the conjugation reaction, excess antibody was removed and the resin blocked with three volumes of 0.1 M ethanolamine. The resin was then washed with thirty column volumes of phosphate buffered saline (PBS).

Twenty-five grams of a single dog spleen were homogenized in 250 ml of buffer containing 0.32 M sucrose in 25 mM Tris-HCl, Ph 8.0, with protease inhibitors. Nuclei and cellular debris were pelleted with centrifugation at 1000 g for 15 minutes. Membranes were pelleted from the supernatant with centrifugation at 100,000 g for 30 minutes. The membrane pellet was resuspended in 200 ml lysis buffer (50 mM NaCl, 50 mM borate, pH 8.0, with 2% NP-40) and incubated for 1 hour on ice. Insoluble material was then pelleted by centrifugation at 100,000 g for 60 minutes. Ten milliliters of the cleared lysate were transferred to a 15 ml polypropylene tube with 0.5 ml Call.8H2- conjugated Affigel 10 resin described above. The tube was incubated overnight at 4°C with rotation and the resin subsequently washed with 50 column volumes

D-PBS. The resin was then transferred to a microfuge tube and boiled for ten minutes in 1 ml Laemmli (non-reducing) sample buffer containing 0.1 M Tris- HC1, pH 6.8, 2% SDS, 20% glycerol and 0.002% bromophenol blue. The resin was pelleted by centrifugation and discarded; the supernatant was treated with 1/15 volume /3-mercaptoethanol (Sigma, St. Louis, MO) and run on a 7% polyacrylamide gel. The separated proteins were transferred to Immobilon PVDF membrane (Millipore, Bedford, MA) as follows.

The gels were washed once in deionized, Millipore-filtered water and equilibrated for 15-45 minutes in 10 mM 3-[cyclohexylamino]-l- propanesulfonic acid (CAPS) transfer buffer, pH 10.5, with 10% methanol.

Immobilon membranes were moistened with methanol, rinsed with filtered water, and equilibrated for 15-30 minutes in CAPS transfer buffer. The initial transfer was carried out using a Biorad transfer apparatus at 70 volts for 3 hours. The Immobilon membrane was removed after transfer and stained in filtered 0.1 % R250 Coomassie stain for 10 minutes. Membranes were destained in 50% methanol/10% acetic acid three times, ten minutes each time. After destaining, the membranes were washed in filtered water and air-dried.

Protein bands of approximately 150 kD, 95 kD, 50 kD and 30 kD were detected. Presumably the 50 kD and 30 kD bands resulted from antibody contamination. N- terminal sequencing was then attempted on both the 150 kD and 95 kD bands, but the 95 kD protein was blocked, preventing sequencing. The protein band of 150 kD was excised from the membrane and directly sequenced with an Applied Biosystems (Foster City, CA) Model 473 A protein sequencer according to the manufacturer's instructions. The resulting amino acid sequence is set in SEQ ID NO: 5 using single letter amino acid designations.

FNLDVEEPMVFQ (SEQ ID NO: 5)

The identified sequence included the FNLD sequence characteristic of subunits of the integrin family [Tamura, et al, J. Cell.Biol 717:1593-1604 (1990)].

From the N-terminal sequence information, three oligonucleotide probes were designed for hybridization: a) "Tommer," a fully degenerate oligonucleotide; b) "Patmer," a partially degenerate oligonucleotide; and c) "Guessmer," a nondegenerate oligonucleotide based on mammalian codon usage. These probes are set out below as SEQ ID NOS: 6, 7 and 8, respectively. Nucleic acid symbols are in accordance with 37 C.F.R. §1.882 for these and all other nucleotide sequences herein.

5 '-TTYAAYYTGGAYGTNGARGARCCNATGGTNTTYCA-3[SEQ ID NO: 6) 5 '-TTCAACCTGGACGTGGAGGAGCCCATGGTGTTCCAA<5EQ ID NO: 7) 5 '-TTCAACCTGGACGTNGAASANCCCATGGTCTTCCAA-φEQ ID NO: 8)

Based on sequencing data, no relevant clones were detected using these oligonucleotides in several low stringency hybridizations to a canine spleen/peripheral blood macrophage cDNA library cloned into λZAP (Stratagene,

La Jolla, CA).

Four other oligonucleotide primers, designated 5 'Deg, 5 'Spec,

3 'Deg and 3 'Spec (as set out in SEQ ID NOS: 9, 10, 11 and 12, respectively, wherein Deg indicates degenerate and Spec indicates non-degenerate) were subsequently designed based on the deduced N-terminal sequence for attempts to amplify canine G-T I sequences by PCR from phage library DNA purified from plate lysates of the Stratagene library described above.

5'-TTYAAYYTNGAYGTNGARGARCC-3' (SEQID NO: 9) 5'-TTYAAYYTGGACGTNGAAGA-3' (SEQID NO: 10)

5'-TGRAANACCATNGGYTC-3' (SEQID NO: 11)

5'-TTGGAAGACCATNGGYTC-3' (SEQIDNO: 12)

The O.TM _! oligonucleotide primers were paired with T3 or T7 vector primers, as set out in SEQ ID NOS: 13 and 14, respectively, which hybridize to sequences flanking the polylinker region in the Bluescript phagemid found in λZAP.

5'-ATTAACCCTCACTAAAG-3' (SEQID NO: 13)

5'-AATACGACTCACTATAG-3' (SEQID NO: 14)

The PCR amplification was carried out in Taq buffer (Boehringer Mannheim, Indianapolis, IN) containing magnesium with 150 ng of library DNA, 1 μg of each primer, 200 μM dNTPs and 2.5 units Taq polymerase (Boehringer Mannheim) and the products were separated by electrophoresis on a 1 % agarose gel in Tris-Acetate-EDTA (TAE) buffer with 0.25 μg/ml ethidium bromide. DNA was transferred to a Hybond (Amersham, Arlington Heights, IL) membrane by wicking overnight in 10X SSPE. After transfer, the immobilized DNA was denatured with 0.5 M NaOH with 0.6 M NaCl, neutralized with 1.0 M Tris-HCl, pH 8.0, in 1.5 M NaCl, and washed with 2X SSPE before UV crosslinking with a Stratalinker (Stratagene) crosslinking apparatus. The membrane was incubated in prehybridization buffer (5X SSPE, 4X Denhardts, 0.8% SDS, 30% formamide) for 2 hr at 50 °C with agitation.

Oligonucleotide probes 5 'Deg, 5 'Spec, 3 'Deg and 3 'Spec (SEQ ID NOS: 9, 10, 11 and 12, respectively) were labeled using a Boehringer Mannheim kinase buffer with 100-300 μCi 7P ³² -dATP and 1-3 units of polynucleotide kinase for 1-3 hr at 37 ^* C. Unincorporated label was removed with Sephadex G-25 fine (Pharmacia, Piscataway, NJ) chromatography using 10 mM

Tris-HCl, pH 8.0, 1 mM EDTA (TE) buffer and the flow-through added directly to the prehybridization solution. Membranes were probed for 16 hr at 42 "C with

agitation and washed repeatedly, with a final stringency wash of IX SSPE/0.1 % SDS at 50° for 15 min. The blot was then exposed to Kodak X-Omat AR film for 1-4 hours at -80 °C.

The oligonucleotides 5 'Deg, 5 'Spec, 3 'Deg and 3 'Spec only hybridized to PCR products from the reactions in which they were used as primers and failed to hybridize as expected to PCR products from the reactions in which they were not used as primers. Thus, it was concluded that none of the PCR products were specific for a- ^ because no product hybridized with all of the appropriate probes.

Example 3

Large Scale Affinity Purification Of Canine o; _τ ^ ₁₁ For Internal Sequencing

In order to provide additional amino acid sequence for primer design, canine a- ^ was purified for internal sequencing. Three sections of frozen spleen (approximately 50 g each) and frozen cells from two partial spleens from adult dogs were used to generate protein for internal sequencing. Fifty grams of spleen were homogenized in 200-300 ml borate buffer with a Waring blender. The homogenized material was diluted with 1 volume of buffer containing 4% NP-40, and the mixture then gently agitated for at least one hour. The resulting lysate was cleared of large debris by centrifugation at 2000 g for 20 min, and then filtered through either a Corning (Corning, NY) prefilter or a

Corning 0.8 micron filter. The lysate was further clarified by filtration through the Corning 0.4 micron filter system.

Splenic lysate and the antibody-conjugated Affigel 10 resin described in Example 2 were combined at a 150: 1 volume ratio in 100 ml aliquots and incubated overnight at 4°C with rocking. The lysate was removed after centrifugation at 1000 g for 5 minutes, combined with more antibody-conjugated Affigel 10 resin and incubated overnight as above. The absorbed resin aliquots were then combined and washed with 50 volumes D-PBS/0.1 % Tween 20 and the

resin transferred to a 50 ml Biorad column. Adsorbed protein was eluted from the resin with 3-5 volumes of 0.1 M glycine (pH 2.5); fractions of approximately 900 μl were collected and neutralized with 100 μl 1 M Tris buffer, pH 8.0. Aliquots of 15 μl were removed from each fraction and boiled in an equal volume of 2X Laemmli sample buffer with 1/15 volume 1 M dithiothreitol (DTT). These samples were electrophoresed on 8% Novex (San Diego, CA) polyacrylamide gels and visualized either by Coomassie stain or by silver stain using a Daiichi kit (Enprotech, Natick, MA) according to the manufacturer's suggested protocol. Fractions which contained the largest amounts of protein were combined and concentrated by vacuum. The remaining solution was diluted by 50% with reducing Laemmli sample buffer and run on 1.5 mm 7% polyacrylamide gels in Tris-glycine/SDS buffer. Protein was transferred from the gels to Immobilon membrane by the procedure described in Example 2 using the Hoefer transfer apparatus. The protein bands corresponding to canine were excised from

10 PVDF membranes and resulted in approximately 47 μg total protein. The bands were destained in 4 ml 50% methanol for 5 minutes, air dried and cut into 1 x 2 mm pieces. The membrane pieces were submerged in 2 ml 95% acetone at 4°C for 30 minutes with occasional vortexing and then air dried. Prior to proteolytic cleavage of the membrane bound protein, 3 mg of cyanogen bromide (CNBr) (Pierce, Rockford, IL) were dissolved in 1.25 ml 70% formic acid. This solution was then added to a tube containing the PVDF membrane pieces and the tube incubated in the dark at room temperature for 24 hours. The supernatant (SI) was then removed to another tube and the membrane pieces washed with 0.25 ml 70% formic acid. This supernatant (S2) was removed and added to the previous supernatant (SI). Two milliliters of Milli Q water were added to the combined supernatants (SI and S2) and the solution lyophilized. The PVDF membrane pieces were dried under nitrogen and extracted again with 1.25 ml 60% acetonitrile, 0.1 % tetrafluoroacetic acid (TFA) at 42 °C for 17 hours.

This supernatant (S3) was removed and the membrane pieces extracted again with 1.0 ml 80% acetonitrile with 0.08% TFA at 42 'C for 1 hour. This supernatant (S4) was combined with the previous supernatants (SI, S2 and S3) and vacuum dried. The dried CNBr fragments were then dissolved in 63 μl 8 M urea,

0.4 M NH ₄ HCO ₃ . The fragments were reduced in 5 μl 45 mM dithiothreitol (DTT) and subsequently incubated at 50 ^* C for 15 minutes. The solution was then cooled to room temperature and the fragments alkylated by adding 5 μl 100 mM iodoacetamide (Sigma, St. Louis, MO). Following a 15 minute incubation at room temperature, the sample was diluted with 187 μl Milli Q water to a final urea concentration of 2.0 M. Trypsin (Worthington, Freehold, NJ) was then added at a ratio of 1:25 (w:w) of enzyme to protein and the protein digested for 24 hours at 37 °C. Digestion was terminated with addition of 30 μl TFA.

The protein fragments were then separated with high performance liquid chromatography (HPLC) on a Waters 625 LC system (Millipore, Milford,

MA) using a 2.1 x 250 mm, 5 micron Vydac C-18 column (Vydac, Hesperia, CA) equilibrated in 0.05% TFA and HPLC water (buffer A). The peptides were eluted with increasing concentration of 80% acetonitrile in 0.04% TFA (buffer B) with a gradient of 38-75% buffer B for 65-95 minutes and 75-98% buffer B for 95-105 minutes. Peptides were fractionated at a flow rate of 0.2 ml/minute and detected at 210 nm.

Following fractionation, the amino acid sequence of the peptides was analyzed by automated Edman degradation performed on an Applied Biosystems Model 437A protein sequencer using the manufacturer's standard cycles and the Model 610A Data Analysis software program, Version 1.2.1. All sequencing reagents were supplied by Applied Biosystems. The amino acid sequences of seven of the eight internal fragments are set out below wherein "X" indicates the identity of the amino acid was not certain.

VFQEXGAGFGQ (SEQ ID NO 15) LYDXVAATGLXQPI (SEQ ID NO 16) PLEYXDVIPQAE (SEQ ID NO 17) FQEGFSXVLX (SEQ ID NO 18) TSPTFIXMSQENVD (SEQ ID NO 19)

LVVGAPLEVVAVXQTGR (SEQ ID NO 20) LDXKPXDTA (SEQ ID NO 21)

Primer Design

One internal amino acid sequence (set out in SEQ ID NO: 22) obtained was then used to design a fully degenerate oligonucleotide primer, designated p4(R) as set out in SEQ ID NO: 23.

FGEQFSE (SEQ ID NO: 22)

5 '-RAANCCYTCYTGRAAACTYTC-3 ' (SEQ ID NO: 23)

Example 4 PCR Cloning Of A Canine Q-TMI Fragment

The 5 ' portion of the canine gene was amplified from double-stranded canine splenic cDNA by PCR.

A. Generation of Double Stranded Canine Spleen cDNA One gram of frozen material from a juvenile dog spleen was ground in liquid nitrogen on dry ice and homogenized in 20 ml RNA-Stat 60 buffer (Tel-

Test B, Inc, Friendswood, TX). Four ml chloroform were added, and the solution extracted by centrifugation at 12,000 g for 15 minutes. RNA was precipitated from the aqueous layer with 10 ml ethanol. Poly A ⁺ RNA was then selected on Dynal Oligo dT Dynabeads (Dynal, Oslo, Norway). Five aliquots of 100 μg total RNA were combined and diluted with an equal volume of 2X binding

buffer (20 mM Tris-HCl, pH 7.5, 1.0 M LiCl, 1 mM EDTA, 0.1 % SDS). RNA was then incubated 5 minutes with the Oligo dT Dynabeads (1.0 ml or 5 mg beads for all the samples). Beads were washed with buffer containing 10 mM Tris-HCl, pH 7.5, 0.15 M LiCl, 1 mM EDTA and 0.1 % SDS, according to the manufacturer's suggested protocol prior to elution of poly A ⁺ mRNA with 2 mM

EDTA, pH 7.5. Double-stranded cDNA was then generated using the eluted poly A ⁺ mRNA and the Boehringer Mannheim cDNA Synthesis Kit according to the manufacturer's suggested protocol.

B. Isolation of a Partial Canine αχ _l cDNA Oligonucleotide primers 5 'Deg (SEQ ID NO: 9) and p4(R) (SEQ

ID NO: 23) were employed in a standard PCR reaction using 150 ng double- stranded cDNA, 500 ng of each primer, 200 μM dNTPs and 1.5 units Taq polymerase (Boehringer Mannheim) in Taq buffer (Boehringer Mannheim) with magnesium. The resulting products (1 μl of the original reaction) were subjected to a second round of PCR with the same primers to increase product yield. This band was eluted from a 1 % agarose gel onto Schleicher & Schuell (Keene, NH) NA45 paper in a buffer containing 10 mM Tris-HCl, pH 8, 1 mM EDTA, 1.5 M NaCl at 65 °C, precipitated, and ϋgated into the pCR ^tm II vector (Invitrogen, San Diego, CA) using the TA cloning kit (Invitrogen) and the manufacturer's suggested protocol. The ligation mixture was transformed by electroporation into

XL-1 Blue bacteria (Stratagene). One clone, 2.7, was determined to contain sequences corresponding to ot- _j n peptide sequences which were not utilized in design of the primers.

Sequencing was performed with an Applied Biosystems 373A DNA sequencer (Foster City, CA) with a Dye-deoxy terminator cycle sequence kit

(ABI) in which fluorescent-labeled dNTPs were incorporated in an asymmetric PCR reaction [McCabe, "Production of Single Stranded DNA by Asymmetric PCR," in PCR Protocols: A Guide to Methods and Applications. Innis, et al.

(eds.) pp. 76-83 Academic Press: New York (1990)] as follows. Samples were held at 96°C for 4 minutes and subjected to 25 cycles of the step sequence: 96°C, for 15 seconds; 50°C for 1 second; 60°C for 4 minutes. Sequence data was automatically down-loaded into sample files on the computer that included chromatogram and text files. The sequence of the entire insert of clone 2.7 is set out in SEQ ID NO: 24.

Attempts to isolate the full length canine α. _j Mi cDNA from the Stratagene library (as described in Example 2) were unsuccessful. Approximately 1 x 10 ⁶ phage plaques were screened by hybridization under low stringency conditions using 30% formamide with clone 2.7 as a probe, but no positive clones resulted. Attempts to amplify relevant sequences downstream from those represented in clone 2.7 using specific oligonucleotides derived from clone 2.7 or degenerate primers based on amino acid sequence from other peptide fragments paired with a degenerate oligonucleotide based on the conserved subunit amino acid motif GFFKR [Tamura, et al , supra] were also unsuccessful.

Example 5 Cloning Of A Putative Human Homolog Of Canine o; _τ ^ _ϊl

To attempt the isolation of a human sequence homologous to canine ^α TMi ^me approximately 1 kb canine ot-γ ^ fragment from clone 2.7 was used as a probe. The probe was generated by PCR under conditions described in

Example 2 using NT2 (as set out in SEQ ID NO: 25) and p4(R) (SEQ ID NO:

23) primers.

5 '-GTNTTYCARGARGAYGG-3 ' (SEQ ID NO: 25)

The PCR product was purified using the Qiagen (Chatsworth, GA) Quick Spin kit and the manufacturer's suggested protocol. The purified DNA (200 ng) was labeled with 200 μCi α ² PdCTP using the Boehringer Mannheim Random Prime

Labelling kit and the manufacturer's suggested protocol. Unincorporated isotope was removed with Sephadex G25 (fine) gravity chromatography. The probe was denatured with 0.2 N NaOH and neutralized with 0.4 M Tris-HCl, pH 8.0, before use. Colony lifts on Hybond filters (Amersham) of a human spleen cDNA library in pCDNA/Amp (Invitrogen, San Diego, CA) were prepared. The filters were initially denatured and neutralized as described in Example 2 and subsequently incubated in a prehybridization solution (8 ml/filter) with 30% formamide at 50 °C with gentle agitation for 2 hours. Labeled probe as described above was added to this solution and incubated with the filters for 14 hours at

42 X. The filters were washed twice in 2X SSC/0.1 % SDS at 37 C and twice in 2X SSC/0.1 % SDS at 50 °C. Final stringency washes were IX SSC/0.1 % SDS, twice at 65 "C (IX SSC is 150 mM NaCl, 15 mM sodium citrate, pH 7.0). Filters were exposed to Kodak X-Omat AR film for six hours with an intensifying screen. Colonies giving signals on duplicate lifts were streaked on LB medium with magnesium (LBM)/carbenicillin plates and incubated overnight at 37 'C. Resulting streaked colonies were lifted with Hybond filters and these filters were treated as above. The filters were hybridized under more stringent conditions with the 1 kb probe from clone 2.7, labeled as previously described, in a 50% formamide hybridization solution at 50 °C for 3 hours. Probed filters were washed with a final stringency of 0.1 X SSC/0.1 % SDS at 65 °C and exposed to Kodak X-Omat AR film for 2.5 hours at -80 'C with an intensifying screen. Positive colonies were identified and cultured in LBM/carbenicillin medium overnight. DNA from the cultures was prepared using the Promega Wizard miniprep kit according to the manufacturer's suggested protocol and the resulting

DNA was sequenced.

The initial screening resulted in 18 positive clones, while the secondary screening under more stringent hybridization conditions produced one positive clone which was designated 19A2. The DNA and deduced amino acid

sequences of the human α _d clone 19 A2 are set out in SEQ ID NOS: 1 and 2, respectively.

Characteristics Of The Human o. _d cDNA and Predicted Polypeptide

Clone 19A2 encompasses the entire coding region for the mature protein, plus 48 bases (16 amino acid residues) of the 5 ' upstream signal sequence and 241 bases of 3 ' untranslated sequence which do not terminate in a polyadenylation sequence. The core molecular weight of the mature protein is predicted to be around 125 kD. The extracellular domain is predicted to encompass approximately amino acid residues 17 through 1108 of SEQ ID NO: 2. This extracellular region is contiguous with about a 20 amino acid region homologous to the human CDllc transmembrane region (residues 1109 through 1128 of SEQ ID NO: 2). The cytoplasmic domain comprises approximately 30 amino acids (about residues 1129 through 1161 of SEQ ID NO: 2). The protein also contains a region (around residues 150 through 352) of approximately 202 amino acids homologous to the I (insertion) domain common to CDlla, CDllb and CDllc [Larson and Springer, supra], α _E [Shaw, et al , J.Biol. Chem. 269:6016-6025 (1994)] and in VLA-1 and VLA-2, [Tamura, et al , supra]. The I domain in other integrins has been shown to participate in ICAM binding [Landis, et al , J. Cell.Biol. 120: 1519-1527 (1993); Diamond, et al , J. Cell.Biol 720: 1031-1043 (1993)], suggesting that α _d may also bind members of the ICAM family of surface molecules. This region has not been demonstrated to exist in any other integrin subunits.

The deduced amino acid sequence of a _d shows approximately 36% identity to that of CDlla, approximately 60% identity to CDl lb and approximately 66 % identity to CD lie. An alignment of amino acid sequences for

(CDl lb SEQ ID NO: 3), CDllc (SEQ ID NO: 4) and _d (SEQ ID NO: 2) is presented in Figure 1.

The cytoplasmic domains of a subunits in β ₂ integrins are typically distinct from one another within the same species, while individual α subunits show high degrees of homology across species boundaries. Consistent with these observations, the cytoplasmic region of α _d differs markedly from CDlla, CDllb, and CDllc except for a membrane proximal GFFKR amino acid sequence which has been shown to be conserved among all α integrins [Rojiani, et al , Biochemistry 30: 9859-9866 (1991)]. Since the cytoplasmic tail region of integrins has been implicated in "inside out" signaling and in avidity regulation [Landis et al, supra], it is possible that α _d interacts with cytosolic molecules distinct from those interacting with CDlla, CDllb, and CDllc, and, as a result, participates in signaling pathways distinct from those involving other β ₂ integrins.

The extracellular domain of a _d contains a conserved DGSGS amino acid sequence adjacent the I-domain; in CDllb, the DGSGS sequence is a metal-binding region required for ligand interaction [Michishita, et al. Cell 72:857-867 (1993)]. Three additional putative cation binding sites in CDllb and

CDllc are conserved in the α _d sequence at amino acids 465-474, 518-527, and 592-600 in clone 19A2 (SEQ ID NO: 1). The a _d I-domain is 36% , 62%, and 57% identical to the corresponding regions in CDlla, CDllb, and CDllc, respectively, and the relatively low sequence homology in this region suggests that α _d may interact with a set of extracellular proteins distinct from proteins with which other known β ₂ integrins interact. Alternatively, the affinity of a _d for known β ₂ integrin ligands, for example, ICAM-1, ICAM-2 and/or ICAM-R, may be distinct from that demonstrated for the other β ₂ integrin/ICAM interactions. [See Example 12.]

Example 6

Northern Analysis of Human _d Expression in Tissues

In order to determine the relative level of expression and tissue specificity of _d , Northern analysis was performed using fragments from clone 19A2 as probes. Approximately 10 μg of total RNA from each of several human tissues or cultured cell lines were loaded on a formaldehyde agarose gel in the presence of 1 μg of ethidium bromide. After electrophoresis at 100 V for 4 hr, the RNA was transferred to a nitrocellulose membrane (Schleicher & Schuell) by wicking in 10X SSC overnight. The membrane was baked 1.5 hr at 80 °C under vacuum. Prehybridization solution containing 50% formamide in 3-(N- morpholino)propane sulfonic acid (MOPS) buffer was used to block the membrane for 3 hr at 42 °C. Fragments of clone 19A2 were labeled with the Boehringer Mannheim Random Prime kit according to the manufacturer's instructions including both αP ² dCTP and α.P ³² dTTP. Unincorporated label was removed on a Sephadex G25 column in TE buffer. The membrane was probed with 1.5 x 10 ⁶ counts per ml of prehybridization buffer. The blot was then washed successively with 2X SSC/0.1 % SDS at room temperature, 2X SSC/0.1 % SDS at 42 °C, 2X SSC/0.1 % SDS at 50°C, IX SSC/0.1 % SDS at 50°C, 0.5X SSC/0.1 % SDS at 50 'C and 0. IX SSC/0.1 % SDS at 50 °C. The blot was then exposed to film for 19 hr.

Hybridization using a BstXI fragment from clone 19A2 (corresponding to nucleotides 2011 to 3388 in SEQ ID NO: 1) revealed a weak signal in the approximately 5 kb range in liver, placenta, thymus, and tonsil total RNA. No signal was detected in kidney, brain or heart samples. The amount of RNA present in the kidney lane was minimal, as determined with ethidium bromide staining.

When using a second fragment of clone 19A2 (encompassing the region from bases 500 to 2100 in SEQ ID NO: 1), RNA transcripts of two different sizes were detected in a human multi-tissue Northern (MTN) blot using

polyA ⁺ RNA (Clontech). An approximately 6.5 kb band was observed in spleen and skeletal muscle, while a 4.5 kb band was detected in lung and peripheral blood leukocytes. The variation in sizes observed could be caused by tissue specific polyadenylation, cross reactivity of the probe with other integrin family members, or hybridization with alternatively spliced mRNAs.

Northern analysis using a third fragment from clone 19A2, spanning nucleotides 2000 to 3100 in SEQ ID NO: 1, gave results consistent with those using the other clone 19A2 fragments.

RNA from three myeloid lineage cell lines was also probed using the fragments corresponding to nucleotides 500 to 2100 and 2000 to 3100 in SEQ

ID NO:l. A THP-1 cell line, previously stimulated with PMA, gave a diffuse signal in the same size range (approximately 5.0 kb), with a slightly stronger intensity than the tissue signals. RNA from unstimulated and DMSO-stimulated HL-60 cells hybridized with the _d probe at the same intensity as the tissue samples, however, PMA treatment seemed to increase the signal intensity. Since

PMA and DMSO drive HL-60 cell differentiation toward monocyte/macrophage and granulocyte pathways, respectively, this result suggests enhanced α _d expression in monocyte/macrophage cell types. U937 cells expressed the _d message and this signal did not increase with PMA stimulation. No band was detected in Molt, Daudi, H9, JY, or Jurkat cells.

Example 7

Transient Expression of Human a _d Constructs

A. Generation of expression constructs

The human clone 19A2 lacks an initiating methionine codon and possibly some of the 5 ' signal sequence. Therefore, in order to generate a human expression plasmid containing 19A2 sequences, two different strategies were used.

In the first, two plasmids were constructed in which signal peptide sequences derived from genes encoding either CDllb or CDllc were spliced into clone

19A2 to generate a chimeric _d sequence. In the second approach, a third plasmid was constructed in which an adenosine base was added at position 0 in clone 19A2 to encode an initiating methionine.

The three plasmids contained different regions which encoded the 5 ' portion of the _d sequence or the chimeric a _d sequence. The o. _d region was

PCR amplified (see conditions in Example 2) with a specific 3 ' primer BamRev (set out below in SEQ ID NO: 26) and one of three 5 ' primers. The three 5 ' primers contained in sequence: (1) identical nonspecific bases at positions 1-6 allowing for digestion, an EcoRI site from positions 7-12 and a consensus Kozak sequence from positions 13-18; (2) a portion of the CDllb (primer ΕR1B) or

CDllc (primer ERIC) signal sequence, or an adenosine (primer ΕR1D); and (3) an additional 15-17 bases specifically overlapping 5 ' sequences from clone 19A2 to allow primer annealing. Primers ΕR1B, ERIC or ΕR1D are set out in SΕQ ID NOS: 27, 28 or 29, respectively, where the initiating methionine codon is underlined and the EcøRI site is double underlined.

5'-CCACTGTCAGGATGCCCGTG-3' (SΕQID NO: 26)

5'-AGTTACGAATTCGCCACCATGGCTCTACGGGTGCT^r6ΕDσDGB^: 27)

5'-AGTTACGAATTCGCCACCATGACTCGGACTGTGCTTT(SΕ(DCπi$rø: 28)

5'-AGTTACGAATTCGCCACCATGACCTTCGGCACTGTQaΞO ID NO: 29)

The resulting PCR product was digested with EcoRI and BamHl.

All three plasmids contained a common second _d region (to be inserted immediately downstream from the 5 ' region described in the previous paragraph) including the 3 ^' end of the α: _d clone. The second α _d region, which

extended from nucleotide 625 into the Xbal site in the vector 3 ' polylinker region of clone 19A2, was isolated by digestion of clone 19A2 with BamΗl and Xbal. Three ligation reactions were prepared in which the 3 ' a _d BamHI/Xbal fragment was ligated to one of the three 5 ' α _d EcoΕl/BamΗL fragments using Boehringer Mannheim ligase buffer and T4 ligase (1 unit per reaction). After a 4 hour incubation at 14 °C, an appropriate amount of vector pcDNA.3 (Invitrogen) digested with EcoRI and Xbal was added to each reaction with an additional unit of ligase. Reactions were allowed to continue for another 14 hours. One tenth of the reaction mixture was then transformed into competent XL-1 Blue cells. The resulting colonies were cultured and the DNA isolated as in Example 5. Digestion with EcoRI identified three clones which were positive for that restriction site, and thus, the engineered signal sequences. The clones were designated pATM.Bl (CDllb/α _d , from primer ΕR1B), pATM.CIO (CDllc/α _d , from primer ERIC) and pATM.D12 (adenosine/ α _d from primer ΕRld). The presence of the appropriate signal sequences in each clone was verified by nucleic acid sequencing.

B. Transfection of COS Cells

Expression from the a _d plasmids discussed above was effected by cotransfection of COS cells with the individual plasmids and a CD 18 expression plasmid, pRC.CDlδ. As a positive control, COS cells were also co-transfected with the plasmid pRC.CD18 and a CDlla expression plasmid, pDC.CDllA.

Cells were passaged in culture medium (DMEM/10%FBS/pen- strep) into 10 cm Corning tissue culture-treated petri dishes at 50% confluency 16 hours prior to transfection. Cells were removed from the plates with Versene buffer (0.5 mM NaEDTA in PBS) without trypsin for all procedures. Before transfection, the plates were washed once with serum-free DMEM. Fifteen micrograms of each plasmid were added to 5 ml transfection buffer (DMEM with 20 μg/ml DEAE-Dextran and 0.5 mM chloroquine) on each plate. After 1.5

hours incubation at 37 °C, the cells were shocked for 1 minute with 5 ml DMEM/10% DMSO. This DMSO solution was then replaced with 10 ml/plate culture medium.

Resulting transfectants were analyzed by ELISA, FACS, and immunoprecipitation as described in Examples 8, 9, and 10.

Example 8

ELISA Analysis of COS Transfectants

In order to determine if the COS cells co-transfected with CD 18 expression plasmid pRC.CDlδ and an _d plasmid expressed a _d on the cell surface in association with CD 18, ELISAs were performed using primary antibodies raised against CD18 (e.g., TS1/18 purified from ATCC HB203). As a positive control, ELISAs were also performed on cells co-transfected with the CD 18 expression plasmid and a CDlla expression plasmid, pDC.CDllA. The primary antibodies in this control included CD 18 antibodies and anti-CD 11a antibodies (e.g., TS1/22 purified from ATCC HB202).

For ELISA, cells from each plate were removed with Versene buffer and transferred to a single 96-well flat-bottomed Corning tissue culture plate. Cells were allowed to incubate in culture media 2 days prior to assay. The plates were then washed twice with 150 μl/well D-PBS/0.5% teleost skin gelatin (Sigma) solution. This buffer was used in all steps except during the development. All washes and incubations were performed at room temperature. The wells were blocked with gelatin solution for 1 hour. Primary antibodies were diluted to 10 μg/ml in gelatin solution and 50 μl were then added to each well. Triplicate wells were set up for each primary antibody. After 1 hour incubation, plates were washed 3X with 150 μl/well gelatin solution. Secondary antibody

(goat anti-mouse Ig/HRP-Fc specific [Jackson, West Grove, PA]) at a 1:3500 dilution was added at 50 μl/well and plates were incubated for 1 hour. After three washes, plates were developed for 20 minutes with 100 μl/well o-

phenyldiamine (OPD) (Sigma) solution (1 mg/ml OPD in citrate buffer) before addition of 50 μl/well 15% sulfuric acid.

Analysis of transfectants in the ELISA format with anti-CD 18 specific antibodies revealed no significant expression above background in cells transfected only with the plasmid encoding CDl 8. Cells co-transfected with plasmid containing CDlla and CD 18 showed an increase in expression over background when analyzed with CD 18 specific antibodies or with reagents specific for CDlla. Further analysis of cells co-transfected with plasmids encoding CD 18 and one of the α _d expression constructs (pATM.ClO or pATM.D12) revealed that cell surface expression of CD 18 was rescued by concomitant expression of α _d . The increase in detectable CD 18 expression in COS cells transfected with pATM.ClO or ρATM.D12 was comparable to that observed in co-transfected CDlla/CD18 positive control cells.

Example 9 FACS Analysis of COS Transfectants

For FACS analysis, cells in petri dishes were fed with fresh culture medium the day after transfection and allowed to incubate 2 days prior to the assay. Transfectant cells were removed from the plates with 3 ml Versene, washed once with 5 ml FACS buffer (DMEM/2% FBS/0.2% sodium azide) and diluted to 500,000 cells/sample in 0.1 ml FACS buffer. Ten microliters of either

1 mg/ml FLTC-conjugated CD18, CDlla, or CDllb specific antibodies (Becton Dickinson) or 800 μg/ml CFSE-conjugated murine 23F2G (anti-CD 18) (ATCC HB11081) were added to each sample. Samples were then incubated on ice for 45 minutes, washed 3X with 5 ml/wash FACS buffer and resuspended in 0.2 ml FACS buffer. Samples were processed on a Becton Dickinson FACscan and the data analyzed using Lysys II software (Becton Dickinson).

COS cells transfected with CD 18 sequences only did not stain for CD 18, CDl la or CDllb. When co-transfected with CDl la CD18, about 15%

of the cells stained with antibodies to CDlla or CD18. All cells transfected with CD 18 and any _d construct resulted in no detectable staining for CDlla and CDllb. The pATM.Bl, pATM.ClO and pATM.D12 groups stained 4%, 13% and 8% positive for CDl 8, respectively. Fluorescence of the positive population in the CDl la/CD 18 group was 4-fold higher than background. In comparison, the co-transfection of α _d constructs with the CD 18 construct produced a positive population that showed a 4- to 7-fold increase in fluorescence intensity over background.

Example 10 Biotin-Labeled Immunoprecipitation of

Human α _d/ CD18 Complexes from Co-transfected COS Cells

Immunoprecipitation was attempted on cells co-transfected with

CD 18 and each of the _d expression plasmids separately described in Example 7 in order to determine if _d could be isolated as part of the aβ heterodimer complex characteristic of integrins.

Transfected cells (1-3 x 10 ⁸ cells/group) were removed from petri dishes with Versene buffer and washed 3 times in 50 ml/group D-PBS. Each sample was labeled with 2 mg Sulpho-NHS Biotin (Pierce, Rockford, IL) for 15 minutes at room temperature. The reaction was quenched by washing 3 times in 50 ml/sample cold D-PBS. Washed cells were resuspended in 1 ml lysis buffer

(1 % NP40, 50 mM Tris-HCl, pH 8.0, 0.2 M NaCl, 2 mM Ca ^{+ +} , 2 mM Mg ^{+ +} , and protease inhibitors) and incubated 15 minutes on ice. Insoluble material was pelleted by centrifugation at 10,000 g for 5 minutes, and the supernatant removed to fresh tubes. In order to remove material non-specifically reactive with mouse immunoglobulin, a pre-clearance step was initially performed. Twenty-five micrograms of mouse immunoglobulin (Cappel, West Chester, PA) was incubated with supernatants at 4°C. After 2.5 hr, 100 μl (25 μg) rabbit anti-mouse Ig conjugated Sepharose (prepared from Protein A Sepharose 4B and rabbit anti- mouse IgG, both from Zymed, San Francisco, CA) was added to each sample;

incubation was continued at 4 ^* C with rocking for 16 hours. Sepharose beads were removed from the supematants by centrifugation. After pre-clearance, the supematants were then treated with 20 μg anti-CD 18 antibody (TS1.18) for 2 hours at 4°C. Antibody /antigen complexes were isolated from supematants by incubation with 100 μl/sample rabbit anti-mouse/Protein A-sepharose preparation described above. Beads were washed 4 times with 10 mM HEPES, 0.2 M NaCl, and 1 % Triton-X 100. Washed beads were pelleted and boiled for 10 minutes in 20 μl 2X Laemmli sample buffer with 2% /3-mercaptoethanol. Samples were centrifuged and run on an 8% prepoured Novex polyacrylamide gel (Novex) at 100 V for 30 minutes. Protein was transferred to nitrocellulose membranes

(Schleicher & Schuell) in TBS-T buffer at 200 mAmps for 1 hour. Membranes were blocked for 2 hr with 3% BSA in TBS-T. Membranes were treated with 1:6000 dilution of Strep-avidin horse radish peroxidase (POD) (Boehringer Mannheim) for 1 hour, followed by 3 washes in TBS-T. The Amersham Enhanced Chemiluminescence kit was then used according to the manufacturer's instructions to develop the blot. The membrane was exposed to Hyperfilm MP (Amersham) for 0.5 to 2 minutes.

Immunoprecipitation of CD 18 complexes from cells transfected with pRC.CD18 and either pATM.Bl, pATM.ClO or pATM.D12 revealed surface expression of a heterodimeric species consisting of approximately 100 kD β chain, consistent with the predicted size of CD 18, and an chain of approximately 150 kD, corresponding to α _d .

Example 11

Stable Transfection of Human a _d in Chinese Hamster Ovary Cells To determine whether _d is expressed on the cell surface as a heterodimer in association with CD18, cDNAs encoding each chain were both transiently and stably transfected into a cell line lacking both α _d and CDl 8.

For these experiments, a _d cDNA was augmented with additional leader sequences and a Kozak consensus sequence, as described in Example 7, and subcloned into expression vector pcDNA3. The final construct, designated pATM.D12, was co-transfected with a modified commercial vector, pDCl.CDlδ encoding human CD 18 into dihydrofolate reductase (DHFR) ^" Chinese hamster ovary (CHO) cells. The plasmid pDCl.CD18 encodes a DHFR ⁺ marker and transfectants can be selected using an appropriate nucleoside-deficient medium. The modifications which resulted in pDCl.CD18 are as follows.

The plasmid pRC/CMV (Invitrogen) is a mammalian expression vector with a cytomegalovirus promoter and ampicillin resistance marker gene.

A DHFR gene from the plasmid pSCl 190-DHFR was inserted into pRC/CMV 5 ' of the SV40 origin of replication. In addition, a poly linker from the 5 ' region of the plasmid pHF2G-DHF was ligated into the pRC/CMV/DHFR construct, 3 ' to the DHFR gene. CD 18 encoding sequences are subsequently cloned into the resulting plasmid between the 5 ' flanking polylinker region and the bovine growth hormone poly A encoding region.

Surface expression of CD 18 was analyzed by flow cytometry using the monoclonal antibody TS1/18. Heterodimer formation detected between a _d and CD 18 in this cell line was consistent with the immunoprecipitation described in Example 10 with transient expression in COS cells.

Example 12

Human α _d binds to ICAM-R in a CD18-dependent fashion

In view of reports that demonstrate interactions between the leukocyte integrins and intercellular adhesion molecules (ICAMs) which mediate cell-cell contact [Hynes, Cell 69: 11-25 (1992)], the ability of CHO cells expressing α _d /CD18 to bind ICAM-1, ICAM-R, or VCAM-1 was assessed by two methods.

In replicate assays, soluble ICAM-1, ICAM-R, or VCAM-1 IgGl fusion proteins were immobilized on plastic and the ability of α _d /CD18 CHO transfected cells to bind the immobilized ligand was determined. Transfected cells were labeled internally with calcein, washed in binding buffer (RPMI with 1 % BSA), and incubated in either buffer only (with or without 10 ng/ml PMA) or buffer with anti-CD18 monoclonal antibodies at 10 μg/ml. Transfected cells were added to 96-well Immulon 4 microtiter plates previously coated with soluble ICAM-1/IgGl, ICAM-R IgGl or VCAM-1/IgGl fusion protein, or bovine serum albumin (BSA) as a negative control. Design of the soluble forms of these adhesion molecules is described and fully disclosed in co-pending and co-owned

U.S. Patent Application Serial No. 08/102,852, filed August 5, 1993. Wells were blocked with 1 % BSA in PBS prior to addition of labeled cells. After washing the plates by immersion in PBS with 0.1 % BSA for 20 minutes, total fluorescence remaining in each well was measured using a Cytofluor 2300 (Millipore, Milford, MA).

In experiments with immobilized ICAMs, α _d /CD 18 co- transfectants consistently showed a 3-5 fold increase in binding to ICAM-R/IgGl wells over BSA coated wells. The specificity and CD18-dependence of this binding was demonstrated by the inhibitory effects of anti-CD 18 antibody TS1/18. The binding of cells transfected with CDl la/CD 18 to ICAM-1/IgGl wells was comparable to the binding observed with BSA coated wells. CDl la/CD18 transfected cells showed a 2-3 fold increase in binding to ICAM-1/IgGl wells only following pretreatment with PMA. PMA treatment of α _d /CD18 transfectants did not affect binding to ICAM-1/IgGl or ICAM-R/IgGl wells. No detectable binding of α _d /CD18 transfectants to VCAM-1/IgGl wells was observed.

Binding of α _d /CD18-transfected cells to soluble ICAM-1/IgGl, ICAM-R/IgGl, or VCAM-1/IgGl fusion proteins was determined by flow cytometry. Approximately one million α _d /CD18-transfected CHO cells (grown in spinner flasks for higher expression) per measurement were suspended in 100 μl

binding buffer (RPMI and 1 % BSA) with or without 10 μg/ml anti-CD 18 antibody. After a 20 minute incubation at room temperature, the cells were washed in binding buffer and soluble ICAM-1/IgGl or ICAM-R/IgGl fusion protein was added to a final concentration of 5 μg/ml. Binding was allowed to proceed for 30 minute at 37°C, after which the cells were washed three times and resuspended in 100 μl binding buffer containing FITC-conjugated sheep anti- human IgGl at a 1:100 dilution. After a 30 minute incubation, samples were washed three times and suspended in 200 μl binding buffer for analysis with a Becton Dickinson FACScan. Approximately 40-50% of the o. _d /CD18 transfectants indicated binding to ICAM-R/IgGl, but no binding to ICAM-1/IgGl or VCAM-1/IgGl proteins. Pretreatment of transfected cells with PMA has no effect on α. _d /CD18 binding to either ICAM-1/IgGl, ICAM-R/IgGl or VCAM-1/IgGl, which was consistent with the immobilized adhesion assay. Binding by ICAM-R was reduced to background levels after treatment of α _d /CD18 transfectants with anti-

CD18 antibody TS1/18.

The collective data from these two binding assays illustrate that α. _d /CD18 binds to ICAM-R and does so preferentially as compared to ICAM-1 and VCAM-1. The α _d /CD18 binding preference for ICAM-R over ICAM-1 is opposite that observed with CDlla/CD18 and CDllb/CD18. Thus modulation of α _d /CD18 binding may be expected to selectively affect normal and pathologic immune function where ICAM-R plays a prominent role. Moreover, results of similar assays, in which antibodies immunospecific for various extracellular domains of ICAM-R were tested for their ability to inhibit binding of ICAM-R to α _d /CD 18 transfectants, indicated that α _d /CD 18 and CD 1 la/CD 18 interact with different domains of ICAM-R.

The failure of CDlla/CD18 to bind ICAM-1/IgGl or ICAM- R/IgGl in solution suggests that the affinity of binding between CDlla/CD18 and ICAM-1 or ICAM-R is too low to permit binding in solution. Detection of

α _d /CD18 binding to ICAM-R/IgGl, however, suggests an unusually high binding affinity.

o; _d Binding to iC3b

Complement component C3 can be proteolytically cleaved to form the complex iC3b, which initiates the altemative pathway of complement activation and leads ultimately to cell-mediated destruction of a target. Both CDllb and CDllc have been implicated in iC3b binding and subsequent phagocytosis of iC3b-coated pai icles. A peptide fragment in the CDl lb I domain has recently been identified as the site of iC3b interaction [Ueda, et al , Proc.Natl.Acad.Sci. (USA) PI: 10680-10684 (1994)]. The region of iC3b binding is highly conserved in CDllb, CDllc, and a _d , suggesting an α _d /iC3b binding interaction.

Binding of α _d to iC3b is performed using transfectants or cell lines naturally expressing a _d (for example, PMA-stimulated HL60 cells) and iC3b- coated sheep red blood cells (sRBC) in a rosette assay [Dana, et al. , J. Clin. Invest.

75: 153-159 (1984)]. The abilities of α _d /CD18 CHO transfectants, VLA4-CHO transfectants (negative control) and PMA-stimulated HL60 cells (positive control) to form rosettes are compared in the presence and absence of an anti-CD 18 monoclonal antibody (for example TS 1/18.1).

Example 13

Screening by Scintillation Proximity Assay

Specific inhibitors of binding between the α _d ligands of the present invention and their binding partners (a _d ligand/anti-ligand pair) may be determined by a variety of means, such as scintillation proximity assay techniques as generally described in U.S. Patent No. 4,271,139, Hart and Greenwald,

Mol.Immunol 72:265-267 (1979), and Hart and Greenwald, J.Nuc.Med. 20: 1062-1065 (1979), each of which is incorporated herein by reference.

Briefly, one member of the a _d ligand/anti-ligand pair is bound to a solid support. A fluorescent agent is also bound to the support. Alternatively, the fluorescent agent may be integrated into the solid support as described in U.S. Patent No. 4,568,649, incorporated herein by reference. The non-support bound member of the α _d ligand/anti-ligand pair is labeled with a radioactive compound that emits radiation capable of exciting the fluorescent agent. When the ligand binds the radiolabeled anti-ligand, the label is brought sufficiently close to the support-bound fluorescer to excite the fluorescer and cause emission of light. When not bound, the label is generally too distant from the solid support to excite the fluorescent agent, and light emissions are low. The emitted light is measured and correlated with binding between the ligand and the anti-ligand. Addition of a binding inhibitor to the sample will decrease the fluorescent emission by keeping the radioactive label from being captured in the proximity of the solid support. Therefore, binding inhibitors may be identified by their effect on fluorescent emissions from the samples. Potential anti-ligands to o; _d may also be identified by similar means.

Example 14

Soluble Human α. _d Expression Constructs

The expression of full-length, soluble human o: _d /CD18 heterodimeric protein provides easily purified material for immunization and binding assays. The advantage of generating soluble protein is that it can be purified from supematants rather than from cell lysates (as with full-length membrane-bound α _d /CD18); recovery in therefore improved and impurities reduced. The soluble a _d expression plasmid was constructed as follows. A nucleotide fragment corresponding to the region from bases 0 to 3161 in SEQ ID NO: 1, cloned into plasmid pATM.D12, was isolated by digestion with Hindlll and Aatll. A PCR fragment corresponding to bases 3130 to 3390 in SEQ ID NO:

1, overlapping the HindlJl/Aatll fragment and containing an addition Mlul restriction site at the 3 ' terminus, was amplified from pATM.D12 with primers sHAD.5 and sHAD.3 set out in SEQ ID NOS: 30 and 31, respectively.

5'-TTGCTGACTGCCTGCAGTTC-3' (SEQIDNO: 30) 5'-GTTCTGACGCGTAATGGCATTGTAGACCTCGTCTTC(SEQIDNO: 31)

The PCR amplification product was digested with AatJ and MM and ligated to the HindHI/AatTL fragment. The resulting product was ligated into Hindlll/Mlul- digested plasmid pDCl.s.

This construct is co-expressed with soluble CD 18 in stably transfected CHO cells, and expression is detected by autoradiographic visualization of immunoprecipitated CD 18 complexes derived from ³⁵ S-methionine labeled cells. The construct is also co-expressed with CD 18 in 293 cells

[Berman, e al , J.Cell.Biochem. 52: 183-195 (1993)].

Soluble Human a _d I Domain Expression Constructs It has previously been reported that the I domain in CDlla can be expressed as an independent structural unit that maintains ligand binding capabilities and antibody recognition [Randi and Hogg, J.Biol Chem. 269: 12395- 12398 (1994); Zhout, et al , J.Biol. Chem. 269: 17075-17079 (1994); Michishita, et al, Cell 72:857-867 (1993)]. To generate a soluble fusion protein comprising the α _d I domain and human IgG4, the a _d I domain is amplified by PCR using primers designed to add flanking BamΑl and Xhol restriction sites to facilitate subcloning. These primers are set out in SEQ ID NOS: 32 and 33 with restriction sites underlined.

5 '-ACGTATGCAGGATCCCATCAAGAGATGGACATCGCTSEO ID NO: 32) 5 '-ACTGCATGTCTCGAGGCTGAAGCCTTCTTGGGACAΕSEq ID NO: 33)

The C nucleotide immediately 3 ' to the BamYΩ. site in SEQ ID NO: 32 corresponds to nucleotide 435 in SEQ ID NO: 1; the G nucleotide 3 ' to the Xhol site in SEQ ID NO: 33 is complementary to nucleotide 1067 in SEQ ID NO: 1. The amplified I domain is digested with the appropriate enzymes, the purified fragment ligated into the mammalian expression vector pDCs and the prokaryotic expression vector pGEX-4T-3 (Pharmacia) and the I domain fragment sequenced. The fusion protein is then expressed in COS, CHO or E.coli cells transfected or transformed with an appropriate expression construct.

Given the affinity of a _d for ICAM-R, expression of the a _d I domain may be of sufficient affinity to be a useful inhibitor of cell adhesion in which a _d participates.

Analysis of Human a _d I Domain/IgG4 Fusion Proteins

Protein was resolved by SDS -PAGE under reducing and non- reducing conditions and visualized by either silver staining or Coomassie staining. Protein was then transferred to Immobilon PVDF membranes and subjected to

Westem blot analysis using anti-human IgG monoclonal antibodies or anti-bovine Ig monoclonal antibodies.

Protein detected was determined to migrate at about 120 kD under non-reducing conditions and at about 45 kD under reducing conditions. Minor bands were also detected on non-reducing gels at approximately 40-50 kD which were reactive with the anti-human, but not anti-bovine, antibodies. A 200 kD minor band was determined to be bovine Ig by Westem blot.

Binding Assays Using I Domain Expression Products

The ability of the I domain to specifically recognize ICAM-R/IgG chimeric protein was tested in an ELISA format. Serial dilutions of α. _d

I domain IgG4 fusion protein (Iα _d /IgG4) in TBS were incubated with ICAM- 1/IgG, ICAM-R/IgG, VCAM-1/IgG, or an irrelevant IgGl myeloma protein

immobilized on Immulon IV RIA/EIA plates. CDlla I domain/IgG chimeric protein and human IgG4/kappa myeloma protein were used as negative controls. Bound IgG4 was detected with the biotinylated anti-IgG4 monoclonal antibody HP6023 followed by addition of strepavidin-peroxidase conjugate and development with substrate o-phenyldiamine.

In repeated assays, no binding of the CDlla/IgG4 protein or the IgG4 myeloma protein was detected with any of the immobilized proteins. The Iα. _d /IgG4 protein did not bind to fish skin gelatin or bovine serum albumin blocking agents, human IgGl, or ICAM-1/IgG. A two to three fold increase in binding signal over background was detected in ICAM-R/IgG protein coated wells using 1-5 μg/ml concentrations of Iα. _d /IgG4 protein. The signal in VCAM-1/IgG protein coated wells was 7-10 fold higher than background. In previous assays, α _d /CD18 transfected CHO cells did not bind VCAM-1/IgG protein, suggesting that VCAM-1 binding may be characteristic of isolated I domain amino acid sequences.

Additional a _d I domain constructs

Additional a _d I domain constructs are generated in the same fashion as the previous construct, but incorporating more amino acids around the a _d I domain. Specific constructs include: i) sequences from exon 5 (amino acids 127- 353 in SEQ ID NO: 2), preceding the current construct, ii) the EF-hand repeats

(amino acids 17-603 in SEQ ID NO: 2) following the I domain, and iii) the alpha chain truncated at the transmembrane region (amino acids 17-1029 in SEQ ID NO: 2), with an IgG4 tail for purification and detection purposes. These constructs are ligated into either the mammalian expression vector pDCSl or the prokaryotic expression vector pGEX-4T-3 (Pharmacia) and the I domain sequenced. The fusion proteins are then be expressed in COS, CHO, or E.coli cells transformed or transfected with an appropriate expression construct. Protein are purified on a ProSepA column (Bioprocessing Limited, Durham, England),

tested for reactivity with the anti-IgG4 monoclonal antibody HP6023 and visualized on polyacrylamide gels with Coomassie staining.

In order to construct an expression plasmid for the entire a _d polypeptide, pATM.D12, described supra, is modified to express an α. _d -IgG4 fusion protein by the following method. IgG4 encoding DNA is isolated from the vector pDCSl by PCR using primers which individually incorporate a 5 ' Aatll restriction site (SEQ ID NO: 89) and a 3 ' Xbal restriction site (SEQ ID NO: 90).

5'-CGCTGTGACGTCAGAGTTGAGTCCAAATATGG-3' (SEQID NO: 89) 5'-GGTGACACTATAGAATAGGGC-3' (SEQID NO: 90)

Plasmid pATM.D12 is digested with Aatll and Xbal, and the appropriately digested and purified IgG4 PCR product ligated into the linear vector.

Examplfi 15

Production of Human α _d -Specific Monoclonal Antibodies

Transiently transfected cells from Example 7 were washed three times in Dulbecco's phosphate buffered saline (D-PBS) and injected at 5 x 10 ⁶ cells/mouse into Balb/c mice with 50 μg/mouse muramyl dipeptidase (Sigma) in

PBS. Mice were injected two more times in the same fashion at two week intervals. The pre-bleed and immunized serum from the mice were screened by FACS analysis as outlined in Example 9 and the spleen from the mouse with the highest reactivity to cells transfected with α _d /CD18 was fused. Hybridoma culture supematants were then screened separately for lack of reactivity against

COS cells transfected with CDl la/CD 18 and for reactivity with cells co- transfected with an a _d expression plasmid and CD 18.

This method resulted in no monoclonal antibodies.

As an altemative for production of monoclonal antibodies, soluble a _d I domain IgG4 fusion protein was affinity purified from supernatant of stably transfected CHO cells and used to immunize Balb/c mice as described above.

Hybridomas were established and supematants from these hybridomas were screened by ELISA for reactivity against a _d I domain fusion protein. Positive cultures were then analyzed for reactivity with full length α _d /CD18 complexes expressed on CHO transfectants.

Mouse 1908 received three initial immunizations of α _d /CD18 transfected CHO cells and two subsequent boosts with soluble α _d /CD18 heterodimer. Two final immunizations included 50 μg/mouse Iα _d /IgG4 fusion protein. The fusion produced 270 IgG-producing wells. Supernatant from 45 wells showed at least 7-fold higher binding to Io. _d /IgG4 fusion protein than to human IgG4 by ELISA. None of the supematants reacted to α _d /CD18 transfected CHO cells as determined by FACS analysis.

To determine whether the supematants were able to recognize integrin alpha subunit proteins in another context, fresh frozen splenic sections

were stained with supematants from 24 of the 45 wells. Three supematants were determined to be positive: one stained large cells in the red pulp, while two others stained scattered cells in the red pulp and also trabeculae.

These supematants were further analyzed by their ability to immunoprecipitate biotinylated CD 18 complexes from either α _d /CD 18 transfected

CHO cells or PMA-stimulated HL60 cells. Fusion wells with supematants that recognized protein in detergent lysates (which should not be as conformationally constrained as protein expressed as heterodimers) were selected for further subcloning. Monoclonal antibodies which recognize protein in detergent may be more useful in immunoprecipitation of heterodimeric complexes from transfectants, tissues, and cell lines.

As another altemative, monoclonal antibodies are generated as follows. Affinity purified o. _d /CD18 heterodimeric protein from detergent lysates of stably transfected CHO cells is used with 50 μg/ml muramyl dipeptidase to immunize Balb/c mice as described above. Mice receive three immunizations before serum reactivity against α _d /CD18 is determined by immunoprecipitation of biotinylated complexes in the CHO transfectants. Hybridomas from positive animals are established according to standard protocols, after which hybridoma cultures are selected by flow cytometry using α _d /CD18 transfectants. CDl la/CD 18 transfectants are utilized to control for CD18-only reactivity.

As another altemative for monoclonal antibody production, Balb/c mice undergo an immunization/immunosuppression protocol designed to reduce reactivity to CHO cell determinants on transfectants used for immunization. This protocol involves immunization with untransfected CHO cells and subsequent killing of CHO-reactive B-cell blasts with cyclophosphamide treatment. After three rounds of immunization and cyclophosphamide treatment are performed, the mice are immunized with o; _d /CD18 CHO transfected cells as described above.

As still another altemative, heterodimeric CD 18 complexes are immunoprecipitated from detergent lysates of whole spleen using an anti-CD 18

monoclonal antibody, following preclearance of CDlla/CD18 and CDllb/CD18. CDl la/CD 18 and CD lib/CD 18 complexes are precleared by affinity chromatography using monoclonal antibodies TS2/4 and Mol, respectively, coupled to a chromatographic resin. The remaining CD 18 complexes are used as an immunogen in Balb/c mice for the first immunization. Three immunizations are given at three week intervals, the initial immunization administered in conjunction with Freund's Complete Adjuvant and the subsequent immunizations with Freund's Incomplete Adjuvant. Serum is assayed for α _d -specific reactivity by immunoprecipitation. Resulting hybridomas are screened by flow cytometry with α: _d /CD18 CHO transfectants.

As another altemative, CD 18 complexes from detergent lysates of PMA stimulated HL60 cells are enriched by preclearance as described above. Other β2 integrins are cleared on the same columns. Immunization with the resulting complexes, hybridoma production, and screening protocols are performed as described supra.

Example 16

Analysis of a _d distribution with polyclonal serum

Tissue distribution of α _d /CD18 was determined using polyclonal antiserum. Antiserum used to stain tissue was obtained from a mouse immunized 3 times with α _d transfected CHO cells (D6.CHO, α _d /CD18) with adjuvant peptide and once with purified α _d /CD18 heterodimer. A final boost included only α _d /CD18 heterodimer. Approximately 100 μl immunized serum was precleared by addition of approximately 10 ⁸ LFA-1 -transfected CHO cells for 2 hours at 4°C. The resulting serum was assayed for a _d reactivity at dilutions of 1/5000, 1/ 10000, 1/20000 and 1/40000 on normal human spleen. The polyclonal antibody was reactive at a dilution of 1/20000, while a 1/40000 dilution stained very weakly.

Once serum was determined to have specific α _d reactivity, it was used to stain various lymphoid and non-lymphoid tissues. Monoclonal antibodies recognizing CD18, CDlla, CDllb, and CDllc were used in the same experiment as controls. Staining of normal spleen sections with a _d polyclonal sera, and monoclonal antibodies to CDlla, CDllb, CDl lc, and CD18 revealed the following results. The pattem observed with a _d polyclonal sera did not display the same pattem of labeling as CDlla, CDllb, CDllc, or CD18. There is a distinct pattem of labeling with some cells located in the marginal zone of the white pulp and a distinct labeling of cells peripheral to the marginal zone. This pattem was not observed with the other antibodies. Individual cells scattered throughout the red pulp were also labeled which may or may not be the same population or subset seen with CDl la and CD 18.

Labeling with CDl lc did display some cells staining in the marginal zone, but the antibody did not show the distinct ring pattem around the white pulp when compared to α _d polyclonal sera, nor did labeling in the red pulp give the same pattem of staining as α _d polyclonal sera.

Therefore, the labeling pattem seen with α. _d polyclonal serum was unique compared to that seen using antibodies to the other β ₂ integrins (CDlla, CDllb, CDllc, and CD18), and suggests that the in vivo distribution of a _d in man is dinstinct from that of other β ₂ integrins.

Example 17

Isolation of Rat cDNA Clones

In view of the existence of both canine and human a _d subunits, attempts were made to isolate homologous genes in other species, including rat (this example) and mouse (Example 17, infra).

A partial sequence of a rat cDNA showing homology to the human o: _d gene was obtained from a rat splenic λgtlO library (Clontech). The library was plated at 2 x 10 ⁴ pfu/plate onto 150 mm LBM/agar plates. The library was

lifted onto Hybond membranes (Amersham), denatured 3 minutes, neutralized 3 minutes and washed 5 minutes with buffers as described in standard protocols [Sambrook, et al, Molecular Cloning: a laboratory manual, p.2.110]. The membranes were placed immediately into a Stratalinker (Stratagene) and the DNA crosslinked using the autocrosslinking setting. The membranes were prehybridized and hybridized in 30% or 50% formamide, for low and high stringency conditions, respectively. Membranes were initially screened with a ³² P-labeled probe generated from the human α _d cDNA, corresponding to bases 500 to 2100 in clone 19A2 (SEQ ID NO: 1). The probe was labeled using Boehringer Mannheim's Random Prime Kit according to manufacturer's suggested protocol. Filters were washed with 2X SSC at 55 °C.

Two clones, designated 684.3 and 705.1, were identified which showed sequence homology to human a , human CDllb, and human CDllc. Both clones aligned to the human α _d gene in the 3 ' region of the gene, starting at base 1871 and extending to base 3012 for clone 684.3, and bases 1551 to 3367 for clone 705.1.

In order to isolate a more complete rat sequence which included the 5 ' region, the same library was rescreened using the same protocol as employed for the initial screening, but using a mouse probe generated from clone A1160 (See Example 17, infra). Single, isolated plaques were selected from the second screening and maintained as single clones on LBM/agar plates. Sequencing primers 434FL and 434FR (SEQ ID NOS: 34 and 35, respectively) were used in a standard PCR protocol to generate DNA for sequencing.

5 '-TATAGACTGCTGGGTAGTCCCCAC-3 ' (SEQ ID NO: 34) 5 '-TGAAGATTGGGGGTAAATAACAGA-3 ' (SEQ ID NO: 35)

DNA from the PCR was purified using a Quick Spin Column (Qiagen) according to manufacturer's suggested protocol.

Two clones, designated 741.4 and 741.11, were identified which overlapped clones 684.3 and 705.1; in the overlapping regions, clones 741.1 and

741.11 were 100% homologous to clones 684.3 and 705.1. A composite rat cDNA having homology to the human α _d gene is set out in SEQ ID NO: 36; the predicted amino acid sequence is set forth in SEQ ID NO: 37.

Cloning of the 5 ' end of Rat a _d

A 5 ' cDNA fragment for the rat a _d gene was obtained using a Clonetech rat spleen RACE cloning kit according to manufacturer's suggested protocol. The gene specific oligonucleotides used were designated 741.11#2R and 741.2#1R (SEQ ID NOS: 59 and 58, respectively).

5 '-CCAAAGCTGGCTGCATCCTCTC-3 ' (SEQ ID NO: 59)

5 '-GGCCTTGCAGCTGGACAATG-3 ' (SEQ ID NO: 58)

Oligo 741.11#2R encompasses base pairs 131-152 in SEQ ID NO: 36, in the reverse orientation and 741.2#1R encompasses bases pairs 696-715 in SEQ ID NO: 36, also in the reverse orientation. A primary PCR was carried out using the 3 '-most oligo, 741.2#1R. A second PCR followed using oligo 741.11#2R and DNA generated from the primary reaction. A band of approximately 300 base pairs was detected on a 1 % agarose gel.

The secondary PCR product was ligated into plasmid pCRTAII (Invitrogen) according to manufacturer's suggested protocol. White (positive) colonies were picked and added to 100 μl LBM containing 1 μl of a 50 mg/ml carbenicillin stock solution and 1 μl M13 K07 phage culture in individual wells in a round bottom 96 well tissue culture plate. The mixture was incubated at 37°C for 30 minutes to one hour. Following the initial incubation period, 100 μl of LBM (containing 1 μl of 50 mg/ml carbenicillin and a 1:250 dilution of a 10

mg/ml kanamycin stock solution) were added and the incubation was continued overnight at 37 °C.

Using a sterile 96 well metal transfer prong, supernatant from the 96 well plate was transferred to four Amersham Hybond nylon filters. The filters were denatured, neutralized and cross linked by standard protocols. The filters were prehybridized in 20 mis of prehybridization buffer (5X SSPE; 5X Denhardts; 1 % SDS; 50 ugs/ml denatured salmon sperm DNA) at 50°C for several hours while shaking.

Oligo probes 741.11#1 and 741.11#1R (SEQ ID NOS: 56 and 57, respectively), encompassing base pairs 86-105 (SEQ ID NO: 36) in the forward and reverse orientation respectively, were labeled as follows.

5'-CCTGTCATGGGTCTAACCTG-3' (SEQ IDNO: 56)

5'-AGGTTAGACCCATGACAGG-3' (SEQ ID NO: 57)

Approximately 65 ng oligo DNA in 12 μl dH ₂ 0 was heated to 65 °C for two minutes. Three μl of 10 mCi/ml 7- ³² P-ATP were added to the tube along with

4 μl 5x Kinase Buffer (Gibco) and 1 μl T4 DNA Kinase (Gibco). The mixture was incubated at 37°C for 30 minutes. Following incubation, 16 μl of each labeled oligo probe were added to the prehybridization buffer and filters and hybridization was continued overnight at 42° C. The filters were washed three times in 5X SSPE; 0.1 % SDS for 5 minutes per wash at room temperature, and autoradiographed for 6 hours. Positive clones were expanded and DNA purified using the Magic Mini Prep Kit (Promega) according to manufacturer's suggested protocol. Clone 2F7 was selected for sequencing and showed 100% homology

- clone 741.11 in the overlapping region. The complete rat a _d nucleic acid .sequence is set out in SEQ ID NO: 54; the amino acid sequence is set out in SEQ

ID NO: 55.

Characteristics of the Rat cDNA and Amino Acid Sequences

Neither nucleic acid nor amino acid sequences have previously been reported for rat a subunits in β ₂ integrins. However sequence comparisons to reported human β ₂ integrin subunits suggests that the isolated rat clone and its predicted amino acid sequence are most closely related to a _d nucleotide and amino acid sequences.

At the nucleic acid level, the isolated rat cDNA clone shows 80% identity in comparison to the human α _d cDNA; 68% identity in comparison to human CDllb; 70% identity in comparison to human CDllc; and 65% identity in comparison to mouse CDllb. No significant identity is found in comparison to human CDlla and to mouse CDlla.

At the amino acid level, the predicted rat polypeptide encoded by the isolated cDNA shows 70% identity in comparison to human o; _d polypeptide;

28% identity in comparison to human CDlla; 58% identity in comparison to human CDllb; 61 % identity in comparison to human CDllc; 28% identity in comparison to mouse CDlla; and 55% identity in comparison to mouse CDllb.

Example 18

Monoclonal Antibodies against Rat a _d I domain/Hu IgG4 Fusion Proteins

In view of the fact that the I domain of human ^ integrins has been demonstrated to participate in ligand binding, it was assumed that the same would be true for rat a _d protein. Monoclonal antibodies immunospecific for the rat α _d I domain may therefore be useful in rat models of human disease states wherein α _d binding is implicated.

Oligos "rat alpha-DI5" (SEQ ID NO: 87) and "rat alpha-DI3" (SEQ ID NO: 88) were generated from the rat α _d sequence corresponding to base pairs

469-493 and base pairs 1101-1125 (in the reverse orientation), respectively, in SEQ ID NO: 54. The oligos were used in a standard PCR reaction to generate a rat α. _d DNA fragment containing the I domain spanning base pairs 459-1125 in

SEQ ID NO: 54. The PCR product was ligated into vector pCRTAII (Invitrogen) according to manufacturer's suggested protocol. A positive colony was selected and expanded for DNA purification using a Qiagen (Chatswoth, GA) Midi Prep kit according to manufacturer's protocol. The DNA was digested with Xhol and BglR in a standard restriction enzyme digest and a 600 base pair band was gel purified which was subsequently ligated into pDCSl/HuIgG4 expression vector. A positive colony was selected, expanded and DNA purified with a Quiagen Maxi Prep Kit.

COS cells were plated at half confluence on 100mm culture dishes and grown overnight at 37°C in 7% CO ₂ . Cells were rinsed once with 5 ml

DMEM. To 5 ml DMEM, 50 μl DEAE-Dextran, 2 μl chloroquine and 15 μg rat α _d I domain/HulgG4 DNA described above was added. The mixture was added to the COS cells and incubated at 37°C for 3 hours. Media was then removed and 5 ml 10% DMSO in CMF-PBS was added for exactly one minute. The cells were gently rinsed once with DMEM. Ten ml DMEM containing 10% FBS was added to the cells and incubation continued overnight at 37°C in 7% CO . The next day, media was replaced with fresh media and incubation continued for three additional days. The media was harvested and fresh media was added to the plate. After three days, the media was collected again and the plates discarded. The procedure was repeated until 2 liters of culture supernatant were collected.

Supernatant collected as described above was loaded onto a Prosep-A column (Bioprocessing Limited) and protein purified as described below.

The column was initially washed with 15 column volumes of Wash Buffer containing 35 mM Tris and 150 mM NaCl, pH 7.5. Supernatant was loaded at a slow rate of less than approximately 60 column volumes per hour. After loading, the column was washed with 15 column volumes of Wash Buffer, 15 column volumes of 0.55 M diethanolamine, pH 8.5, and 15 column volumes

50 mM citric acid, pH 5.0. Protein was eluted with 50 mM citric acid, pH 3.0. Protein was neutralized with 1.0 M Tris, pH 8.0, and dialyzed in sterile PBS.

The rat a _d I domain protein was analyzed as described in Example 14. The detected protein migrated in the same manner as observed with human I domain protein.

Immunization Protocol

Mice were individually immunized with 50 μg purified rat α I domain/HulgG4 fusion protein previously emulsified in an equal volume of Freunds Complete Adjuvant (FCA) (Sigma). Approximately 200 μl of the antigen/adjuvant preparation was injected at 4 sites in the back and flanks of each of the mice. Two weeks later the mice were boosted with an injection of 100 μl rat a _d I domain/HuIgG4 antigen (50 μg/mouse) previously emulsified in an equal volume of Freunds Incomplete Adjuvant (FIA). After two additional weeks, the mice were boosted with 50 μg antigen in 200 μl PBS injected intravenously. To evaluate serum titers in the immunized mice, retro-orbital bleeds were performed on the animals ten days following the third immunization. The blood was allowed to clot and serum isolated by centrifugation. The serum was used in an immunoprecipitation on biotinylated (BIP) rat splenocytes. Serum from each mouse immunoprecipitated protein bands of expected molecular weight for rat α _d and rat CD18. One mouse was selected for the fusion and was boosted a fourth time as described above for the third boost.

The hybridoma supematants were screened by antibody capture, described as follows. Immulon 4 plates (Dynatech, Cambridge, Massachusetts) were coated at 4°C with 50 μl/well goat anti-mouse IgA, IgG or IgM (Organon Teknika) diluted 1:5000 in 50 mM carbonate buffer, pH 9.6. Plates were washed

3X with PBS containing 0.05% Tween 20 (PBST) and 50 μl culture supernatant was added. After incubation at 37°C for 30 minutes, and washing as described above, 50 μl horseradish peroxidase-conjugated goat anti-mouse IgG9(Fc)

(Jackson ImmunoResearch, West Grove, Pennsylvania) diluted 1:3500 in PBST was added. Plates were incubated as described above and washed 4X with PBST. Immediately thereafter, lOOμl substrate, containing 1 mg/ml o-phenylene diamine (Sigma) and 0.1 μl/ml 30% H ₂ O ₂ in 100 mM citrate, pH4.5, was added. The color reaction was stopped after 5 minutes with the addition of 50 μl 15 % H ₂ SO ₄ .

Absorbance at 490 nm was read on a Dynatech plate reader.

Supernatant from antibody-containing wells was also analyzed by

ELISA with immobilized rat a _d I domain/HulgG4 fusion protein. An ELISA with

HuIgG4 antibody coated plates served as a control for reactivity against the IgG fusion partner. Positive wells were selected for further screening by BIP on rat splenocyte lysates using techniques described below.

Biotinylation of Cell Surface Antigens

Rats were sacrificed by asphyxiation with CO ₂ and spleens were removed using standard surgical techniques. Splenocytes were harvested by gently pushing the spleen through a wire mesh with a 3 cc syringe plunger in 20 mis RPMI. Cells were collected into a 50 ml conical tube and washed in the appropriate buffer.

Cells were washed three times in cold D-PBS and resuspended at a density of 10 ⁸ to 10 ⁹ cells in 40 ml PBS. Four mg of NHS-Biotin (Pierce) was added to the cell suspension and the reaction was allowed to continue for exactly

15 minutes at room temperature. The cells were pelleted and washed three times in cold D-PBS.

Cell Lysates

Cells were resuspended at a density of 10 ⁸ cells/ ml in cold lysis Buffer (1 % NP40; 50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 2 mM CaCl; 2 mM

MgCl; 1:100 solution of pepstain, leupeptine, and aprotinin, added just before adding to cells; and 0.0001 g PMSF crystals, added just before adding to cells).

Lysates were vortexed for approximately 30 seconds, incubated for 5 minute at room temperature, and further incubated for 15 minutes on ice. Lysates were centrifuged for 10 minutes at 10,000 xg to pellet the insoluble material. Supernatant was collected into a new tube and stored at between 4°C and -20° C.

Immunoprecipitation

One ml cell lysate was precleared by incubation with 200 μl of a protein A sepharose slurry (Zymed) ovemight at 4°C. Precleared lysate was aliquoted into Eppendorf tubes at 50 μl/tube for each antibody to be tested. Twenty-five μl of polyclonal semm or 100 to 500 μl of monoclonal antibody supernatant were added to the precleared lysates and the resulting mixture incubated for 2 hours at 4°C with rotation. One hundred μl rabbit anti-mouse IgG (Jackson) bound to protein A sepharose beads in a PBS slurry was then added and incubation continued for 30 minutes at room temperature with rotation. Beads were pelleted with gentle centrifugation, and washed three times with cold Wash Buffer (10 mM HEPES; 0.2 M NaCl; 1 % Trition X-100). Supernatant was removed by aspiration, and 20 μl 2X SDS sample buffer containing 10% /3-mercaptoethanol was added. The sample was boiled for 2 minutes in a water bath, and the sample loaded onto a 5% SDS PAGE gel. Following separation, the proteins were transferred to nitrocellulose at constant current ovemight. The nitrocellulose filters were blocked with 3% BSA in TBS-T for 1 hour at room temperature and the blocking buffer was removed. A 1:6000 dilution of Strepavidin-HRP conjugate (Jackson) in 0.1 % BSA TBS-T was added and incubation continued for 30 minutes at room temperature. Filters were washed three times for 15 minutes each with TBS-T and autoradiographed using Amersham's ECL kit according to manufacturer's suggested protocol.

Example 19

Isolation of Mouse cDNA Clones

Isolation of a mouse a _d homolog was attempted.

Cross-species hybridization was performed using two PCR- generated probes: a 1.5 kb fragment corresponding to bases 522 to 2047 from human clone 19A2 (SEQ ID NO: 1), and a 1.0 kb rat fragment which corresponds to bases 1900 to 2900 in human clone 19A2 (SEQ ID NO: 1). The human probe was generated by PCR using primer pairs designated ATM-2 and 9-10.1 set out in SEQ ID NOS: 38 and 39, respectively; the rat probe was generated using primer pairs 434L and 434R, set out in SEQ ID NOS: 34 and 35, respectively.

Samples were incubated at 94°C for 4 minutes and subjected to 30 cycles of the temperature step sequence: 94 °C; 50 °C 2 minutes; 72 °C, 4 minutes.

5 '-GTCCAAGCTGTCATGGGCCAG-3 ' (SEQ ID NO: 38)

5 '-GTCCAGCAGACTGAAGAGCACGG-3 ' (SEQ ID NO: 39)

The PCR products were purified using the Qiagen Quick Spin kit according to manufacturer's suggested protocol, and approximately 180 ng DNA was labeled with 200 μCi [ ³² P]-dCTP using a Boehringer Mannheim Random Primer Labeling kit according to manufacturer's suggested protocol. Unincorporated isotope was removed using a Centri-sep Spin Column (Princeton Separations, Adelphia, NJ) according to manufacturer's suggested protocol. The probes were denatured with 0.2 N NaOH and neutralized with 0.4 M Tris-HCl, pH 8.0, before use.

A mouse thymic oligo dT-primed cDNA library in lambda ZAP II (Stratagene) was plated at approximately 30,000 plaques per 15 cm plate. Plaque lifts on nitrocellulose filters (Schleicher & Schuell, Keene, NH) were incubated at 50 °C with agitation for 1 hour in a prehybridization solution (8 ml/lift) containing 30% formamide. Labeled human and rat probes were added to the

prehybridization solution and incubation continued ovemight at 50°C. Filters were washed twice in 2X SSC/0.1 % at room temperature, once in 2X SSC/0.1 % SDS at 37°C, and once in 2X SSC/0.1 % SDS at 42°C. Filters were exposed on Kodak X-Omat AR film at -80°C for 27 hours with an intensifying screen. Four plaques giving positive signals on duplicate lifts were restieaked on LB medium with magnesium (LBM)/carbenicillin (100 mg/ml) plates and incubated ovemight at 37° C. The phage plaques were lifted with Hybond filters (Amersham), probed as in the initial screen, and exposed on Kodak X-Omat AR film for 24 hours at -80 °C with an intensifying screen. Twelve plaques giving positive signals were transferred into low

Mg ^{+ +} phage diluent containing 10 mM Tris-HCl and 1 mM MgCl ₂ . Insert size was determined by PCR amplification using T3 and T7 primers (SEQ ID NOS: 13 and 14, respectively) and the following reaction conditions. Samples were incubated at 94 °C for 4 minutes and subjected to 30 cycles of the temperature step sequence: 94°C, for 15 seconds; 50°C, for 30 seconds; and 72°C for 1 minute.

Six samples produced distinct bands that ranged in size from 300 bases to 1 kb. Phagemids were released via co-infection with helper phage and recircularized to generate Bluescript SK ^" (Stratagene). The resulting colonies were cultured in LBM/carbenicillin (100 mg/ml) ovemight. DNA was isolated with a Promega Wizard miniprep kit (Madison, WI) according to manufacturer's suggested protocol. EcoRI restriction analysis of purified DNA confirmed the molecular weights which were detected using PCR. Insert DNA was sequenced with M13 and M13 reverse.1 primers set out in SΕQ ID NOS: 40 and 41, respectively.

5 '-TGTAAAACGACGGCCAGT-3 ' (SΕQ ID NO: 40)

5 '-GGAAACAGCTATGACCATG-3 ' (SΕQ ID NO: 41)

Sequencing was performed as described in Example 4. Of the six clones, only two, designated 10.3-1 and 10.5-2, provided sequence information and were identical 600 bp fragments. The 600 bp sequence was 68% identical to a corresponding region of human a _d , 40% identical to human CDlla, 58% identical to human CDllc, and 54% identical to mouse

CDllb. This 600 bp fragment was then utilized to isolate a more complete cDNA encoding a putative mouse α _d homolog .

A mouse splenic cDNA library (oligo dT and random-primed) in lambda Zap II (Stratagene) was plated at 2.5 x 10 ⁴ phage/15 cm LBM plate. Plaques were lifted on Hybond nylon transfer membranes (Amersham), denatured with 0.5 M NaOH/1.5 M NaCl, neutralized with 0.5 M Tris Base/1.5 M NaCl/11.6 HC1, and washed in 2X SSC. The DNA was cross-linked to filters by ultraviolet irradiation.

Approximately 500,000 plaques were screened using probes 10.3-1 and 10.5-2 previously labeled as described supra. Probes were added to a prehybridization solution and incubated ovemight at 50 °C. The filters were washed twice in 2X SSC/0.1 % SDS at room temperature, once in 2X SSC/0.1 % SDS at 37°C, and once in 2X SSC/0.1 % SDS at 42°C. Filters were exposed on Kodak X-Omat AR film for 24 hours at -80°C with an intensifying screen. Fourteen plaques giving positive signals on duplicate lifts were subjected to a secondary screen identical to that for the initial screen except for additional final high stringency washes in 2X SSC/ 0.1 % SDS at 50°C, in 0.5X SSC/0.1 % SDS at 50°C, and at 55°C in 0.2X SSC/0.1 % SDS. The filters were exposed on Kodak X-Omat AR film at -80 °C for 13 hours with an intensifying screen. Eighteen positive plaques were transferred into low Mg ^{+ +} phage diluent and insert size determined by PCR amplification as described above. Seven of the samples gave single bands that ranged in size from 600 bp to 4 kb. EcoRI restriction analysis of purified DNA confirmed the sizes observed from

PCR and the DNA was sequenced with primers M13 and M13 reverse.1 (SEQ ID NOS: 40 and 41, respectively).

One clone designated B3800 contained a 4 kb insert which corresponded to a region 200 bases downstream of the 5 ' end of the human α _d 19A2 clone and includes 553 bases of a 3 ' untranslated region. Clone B3800 showed 77% identity to a corresponding region of human a _d , 44% identity to a corresponding region of human CDlla, 59% identity to a corresponding region of human CDllc, and 51 % identity to a corresponding region of mouse CDllb. The second clone A1160 was a 1.2 kb insert which aligned to the 5 ' end of the coding region of human a _d approximately 12 nucleic acids downstream of the initiating methionine. Clone A1160 showed 75% identity to a corresponding region of human _d , 46% identity to a corresponding region of human CDlla, 62% identity to a corresponding region of human CDl lc, and 66% identity to a corresponding region of mouse CDllb. Clone A1160, the fragment closer to the 5 ' end of human clone

19A2, is 1160 bases in length, and shares a region of overlap with clone B3800 starting at base 205 and continuing to base 1134. Clone Al 160 has a 110-base insertion (bases 704-814 of clone A1160) not present in the overlapping region of clone B3800. This insertion occurs at a probable exon-intron boundary [Fleming, et al , J. Immunol 750:480-490 (1993)] and was removed before subsequent ligation of clones A1160 and B3800.

Rapid Amplification of 5 ' cDNA End of the Putative Mouse α. _d Clone

RACE PCR [Frohman, "RACE: Rapid Amplification of cDNA Ends," in PCR Protocols: A Guide to Methods and Applications. Innis, et al (eds.) pp. 28-38, Academic Press:New York (1990)] was used to obtain missing

5 ' sequences of the putative mouse α. _d clone, including 5 ' untranslated sequence and initiating methionine. A mouse splenic RACE-Ready kit (Clontech, Palo Alto, CA) was used according to the manufacturer's suggested protocol. Two

antisense, gene-specific primers, A1160 RACEl-primary and A1160 RACE2- nested (SEQ ID NOS: 42 and 43), were designed to perform primary and nested PCR.

5 '-GGACATGTTCACTGCCTCTAGG-3 ' (SEQ ID NO: 42) 5 '-GGCGGACAGTCAGACGACTGTCCTG-3 ' (SEQ ID NO: 43)

The primers, SEQ ID NOS: 42 and 43, correspond to regions starting 302 and 247 bases from the 5 ' end, respectively. PCR was performed as described, supra, using the 5 ' anchor primer (SEQ ID NO: 44) and mouse spleen cDNA supplied with the kit.

5 '-CTGGTTCGGCCCACCTCTGAAGGTTCCAGAATCGA JSE^BID NO: 44)

Electrophoresis of the PCR product revealed a band approximately 280 bases in size, which was subcloned using a TA cloning kit (Invitrogen) according to manufacturer's suggested protocol. Ten resulting colonies were cultured, and the DNA isolated and sequenced. An additional 60 bases of 5 ' sequence were identified by this method, which correspond to bases 1 to 60 in SEQ ID NO: 45.

Characteristics of the Mouse cDNA and Predicted Amino Acid Sequence

A composite sequence of the mouse cDNA encoding a putative homolog of human α _d is set out in SEQ ID NO: 45. Although homology between the external domains of the human and mouse clones is high, homology between the cytoplasmic domains is only 30%. The observed variation may indicate C- terminal functional differences between the human and mouse proteins. Alternatively, the variation in the cytoplasmic domains may result from splice variation, or may indicate the existence of an additional β ₂ integrin gene(s).

At the amino acid level, the mouse cDNA predicts a protein (SEQ ID NO: 46) with 28% identity to mouse CDlla, 53% identity to mouse CDllb, 28% identity to human CDlla, 55% identity to human CDllb, 59% identity to human CDllc, and 70% identity to human α _d . Comparison of the amino acid sequences of the cytoplasmic domains of human α _d and the putative mouse homolog indicates regions of the same length, but having divergent primary structure. Similar sequence length in these regions suggests species variation rather than splice variant forms. When compared to the predicted rat polypeptide, Example 16, supra, mouse and rat cytoplasmic domains show greater than 60% identity.

Example 20

Isolation of additional mouse αd cDNA clones for sequence verification

In order to verify the nucleic and amino acids sequences describe in Example 19 for mouse a _d , additional mouse sequences were isolated for the purposes of confirmation.

Isolation of mouse cDNA by hybridization with two homologous α. _d probes (3 ' and 5 ') was performed using both a mouse splenic random primed library and an oligo dT-primed cDNA library in lambda ZAP II (Strategene).

The library was plated at 5 x 10 ⁵ phage per 15 cm LBM plate. Plaques were lifted on Hybond nylon membranes (Amersham), and the membranes were denatured (0.5 M NaOH/1.5 M NaCl), neutralized (0.5 M Tris Base/ 1.5 M NaCl

/ 11.6 M HC1) and washed (2X SSC salt solution). DNA was cross-lined to filters by ultraviolet irradiation.

Probes were generated using primers described below in a PCR reaction under the following conditions. Samples were held at 94 °C for 4 minutes and then run through 30 cycles of the temperature step sequence (94 °C for 15 seconds; 50°C for 30 seconds; 72°C for 1 minute in a Perkin-Elmer 9600 thermocycler).

The 3 ' probe was approximately 900 bases long and spanned a region from nucleotides 2752 to 3651 (in SEQ ID NO: 1) (5 ' -* 3 ') and was produced with primers ll.b-l/2FORll and ll.b-l/2REV2 as shown in SEQ ID NOS: 69 and 74, respectively. This probe was used in a first set of lifts. The 5 ' probe was approximately 800 bases long and spanned a region from nucleotides 149 to 946 (in SEQ ID NO: 1) (5 ' → 3 ') and was produced with primers ll.b-l/2FORl and ll.a-1/lREVl as shown in SEQ ID NOS: 50 and 85, respectively). This probe was used in a second set of lifts.

In a third set of lifts, both probes described above were used together on the same plates.

Approximately 500,000 plaques were screened using the two probes from above which were labeled in the same way as described in Example 17. Labeled probes were added to a prehybridization solution, containing 45% formamide, and incubated ovemight at 50°C. Filters were washed twice in 2X SSC/0.1 % SDS at room temperature (22°C). A final wash was carried out in 2X

SSC/0.1 % SDS at 50°C. Autoradiography was for 19 hours at -80°C on Kodak X-Omat AR film with an intensifying screen.

Thirteen plaques giving positive signals on at least duplicate lifts were subjected to a secondary screen performed as described for the initial screen except that both the 3 ' and 5 ' labeled probes were used for hybridization and an additional final wash was incorporated using 2X SSC/0.1 % SDS at 65 'C.

Autoradiography was performed as described above for 2.5 hours.

Thirteen plaques (designated MS2P1 through MS2P13) giving positive signals were transferred into low Mg ^{+ +} phage diluent. Insert size was determined by PCR amplification (Perkin-Elmer 9600 thermocycler) using T3 and

T7 primers which anneal to Bluescript phagemid in ZAP II (sequence previously described) under the same conditions shown above. Band sizes ranged from 500 bases to 4Kb. Phagemids were isolated, prepared, and sequenced with M13 and M13 reverse.1 primers (SEQ ID NOS: 40 and 41, respectively). Five of the

thirteen clones; MS2P-3, MS2P-6, MS2P-9, MS2P-12, and MS2P-13, were sequenced, and together, represented a region from approximately base 200 at the 5 ' end to about 300 bases past a first stop codon at the 3 ' end.

Automated sequencing was performed as described in Example 4 by first using M13 and M13 reverse.1 primers (SEQ ID NOS: 40 and 41, respectively) to sequence the ends of each clone and to determine its position relative to construct #17 (SEQ ID NO: 45). Each clone was then completely sequenced using the appropriate primers (listed below) for that particular region.

ll.b-l/2FORl 5 '-GCAGCCAGCTTCGGACAGAC-3 ' (SEQ ID NO: 50) ll.a-l/lFOR2 5 '-CCGCCTGCCACTGGCGTGTGC-3 ' (SEQ ID NO: 60) ll.a-l/lFOR3 5 '-CCCAGATGAAGGACTTCGTCAA-3 ' (SEQ ID NO: 61) ll.b-l/2FOR4 5 '-GCTGGGATCATTCGCTATGC-3 ' (SEQ ID NO: 62)

11.b-l/2FOR5 5 '-CAATGGATGGACCAGTTCTGG-3 ' (SEQ ID NO: 63) ll.b-l/2FOR6 5 '-CAGATCGGCTCCTACTTTGG-3 ' (SEQ ID NO: 64) l l.b-l/2FOR7 5 '-CATGGAGCCTCGAGACAGG-3 ' (SEQ ID NO: 65)

11.b-l/2FOR8 5 '-CCACTGTCCTCGAAGCTGGAG-3 ' (SEQ ID NO: 66) ll.b-l/2FOR9 5 '-CTTCGTCCTGTGCTGGCTGTGGGCTC-3

(SEQ ID NO: 67)

11.b-l/2FOR10 5 '-CGCCTGGCATGTGAGGCTGAG-3 ' (SEQ ID NO: 68) ll.b-l/2FORll 5 '-CCGTGATCAGTAGGCAGGAAG-3 ' (SEQ ID NO: 69) ll.b-l/2FOR12 5 -GTCACAGAGGGAACCTCC-3 ' (SEQ ID NO: 70) ll.b-l/2FOR13 5 ^' -GCTCCTGAGTGAGGCTGAAATCA-3 '(SEQ ID NO: 71) ll.b-l/2FOR14 5 '-GAGATGCTGGATCTACCATCTGC-3 ' (SEQ ID NO: 72)

11.b-l/2FOR15 5 ^' -CTGAGCTGGGAGATTTTTATGG-3 ' (SEQ ID NO: 73) l l.b-l/2REV2 5 '-GTGGATCAGCACTGAAATCTG-3 ' (SEQ ID NO: 74)

11.b-l/2REV3 5 ^' -CGTTTGAAGAAGCCAAGCTTG-3 ' (SEQ ID NO: 75)

1 l.b-l/2REV4 5 '-CACAGCGGAGGTGCAGGCAG-3 ' (SEQ ID NO: 76) ll.b-l/2REV5 5 ^' -CTCACTGCTTGCGCTGGC-3 ' (SEQ ID NO: 77)

ll.b-l/2REV6 5 '-CGGTAAGATAGCTCTGCTGG-3 ' (SEQ ID NO: 78

1 l.b-l/2REV7 5 '-GAGCCCACAGCCAGCACAGG-3 ' (SEQ ID NO: 79; ll.b-l/2REV8 5 '-GATCCAACGCCAGATCATACC-3 ' (SEQ ID NO: 80; ll.b-l/2REV9 5 '-CACGGCCAGGTCCACCAGGC-3 ' (SEQ ID NO: 81 ll.b-l/2REV10 5 '-CACGTCCCCTAGCACTGTCAG-3 ' (SEQ ID NO: 82 ll.b-l/2REVll 5 '-CCATGTCCACAGAACAGAGAG-3 ' (SEQ ID NO: 51 ll.b-l/2REV12 5 '-TTGACGAAGTCCTTCATCTGGG-3 ' (SEQ ID NO: 83 ll.b-l/2REV13 5 '-GAACTGCAAGCTGGAGCCCAG-3 ' (SEQ ID NO: 84

Il.a-1/1REV1 5 '-CTGGATGCTGCGAAGTGCTAC-3 ' (SEQ ID NO: 85 Il.a-1/1REV2 5 '-GCCTTGGAGCTGGACGATGGC-3 ' (SEQ ID NO: 86;

Sequences were edited, aligned, and compared to a previously isolated mouse a _d sequence (constmct #17, SEQ ID NO: 45).

Alignment of the new sequences revealed an 18 base deletion in constmct #17 beginning at nucleotide 2308; the deletion did not cause a shift in the reading frame. Clone MS2P-9, sequenced as described above, also revealed the same 18 base deletion. The deletion has been observed to occur in 50% of mouse clones that include the region but has not been detected in rat or human α _d clones. The eighteen base deletion is characterized by a 12 base palindromic sequence AAGCAGGAGCTCCTGTGT (SEQ ID NO: 91). This inverted repeat in the nucleic acid sequence is self-complementary and may form a loop out, causing cleavage during reverse transcription. The mouse α _d sequence which includes the additional 18 bases is set forth in SEQ ID NO: 52; the deduced amino acid sequence is set forth in SEQ ID NO: 53.

Example 21

In situ hybridizations in Mouse

Tissue distribution was then determined for mouse a _d in order to provide a comparison to that in humans, described in Example 6. A single stranded 200 bp mRNA probe was generated from a DNA template, corresponding to nucleotides 3460 to 3707 in the cytoplasmic tail region of the murine cDNA, by in vitro RNA transcription incorporating ³⁵ S-UTP (Amersham).

Whole mouse embryos (harvested at days 11-18 after fertilization) and various mouse tissues, including spleen, kidney, liver, intestine, and thymus, were hybridized in situ with the radiolabeled single-stranded mRNA probe.

Tissues were sectioned at 6 μm thickness, adhered to Vectabond

(Vector Laboratories, Inc., Burlingame, CA) coated slides, and stored at -70° C.

Prior to use, slides were removed from -70°C and placed at 50°C for approximately 5 minutes. Sections were fixed in 4% paraformaldehyde for 20 minutes at 4°C, dehydrated with an increasing ethanol gradient (70-95-100%) for

1 minute at 4°C at each concentration, and air dried for 30 minutes at room temperature. Sections were denatured for 2 minutes at 70°C in 70% formamide/2X SSC, rinsed twice in 2X SSC, dehydrated with the ethanol gradient described supra and air dried for 30 minutes. Hybridization was carried out ovemight (12-16 hours) at 55°C in a solution containing ³⁵ S-labeled riboprobes at 6 x 10 ⁵ cpm/section and diethylpyrocarbonate (DEPC)-treated water to give a final concentration of 50% formamide, 0.3 M NaCl, 20 mM Tris-HCl, pH 7.5, 10% dextran sulfate, IX Denhardt's solution, 100 mM dithiothreitol (DTT) and 5 mM EDTA. After hybridization, sections were washed for 1 hour at room temperature in 4X SSC/10 mM DTT, 40 minutes at 60°C in 50% formamide/2X SSC/10 mM DTT, 30 minutes at room temperature in 2X SSC, and 30 minutes at room temperature in 0.1X SSC. The sections were dehydrated, air dried for

2 hours, coated with Kodak NTB2 photographic emulsion, air dried for 2 hours,

developed (after storage at 4°C in complete darkness) and counterstained with hematoxylin/eosin .

Spleen tissue showed a strong signal primarily in the red pulp. This pattem is consistent with that of tissue macrophage distribution in the spleen, but does not exclude other cell types.

Example 22

Generation of Mouse Expression Constructs

In order to construct an expression plasmid including mouse cDNA sequences exhibiting homology to human a _d , inserts from clones A1160 and B3800 were ligated. Prior to this ligation, however, a 5 ' leader sequence, including an initiating methionine, was added to clone A1160. A primer designated "5 ' PCR leader" (SEQ ID NO: 47) was designed to contain: (1) identical nonspecific bases at positions 1-6 allowing for digestion; (2) a BamBI site (underlined in SEQ ID NO: 47) from positions 7-12 to facilitate subcloning into an expression vector; (3) a consensus Kozak sequence from positions 13-18,

(4) a signal sequence including a codon for an initiating methionine (bold in SEQ ID NO: 47), and (5) an additional 31 bases of specifically overlapping 5 ' sequence from clone A1160 to allow primer annealing. A second primer designated "3 ^' end frag" (SEQ ID NO: 48) was used with primer "5 ' PCR leader" to amplify the insert from clone A1160.

5 '-AGTTACGGATCCGGCACCATGAC-

-CTTCGGCACTGTGATCCTCCTGTGTG-3 ' (SEQ ID NO: 47)

5 '-GCTGGACGATGGCATCCAC-3 ' (SEQ ID NO: 48)

The resulting PCR product did not digest with BamHl, suggesting that an insufficient number of bases preceded the restriction site, prohibiting

recognition by the enzyme. The length of the "tail" sequence preceding the BamHl site in the 5 ' primer (SEQ ID NO: 47) was increased and PCR was repeated on the amplification product from the first PCR. A 5 ' primer, designated mAD.5 '.2 (SEQ ID NO: 49), was designed with additional nonspecific bases at positions 1-4 and an additional 20 bases specifically overlapping the previously employed "5 ' PCR leader" primer sequences.

5 '-GTAGAGTTACGGATCCGGCACCAT-3 ' (SEQ ID NO: 49)

Primers "mAD.5 '.2" and "3 ' end frag" were used together in PCR with the product from the first amplification as template. A resulting secondary PCR product was subcloned into plasmid pCRtmll (Invitrogen) according to manufacturer's suggested protocol and transformed into competent One shot cells (Invitrogen). One clone containing the PCR product was identified by restriction enzyme analysis using BamHl and EcoRI and sequenced. After the sequence was verified, the insert was isolated by digestion with BamHl and EcoRI and gel purified.

The insert from clone B3800 was isolated by digestion with EcoRI and NotI, gel purified, and added to a ligation reaction which included the augmented A1160 itamHI/EcoRI fragment. Ligation was allowed to proceed for 14 hours at 14°C. Vector pcDΝA.3 (Invitrogen), digested with BamHl and NotI, was added to the ligation reaction with additional ligase and the reaction was continued for another 12 hours. An aliquot of the reaction mixture was transformed into competent E. coli cells, the resulting colonies cultured, and one positive clone identified by PCR analysis with the primers ll.b-l/2FORl and l l.b-l/2RΕVl l (SEQ ID ΝOS: 50 and 51, respectively). These primers bridge the A1160 and B3800 fragments, therefore detection of an amplification product indicates the two fragments were ligated. The sequence of the positive clone was

- 70 - thymidine kinase encoding cassettes. Further analysis of this clone with an I domain probe (corresponding to nucleotides 454-1064 in SEQ ID NO: 45) indicated that the clone did not contain I domain encoding sequences.

Using the same I domain probe, the λFIXII genomic library was rescreened. Initially, six positive clones were detected, one of which remained positive upon secondary screening. DNA isolated from this clone reacted strongly in Southem analysis with an I domain probe. No reactivity was detected using the original 750 bp probe, however, indicating that this clone included regions 5 ' to nucleotides 1985-2773 of SEQ ID NO: 45.. Alternatively, the lack of hybridization to the 750 bp probe may have suggested that the clone was another member of the integrin family of proteins. To determine if this explanation was plausible, the 13 kb insert was subcloned into pBluescript SKII ⁺ . Purified DNA was sequenced using primers corresponding to a _d I domain nucleic acid sequences 441-461, 591-612, 717-739, and reverse 898-918 in SEQ ID NO: 52. Sequence information was obtained using only the first 4441-4461 primer, and only the 5 '-most exon of the I domain was efficiently amplified. The remainder of the I domain was not amplified. The resulting clone therefore comprised exon 6 of the mouse α. _d gene, and intronic sequences to the 3 ' and 5 ' end of the exon. Exon 7 was not represented in the clone. After sequencing, a construct is generated containing neomycin resistance and thymidine kinase genes.

The neomycin resistance (neo ^1" ) gene is inserted into the resulting plasmid in a manner that interrupts the protein coding sequence of the genomic mouse DNA. The resulting plasmid therefore contains a neo ^r gene within the mouse genomic DNA sequences, all of which are positioned within a thymidine kinase encoding region. Plasmid construction in this manner is required to favor homologous recombination over random recombination [Chisaka, et al. , Nature 555:516-520 (1992)].

- 69 - verified with the primers set out in SEQ ID NOS: 50 and 51, which amplify from base 100 to 1405 after the initiating methionine.

Example 23

Construction of a Knock-out Mouse In order to more accurately assess the immunological role of the protein encoded by the putative mouse a _d cDNA, a "knock-out" mouse is designed wherein the genomic DNA sequence encoding the putative a _d homolog is dismpted by homologous recombination. The significance of the protein encoded by the dismpted gene is thereby assessed by the absence of the encoded protein. Generation of "knock-out" mice is described in Deng, et al ,

MoL CelLBiol. 75:2134-2140 (1993).

Design of such a mouse begins with construction of a plasmid containing sequences to be "knocked out" by homologous recombination events. A 750 base pair fragment of the mouse cDNA (corresponding to nucleotides 1985 to 2733 in SEQ ID NO: 45) was used to identify a mouse genomic sequence encoding the putative mouse a _d homolog from a λFIXII genomic library. Primary screening resulted in 14 positive plaques, seven of which were confirmed by secondary screening. Liquid lysates were obtained from two of the plaques giving the strongest signal and the λ DNA was isolated by conventional methods. Restriction mapping and Southem analysis confirmed the authenticity of one clone, designated 14-1, and the insert DNA was isolated by digestion with NotI. This fragment was cloned into Bluescript SKII ⁺ .

In order to identify a restriction fragment of approximately 9 to 14 kb, a length reported to optimize the probability of homologous recombination events, Southem hybridization was performed with the 750 bp cDΝA probe.

Prior to hybridization, a restriction map was constructed for clone 14-1. A 12 kb fragment was identified as a possible candidate and this fragment was subcloned into pBluescript SKII ⁺ in a position wherein the mouse DΝA is flanked by

- 72 -

10 ⁶ dpm/ml. Hybridization was carried out at 42°C for 16-18 hours. Filters were washed extensively in 2X SSPE/0.1 % SDS at room temperature and exposed to X-ray film to visualize any hybridizing plaques.

Two clones with significant sequence homology to human α _d were identified. Clone #2 was approximately 800 bp in length and mapped to the 5 ' end of human α _d . Clone #2 includes an initiating methionine and complete leader sequence. Clone #7 was approximately 1.5 kb and includes an initiating methionine. The 5 ' end of clone #7 overlapped that of clone #2, while the 3 ' sequences terminated at a point beyond the I domain sequences. Internal sequencing of clone #7 is performed using the nested deletions sequencing technique.

The predicted N terminal amino acid sequence for rabbit α. _d as determined from clones #2 and #7 indicated a protein with 73% identity with human a _d , 65% identity with mouse a _d , and 58% identity with mouse CDllb, human CDllb, and human CDllc. The nucleic acid sequence for clone #2 is set out in SEQ ID NO: 92; the predicted amino acid sequence is set out in SEQ ID NO: 93

Isolation of a full length rabbit a _d cDNA is carried out using labeled rabbit fragment, clone # 7, and rescreening the cDNA library from which the fragment was derived.

Isolation of a rabbit α _d clone allows expression of the protein, either on the surface of transfectants or as a soluble full length or truncated form. This protein is then used as an immunogen for the production of monoclonal antibodies for use in rabbit models of human disease states.

- 71 -

Example 24

Cloning of Rabbit a _d - Construction and Screening of the Rabbit cDNA Library Identification of human α _d homologs in rats and mice led to the investigation of the existence of a rabbit homolog which would be useful in rabbit models of human disease states described infra.

Poly A ⁺ RNA was prepared from a whole rabbit spleen using an Invitrogen FastTrack kit (San Diego, CA) according to manufacturer's suggested protocol and reagents supplied with the kit. From 1.65 g tissue, 73 μg poly A ⁺ RNA were isolated. The rabbit spleen RNA was used to construct a ZAP Express cDNA library using a kit from Stratagene (La Jolla, CA). Resulting cDNA was directionally cloned into EcoRI and XTzoI sites in the lambda arms of a pBK-CMV phagemid vector. Gigapack II Gold (Stratagene) was used to package the lambda arms into phage particles. The resulting library titer was estimated to be approximately 8 x 10 ⁵ particles, with an average insert size of 1.2 kb.

The library was amplified once by plating for confluent plaque growth and cell lysate was collected. The amplified library was plated at approximately 30,000 plaque forming units (pfu) per 150 mm plate with E. coli and the resulting mixture incubated for 12-16 hrs at 37°C to allow plaque formation. Phage DNA was transferred onto Hybond N ⁺ nylon membranes

(Amersham, Arlington Heights, Illinois). The membranes were hybridized with a mixture of two random primed radiolabeled mouse a _d PCR DNA probes. The first probe was generated from a PCR product spanning nucleotides 149-946 in SΕQ ID NO: 52. The second probe was from a PCR product spanning nucleotides 2752-3651 in SΕQ ID NO: 52. Probes were labeled by random priming (Boehringer Mannheim Random Primed DNA Labeling Kit) and the reaction mixture was passed over a Sephadex G-50 column to remove unincorporated nucleotides. The hybridization solution was composed of 5X SSPΕ, 5X Denhardts, 1 % SDS, 40% Formamide and the labeled probes at 1 x

- 74 - functions which damage normal host tissue through either specific autoimmune responses or as a result of bystander cell damage.

Disease states in which there is evidence of macrophages playing a significant role in the disease process include multiple sclerosis, arthritis, graft atherosclerosis, some forms of diabetes and inflammatory bowel disease. Animal models, discussed below, have been shown to reproduce many of the aspects of these human disorders. Inhibitors of a _d function are tested in these model systems to determine if the potential exists for treating the corresponding human diseases.

A. Graft Arteriosclerosis

Cardiac transplantation is now the accepted form of therapeutic intervention for some types of end-state heart disease. As the use of cyclosporin A has increased one year survival rates to 80%, the development of progressive graft arteriosclerosis has emerged as the leading cause of death in cardiac transplants surviving beyond the first year. Recent studies have found that the incidence of significant graft arteriosclerosis 3 years following a cardiac transplant is in the range of 36-44% [Adams, et al , Transplantation 55: 1115-1119 (1992); Adams, et al , Transplantation 56:794-799 (1993)].

Graft arteriosclerosis typically consists of diffuse, occlusive, intimal lesions which affect the entire coronary vessel wall, and are often accompanied by lipid deposition. While the pathogenesis of graft arteriosclerosis remains unknown, it is presumably linked to histocompatibility differences between donor and recipient, and is immunologic in nature. Histologically, the areas of intimal thickening are composed primarily of macrophages, although T cells are occasionally seen. It is therefore possible that macrophages expressing a _d may play a significant role in the induction and/or development of graft arteriosclerosis. In such a case, monoclonal antibodies or small molecule inhibitors (for example, soluble ICAM-R) of a _d function could be given

- 73 - Example 25

Animal Models For Determining α. _d Therapeutic Utility

Immunohistologic data in dog and in situ hybridization in rats and mice has determined that in spleen α _d is expressed primarily by macrophages present in red pulp and in lymph nodes, α _d is found in medullary cords and sinuses. The expression pattem is remarkably similar to what has been reported for two murine antigens defined by the monoclonal antibodies F4/80 and SK39. While biochemical characterization of these murine antigens has demonstrated that they are distinct from a _d , it is highly probably that α _d defines the same macrophage subset as the murine F4/80 and SK39 antigens.

In mouse, SK39-positive macrophages have been identified in splenic red pulp where they may participate in the clearance of foreign materials from circulation, and in medulla of lymph nodes [Jutila, et al, J.Leukocyte Biol 54:30-39 (1993)]. SK39-positive macrophages have also been reported at sites of both acute and chronic inflammation. Furthermore, monocytes recruited to thioglycolate-inflamed peritoneal cavities also express the SK39 antigen. Collectively, these findings suggest that, if SK39 ⁺ cells are also a _d ⁺ , then these cells are responsible for the clearance of foreign materials in the spleen and participate in inflammation where macrophages play a significant role. While the function of a _d remains unclear, other more well characterized β ₂ integrins have been shown to participate in a wide variety of adhesion events that facilitate cell migration, enhance phagocytosis, and promote cell-cell interactions, events which all lead to upregulation of inflammatory processes. Therefore, it is highly plausible that interfering with the normal a _d function may also interfere with inflammation where macrophages play a significant role. Such an anti-inflammatory effect could result from: i) blocking macrophage recruitment to sites of inflammation, ii) preventing macrophage activation at the site of inflammation or iii) interfering with macrophage effector

- 76 - lumenal surface of the ascending aorta [Rosenfeld, et al, Arteriosclerosis 7:9-23 (1987); Rosenfeld, et al, Arteriosclerosis 7:24-34 (1987)]. The atherosclerotic lesions seen in these rabbits are simmer to those in humans. Lesions contain large numbers of T cells, most of which express CD45RO, a marker associated with memory T cells. Approximately half of the infiltrating T cells also express

MHC class II antigen and some express the IL-2 receptor suggesting that many of the cells are in an activated state.

One feature of the atherosclerotic lesions found in cholesterol fed rabbits, but apparently absent in rodent models, is the accumulation of foam cell- rich lesions. Foam cell macrophages are believed to result from the uptake of oxidized low-density lipoprotein (LDL) by specific receptors. Oxidized LDL particles have been found to be toxic for some cell types including endothelial cells and smooth muscle cells. The uptake of potentially toxic, oxidized LDL particles by macrophages serves as an irritant and drives macrophage activation, contributing to the inflammation associated with atherosclerotic lesions.

Once monoclonal antibodies have been generated to rabbit a _d , cholesterol fed rabbits are treated. Treatments include prophylactic administration of α. _d monoclonal antibodies or small molecule inhibitors, to demonstrate that a _d ⁺ macrophages are involved in the disease process. Additional studies would demonstrate that monoclonal antibodies to α _d or small molecule inhibitors are capable of reversing vessel damage detected in rabbits fed an atherogenic diet.

C. Insulin-dependent Diabetes

BB rats spontaneously develop insulin-dependent diabetes at 70-150 days of age. Using immunohistochemistry, MHC class II ⁺ , ED1 ⁺ macrophages can be detected infiltrating the islets early in the disease. Many of the macrophages appear to be engaged in phagocytosis of cell debris or normal cells.

As the disease progresses, larger numbers of macrophages are found infiltrating

- 75 - prophylactically to individuals who received heart transplants and are at risk of developing graft arteriosclerosis.

Although atherosclerosis in heart transplants presents the greatest threat to life, graft arteriosclerosis is also seen in other solid organ transplants, including kidneys and livers. Therapeutic use of α _d blocking agents could prevent graft arteriosclerosis in other organ transplants and reduce complications resulting from graft failure.

One model for graft arteriosclerosis in the rat involves heterotopic cardiac allografts transplanted across minor histocompatibility barriers. When Lewis cardiac allografts are transplanted into MHC class I and II compatible F-

344 recipients, 80% of the allografts survive at least 3 weeks, while 25% of the grafts survive indefinitely. During this low-grade graft rejection, arteriosclerosis lesions form in the donor heart. Arterial lesions in 120 day old allografts typically have diffuse fibrotic intimal thickening indistinguishable in appearance from graft arteriosclerosis lesions found in rejecting human cardiac allografts.

Rats are transplanted with hearts mismatched at minor histocompatibility antigens, for example Lewis into F-344. Monoclonal antibodies specific for rat a _d or small molecule inhibitors of a _d are given periodically to transplant recipients. Treatment is expected to reduce the incidence of graft arteriosclerosis in non-rejecting donor hearts. Treatment of rats with a _d monoclonal antibodies or small molecule inhibitors may not be limited to prophylactic treatments. Blocking α _d function is also be expected to reduce macrophage mediated inflammation and allow reversal of arterial damage in the graft.

B. Atherosclerosis in Rabbits Fed Cholesterol

Rabbits fed an atherogenic diet containing a cholesterol supplement for approximately 12-16 weeks develop intimal lesions that cover most of the

the islets, although significant numbers of T cells, and later B cells, also appear to be recruited to the site [Hanenberg, et al , Diabetologia 52:126-134 (1989)]. Development of diabetes in BB rats appears to depend on both early macrophage infiltration and subsequent T cells recruitment. Treatment of BB rats with silica particles, which are toxic to macrophages, has been effective in blocking the early macrophage infiltration of the islets. In the absence of early macrophage infiltration, subsequent tissue damage by an autoaggressive lymphocyte population fails to occur. Administration of monoclonal antibody OX- 19 (specific for rat CD5) or monoclonal antibody OX-8 (specific for rat CD8), which block the T cell-associated phase of the disease, is also effective in suppressing the development of diabetes.

The central role of macrophages in the pathology of this model makes it attractive for testing inhibitors of a _d function. Rats genetically predisposed to the development of insulin-dependent diabetes are treated with monoclonal antibodies to α. _d or small molecule inhibitors and evaluated for the development of the disease. Preventing or delaying clinical onset is evidence that α _d plays a pivotal role in macrophage damage to the islet cells.

D. Inflammatory Bowel Disease (Crohn's Disease, Ulcerative Colitis) Animal models used in the study of inflammatory bowel disease

(IBD) are generally elicited by intrarectal administration of noxious irritants (e.g. acetic acid or trinitrobenzene sulfonic acid/ethanol). Colonic inflammation induced by these agents is the result of chemical or metabolic injury and lacks the chronic and spontaneously relapsing inflammation associated with human IBD. However, a recently described model using subserosal injections of purified peptidoglycan-polysaccharide (PG-PS) polymers from either group A or group D streptococci appears to be a more physiologically relevant model for human IBD [Yamada, et al, Gastroenterolgy 104:759-771 (1993)].

In this model PG-PS is injected into the subserosal layer of the distal colon. The resulting inflammatory response is biphasic with an initial acute episode three days after injection, which is followed by a spontaneous chronic phase three to four weeks later. The late phase response is granulomatous in nature, and results in colonic thickening, adhesions, colonic nodules and mucosal lesions. In addition to mucosal injury, PG-PS colitis frequently leads to arthritis anemia and granulomatous hepatitis. The extraintestinal manifestations of the disease make the model attractive for studying Crohn's colitis in that a significant number of patients with active Crohn's disease suffer from arthritic joint disease and hepatobillary inflammation.

Granulomatous lesions are the result of chronic inflammation which leads to the recruitment and subsequent activation of cells of the monocyte/macrophage lineage. Presence of granulomatous lesions in Crohn's disease and the above animal model make this an attractive clinical target for a _d monoclonal antibodies or other inhibitors of a _d function. Inhibitors of a _d function are expected to block the formation of lesions associated with IBD or even reverse tissue damage seen in the disease.

E. Arthritis

Arthritis appears to be a multi-factorial disease process involving a variety of inflammatory cell types including neutrophils, T lymphocytes and phagocytic macrophages. Although a variety of arthritis models exist, preparations of streptococcal cell wall proteoglycan produce a disorder most similar to the human disease.

In rats, streptococcal cell wall induces inflammation of peripheral joints characterized by repeated episodes of disease progression followed by remission and eventually resulting in joint destruction over a period of several months [Cromartie, et al , J.Exp.Med. 746: 1585-1602 (1977); Schwab et al ,

Infection and Immunity 5 :4436-4442 (1991)]. During the chronic phase of the

disease, mononuclear phagocytes or macrophages are believed to play a major role in destruction of the synovium. Furthermore, agents which suppress the recmitment of macrophages into the synovium effectively reduce the inflammation and pathology characteristic of arthritis. A central role for the macrophage in synovium destruction that leads to arthritis predicts that monoclonal antibodies to a _d or inhibitors of α _d function may have therapeutic potential in the treatment of this disease. As in other models previously described, o. _d monoclonal antibodies or small molecule inhibitors administered prophylactically are expected to block or moderate joint inflammation and prevent destruction of the synovium. Agents that interfere with o! _d function may also moderate ongoing inflammation by preventing the recmitment of additional macrophages to the joint or blocking macrophage activation. The net result would be to reverse ongoing destruction of the joint and facilitate tissue repair.

F. Multiple Sclerosis

Although pathogenesis of multiple sclerosis (MS) remains unclear, it is generally accepted that the disease is mediated by CD4 ⁺ T cells which recognize autoantigens in the central nervous system and initiate an inflammatory cascade. The resulting immune response results in the recmitment of additional inflammatory cells, including activated macrophages which contribute to the disease. Experimental autoimmune encephalomyelitis (EAE) is an animal model which reproduces some aspects of MS. Recently, monoclonal antibodies reactive with CDllb/CD18 [Huitinga, et al , Eur.J .Immunol 25:709-715 (1993)] present on inflammatory macrophages have been shown to block both clinical and histologic disease. The results suggest that monoclonal antibodies or small molecule inhibitors to a _d are likely to be effective in blocking the inflammatory response in EAE. Such agents also have important therapeutic applications in the treatment of MS.

Numerous modifications and variations in the invention as set forth in the above illustrative examples are expected to occur to those skilled in the art. Consequently only such limitations as appear in the appended claims should be placed on the invention.

THIS PAGE WAS NOT FURNISHED UPON FILING THE INTERNATIONAL APPLICATION

( ix ) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 3..3485

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

TG ACC TTC GGC ACT GTG CTT CTT CTG AGT GTC CTG GCT TCT TAT CAT 47

Thr Phe Gly Thr Val Leu Leu Leu Ser Val Leu Ala Ser Tyr His 1 5 10 15

GGA TTC AAC CTG GAT GTG GAG GAG CCT ACG ATC TTC CAG GAG GAT GCA 95 Gly Phe Asn Leu Asp Val Glu Glu Pro Thr lie Phe Gin Glu Asp Ala 20 25 30

GGC GGC TTT GGG CAG AGC GTG GTG CAG TTC GGT GGA TCT CGA CTC GTG 143 Gly Gly Phe Gly Gin Ser Val Val Gin Phe Gly Gly Ser Arg Leu Val 35 40 45

GTG GGA GCA CCC CTG GAG GTG GTG GCG GCC AAC CAG ACG GGA CGG CTG 191 Val Gly Ala Pro Leu Glu Val Val Ala Ala Asn Gin Thr Gly Arg Leu 50 55 60

TAT GAC TGC GCA GCT GCC ACC GGC ATG TGC CAG CCC ATC CCG CTG CAC 239 Tyr Asp Cys Ala Ala Ala Thr Gly Met Cys Gin Pro lie Pro Leu His 65 70 75

ATC CGC CCT GAG GCC GTG AAC ATG TCC TTG GGC CTG ACC CTG GCA GCC 287 lie Arg Pro Glu Ala Val Asn Met Ser Leu Gly Leu Thr Leu Ala Ala 80 85 90 95

TCC ACC AAC GGC TCC CGG CTC CTG GCC TGT GGC CCG ACC CTG CAC AGA 335 Ser Thr Asn Gly Ser Arg Leu Leu Ala Cys Gly Pro Thr Leu His Arg 100 105 110

GTC TGT GGG GAG AAC TCA TAC TCA AAG GGT TCC TGC CTC CTG CTG GGC 383 Val Cys Gly Glu Asn Ser Tyr Ser Lys Gly Ser Cys Leu Leu Leu Gly 115 120 ^' 125

TCG CGC TGG GAG ATC ATC CAG ACA GTC CCC GAC GCC ACG CCA GAG TGT 431 Ser Arg Trp Glu lie lie Gin Thr Val Pro Asp Ala Thr Pro Glu Cys 130 135 140

CCA CAT CAA GAG ATG GAC ATC GTC TTC CTG ATT GAC GGC TCT GGA AGC 479 Pro His Gin Glu Met Asp lie Val Phe Leu lie Asp Gly Ser Gly Ser 145 150 155

ATT GAC CAA AAT GAC TTT AAC CAG ATG AAG GGC TTT GTC CAA GCT GTC 527 lie Asp Gin Asn Asp Phe Asn Gin Met Lys Gly Phe Val Gin Ala Val 160 165 170 175

ATG GGC CAG TTT GAG GGC ACT GAC ACC CTG TTT GCA CTG ATG CAG TAC 575 Met Gly Gin Phe Glu Gly Thr Asp Thr Leu Phe Ala Leu Met Gin Tyr 180 185 190

TCA AAC CTC CTG AAG ATC CAC TTC ACC TTC ACC CAA TTC CGG ACC AGC 623 Ser Asn Leu Leu Lys lie His Phe Thr Phe Thr Gin Phe Arg Thr Ser 195 200 205

CCG AGC CAG CAG AGC CTG GTG GAT CCC ATC GTC CAA CTG AAA GGC CTG 671 Pro Ser Gin Gin Ser Leu Val Asp Pro lie Val Gin Leu Lys Gly Leu 210 215 220

ACG TTC ACG GCC ACG GGC ATC CTG ACA GTG GTG ACA CAG CTA TTT CAT 719 Thr Phe Thr Ala Thr Gly lie Leu Thr Val Val Thr Gin Leu Phe His 225 230 235

CAT AAG AAT GGG GCC CGA AAA AGT GCC AAG AAG ATC CTC ATT GTC ATC 767 His Lys Asn Gly Ala Arg Lys Ser Ala Lys Lys lie Leu lie Val lie 240 245 250 255

ACA GAT GGG CAG AAG TAC AAA GAC CCC CTG GAA TAC AGT GAT GTC ATC 815 Thr Asp Gly Gin Lys Tyr Lys Asp Pro Leu Glu Tyr Ser Asp Val lie 260 265 270

CCC CAG GCA GAG AAG GCT GGC ATC ATC CGC TAC GCT ATC GGG GTG GGA 863 Pro Gin Ala Glu Lys Ala Gly lie lie Arg Tyr Ala lie Gly Val Gly 275 280 285

CAC GCT TTC CAG GGA CCC ACT GCC AGG CAG GAG CTG AAT ACC ATC AGC 911 His Ala Phe Gin Gly Pro Thr Ala Arg Gin Glu Leu Asn Thr lie Ser 290 295 300

TCA GCG CCT CCG CAG GAC CAC GTG TTC AAG GTG GAC AAC TTT GCA GCC 959 Ser Ala Pro Pro Gin Asp His Val Phe Lys Val Asp Asn Phe Ala Ala 305 310 315

CTT GGC AGC ATC CAG AAG CAG CTG CAG GAG AAG ATC TAT GCA GTT GAG 1007 Leu Gly Ser lie Gin Lys Gin Leu Gin Glu Lys lie Tyr Ala Val Glu 320 325 330 335

GGA ACC CAG TCC AGG GCA AGC AGC TCC TTC CAG CAC GAG ATG TCC CAA 1055 Gly Thr Gin Ser Arg Ala Ser Ser Ser Phe Gin His Glu Met Ser Gin 340 345 350

GAA GGC TTC AGC ACA GCC CTC ACA ATG GAT GGC CTC TTC CTG GGG GCT 1103 Glu Gly Phe Ser Thr Ala Leu Thr Met Asp Gly Leu Phe Leu Gly Ala 355 360 365

GTG GGG AGC TTT AGC TGG TCT GGA GGT GCC TTC CTG TAT CCC CCA AAT 1151 Val Gly Ser Phe Ser Trp Ser Gly Gly Ala Phe Leu Tyr Pro Pro Asn 370 375 380

ATG AGC CCC ACC TTC ATC AAC ATG TCT CAG GAG AAT GTG GAC ATG AGG 1199 Met Ser Pro Thr Phe lie Asn Met Ser Gin Glu Asn Val Asp Met Arg 385 390 395

GAC TCT TAC CTG GGT TAC TCC ACC GAG CTA GCC CTG TGG AAG GGG GTA 1247 Asp Ser Tyr Leu Gly Tyr Ser Thr Glu Leu Ala Leu Trp Lys Gly Val 400 405 410 415

CAG AAC CTG GTC CTG GGG GCC CCC CGC TAC CAG CAT ACC GGG AAG GCT 1295 Gin Asn Leu Val Leu Gly Ala Pro Arg Tyr Gin His Thr Gly Lys Ala 420 425 430

GTC ATC TTC ACC CAG GTG TCC AGG CAA TGG AGG AAG AAG GCC GAA GTC 1343 Val lie Phe Thr Gin Val Ser Arg Gin Trp Arg Lys Lys Ala Glu Val 435 440 445

ACA GGG ACG CAG ATC GGC TCC TAC TTC GGG GCC TCC CTC TGC TCC GTG 1391 Thr Gly Thr Gin lie Gly Ser Tyr Phe Gly Ala Ser Leu Cys Ser Val 450 455 460

GAT GTG GAC AGC GAT GGC AGC ACC GAC CTG ATC CTC ATT GGG GCC CCC 1439

Asp Val Asp Ser Asp Gly Ser Thr Asp Leu He Leu He Gly Ala Pro 465 470 475

CAT TAC TAT GAG CAG ACC CGA GGG GGC CAG GTG TCC GTG TGT CCC TTG 1487

His Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys Pro Leu 480 485 490 495

CCT AGG GGG CAG AGG GTG CAG TGG CAG TGT GAC GCT GTT CTC CGT GGT 1535

Pro Arg Gly Gin Arg Val Gin Trp Gin Cys Asp Ala Val Leu Arg Gly 500 505 510

GAG CAG GGC CAC CCC TGG GGC CGC TTT GGG GCA GCC CTG ACA GTG TTG 1583

Glu Gin Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu 515 520 525

GGG GAT GTG AAT GAG GAC AAG CTG ATA GAC GTG GCC ATT GGG GCC CCG 1631

Gly Asp Val Asn Glu Asp Lys Leu He Asp Val Ala He Gly Ala Pro 530 535 540

GGA GAG CAG GAG AAC CGG GGT GCT GTC TAC CTG TTT CAC GGA GCC TCA 1679

Gly Glu Gin Glu Asn Arg Gly Ala Val Tyr Leu Phe His Gly Ala Ser 545 550 555

GAA TCC GGC ATC AGC CCC TCC CAC AGC CAG CGG ATT GCC AGC TCC CAG 1727

Glu Ser Gly He Ser Pro Ser His Ser Gin Arg He Ala Ser Ser Gin

560 565 570 575

CTC TCC CCC AGG CTG CAG TAT TTT GGG CAG GCG CTG AGT GGG GGT CAG 1775

Leu Ser Pro Arg Leu Gin Tyr Phe Gly Gin Ala Leu Ser Gly Gly Gin 580 585 590

GAC CTC ACC CAG GAT GGA CTG ATG GAC CTG GCC GTG GGG GCC CGG GGC 1823

Asp Leu Thr Gin Asp Gly Leu Met Asp Leu Ala Val Gly Ala Arg Gly 595 600 605

CAG GTG CTC CTG CTC AGG AGT CTG CCG GTG CTG AAA GTG GGG GTG GCC 1871

Gin Val Leu Leu Leu Arg Ser Leu Pro Val Leu Lys Val Gly Val Ala 610 615 620

ATG AGA TTC AGC CCT GTG GAG GTG GCC AAG GCT GTG TAC CGG TGC TGG 1919

Met Arg Phe Ser Pro Val Glu Val Ala Lys Ala Val Tyr Arg Cys Trp 625 630 635

GAA GAG AAG CCC AGT GCC CTG GAA GCT GGG GAC GCC ACC GTC TGT CTC 1967

Glu Glu Lys Pro Ser Ala Leu Glu Ala Gly Asp Ala Thr Val Cys Leu

640 645 650 655

ACC ATC CAG AAA AGC TCA CTG GAC CAG CTA GGT GAC ATC CAA AGC TCT 2015

Thr He Gin Lys Ser Ser Leu Asp Gin Leu Gly Asp He Gin Ser Ser

660 665 670

GTC AGG TTT GAT CTG GCA CTG GAC CCA GGT CGT CTG ACT TCT CGT GCC 2063

Val Arg Phe Asp Leu Ala Leu Asp Pro Gly Arg Leu Thr Ser Arg Ala 675 680 685

ATT TTC AAT GAA ACC AAG AAC CCC ACT TTG ACT CGA AGA AAA ACC CTG 2111

He Phe Asn Glu Thr Lys Asn Pro Thr Leu Thr Arg Arg Lys Thr Leu 690 695 700

GGA CTG GGG ATT CAC TGT GAA ACC CTG AAG CTG CTT TTG CCA GAT TGT 2159 Gly Leu Gly He His Cys Glu Thr Leu Lys Leu Leu Leu Pro Asp Cys 705 710 715

GTG GAG GAT GTG GTG AGC CCC ATC ATT CTG CAC CTC AAC TTC TCA CTG 2207 Val Glu Asp Val Val Ser Pro He He Leu His Leu Asn Phe Ser Leu 720 725 730 735

GTG AGA GAG CCC ATC CCC TCC CCC CAG AAC CTG CGT CCT GTG CTG GCC 2255 Val Arg Glu Pro He Pro Ser Pro Gin Asn Leu Arg Pro Val Leu Ala 740 745 750

GTG GGC TCA CAA GAC CTC TTC ACT GCT TCT CTC CCC TTC GAG AAG AAC 2303 Val Gly Ser Gin Asp Leu Phe Thr Ala Ser Leu Pro Phe Glu Lys Asn 755 760 765

TGT GGG CAA GAT GGC CTC TGT GAA GGG GAC CTG GGT GTC ACC CTC AGC 2351 Cys Gly Gin Asp Gly Leu Cys Glu Gly Asp Leu Gly Val Thr Leu Ser 770 775 780

TTC TCA GGC CTG CAG ACC CTG ACC GTG GGG AGC TCC CTG GAG CTC AAC 2399 Phe Ser Gly Leu Gin Thr Leu Thr Val Gly Ser Ser Leu Glu Leu Asn 785 790 795

GTG ATT GTG ACT GTG TGG AAC GCA GGT GAG GAT TCC TAC GGA ACC GTG 2447 Val He Val Thr Val Trp Asn Ala Gly Glu Asp Ser Tyr Gly Thr Val 800 805 810 815

GTC AGC CTC TAC TAT CCA GCA GGG CTG TCG CAC CGA CGG GTG TCA GGA 2495 Val Ser Leu Tyr Tyr Pro Ala Gly Leu Ser His Arg Arg Val Ser Gly 820 825 830

GCC CAG AAG CAG CCC CAT CAG AGT GCC CTG CGC CTG GCA TGT GAG ACA 2543 Ala Gin Lys Gin Pro His Gin Ser Ala Leu Arg Leu Ala Cys Glu Thr 835 840 845

GTG CCC ACT GAG GAT GAG GGC CTA AGA AGC AGC CGC TGC AGT GTC AAC 2591 Val Pro Thr Glu Asp Glu Gly Leu Arg Ser Ser Arg Cys Ser Val Asn 850 855 860

CAC CCC ATC TTC CAT GAG GGC TCT AAC GGC ACC TTC ATA GTC ACA TTC 2639 His Pro He Phe His Glu Gly Ser Asn Gly Thr Phe He Val Thr Phe 865 870 875

GAT GTC TCC TAC AAG GCC ACC CTG GGA GAC AGG ATG CTT ATG AGG GCC 2687 Asp Val Ser Tyr Lys Ala Thr Leu Gly Asp Arg Met Leu Met Arg Ala 880 885 890 895

AGT GCA AGC AGT GAG AAC AAT AAG GCT TCA AGC AGC AAG GCC ACC TTC 2735 Ser Ala Ser Ser Glu Asn Asn Lys Ala Ser Ser Ser Lys Ala Thr Phe 900 905 910

CAG CTG GAG CTC CCG GTG AAG TAT GCA GTC TAC ACC ATG ATC AGC AGG 2783 Gin Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Thr Met He Ser Arg 915 920 925

CAG GAA GAA TCC ACC AAG TAC TTC AAC TTT GCA ACC TCC GAT GAG AAG 2831 Gin Glu Glu Ser Thr Lys Tyr Phe Asn Phe Ala Thr Ser Asp Glu Lys 930 935 940

AAA ATG AAA GAG GCT GAG CAT CGA TAC CGT GTG AAT AAC CTC AGC CAG 2879 Lys Met Lys Glu Ala Glu His Arg Tyr Arg Val Asn Asn Leu Ser Gin 945 950 955

CGA GAT CTG GCC ATC AGC ATT AAC TTC TGG GTT CCT GTC CTG CTG AAC 2927 Arg Asp Leu Ala He Ser He Asn Phe Trp Val Pro Val Leu Leu Asn 960 965 970 975

GGG GTG GCT GTG TGG GAT GTG GTC ATG GAG GCC CCA TCT CAG AGT CTC 2975 Gly Val Ala Val Trp Asp Val Val Met Glu Ala Pro Ser Gin Ser Leu 980 985 990

CCC TGT GTT TCA GAG AGA AAA CCT CCC CAG CAT TCT GAC TTC CTG ACC 3023 Pro Cys Val Ser Glu Arg Lys Pro Pro Gin His Ser Asp Phe Leu Thr 995 1000 1005

CAG ATT TCA AGA AGT CCC ATG CTG GAC TGC TCC ATT GCT GAC TGC CTG 3071 Gin He Ser Arg Ser Pro Met Leu Asp Cys Ser He Ala Asp Cys Leu 1010 1015 1020

CAG TTC CGC TGT GAC GTC CCC TCC TTC AGC GTC CAG GAG GAG CTG GAT 3119 Gin Phe Arg Cys Asp Val Pro Ser Phe Ser Val Gin Glu Glu Leu Asp 1025 1030 1035

TTC ACC CTG AAG GGC AAT CTC AGT TTC GGC TGG GTC CGC GAG ACA TTG 3167 Phe Thr Leu Lys Gly Asn Leu Ser Phe Gly Trp Val Arg Glu Thr Leu 1040 1045 1050 1055

CAG AAG AAG GTG TTG GTC GTG AGT GTG GCT GAA ATT ACG TTC GAC ACA 3215 Gin Lys Lys Val Leu Val Val Ser Val Ala Glu He Thr Phe Asp Thr 1060 1065 1070

TCC GTG TAC TCC CAG CTT CCA GGA CAG GAG GCA TTT ATG AGA GCT CAG 3263 Ser Val Tyr Ser Gin Leu Pro Gly Gin Glu Ala Phe Met Arg Ala Gin 1075 1080 1085

ATG GAG ATG GTG CTA GAA GAA GAC GAG GTC TAC AAT GCC ATT CCC ATC 3311 Met Glu Met Val Leu Glu Glu Asp Glu Val Tyr Asn Ala He Pro He 1090 1095 1100

ATC ATG GGC AGC TCT GTG GGG GCT CTG CTA CTG CTG GCG CTC ATC ACA 3359 He Met Gly Ser Ser Val Gly Ala Leu Leu Leu Leu Ala Leu He Thr 1105 1110 1115

GCC ACA CTG TAC AAG CTT GGC TTC TTC AAA CGC CAC TAC AAG GAA ATG 3407 Ala Thr Leu Tyr Lys Leu Gly Phe Phe Lys Arg His Tyr Lys Glu Met 1120 1125 1130 1135

CTG GAG GAC AAG CCT GAA GAC ACT GCC ACA TTC AGT GGG GAC GAT TTC 3455 Leu Glu Asp Lys Pro Glu Asp Thr Ala Thr Phe Ser Gly Asp Asp Phe 1140 1145 1150

AGC TGT GTG GCC CCA AAT GTG CCT TTG TCC TAATAATCCA CTTTCCTGTT 3505 Ser Cys Val Ala Pro Asn Val Pro Leu Ser 1155 1160

TATCTCTACC ACTGTGGGCT GGACTTGCTT GCAACCATAA ATCAACTTAC ATGGAAACAA 3566

CTTCTGCATA GATCTGCACT GGCCTAAGCA ACCTACCAGG TGCTAAGCAC CTTCTCGGAG 3625

AGATAGAGAT TGTAATGTTT TTACATATCT GTCCATCTTT TTCAGCAATG ACCCACTTTT 3685

TACAGAAGCA GGCATGGTGC CAGCATAAAT TTTCATATGC T 3726

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1161 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Thr Phe Gly Thr Val Leu Leu Leu Ser Val Leu Ala Ser Tyr His Gly 1 5 10 15

Phe Asn Leu Asp Val Glu Glu Pro Thr He Phe Gin Glu Asp Ala Gly 20 25 30

Gly Phe Gly Gin Ser Val Val Gin Phe Gly Gly Ser Arg Leu Val Val 35 40 45

Gly Ala Pro Leu Glu Val Val Ala Ala Asn Gin Thr Gly Arg Leu Tyr 50 55 60

Asp Cys Ala Ala Ala Thr Gly Met Cys Gin Pro He Pro Leu His He 65 70 75 80

Arg Pro Glu Ala Val Asn Met Ser Leu Gly Leu Thr Leu Ala Ala Ser 85 90 95

Thr Asn Gly Ser Arg Leu Leu Ala Cys Gly Pro Thr Leu His Arg Val 100 105 110

Cys Gly Glu Asn Ser Tyr Ser Lys Gly Ser Cys Leu Leu Leu Gly Ser 115 120 125

Arg Trp Glu He He Gin Thr Val Pro Asp Ala Thr Pro Glu Cys Pro 130 135 140

His Gin Glu Met Asp He Val Phe Leu He Asp Gly Ser Gly Ser He 145 150 155 160

Asp Gin Asn Asp Phe Asn Gin Met Lys Gly Phe Val Gin Ala Val Met 165 170 175

Gly Gin Phe Glu Gly Thr Asp Thr Leu Phe Ala Leu Met Gin Tyr Ser 180 185 190

Asn Leu Leu Lys He His Phe Thr Phe Thr Gin Phe Arg Thr Ser Pro 195 200 205

Ser Gin Gin Ser Leu Val Asp Pro He Val Gin Leu Lys Gly Leu Thr 210 215 220

Phe Thr Ala Thr Gly He Leu Thr Val Val Thr Gin Leu Phe His His 225 230 235 240

Lys Asn Gly Ala Arg Lys Ser Ala Lys Lys He Leu He Val He Thr 245 250 255

Asp Gly Gin Lys Tyr Lys Asp Pro Leu Glu Tyr Ser Asp Val He Pro 260 265 270

Gin Ala Glu Lys Ala Gly He He Arg Tyr Ala He Gly Val Gly His 275 280 285

Ala Phe Gin Gly Pro Thr Ala Arg Gin Glu Leu Asn Thr He Ser Ser 290 295 300

Ala Pro Pro Gin Asp His Val Phe Lys Val Asp Asn Phe Ala Ala Leu 305 310 315 320

Gly Ser He Gin Lys Gin Leu Gin Glu Lys He Tyr Ala Val Glu Gly 325 330 335

Thr Gin Ser Arg Ala Ser Ser Ser Phe Gin His Glu Met Ser Gin Glu 340 345 350

Gly Phe Ser Thr Ala Leu Thr Met Asp Gly Leu Phe Leu Gly Ala Val 355 360 365

Gly Ser Phe Ser Trp Ser Gly Gly Ala Phe Leu Tyr Pro Pro Asn Met 370 375 380

Ser Pro Thr Phe He Asn Met Ser Gin Glu Asn Val Asp Met Arg Asp 385 390 395 400

Ser Tyr Leu Gly Tyr Ser Thr Glu Leu Ala Leu Trp Lys Gly Val Gin 405 410 415

Asn Leu Val Leu Gly Ala Pro Arg Tyr Gin His Thr Gly Lys Ala Val 420 425 430

He Phe Thr Gin Val Ser Arg Gin Trp Arg Lys Lys Ala Glu Val Thr 435 440 445

Gly Thr Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys Ser Val Asp 450 455 460

Val Asp Ser Asp Gly Ser Thr Asp Leu He Leu He Gly Ala Pro His 465 470 475 480

Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys Pro Leu Pro 485 490 495

Arg Gly Gin Arg Val Gin Trp Gin Cys Asp Ala Val Leu Arg Gly Glu 500 505 510

Gin Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu Gly 515 520 525

Asp Val Asn Glu Asp Lys Leu He Asp Val Ala He Gly Ala Pro Gly 530 535 540

Glu Gin Glu Asn Arg Gly Ala Val Tyr Leu Phe His Gly Ala Ser Glu 545 550 555 560

Ser Gly He Ser Pro Ser His Ser Gin Arg He Ala Ser Ser Gin Leu 565 570 575

Ser Pro Arg Leu Gin Tyr Phe Gly Gin Ala Leu Ser Gly Gly Gin Asp 580 585 590

Leu Thr Gin Asp Gly Leu Met Asp Leu Ala Val Gly Ala Arg Gly Gin 595 600 605

Val Leu Leu Leu Arg Ser Leu Pro Val Leu Lys Val Gly Val Ala Met 610 615 620

Arg Phe Ser Pro Val Glu Val Ala Lys Ala Val Tyr Arg Cys Trp Glu 625 630 635 640

Glu Lys Pro Ser Ala Leu Glu Ala Gly Asp Ala Thr Val Cys Leu Thr 645 650 655

He Gin Lys Ser Ser Leu Asp Gin Leu Gly Asp He Gin Ser Ser Val 660 665 670

Arg Phe Asp Leu Ala Leu Asp Pro Gly Arg Leu Thr Ser Arg Ala He 675 680 685

Phe Asn Glu Thr Lys Asn Pro Thr Leu Thr Arg Arg Lys Thr Leu Gly 690 695 700

Leu Gly He His Cys Glu Thr Leu Lys Leu Leu Leu Pro Asp Cys Val 705 710 715 720

Glu Asp Val Val Ser Pro He He Leu His Leu Asn Phe Ser Leu Val 725 730 735

Arg Glu Pro He Pro Ser Pro Gin Asn Leu Arg Pro Val Leu Ala Val 740 745 750

Gly Ser Gin Asp Leu Phe Thr Ala Ser Leu Pro Phe Glu Lys Asn Cys 755 760 765

Gly Gin Asp Gly Leu Cys Glu Gly Asp Leu Gly Val Thr Leu Ser Phe 770 775 780

Ser Gly Leu Gin Thr Leu Thr Val Gly Ser Ser Leu Glu Leu Asn Val 785 790 795 800

He Val Thr Val Trp Asn Ala Gly Glu Asp Ser Tyr Gly Thr Val Val 805 810 815

Ser Leu Tyr Tyr Pro Ala Gly Leu Ser His Arg Arg Val Ser Gly Ala 820 825 830

Gin Lys Gin Pro His Gin Ser Ala Leu Arg Leu Ala Cys Glu Thr Val 835 840 845

Pro Thr Glu Asp Glu Gly Leu Arg Ser Ser Arg Cys Ser Val Asn His 850 855 860

Pro He Phe His Glu Gly Ser Asn Gly Thr Phe He Val Thr Phe Asp 865 870 875 880

Val Ser Tyr Lys Ala Thr Leu Gly Asp Arg Met Leu Met Arg Ala Ser 885 890 895

Ala Ser Ser Glu Asn Asn Lys Ala Ser Ser Ser Lys Ala Thr Phe Gin 900 905 910

Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Thr Met He Ser Arg Gin 915 920 925

Glu Glu Ser Thr Lys Tyr Phe Asn Phe Ala Thr Ser Asp Glu Lys Lys 930 935 940

Met Lys Glu Ala Glu His Arg Tyr Arg Val Asn Asn Leu Ser Gin Arg 945 950 955 960

Asp Leu Ala He Ser He Asn Phe Trp Val Pro Val Leu Leu Asn Gly 965 970 975

Val Ala Val Trp Asp Val Val Met Glu Ala Pro Ser Gin Ser Leu Pro 980 985 990

Cys Val Ser Glu Arg Lys Pro Pro Gin His Ser Asp Phe Leu Thr Gin 995 1000 1005

He Ser Arg Ser Pro Met Leu Asp Cys Ser He Ala Asp Cys Leu Gin 1010 1015 1020

Phe Arg Cys Asp Val Pro Ser Phe Ser Val Gin Glu Glu Leu Asp Phe 1025 1030 1035 1Q40

Thr Leu Lys Gly Asn Leu Ser Phe Gly Trp Val Arg Glu Thr Leu Gin 1045 1050 1055

Lys Lys Val Leu Val Val Ser Val Ala Glu He Thr Phe Asp Thr Ser 1060 1065 1070

Val Tyr Ser Gin Leu Pro Gly Gin Glu Ala Phe Met Arg Ala Gin Met 1075 1080 1085

Glu Met Val Leu Glu Glu Asp Glu Val Tyr Asn Ala He Pro He He 1090 1095 1100

Met Gly Ser Ser Val Gly Ala Leu Leu Leu Leu Ala Leu He Thr Ala 1105 1110 1115 1120

Thr Leu Tyr Lys Leu Gly Phe Phe Lys Arg His Tyr Lys Glu Met Leu 1125 1130 1135

Glu Asp Lys Pro Glu Asp Thr Ala Thr Phe Ser Gly Asp Asp Phe Ser 1140 1145 1150

Cys Val Ala Pro Asn Val Pro Lys Ser 1155 1160

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1153 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

Met Ala Leu Arg Val Leu Leu Leu Thr Ala Leu Thr Leu Cys His Gly 1 5 10 15

Phe Asn Leu Asp Thr Glu Asn Ala Met Thr Phe Gin Glu Asn Ala Arg 20 25 30

Gly Phe Gly Gin Ser Val Val Gin Leu Gin Gly Ser Arg Val Val Val 35 40 45

Gly Ala Pro Gin Glu He Val Ala Ala Asn Gin Arg Gly Ser Leu Tyr 50 55 60

Gin Cys Asp Tyr Ser Thr Gly Ser Cys Glu Pro He Arg Leu Gin Val 65 70 75 80

Pro Val Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu Ala Ala Thr 85 90 95

Thr Ser Pro Pro Gin Leu Leu Ala Cys Gly Pro Thr Val His Gin Thr 100 105 110

Cys Ser Glu Asn Thr Tyr Val Lys Gly Leu Cys Phe Leu Phe Gly Ser 115 120 125

Asn Leu Arg Gin Gin Pro Gin Lys Phe Pro Glu Ala Leu Arg Gly Cys 130 135 140

Pro Gin Glu Asp Ser Asp He Ala Phe Leu He Asp Gly Ser Gly Ser 145 150 155 160

He He Pro His Asp Phe Arg Arg Met Lys Glu Phe Val Ser Thr Val 165 170 175

Met Glu Gin Leu Lys Lys Ser Lys Thr Leu Phe Ser Leu Met Gin Tyr 180 185 190

Ser Glu Glu Phe Arg He His Phe Thr Phe Lys Glu Phe Gin Asn Asn 195 200 205

Pro Asn Pro Arg Ser Leu Val Lys Pro He Thr Gin Leu Leu Gly Arg 210 215 220

Thr His Thr Ala Thr Gly He Arg Lys Val Val Arg Glu Leu Phe Asn 225 230 235 240

He Thr Asn Gly Ala Arg Lys Asn Ala Phe Lys He Leu Val Val He 245 250 255

Thr Asp Gly Glu Lys Phe Gly Asp Pro Leu Gly Tyr Glu Asp Val He 260 265 270

Pro Glu Ala Asp Arg Glu Gly Val He Arg Tyr Val He Gly Val Gly 275 280 285

Asp Ala Phe Arg Ser Glu Lys Ser Arg Gin Glu Leu Asn Thr He Ala 290 295 300

Ser Lys Pro Pro Arg Asp His Val Phe Gin Val Asn Asn Phe Glu Ala 305 310 315 320

Leu Lys Thr He Gin Asn Gin Leu Arg Glu Lys He Phe Ala He Glu 325 330 335

Gly Thr Gin Thr Gly Ser Ser Ser Ser Phe Glu His Glu Met Ser Gin 340 345 350

Glu Gly Phe Ser Ala Ala He Thr Ser Asn Gly Pro Leu Leu Ser Thr 355 360 365

Val Gly Ser Tyr Asp Trp Ala Gly Gly Val Phe Leu Tyr Thr Ser Lys 370 375 380

Glu Lys Ser Thr Phe He Asn Met Thr Arg Val Asp Ser Asp Met Asn 385 390 395 400

Asp Ala Tyr Leu Gly Tyr Ala Ala Ala He He Leu Arg Asn Arg Val 405 410 415

Gin Ser Leu Val Leu Gly Ala Pro Arg Tyr Gin His He Gly Leu Val 420 425 430

Ala Met Phe Arg Gin Asn Thr Gly Met Trp Glu Ser Asn Ala Asn Val 435 440 445

Lys Gly Thr Gin He Gly Ala Tyr Phe Gly Ala Ser Leu Cys Ser Val 450 455 460

Asp Val Asp Ser Asn Gly Ser Thr Asp Leu Val Leu He Gly Ala Pro 465 470 475 480

His Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys Pro Leu 485 490 495

Pro Arg Gly Gin Arg Ala Arg Trp Gin Cys Asp Ala Val Leu Tyr Gly 500 505 510

Glu Gin Gly Gin Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu 515 520 525

Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Ala He Gly Ala Pro 530 535 540

Gly Glu Glu Asp Asn Arg Gly Ala Val Tyr Leu Phe His Gly Thr Ser 545 550 555 560

Gly Ser Gly He Ser Pro Ser His Ser Gin Arg He Ala Gly Ser Lys 565 570 575

Leu Ser Pro Arg Leu Gin Tyr Phe Gly Gin Ser Leu Ser Gly Gly Gin 580 585 590

Asp Leu Thr Met Asp Gly Leu Val Asp Leu Thr Val Gly Ala Gin Gly 595 600 605

His Val Leu Leu Leu Arg Ser Gin Pro Val Leu Arg Val Lys Ala He 610 615 620

Met Glu Phe Asn Pro Arg Glu Val Ala Arg Asn Val Phe Glu Cys Asn 625 630 635 640

Asp Gin Val Val Lys Gly Lys Glu Ala Gly Glu Val Arg Val Cys Leu 645 650 655

His Val Gin Lys Ser Thr Arg Asp Arg Leu Arg Glu Gly Gin He Gin 660 665 670

Ser Val Val Thr Tyr Asp Leu Ala Leu Asp Ser Gly Arg Pro His Ser 675 680 685

Arg Ala Val Phe Asn Glu Thr Lys Asn Ser Thr Arg Arg Gin Thr Gin 690 695 700

Val Leu Gly Leu Thr Gin Thr Cys Glu Thr Leu Lys Leu Gin Leu Pro 705 710 715 720

Asn Cys He Glu Asp Pro Val Ser Pro He Val Leu Arg Leu Asn Phe 725 730 735

Ser Leu Val Gly Thr Pro Leu Ser Ala Phe Gly Asn Leu Arg Pro Val 740 745 750

Leu Ala Glu Asp Ala Gin Arg Leu Phe Thr Ala Leu Phe Pro Phe Glu 755 760 765

Lys Asn Cys Gly Asn Asp Asn He Cys Gin Asp Asp Leu Ser He Thr 770 775 780

Phe Ser Phe Met Ser Leu Asp Cys Leu Val Val Gly Gly Pro Arg Glu 785 790 795 800

Phe Asn Val Thr Val Thr Val Arg Asn Asp Gly Glu Asp Ser Tyr Arg 805 810 815

Thr Gin Val Thr Phe Phe Phe Pro Leu Asp Leu Ser Tyr Arg Lys Val 820 825 830

Ser Thr Leu Gin Asn Gin Arg Ser Gin Arg Ser Trp Arg Leu Ala Cys 835 840 845

Glu Ser Ala Ser Ser Thr Glu Val Ser Gly Ala Leu Lys Ser Thr Ser 850 855 860

Cys Ser He Asn His Pro He Phe Pro Glu Asn Ser Glu Val Thr Phe 865 870 875 880

Asn He Thr Phe Asp Val Asp Ser Lys Ala Ser Leu Gly Asn Lys Leu 885 890 895

Leu Leu Lys Ala Asn Val Thr Ser Glu Asn Asn Met Pro Arg Thr Asn 900 905 910

Lys Thr Glu Phe Gin Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr Met 915 920 925

Val Val Thr Ser His Gly Val Ser Thr Lys Tyr Leu Asn Phe Thr Ala 930 935 940

Ser Glu Asn Thr Ser Arg Val Met Gin His Gin Tyr Gin Val Ser Asn 945 950 955 960

Leu Gly Gin Arg Ser Leu Pro He Ser Leu Val Phe Leu Val Pro Val 965 970 975

Arg Leu Asn Gin Thr Val He Trp Asp Arg Pro Gin Val Thr Phe Ser 980 985 990

Glu Asn Leu Ser Ser Thr Cys His Thr Lys Glu Arg Leu Pro Ser His 995 1000 1005

Ser Asp Phe Leu Ala Glu Leu Arg Lys Ala Pro Val Val Asn Cys Ser 1010 1015 1020

He Ala Val Cys Gin Arg He Gin Cys Asp He Pro Phe Phe Gly He 1025 1030 1035 1040

Gin Glu Glu Phe Asn Ala Thr Leu Lys Gly Asn Leu Ser Phe Asp Trp 1045 1050 1055

Tyr He Lys Thr Ser His Asn His Leu Leu He Val Ser Thr Ala Glu 1060 1065 1070

He Leu Phe Asn Asp Ser Val Phe Thr Leu Leu Pro Gly Gin Gly Ala 1075 1080 1085

Phe Val Arg Ser Gin Thr Glu Thr Lys Val Glu Pro Phe Glu Val Pro 1090 1095 1100

Asn Pro Leu Pro Leu He Val Gly Ser Ser Val Gly Gly Leu Leu Leu 1105 1110 1115 1120

Leu Ala Leu He Thr Ala Ala Leu Tyr Lys Leu Gly Phe Phe Lys Arg 1125 1130 1135

Gin Tyr Lys Asp Met Met Ser Glu Gly Gly Pro Pro Gly Ala Glu Pro 1140 1145 1150

Gin

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1163 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:

Met Thr Arg Thr Arg Ala Ala Leu Leu Leu Phe Thr Ala Leu Ala Thr 1 5 10 15

Ser Leu Gly Phe Asn Leu Asp Thr Glu Glu Leu Thr Ala Phe Arg Val 20 25 30

Asp Ser Ala Gly Phe Gly Asp Ser Val Val Gin Tyr Ala Asn Ser Trp 35 40 45

Val Val Val Gly Ala Pro Gin Lys He He Ala Ala Asn Gin He Gly 50 55 60

Gly Leu Tyr Gin Cys Gly Tyr Ser Thr Gly Ala Cys Glu Pro He Gly 65 70 75 80

Leu Gin Val Pro Pro Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu 85 90 95

Ala Ser Thr Thr Ser Pro Ser Gin Leu Leu Ala Cys Gly Pro Thr Val 100 105 110

His His Glu Cys Gly Arg Asn Met Tyr Leu Thr Gly Leu Cys Phe Leu 115 120 125

Leu Gly Pro Thr Gin Leu Thr Gin Arg Leu Pro Val Ser Arg Gin Glu 130 135 140

Cys Pro Arg Gin Glu Gin Asp He Val Phe Leu He Asp Gly Ser Gly 145 150 155 160

Ser He Ser Ser Arg Asn Phe Ala Thr Met Met Asn Phe Val Arg Ala 165 170 175

Val He Ser Gin Phe Gin Arg Pro Ser Thr Gin Phe Ser Leu Met Gin 180 185 190

Phe Ser Asn Lys Phe Gin Thr His Phe Thr Phe Glu Glu Phe Arg Arg 195 200 205

Thr Ser Asn Pro Leu Ser Leu Leu Ala Ser Val His Gin Leu Gin Gly 210 215 220

Phe Thr Tyr Thr Ala Thr Ala He Gin Asn Val Val His Arg Leu Phe 225 230 235 240

His Ala Ser Tyr Gly Ala Arg Arg Asp Ala He Lys He Leu He Val 245 250 255

He Thr Asp Gly Lys Lys Glu Gly Asp Ser Leu Asp Tyr Lys Asp Val 260 265 270

He Pro Met Ala Asp Ala Ala Gly He He Arg Tyr Ala He Gly Val 275 280 285

Gly Leu Ala Phe Gin Asn Arg Asn Ser Trp Lys Glu Leu Asn Asp He 290 295 300

Ala Ser Lys Pro Ser Gin Glu His He Phe Lys Val Glu Asp Phe Asp 305 310 315 320

Ala Leu Lys Asp He Gin Asn Gin Leu Lys Glu Lys He Phe Ala He 325 330 335

Glu Gly Thr Glu Thr He Ser Ser Ser Ser Phe Glu Leu Glu Met Ala 340 345 350

Gln Glu Gly Phe Ser Ala Val Phe Thr Pro Asp Gly Pro Val Leu Gly 355 360 365

Ala Val Gly Ser Phe Thr Trp Ser Gly Gly Ala Phe Leu Tyr Pro Pro 370 375 380

Asn Met Ser Pro Thr Phe He Asn Met Ser Gin Glu Asn Val Asp Met 385 390 395 400

Arg Asp Ser Tyr Leu Gly Tyr Ser Thr Glu Leu Ala Leu Trp Lys Gly 405 410 415

Val Gin Ser Leu Val Leu Gly Ala Pro Arg Tyr Gin His He Gly Lys 420 425 430

Ala Val He Phe He Gin Val Ser Arg Gin Trp Arg Met Lys Ala Glu 435 440 445

Val He Gly Thr Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys Ser 450 455 460

Val Asp Val Asp Thr Asp Gly Ser Thr Asp Leu Val Leu He Gly Ala 465 470 475 480

Pro His Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Cys Pro 485 490 495

Leu Pro Arg Gly Trp Arg Arg Trp Trp Cys Asp Ala Val Leu Tyr Gly 500 505 510

Glu Gin Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu 515 520 525

Gly Asp Val Asn Gly Asp Lys Leu Thr Asp Val Val He Gly Ala Pro 530 535 540

Gly Glu Glu Glu Asn Arg Gly Ala Val Tyr Leu Phe His Gly Val Leu 545 550 555 560

Gly Pro Ser He Ser Pro Ser His Ser Gin Arg He Ala Gly Ser Gin 565 570 575

Leu Ser Ser Arg Leu Gin Tyr Phe Gly Gin Ala Leu Ser Gly Gly Gin 580 585 590

Asp Leu Thr Gin Asp Gly Leu Val Asp Leu Ala Val Gly Ala Arg Gly 595 600 605

Gin Val Leu Leu Leu Arg Thr Arg Pro Val Leu Trp Val Gly Val Ser 610 615 620

Met Gin Phe He Pro Ala Glu He Pro Arg Ser Ala Phe Glu Cys Arg 625 630 635 640

Glu Gin Val Val Ser Glu Gin Thr Leu Val Gin Ser Asn He Cys Leu 645 650 655

Tyr He Asp Lys Arg Ser Lys Asn Leu Leu Gly Ser Arg Asp Leu Gin 660 665 670

Ser Ser Val Thr Leu Asp Leu Ala Leu Ala Pro Gly Arg Leu Ser Pro 675 680 685

Arg Ala He Phe Gin Glu Thr Lys Asn Arg Ser Leu Ser Arg Val Arg 690 695 700

Val Leu Gly Leu Lys Ala His Cys Glu Asn Phe Asn Leu Leu Leu Pro 705 710 715 720

Ser Cys Val Glu Asp Ser Val He Pro He He Leu Arg Leu Asn Phe 725 730 735

Thr Leu Val Gly Lys Pro Leu Leu Ala Phe Arg Asn Leu Arg Pro Met 740 745 750

Leu Ala Ala Leu Ala Gin Arg Tyr Phe Thr Ala Ser Leu Pro Phe Glu 755 760 765

Lys Asn Cys Gly Ala Asp His He Cys Gin Asp Asn Leu Gly He Ser 770 775 780

Phe Ser Phe Pro Gly Leu Lys Ser Leu Leu Val Gly Ser Asn Leu Glu 785 790 795 800

Leu Asn Ala Glu Val Met Val Trp Asn Asp Gly Glu Asp Ser Tyr Gly 805 810 815

Thr Thr He Thr Phe Ser His Pro Ala Gly Leu Ser Tyr Arg Tyr Val 820 825 830

Ala Glu Gly Gin Lys Gin Gly Gin Leu Arg Ser Leu His Leu Thr Cys 835 840 845

Cys Ser Ala Pro.Val Gly Ser Gin Gly Thr Trp Ser Thr Ser Cys Arg 850 855 860

He Asn His Leu He Phe Arg Gly Gly Ala Gin He Thr Phe Leu Ala 865 870 875 880

Thr Phe Asp Val Ser Pro Lys Ala Val Gly Leu Asp Arg Leu Leu Leu 885 890 895

He Ala Asn Val Ser Ser Glu Asn Asn He Pro Arg Thr Ser Lys Thr 900 905 910

He Phe Gin Leu Glu Leu Pro Val Lys Tyr Ala Val Tyr He Val Val 915 920 925

Ser Ser His Glu Gin Phe Thr Lys Tyr Leu Asn Phe Ser Glu Ser Glu 930 935 940

Glu Lys Glu Ser His Val Ala Met His Arg Tyr Gin Val Asn Asn Leu 945 950 955 960

Gly Gin Arg Asp Leu Pro Val Ser He Asn Phe Trp Val Pro Val Glu 965 970 975

Leu Asn Gin Glu Ala Val Trp Met Asp Val Glu Val Ser His Pro Gin 980 985 990

Asn Pro Ser Leu Arg Cys Ser Ser Glu Lys He Ala Pro Pro Ala Ser 995 1000 1005

Asp Phe Leu Ala His He Gin Lys Asn Pro Val Leu Asp Cys Ser He 1010 1015 1020

Ala Gly Cys Leu Arg Phe Arg Cys Asp Val Pro Ser Phe Ser Val Gin 1025 1030 1035 1040

Glu Glu Leu Asp Phe Thr Leu Lys Gly Asn Leu Ser Phe Gly Trp Val 1045 1050 1055

Arg Gin He Leu Gin Lys Lys Val Ser Val Val Ser Val Ala Glu He 1060 1065 1070

He Phe Asp Thr Ser Val Tyr Ser Gin Leu Pro Gly Gin Glu Ala Phe 1075 1080 1085

Met Arg Ala Gin Thr He Thr Val Leu Glu Lys Tyr Lys Val His Asn 1090 1095 1100

Pro He Pro Leu He Val Gly Ser Ser He Gly Gly Leu Leu Leu Leu 1105 1110 1115 1120

Ala Leu He Thr Ala Val Leu Tyr Lys Val Gly Phe Phe Lys Arg Gin 1125 1130 1135

Tyr Lys Glu Met Met Glu Glu Ala Asn Gly Gin He Ala Pro Glu Asn 1140 1145 1150

Gly Thr Gin Thr Pro Ser Pro Pro Ser Glu Lys 1155 1160

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Phe Asn Leu Asp Val Glu Glu Pro Met Val Phe Gin 1 5 10

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 35 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: TTYAAYYTGG AYGTNGARGA RCCNATGGTN TTYCA 35

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: TTCAACCTGG ACGTGGAGGA GCCCATGGTG TTCCAA 36

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TTCAACCTGG ACGTNGAASA NCCCATGGTC TTCCAA 36

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: TTYAAYYTNG AYGTNGARGA RCC 23

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: TTYAAYYTGG ACGTNGAAGA 20

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: TGRAANACCA TNGGYTC 17 (2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: TTGGAAGACC ATNGGYTC 18 (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: ATTAACCCTC ACTAAAG 17

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: AATACGACTC ACTATAG 17

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 11 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Val Phe Gin Glu Xaa Gly Ala Gly Phe Gly Gin 1 5 10

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Leu Tyr Asp Xaa Val Ala Ala Thr Gly Leu Xaa Gin Pro He 1 5 10

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 12 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Pro Leu Glu Tyr Xaa Asp Val He Pro Gin Ala Glu 1 5 10

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 10 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Phe Gin Glu Gly Phe Ser Xaa Val Leu Xaa 1 5 10

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Thr Ser Pro Thr Phe He Xaa Met Ser Gin Glu Asn Val Asp 1 5 10

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Leu Val Val Gly Ala Pro Leu Glu Val Val Ala Val Xaa Gin Thr Gly 1 5 10 15

Arg

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Leu Asp Xaa Lys Pro Xaa Asp Thr Ala 1 5

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 7 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

Phe Gly Glu Gin Phe Ser Glu 1 5

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: RAANCCYTCY TGRAAACTYT C 21

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1006 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

TTCAACCTGG ACGTGGAGGA GCCCATGGTG TTCAAGAGGA TGGAGCTGGC TTTGGACAGA 60

GCGTGGCCCA GCTTGGCGGA TCTAGACTCG TGGTGGGAGC CCCCCTGGAG GTGGTGGCGG 120

TCAACCAAAC AGGAAGGTTG TATGACTGTG TGGCTGCCAC TGGCCTTGTC AACCCATACC 180

CCTGCACACA CCCCCAGATG CTGTGAACAT GTCCCTGGGT CTGTCCCTGT CAGCCGCCGC 240

CAGTCGCCCC TGGCTGCTGG CCTGTGGCCC AACCATGCAC AGAGCCTGTG GGGAGAATAT 300

GTATGCAGAA GGCTTTTGCC TCCTGTTGGA CTCCCATCTG CAGACCATTT GGACAGTACC 360

TGCTGCCCTA CCAGAGTGTC CAAGTCAAGA GATGGACATT GTCTTCCTGA TTGATGGTTC 420

TGGCAGTATG AGCAAAGTGA CTTTAAACAA ATGAAGGATT TGTGAGAGCT GTGATGGGAC 480

AGTTTGAGGG CACCCAAACC CTGTTCTCAC TGATACAGTA TCCCACCTCC CTGAAGATCC 540

ACTTCACCTT CACGCAATTC CAGAGCAGCT GGAACCCTCT GAGCCTGGTG GATCCCATTG 600

TCCAACTGGA CGGCCTGACA TATACAGCCA CGGGCATCCG GAAAGTGGTG GAGGAACTGT 660

TTCATAGTAA GAATGGGGCC CGTAAAAGTG CCAAGAAGAT CCTCATTGTC ATCACAGATG 720

GCAAAAATAC AAAGACCCCC TGGAGTACGA GGACGTATCC CCAGGCAGAG AGAGCGGATC 780

ATCCGCTATG CCATTGGGGT GGGAGATGCT TTCTGGAAAC CCAGTGCCAA GCAGGAGCTG 840

GACAACATTG GCTCAGAGCC GGCTCAGGAC CATGTGTTCA GGGTGGACAA CTTTGCAGCA 900

CTCAGCAGCA TCCAGGAGCA GCTGCAGGAG AAGATCTTTG CACTCGAAGG AACCCAGTCG 960

ACGACAAGTA GCTCTTTCCA ACATGAGATG TTCCAAGAAG GGTTCA 1006 (2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 17 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: GTNTTYCARG ARGAYGG 17

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CCACTGTCAG GATGCCCGTG 20

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: AGTTACGAAT TCGCCACCAT GGCTCTACGG GTGCTTCTTC TG 42

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 42 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: AGTTACGAAT TCGCCACCAT GACTCGGACT GTGCTTCTTC TG 42

(2) INFORMATION FOR SEQ ID NO:29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: AGTTACGAAT TCGCCACCAT GACCTTCGGC ACTGTG 36

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

( i) SEQUENCE DESCRIPTION: SEQ ID NO:30: TTGCTGACTG CCTGCAGTTC 20

(2) INFORMATION FOR SEQ ID NO:31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GTTCTGACGC GTAATGGCAT TGTAGACCTC GTCTTC 36

(2) INFORMATION FOR SEQ ID NO:32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: ACGTATGCAG GATCCCATCA AGAGATGGAC ATCGCT 36

(2) INFORMATION FOR SEQ ID NO:33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 37 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: ACTGCATGTC TCGAGGCTGA AGCCTTCTTG GGACATC 37

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: TATAGACTGC TGGGTAGTCC CCAC 24

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: TGAAGATTGG GGGTAAATAA CAGA 24

(2) INFORMATION FOR SEQ ID NO:36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3528 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..3456

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:

GGC TGG GCC CTG GCT TCC TGT CAT GGG TCT AAC CTG GAT GTG GAG GAA 48 Gly Trp Ala Leu Ala Ser Cys His Gly Ser Asn Leu Asp Val Glu Glu 1 5 10 15

CCC ATC GTG TTC AGA GAG GAT GCA GCC AGC TTT GGA CAG ACT GTG GTG 96 Pro lie Val Phe Arg Glu Asp Ala Ala Ser Phe Gly Gin Thr Val Val 20 25 30

CAG TTT GGT GGA TCT CGA CTC GTG GTG GGA GCC CCT CTG GAG GCG GTG 144 Gin Phe Gly Gly Ser Arg Leu Val Val Gly Ala Pro Leu Glu Ala Val 35 40 45

GCA GTC AAC CAA ACA GGA CGG TTG TAT GAC TGT GCA CCT GCC ACT GGC 192 Ala Val Asn Gin Thr Gly Arg Leu Tyr Asp Cys Ala Pro Ala Thr Gly 50 55 60

ATG TGC CAG CCC ATC GTA CTG CGC AGT CCC CTA GAG GCA GTG AAC ATG 240 Met Cys Gin Pro lie Val Leu Arg Ser Pro Leu Glu Ala Val Asn Met 65 70 75 80

TCC CTG GGC CTG TCT CTG GTG ACT GCC ACC AAT AAC GCC CAG TTG CTG 288 Ser Leu Gly Leu Ser Leu Val Thr Ala Thr Asn Asn Ala Gin Leu Leu 85 90 95

GCT TGT GGT CCA ACT GCA CAG AGA GCT TGT GTG AAG AAC ATG TAT GCG 336 Ala Cys Gly Pro Thr Ala Gin Arg Ala Cys Val Lys Asn Met Tyr Ala 100 105 110

AAA GGT TCC TGC CTC CTT CTC GGC TCC AGC TTG CAG TTC ATC CAG GCA 384 Lys Gly Ser Cys Leu Leu Leu Gly Ser Ser Leu Gin Phe lie Gin Ala 115 120 125

GTC CCT GCC TCC ATG CCA GAG TGT CCA AGA CAA GAG ATG GAC ATT GCT 432 Val Pro Ala Ser Met Pro Glu Cys Pro Arg Gin Glu Met Asp lie Ala 130 135 140

TTC CTG ATT GAT GGT TCT GGC AGC ATT AAC CAA AGG GAC TTT GCC CAG 480 Phe Leu lie Asp Gly Ser Gly Ser lie Asn Gin Arg Asp Phe Ala Gin 145 150 155 160

ATG AAG GAC TTT GTC AAA GCT TTG ATG GGA GAG TTT GCG AGC ACC AGC 528 Met Lys Asp Phe Val Lys Ala Leu Met Gly Glu Phe Ala Ser Thr Ser 165 170 175

ACC TTG TTC TCC CTG ATG CAA TAC TCG AAC ATC CTG AAG ACC CAT TTT 576 Thr Leu Phe Ser Leu Met Gin Tyr Ser Asn lie Leu Lys Thr His Phe 180 185 190

ACC TTC ACT GAA TTC AAG AAC ATC CTG GAC CCT CAG AGC CTG GTG GAT 624 Thr Phe Thr Glu Phe Lys Asn lie Leu Asp Pro Gin Ser Leu Val Asp 195 200 205

CCC ATT GTC CAG CTG CAA GGC CTG ACC TAC ACA GCC ACA GGC ATC CGG 672 Pro lie Val Gin Leu Gin Gly Leu Thr Tyr Thr Ala Thr Gly lie Arg 210 215 220

ACA GTG ATG GAA GAG CTA TTT CAT AGC AAG AAT GGG TCC CGT AAA AGT 720 Thr Val Met Glu Glu Leu Phe His Ser Lys Asn Gly Ser Arg Lys Ser 225 230 235 240

GCC AAG AAG ATC CTC CTT GTC ATC ACA GAT GGG CAG AAA TAC AGA GAC 768 Ala Lys Lys lie Leu Leu Val lie Thr Asp Gly Gin Lys Tyr Arg Asp 245 250 255

CCC CTG GAG TAT AGT GAT GTC ATT CCC GCC GCA GAC AAA GCT GGC ATC 816 Pro Leu Glu Tyr Ser Asp Val lie Pro Ala Ala Asp Lys Ala Gly lie 260 265 270

ATT CGT TAT GCT ATT GGG GTG GGA GAT GCC TTC CAG GAG CCC ACT GCC 864 lie Arg Tyr Ala lie Gly Val Gly Asp Ala Phe Gin Glu Pro Thr Ala 275 280 285

CTG AAG GAG CTG AAC ACC ATT GGC TCA GCT CCC CCA CAG GAC CAC GTG 912 Leu Lys Glu Leu Asn Thr lie Gly Ser Ala Pro Pro Gin Asp His Val 290 295 300

TTC AAG GTA GGC AAC TTT GCA GCA CTT CGC AGC ATC CAG AGG CAA CTT 960 Phe Lys Val Gly Asn Phe Ala Ala Leu Arg Ser lie Gin Arg Gin Leu 305 310 315 320

CAG GAG AAA ATC TTC GCC ATT GAG GGA ACT CAA TCA AGG TCA AGT AGT 1008 Gin Glu Lys lie Phe Ala lie Glu Gly Thr Gin Ser Arg Ser Ser Ser 325 330 335

TCC TTT CAG CAC GAG ATG TCA CAA GAA GGT TTC AGT TCA GCT CTC ACA 1056 Ser Phe Gin His Glu Met Ser Gin Glu Gly Phe Ser Ser Ala Leu Thr 340 345 350

TCG GAT GGA CCC GTT CTG GGG GCC GYG GGA AGC TTC AGC TGG TCC GGA 1104 Ser Asp Gly Pro Val Leu Gly Ala Xaa Gly Ser Phe Ser Trp Ser Gly 355 360 365

GGT GCC TTC TTA TAT CCC CCA AAT ACG AGA CCC ACC TTT ATC AAC ATG 1152 Gly Ala Phe Leu Tyr Pro Pro Asn Thr Arg Pro Thr Phe lie Asn Met 370 375 380

TCT CAG GAG AAT GTG GAC ATG AGA GAC TCC TAC CTG GGT TAC TCC ACC 1200 Ser Gin Glu Asn Val Asp Met Arg Asp Ser Tyr Leu Gly Tyr Ser Thr 385 390 395 400

GCA GTG GCC TTT TGG AAG GGG GTT CAC AGC CTG ATC CTG GGG GCC CCG 1248 Ala Val Ala Phe Trp Lys Gly Val His Ser Leu lie Leu Gly Ala Pro 405 410 415

CGT CAC CAG CAC ACG GGG AAG GTT GTC ATC TTT ACC CAG GAA GCC AGG 1296 Arg His Gin His Thr Gly Lys Val Val He Phe Thr Gin Glu Ala Arg 420 425 430

CAT TGG AGG CCC AAG TCT GAA GTC AGA GGG ACA CAG ATC GGC TCC TAC 1344 His Trp Arg Pro Lys Ser Glu Val Arg Gly Thr Gin He Gly Ser Tyr 435 440 445

TTC GGG GCC TCT CTC TGT TCT GTG GAC GTG GAT AGA GAT GGC AGC ACY 1392 Phe Gly Ala Ser Leu Cys Ser Val Asp Val Asp Arg Asp Gly Ser Xaa 450 455 460

GAC CTG GTC CTG ATC GGA GCC CCC CAT TAC TAT GAG CAG ACC CGA GGG 1440 Asp Leu Val Leu He Gly Ala Pro His Tyr Tyr Glu Gin Thr Arg Gly 465 470 475 480

GGG CAG GTC TCA GTG TKC CCC GTG CCC GGT GTG AGG GGC AGG TGG CAG 1488 Gly Gin Val Ser Val Xaa Pro Val Pro Gly Val Arg Gly Arg Trp Gin 485 490 495

TGT GAG GCC ACC CTC CAC GGG GAG CAG GRC CAT CCT TGG GGC CGC TTT 1536 Cys Glu Ala Thr Leu His Gly Glu Gin Xaa His Pro Trp Gly Arg Phe 500 505 510

GGG GTG GCT CTG ACA GTG CTG GGG GAC GTA AAC GGG GAC AAT CTG GCA 1584 Gly Val Ala Leu Thr Val Leu Gly Asp Val Asn Gly Asp Asn Leu Ala 515 520 525

GAC GTG GCT ATT GGT GCC CCT GGA GAG GAG GAG AGC AGA GGT GCT GTC 1632 Asp Val Ala He Gly Ala Pro Gly Glu Glu Glu Ser Arg Gly Ala Val 530 535 540

TAC ATA TTT CAT GGA GCC TCG AGA CTG GAG ATC ATG CCC TCA CCC AGC 1680 Tyr He Phe His Gly Ala Ser Arg Leu Glu He Met Pro Ser Pro Ser 545 550 555 560

CAG CGG GTC ACT GGC TCC CAG CTC TCC CTG AGA CTG CAG TAT TTT GGG 1728 Gin Arg Val Thr Gly Ser Gin Leu Ser Leu Arg Leu Gin Tyr Phe Gly 565 570 575

CAG TCA TTG AGT GGG GGT CAG GAC CTT ACA CAG GAT GGC CTG GTG GAC 1776 Gin Ser Leu Ser Gly Gly Gin Asp Leu Thr Gin Asp Gly Leu Val Asp 580 585 590

CTG GCC GTG GGA GCC CAG GGG CAC GTA CTG CTG CTC AGG AGT CTG CCT 1824 Leu Ala Val Gly Ala Gin Gly His Val Leu Leu Leu Arg Ser Leu Pro 595 600 605

CTG CTG AAA GTG GAG CTC TCC ATA AGA TTC GCC CCC ATG GAG GTG GCA 1872 Leu Leu Lys Val Glu Leu Ser He Arg Phe Ala Pro Met Glu Val Ala 610 615 620

AAG GCT GTG TAC CAG TGC TGG GAA AGG ACT CCC ACT GTC CTC GAA GCT 1920 Lys Ala Val Tyr Gin Cys Trp Glu Arg Thr Pro Thr Val Leu Glu Ala 625 630 635 640

GGA GAG GCC ACT GTC TGT CTC ACT GTC CAC AAA GGC TCA CCT GAC CTG 1968 Gly Glu Ala Thr Val Cys Leu Thr Val His Lys Gly Ser Pro Asp Leu 645 650 655

TTA GGT AAT GTC CAA GGC TCT GTC AGG TAT GAT CTG GCG TTA GAT CCG 2016 Leu Gly Asn Val Gin Gly Ser Val Arg Tyr Asp Leu Ala Leu Asp Pro 660 665 670

GGC CGC CTG ATT TCT CGT GCC ATT TTT GAT GAG ACT AAG AAC TGC ACT 2064 Gly Arg Leu He Ser Arg Ala He Phe Asp Glu Thr Lys Asn Cys Thr 675 680 685

TTG ACG GGA AGG AAG ACT CTG GGG CTT GGT GAT CAC TGC GAA ACA GTG 2112 Leu Thr Gly Arg Lys Thr Leu Gly Leu Gly Asp His Cys Glu Thr Val 690 695 700

AAG CTG CTT TTG CCG GAC TGT GTG GAA GAT GCA GTG AGC CCT ATC ATC 2160 Lys Leu Leu Leu Pro Asp Cys Val Glu Asp Ala Val Ser Pro He He 705 710 715 720

CTG CGC CTC AAC TTT TCC CTG GTG AGA GAC TCT GCT TCA CCC AGG AAC 2208 Leu Arg Leu Asn Phe Ser Leu Val Arg Asp Ser Ala Ser Pro Arg Asn 725 730 735

CTG CAT CCT GTG CTG GCT GTG GGC TCA CAA GAC CAC ATA ACT GCT TCT 2256 Leu His Pro Val Leu Ala Val Gly Ser Gin Asp His He Thr Ala Ser 740 745 750

CTG CCG TTT GAG AAG AAC TGT AAG CAA GAA CTC CTG TGT GAG GGG GAC 2304 Leu Pro Phe Glu Lys Asn Cys Lys Gin Glu Leu Leu Cys Glu Gly Asp 755 760 765

CTG GGC ATC AGC TTT AAC TTC TCA GGC CTG CAG GTC TTG GTG GTG GGA 2352 Leu Gly He Ser Phe Asn Phe Ser Gly Leu Gin Val Leu Val Val Gly 770 775 780

- Ill -

GGC TCC CCA GAG CTC ACT GTG ACA GTC ACT GTG TGG AAT GAG GGT GAG 2400 Gly Ser Pro Glu Leu Thr Val Thr Val Thr Val Trp Asn Glu Gly Glu 785 790 795 800

GAC AGC TAT GGA ACT TTA GTC AAG TTC TAC TAC CCA GCA GGG CTA TCT 2448 Asp Ser Tyr Gly Thr Leu Val Lys Phe Tyr Tyr Pro Ala Gly Leu Ser 805 810 815

TAC CGA CGG GTA ACA GGG ACT CAG CAA CCT CAT CAG TAC CCA CTA CGC 2496 Tyr Arg Arg Val Thr Gly Thr Gin Gin Pro His Gin Tyr Pro Leu Arg 820 825 830

TTG GCC TGT GAG GCT GAG CCC GCT GCC CAG GAG GAC CTG AGG AGC AGC 2544 Leu Ala Cys Glu Ala Glu Pro Ala Ala Gin Glu Asp Leu Arg Ser Ser 835 840 845

AGC TGT AGC ATT AAT CAC CCC ATC TTC CGA GAA GGT GCA AAG ACC ACC 2592 Ser Cys Ser He Asn His Pro He Phe Arg Glu Gly Ala Lys Thr Thr 850 855 860

TTC ATG ATC ACA TTC GAT GTC TCC TAC AAG GCC TTC CTA GGA GAC AGG 2640 Phe Met He Thr Phe Asp Val Ser Tyr Lys Ala Phe Leu Gly Asp Arg 865 870 875 880

TTG CTT CTG AGG GCC AAA GCC AGC AGT GAG AAT AAT AAG CCT GAT ACC 2688 Leu Leu Leu Arg Ala Lys Ala Ser Ser Glu Asn Asn Lys Pro Asp Thr 885 890 895

AAC AAG ACT GCC TTC CAG CTG GAG CTC CCA GTG AAG TAC ACC GTC TAT 2736 Asn Lys Thr Ala Phe Gin Leu Glu Leu Pro Val Lys Tyr Thr Val Tyr 900 905 910

ACC CTG ATC AGT AGG CAA GAA GAT TCC ACC AAC CAT GTC AAC TTT TCA 2784 Thr Leu He Ser Arg Gin Glu Asp Ser Thr Asn His Val Asn Phe Ser 915 920 925

TCT TCC CAC GGG GGG AGA AGG CAA GAA GCC GCA CAT CGC TAT CGT GTG 2832 Ser Ser His Gly Gly Arg Arg Gin Glu Ala Ala His Arg Tyr Arg Val 930 935 940

AAT AAC CTG AGT CCA CTG AAG CTG GCC GTC AGA GTT AAC TTC TGG GTC 2880 Asn Asn Leu Ser Pro Leu Lys Leu Ala Val Arg Val Asn Phe Trp Val 945 950 955 960

CCT GTC CTT CTG AAC GGT GTG GCT GTG TGG GAC GTG ACT CTG AGC AGC 2928 Pro Val Leu Leu Asn Gly Val Ala Val Trp Asp Val Thr Leu Ser Ser 965 970 975

CCA GCA CAG GGT GTC TCC TGC GTG TCC CAG ATG AAA CCT CCT CAG AAT 2976 Pro Ala Gin Gly Val Ser Cys Val Ser Gin Met Lys Pro Pro Gin Asn 980 985 990

CCC GAC TTT CTG ACC CAG ATT CAG AGA CGT TCT GTG CTG GAC TGC TCC 3024 Pro Asp Phe Leu Thr Gin He Gin Arg Arg Ser Val Leu Asp Cys Ser 995 1000 1005

ATT GCT GAC TGC CTG CAC TCC CGC TGT GAC ATC CCC TCC TTG GAC ATC 3072 He Ala Asp Cys Leu His Ser Arg Cys Asp He Pro Ser Leu Asp He 1010 1015 1020

CAG GAT GAA CTT GAC TTC ATT CTG AGG GGC AAC CTC AGC TTC GGC TGG 3120 Gin Asp Glu Leu Asp Phe He Leu Arg Gly Asn Leu Ser Phe Gly Trp 1025 1030 1035 1040

GTC AGT CAG ACA TTG CAG GAA AAG GTG TTG CTT GTG AGT GAG GCT GAA 3168 Val Ser Gin Thr Leu Gin Glu Lys Val Leu Leu Val Ser Glu Ala Glu 1045 1050 1055

ATC ACT TTC GAC ACA TCT GTG TAC TCC CAG CTG CCA GGA CAG GAG GCA 3216 He Thr Phe Asp Thr Ser Val Tyr Ser Gin Leu Pro Gly Gin Glu Ala 1060 1065 1070

TTT CTG AGA GCC CAG GTG GAG ACA ACG TTA GAA GAA TAC GTG GTC TAT 3264 Phe Leu Arg Ala Gin Val Glu Thr Thr Leu Glu Glu Tyr Val Val Tyr 1075 1080 1085

GAG CCC ATC TTC CTC GTG GCG GGC AGC TCG GTG GGA GGT CTG CTG TTA 3312 Glu Pro He Phe Leu Val Ala Gly Ser Ser Val Gly Gly Leu Leu Leu 1090 1095 1100

CTG GCT CTC ATC ACA GTG GTA CTG TAC AAG CTT GGC TYC TYC AAA CGT 3360 Leu Ala Leu He Thr Val Val Leu Tyr Lys Leu Gly Xaa Xaa Lys Arg 1105 1110 1115 1120

CAG TAC AAA GAA ATG CTG GAC GGC AAG GCT GCA GAT CCT GTC ACA GCC 3408 Gin Tyr Lys Glu Met Leu Asp Gly Lys Ala Ala Asp Pro Val Thr Ala 1125 1130 1135

GGC CAG GCA GAT TTC GGC TGT GAG ACT CCT CCA TAT CTC GTG AGC TAGGAATCCA 3463 Gly Gin Ala Aεp Phe Gly Cys Glu Thr Pro Pro Tyr Leu Val Ser 1140 1145 1150

CTCTCCTGCC TATCTCTGNA ATGAAGATTG GTCCTGCCTA TGAGTCTACT GGCATGGGAA 3523

CGAGT 3528

(2) INFORMATION FOR SEQ ID NO:37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1151 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:

Gly Trp Ala Leu Ala Ser Cys His Gly Ser Asn Leu Asp Val Glu Glu 1 5 10 15

Pro He Val Phe Arg Glu Asp Ala Ala Ser Phe Gly Gin Thr Val Val 20 25 30

Gin Phe Gly Gly Ser Arg Leu Val Val Gly Ala Pro Leu Glu Ala Val 35 40 45

Ala Val Asn Gin Thr Gly Arg Leu Tyr Asp Cys Ala Pro Ala Thr Gly 50 55 60

Met Cys Gin Pro He Val Leu Arg Ser Pro Leu Glu Ala Val Asn Met 65 70 75 80

Ser Leu Gly Leu Ser Leu Val Thr Ala Thr Asn Asn Ala Gin Leu Leu 85 90 95

Ala Cys Gly Pro Thr Ala Gin Arg Ala Cys Val Lys Asn Met Tyr Ala 100 105 110

Lys Gly Ser Cys Leu Leu Leu Gly Ser Ser Leu Gin Phe He Gin Ala 115 120 125

Val Pro Ala Ser Met Pro Glu Cys Pro Arg Gin Glu Met Asp He Ala 130 135 140

Phe Leu He Asp Gly Ser Gly Ser He Asn Gin Arg Asp Phe Ala Gin 145 150 155 160

Met Lys Asp Phe Val Lys Ala Leu Met Gly Glu Phe Ala Ser Thr Ser 165 170 175

Thr Leu Phe Ser Leu Met Gin Tyr Ser Asn He Leu Lys Thr His Phe 180 185 190

Thr Phe Thr Glu Phe Lys Asn He Leu Asp Pro Gin Ser Leu Val Asp 195 200 205

Pro He Val Gin Leu Gin Gly Leu Thr Tyr Thr Ala Thr Gly He Arg 210 215 220

Thr Val Met Glu Glu Leu Phe His Ser Lys Asn Gly Ser Arg Lys Ser 225 230 235 240

Ala Lys Lys He Leu Leu Val He Thr Asp Gly Gin Lys Tyr Arg Asp 245 250 255

Pro Leu Glu Tyr Ser Asp Val He Pro Ala Ala Asp Lys Ala Gly He 260 265 270

He Arg Tyr Ala He Gly Val Gly Asp Ala Phe Gin Glu Pro Thr Ala 275 280 285

Leu Lys Glu Leu Asn Thr He Gly Ser Ala Pro Pro Gin Asp His Val 290 295 300

Phe Lys Val Gly Asn Phe Ala Ala Leu Arg Ser He Gin Arg Gin Leu 305 310 315 320

Gin Glu Lys He Phe Ala He Glu Gly Thr Gin Ser Arg Ser Ser Ser 325 330 335

Ser Phe Gin His Glu Met Ser Gin Glu Gly Phe Ser Ser Ala Leu Thr 340 345 350

Ser Asp Gly Pro Val Leu Gly Ala Xaa Gly Ser Phe Ser Trp Ser Gly 355 360 365

Gly Ala Phe Leu Tyr Pro Pro Asn Thr Arg Pro Thr Phe He Asn Met 370 375 380

Ser Gin Glu Asn Val Asp Met Arg Asp Ser Tyr Leu Gly Tyr Ser Thr 385 390 395 400

Ala Val Ala Phe Trp Lys Gly Val His Ser Leu He Leu Gly Ala Pro 405 410 415

Arg His Gin His Thr Gly Lys Val Val He Phe Thr Gin Glu Ala Arg 420 425 430

His Trp Arg Pro Lys Ser Glu Val Arg Gly Thr Gin He Gly Ser Tyr 435 440 445

Phe Gly Ala Ser Leu Cys Ser Val Asp Val Asp Arg Asp Gly Ser Xaa 450 455 460

Asp Leu Val Leu He Gly Ala Pro His Tyr Tyr Glu Gin Thr Arg Gly 465 470 475 480

Gly Gin Val Ser Val Xaa Pro Val Pro Gly Val Arg Gly Arg Trp Gin 485 490 495

Cys Glu Ala Thr Leu His Gly Glu Gin Xaa His Pro Trp Gly Arg Phe 500 505 510

Gly Val Ala Leu Thr Val Leu Gly Asp Val Asn Gly Asp Asn Leu Ala 515 520 * 525

Asp Val Ala He Gly Ala Pro Gly Glu Glu Glu Ser Arg Gly Ala Val 530 535 540

Tyr He Phe His Gly Ala Ser Arg Leu Glu He Met Pro Ser Pro Ser 545 550 555 560

Gin Arg Val Thr Gly Ser Gin Leu Ser Leu Arg Leu Gin Tyr Phe Gly 565 570 575

Gin Ser Leu Ser Gly Gly Gin Asp Leu Thr Gin Asp Gly Leu Val Asp 580 585 590

Leu Ala Val Gly Ala Gin Gly His Val Leu Leu Leu Arg Ser Leu Pro 595 600 605

Leu Leu Lys Val Glu Leu Ser He Arg Phe Ala Pro Met Glu Val Ala 610 615 620

Lys Ala Val Tyr Gin Cys Trp Glu Arg Thr Pro Thr Val Leu Glu Ala 625 630 635 640

Gly Glu Ala Thr Val Cys Leu Thr Val His Lys Gly Ser Pro Asp Leu 645 650 655

Leu Gly Asn Val Gin Gly Ser Val Arg Tyr Asp Leu Ala Leu Asp Pro 660 665 670

Gly Arg Leu He Ser Arg Ala He Phe Asp Glu Thr Lys Asn Cys Thr 675 680 685

Leu Thr Gly Arg Lys Thr Leu Gly Leu Gly Asp His Cys Glu Thr Val 690 695 700

Lys Leu Leu Leu Pro Asp Cys Val Glu Asp Ala Val Ser Pro He He 705 710 715 720

Leu Arg Leu Asn Phe Ser Leu Val Arg Asp Ser Ala Ser Pro Arg Asn 725 730 735

Leu His Pro Val Leu Ala Val Gly Ser Gin Asp His He Thr Ala Ser 740 745 750

Leu Pro Phe Glu Lys Asn Cys Lys Gin Glu Leu Leu Cys Glu Gly Asp 755 760 765

Leu Gly He Ser Phe Asn Phe Ser Gly Leu Gin Val Leu Val Val Gly 770 775 780

Gly Ser Pro Glu Leu Thr Val Thr Val Thr Val Trp Asn Glu Gly Glu 785 790 795 800

Asp Ser Tyr Gly Thr Leu Val Lys Phe Tyr Tyr Pro Ala Gly Leu Ser 805 810 815

Tyr Arg Arg Val Thr Gly Thr Gin Gin Pro His Gin Tyr Pro Leu Arg 820 825 830

Leu Ala Cys Glu Ala Glu Pro Ala Ala Gin Glu Asp Leu Arg Ser Ser 835 840 845

Ser Cys Ser He Asn His Pro He Phe Arg Glu Gly Ala Lys Thr Thr 850 855 860

Phe Met He Thr Phe Asp Val Ser Tyr Lys Ala Phe Leu Gly Asp Arg 865 870 875 880

Leu Leu Leu Arg Ala Lys Ala Ser Ser Glu Asn Asn Lys Pro Asp Thr 885 890 895

Asn Lys Thr Ala Phe Gin Leu Glu Leu Pro Val Lys Tyr Thr Val Tyr 900 905 910

Thr Leu He Ser Arg Gin Glu Asp Ser Thr Asn His Val Asn Phe Ser 915 920 925

Ser Ser His Gly Gly Arg Arg Gin Glu Ala Ala His Arg Tyr Arg Val 930 935 940

Asn Asn Leu Ser Pro Leu Lys Leu Ala Val Arg Val Asn Phe Trp Val 945 950 955 960

Pro Val Leu Leu Asn Gly Val Ala Val Trp Asp Val Thr Leu Ser Ser 965 970 975

Pro Ala Gin Gly Val Ser Cys Val Ser Gin Met Lys Pro Pro Gin Asn 980 985 990

Pro Asp Phe Leu Thr Gin He Gin Arg Arg Ser Val Leu Asp Cys Ser 995 1000 1005

He Ala Asp Cys Leu His Ser Arg Cys Asp He Pro Ser Leu Asp He 1010 1015 1020

Gln Asp Glu Leu Asp Phe He Leu Arg Gly Asn Leu Ser Phe Gly Trp 1025 1030 1035 1040

Val Ser Gin Thr Leu Gin Glu Lys Val Leu Leu Val Ser Glu Ala Glu 1045 1050 1055

He Thr Phe Asp Thr Ser Val Tyr Ser Gin Leu Pro Gly Gin Glu Ala 1060 1065 1070

Phe Leu Arg Ala Gin Val Glu Thr Thr Leu Glu Glu Tyr Val Val Tyr 1075 1080 1085

Glu Pro He Phe Leu Val Ala Gly Ser Ser Val Gly Gly Leu Leu Leu 1090 1095 1100

Leu Ala Leu He Thr Val Val Leu Tyr Lys Leu Gly Xaa Xaa Lys Arg 1105 1110 1115 1120

Gin Tyr Lys Glu Met Leu Asp Gly Lys Ala Ala Asp Pro Val Thr Ala 1125 1130 1135

Gly Gin Ala Asp Phe Gly Cys Glu Thr Pro Pro Tyr Leu Val Ser 1140 1145 1150

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: GTCCAAGCTG TCATGGGCCA G 21

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: GTCCAGCAGA CTGAAGAGCA CGG 23

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: TGTAAAACGA CGGCCAGT 18

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: GGAAACAGCT ATGACCATG 19

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: GGACATGTTC ACTGCCTCTA GG 22

(2) INFORMATION FOR SEQ ID NO:43:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: GGCGGACAGT CAGACGACTG TCCTG 25

(2) INFORMATION FOR SEQ ID NO:44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 38 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CTGGTTCGGC CCACCTCTGA AGGTTCCAGA ATCGATAG 38

(2) INFORMATION FOR SEQ ID NO:45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3519 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 52..3519

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:

GCTTTCTGAA GGTTCCAGAA TCGATAGTGA ATTCGTGGGC ACTGCTCAGA T ATG GTC 57

Met Val

1

CGT GGA GTT GTG ATC CTC CTG TGT GGC TGG GCC CTG GCT TCC TGT CAT 105 Arg Gly Val Val He Leu Leu Cys Gly Trp Ala Leu Ala Ser Cys His 5 10 15

GGG TCT AAC CTG GAT GTG GAG AAG CCC GTC GTG TTC AAA GAG GAT GCA 153 Gly Ser Asn Leu Asp Val Glu Lys Pro Val Val Phe Lys Glu Asp Ala 20 25 30

GCC AGC TTC GGA CAG ACT GTG GTG CAG TTT GGT GGA TCT CGA CTC GTG 201 Ala Ser Phe Gly Gin Thr Val Val Gin Phe Gly Gly Ser Arg Leu Val 35 40 45 50

GTG GGA GCC CCT CTG GAG GCG GTG GCA GTC AAC CAA ACA GGA CAG TCG 249 Val Gly Ala Pro Leu Glu Ala Val Ala Val Asn Gin Thr Gly Gin Ser 55 60 65

TCT GAC TGT CCG CCT GCC ACT GGC GTG TGC CAG CCC ATC TTA CTG CAC 297 Ser Asp Cys Pro Pro Ala Thr Gly Val Cys Gin Pro He Leu Leu His 70 75 80

ATT CCC CTA GAG GCA GTG AAC ATG TCC CTG GGC CTG TCT CTG GTG GCT 345 He Pro Leu Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu Val Ala 85 90 95

GAC ACC AAT AAC TCC CAG TTG CTG GCT TGT GGT CCA ACT GCA CAG AGA 393 Asp Thr Asn Asn Ser Gin Leu Leu Ala Cys Gly Pro Thr Ala Gin Arg 100 105 110

GCT TGT GCA AAG AAC ATG TAT GCA AAA GGT TCC TGC CTC CTT CTG GGC 441 Ala Cys Ala Lys Asn Met Tyr Ala Lys Gly Ser Cys Leu Leu Leu Gly 115 120 125 130

TCC AGC TTG CAG TTC ATC CAG GCA ATC CCT GCT ACC ATG CCA GAG TGT 489 Ser Ser Leu Gin Phe He Gin Ala He Pro Ala Thr Met Pro Glu Cys 135 140 145

CCA GGA CAA GAG ATG GAC ATT GCT TTC CTG ATT GAT GGC TCC GGC AGC 537 Pro Gly Gin Glu Met Asp He Ala Phe Leu He Asp Gly Ser Gly Ser 150 155 160

ATT GAT CAA AGT GAC TTT ACC CAG ATG AAG GAC TTC GTC AAA GCT TTG 585 He Asp Gin Ser Asp Phe Thr Gin Met Lys Asp Phe Val Lys Ala Leu 165 170 175

ATG GGC CAG TTG GCG AGC ACC AGC ACC TCG TTC TCC CTG ATG CAA TAC 633 Met Gly Gin Leu Ala Ser Thr Ser Thr Ser Phe Ser Leu Met Gin Tyr 180 185 190

TCA AAC ATC CTG AAG ACT CAT TTT ACC TTC ACG GAA TTC AAG AGC AGC 681 Ser Asn He Leu Lys Thr His Phe Thr Phe Thr Glu Phe Lys Ser Ser 195 200 205 210

CTG AGC CCT CAG AGC CTG GTG GAT GCC ATC GTC CAG CTC CAA GGC CTG 729 Leu Ser Pro Gin Ser Leu Val Asp Ala He Val Gin Leu Gin Gly Leu 215 220 225

ACG TAC ACA GCC TCG GGC ATC CAG AAA GTG GTG AAA GAG CTA TTT CAT 777 Thr Tyr Thr Ala Ser Gly He Gin Lys Val Val Lys Glu Leu Phe His 230 235 240

AGC AAG AAT GGG GCC CGA AAA AGT GCC AAG AAG ATA CTA ATT GTC ATC 825 Ser Lys Asn Gly Ala Arg Lys Ser Ala Lys Lys He Leu He Val He 245 250 255

ACA GAT GGG CAG AAA TTC AGA GAC CCC CTG GAG TAT AGA CAT GTC ATC 873 Thr Asp Gly Gin Lys Phe Arg Asp Pro Leu Glu Tyr Arg His Val He 260 265 270

CCT GAA GCA GAG AAA GCT GGG ATC ATT CGC TAT GCT ATA GGG GTG GGA 921 Pro Glu Ala Glu Lys Ala Gly He He Arg Tyr Ala He Gly Val Gly 275 280 285 290

GAT GCC TTC CGG GAA CCC ACT GCC CTA CAG GAG CTG AAC ACC ATT GGC 969 Asp Ala Phe Arg Glu Pro Thr Ala Leu Gin Glu Leu Asn Thr He Gly 295 300 305

TCA GCT CCC TCG CAG GAC CAC GTG TTC AAG GTG GGC AAT TTT GTA GCA 1017 Ser Ala Pro Ser Gin Asp His Val Phe Lys Val Gly Asn Phe Val Ala 310 315 320

CTT CGC AGC ATC CAG CGG CAA ATT CAG GAG AAA ATC TTT GCC ATT GAA 1065 Leu Arg Ser He Gin Arg Gin He Gin Glu Lys He Phe Ala He Glu 325 330 335

GGA ACC GAA TCA AGG TCA AGT AGT TCC TTT CAG CAC GAG ATG TCA CAA 1113 Gly Thr Glu Ser Arg Ser Ser Ser Ser Phe Gin His Glu Met Ser Gin 340 345 350

GAA GGT TTC AGC TCA GCT CTC TCA ATG GAT GGA CCA GTT CTG GGG GCT 1161 Glu Gly Phe Ser Ser Ala Leu Ser Met Asp Gly Pro Val Leu Gly Ala 355 360 365 370

GTG GGA GGC TTC AGC TGG TCT GGA GGT GCC TTC TTG TAC CCC TCA AAT 1209 Val Gly Gly Phe Ser Trp Ser Gly Gly Ala Phe Leu Tyr Pro Ser Asn 375 380 385

ATG AGA TCC ACC TTC ATC AAC ATG TCT CAG GAG AAC GAG GAT ATG AGG 1257 Met Arg Ser Thr Phe He Asn Met Ser Gin Glu Asn Glu Asp Met Arg 390 395 400

GAC GCT TAC CTG GGT TAC TCC ACC GCA CTG GCC TTT TGG AAG GGG GTC 1305 Asp Ala Tyr Leu Gly Tyr Ser Thr Ala Leu Ala Phe Trp Lys Gly Val 405 410 415

CAC AGC CTG ATC CTG GGG GCC CCT CGC CAC CAG CAC ACG GGG AAG GTT 1353 His Ser Leu He Leu Gly Ala Pro Arg His Gin His Thr Gly Lys Val 420 425 430

GTC ATC TTT ACC CAG GAA TCC AGG CAC TGG AGG CCC AAG TCT GAA GTC 1401 Val He Phe Thr Gin Glu Ser Arg His Trp Arg Pro Lys Ser Glu Val 435 440 445 450

AGA GGG ACA CAG ATC GGC TCC TAC TTT GGG GCA TCT CTC TGT TCT GTG 1449 Arg Gly Thr Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys Ser Val 455 460 465

GAC ATG GAT AGA GAT GGC AGC ACT GAC CTG GTC CTG ATT GGA GTC CCC 1497 Asp Met Asp Arg Asp Gly Ser Thr Asp Leu Val Leu He Gly Val Pro 470 475 480

CAT TAC TAT GAG CAC ACC CGA GGG GGG CAG GTG TCG GTG TGC CCC ATG 1545 His Tyr Tyr Glu His Thr Arg Gly Gly Gin Val Ser Val Cys Pro Met 485 490 495

CCT GGT GTG AGG AGC AGG TGG CAT TGT GGG ACC ACC CTC CAT GGG GAG 1593 Pro Gly Val Arg Ser Arg Trp His Cys Gly Thr Thr Leu His Gly Glu 500 505 510

CAG GGC CAT CCT TGG GGC CGC TTT GGG GCG GCT CTG ACA GTG CTA GGG 1641 Gin Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val Leu Gly 515 520 525 530

GAC GTG AAT GGG GAC AGT CTG GCG GAT GTG GCT ATT GGT GCA CCC GGA 1689 Asp Val Asn Gly Asp Ser Leu Ala Asp Val Ala He Gly Ala Pro Gly 535 540 545

GAG GAG GAG AAC AGA GGT GCT GTC TAC ATA TTT CAT GGA GCC TCG AGA 1737 Glu Glu Glu Asn Arg Gly Ala Val Tyr He Phe His Gly Ala Ser Arg 550 555 560

CAG GAC ATC GCT CCC TCG CCT AGC CAG CGG GTC ACT GGC TCC CAG CTC 1785 Gin Asp He Ala Pro Ser Pro Ser Gin Arg Val Thr Gly Ser Gin Leu 565 570 575

TTC CTG AGG CTC CAA TAT TTT GGG CAG TCA TTA AGT GGG GGT CAG GAC 1833 Phe Leu Arg Leu Gin Tyr Phe Gly Gin Ser Leu Ser Gly Gly Gin Asp 580 585 590

CTT ACA CAG GAT GGC CTG GTG GAC CTG GCC GTG GGA GCC CAG GGG CAC 1881 Leu Thr Gin Asp Gly Leu Val Asp Leu Ala Val Gly Ala Gin Gly His 595 600 605 610

GTG CTG CTG CTT AGG AGT CTG CCT TTG CTG AAA GTG GGG ATC TCC ATT 1929 Val Leu Leu Leu Arg Ser Leu Pro Leu Leu Lys Val Gly He Ser He 615 620 625

AGA TTT GCC CCC TCA GAG GTG GCA AAG ACT GTG TAC CAG TGC TGG GGA 1977 Arg Phe Ala Pro Ser Glu Val Ala Lys Thr Val Tyr Gin Cys Trp Gly 630 635 640

AGG ACT CCC ACT GTC CTC GAA GCT GGA GAG GCC ACC GTC TGT CTC ACT 2025 Arg Thr Pro Thr Val Leu Glu Ala Gly Glu Ala Thr Val Cys Leu Thr 645 650 655

GTC CGC AAA GGT TCA CCT GAC CTG TTA GGT GAT GTC CAA AGC TCT GTC 2073 Val Arg Lys Gly Ser Pro Asp Leu Leu Gly Asp Val Gin Ser Ser Val 660 665 670

AGG TAT GAT CTG GCG TTG GAT CCG GGC CGT CTG ATT TCT CGT GCC ATT 2121 Arg Tyr Asp Leu Ala Leu Asp Pro Gly Arg Leu He Ser Arg Ala He 675 680 685 690

TTT GAT GAG ACG AAG AAC TGC ACT TTG ACC CGA AGG AAG ACT CTG GGG 2169 Phe Asp Glu Thr Lys Asn Cys Thr Leu Thr Arg Arg Lys Thr Leu Gly 695 700 705

CTT GGT GAT CAC TGC GAA ACA ATG AAG CTG CTT TTG CCA GAC TGT GTG 2217 Leu Gly Asp His Cys Glu Thr Met Lys Leu Leu Leu Pro Asp Cys Val 710 715 720

GAG GAT GCA GTG ACC CCT ATC ATC CTG CGC CTT AAC TTA TCC CTG GCA 2265 Glu Asp Ala Val Thr Pro He He Leu Arg Leu Asn Leu Ser Leu Ala 725 730 735

GGG GAC TCT GCT CCA TCC AGG AAC CTT CGT CCT GTG CTG GCT GTG GGC 2313 Gly Asp Ser Ala Pro Ser Arg Asn Leu Arg Pro Val Leu Ala Val Gly 740 745 750

TCA CAA GAC CAT GTA ACA GCT TCT TTC CCG TTT GAG AAG AAC TGT GAG 2361 Ser Gin Asp His Val Thr Ala Ser Phe Pro Phe Glu Lys Asn Cys Glu 755 760 765 770

GGG AAC CTG GGC GTC AGC TTC AAC TTC TCA GGC CTG CAG GTC TTG GAG 2409 Gly Asn Leu Gly Val Ser Phe Asn Phe Ser Gly Leu Gin Val Leu Glu 775 780 785

GTA GGA AGC TCC CCA GAG CTC ACT GTG ACA GTA ACA GTT TGG AAT GAG 2457 Val Gly Ser Ser Pro Glu Leu Thr Val Thr Val Thr Val Trp Asn Glu 790 795 800

GGT GAG GAC AGC TAT GGA ACC TTA ATC AAG TTC TAC TAC CCA GCA GAG 2505 Gly Glu Asp Ser Tyr Gly Thr Leu He Lys Phe Tyr Tyr Pro Ala Glu 805 810 815

CTA TCT TAC CGA CGG GTG ACA AGA GCC CAG CAA CCT CAT CCG TAC CCA 2553 Leu Ser Tyr Arg Arg Val Thr Arg Ala Gin Gin Pro His Pro Tyr Pro 820 825 830

CTA CGC CTG GCA TGT GAG GCT GAG CCC ACG GGC CAG GAG AGC CTG AGG 2601 Leu Arg Leu Ala Cys Glu Ala Glu Pro Thr Gly Gin Glu Ser Leu Arg 835 840 845 850

AGC AGC AGC TGT AGC ATC AAT CAC CCC ATC TTC CGA GAA GGT GCC AAG 2649 Ser Ser Ser Cys Ser He Asn His Pro He Phe Arg Glu Gly Ala Lys 855 860 865

GCC ACC TTC ATG ATC ACA TTT GAT GTC TCC TAC AAG GCC TTC CTG GGA 2697 Ala Thr Phe Met He Thr Phe Asp Val Ser Tyr Lys Ala Phe Leu Gly 870 875 880

GAC AGG TTG CTT CTG AGG GCC AGC GCA AGC AGT GAG AAT AAT AAG CCT 2745 Asp Arg Leu Leu Leu Arg Ala Ser Ala Ser Ser Glu Asn Asn Lys Pro 885 890 895

GAA ACC AGC AAG ACT GCC TTC CAG CTG GAG CTT CCG GTG AAG TAC ACG 2793 Glu Thr Ser Lys Thr Ala Phe Gin Leu Glu Leu Pro Val Lys Tyr Thr 900 905 910

GTC TAT ACC GTG ATC AGT AGG CAG GAA GAT TCT ACC AAG CAT TTC AAC 2841 Val Tyr Thr Val He Ser Arg Gin Glu Asp Ser Thr Lys His Phe Asn 915 920 925 930

TTC TCA TCT TCC CAC GGG GAG AGA CAG AAA GAG GCC GAA CAT CGA TAT 2889 Phe Ser Ser Ser His Gly Glu Arg Gin Lys Glu Ala Glu His Arg Tyr 935 940 945

CGT GTG AAT AAC CTG AGT CCA TTG ACG CTG GCC ATC AGC GTT AAC TTC 2937 Arg Val Asn Asn Leu Ser Pro Leu Thr Leu Ala He Ser Val Asn Phe 950 955 960

TGG GTC CCC ATC CTT CTG AAT GGT GTG GCC GTG TGG GAT GTG ACT CTG 2985 Trp Val Pro He Leu Leu Asn Gly Val Ala Val Trp Asp Val Thr Leu 965 970 975

AGG AGC CCA GCA CAG GGT GTC TCC TGT GTG TCA CAG AGG GAA CCT CCT 3033 Arg Ser Pro Ala Gin Gly Val Ser Cys Val Ser Gin Arg Glu Pro Pro 980 985 990

CAA CAT TCC GAC CTT CTG ACC CAG ATC CAA GGA CGC TCT GTG CTG GAC 3081 Gin His Ser Asp Leu Leu Thr Gin He Gin Gly Arg Ser Val Leu Asp 995 1000 1005 1010

TGC GCC ATC GCC GAC TGC CTG CAC CTC CGC TGT GAC ATC CCC TCC TTG 3129 Cys Ala He Ala Asp Cys Leu His Leu Arg Cys Asp He Pro Ser Leu 1015 1020 1025

GGC ACC CTG GAT GAG CTT GAC TTC ATT CTG AAG GGC AAC CTC AGC TTC 3177 Gly Thr Leu Asp Glu Leu Asp Phe He Leu Lys Gly Asn Leu Ser Phe 1030 1035 1040

GGC TGG ATC AGT CAG ACA TTG CAG AAA AAG GTG TTG CTC CTG AGT GAG 3225 Gly Trp He Ser Gin Thr Leu Gin Lys Lys Val Leu Leu Leu Ser Glu 1045 1050 1055

GCT GAA ATC ACA TTC AAC ACA TCT GTG TAT TCC CAG CTG CCG GGA CAG 3273 Ala Glu He Thr Phe Asn Thr Ser Val Tyr Ser Gin Leu Pro Gly Gin 1060 1065 1070

GAG GCA TTT CTG AGA GCC CAG GTG TCA ACG ATG CTA GAA GAA TAC GTG 3321 Glu Ala Phe Leu Arg Ala Gin Val Ser Thr Met Leu Glu Glu Tyr Val 1075 1080 1085 1090

GTC TAT GAG CCC GTC TTC CTC ATG GTG TTC AGC TCA GTG GGA GGT CTG 3369 Val Tyr Glu Pro Val Phe Leu Met Val Phe Ser Ser Val Gly Gly Leu 1095 1100 1105

CTG TTA CTG GCT CTC ATC ACT GTG GCG CTG TAC AAG CTT GGC TTC TTC 3417 Leu Leu Leu Ala Leu He Thr Val Ala Leu Tyr Lys Leu Gly Phe Phe 1110 1115 1120

AAA CGT CAG TAT AAA GAG ATG CTG GAT CTA CCA TCT GCA GAT CCT GAC 3465 Lys Arg Gin Tyr Lys Glu Met Leu Asp Leu Pro Ser Ala Asp Pro Asp 1125 1130 1135

CCA GCC GGC CAG GCA GAT TCC AAC CAT GAG ACT CCT CCA CAT CTC ACG 3513 Pro Ala Gly Gin Ala Asp Ser Asn His Glu Thr Pro Pro His Leu Thr 1140 1145 1150

TCC TAG 3519

Ser

1155

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1155 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

Met Val Arg Gly Val Val He Leu Leu Cys Gly Trp Ala Leu Ala Ser 1 5 10 15

Cys His Gly Ser Asn Leu Asp Val Glu Lys Pro Val Val Phe Lys Glu 20 25 30

Asp Ala Ala Ser Phe Gly Gin Thr Val Val Gin Phe Gly Gly Ser Arg 35 40 45

Leu Val Val Gly Ala Pro Leu Glu Ala Val Ala Val Asn Gin Thr Gly 50 55 60

Gin Ser Ser Asp Cys Pro Pro Ala Thr Gly Val Cys Gin Pro He Leu 65 70 75 80

Leu His He Pro Leu Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu 85 90 95

Val Ala Asp Thr Asn Asn Ser Gin Leu Leu Ala Cys Gly Pro Thr Ala 100 105 110

Gln Arg Ala Cys Ala Lys Asn Met Tyr Ala Lys Gly Ser Cys Leu Leu 115 120 125

Leu Gly Ser Ser Leu Gin Phe He Gin Ala He Pro Ala Thr Met Pro 130 135 140

Glu Cys Pro Gly Gin Glu Met Asp He Ala Phe Leu He Asp Gly Ser 145 150 155 160

Gly Ser He Asp Gin Ser Asp Phe Thr Gin Met Lys Asp Phe Val Lys 165 170 175

Ala Leu Met Gly Gin Leu Ala Ser Thr Ser Thr Ser Phe Ser Leu Met 180 185 190

Gin Tyr Ser Asn He Leu Lys Thr His Phe Thr Phe Thr Glu Phe Lys 195 200 205

Ser Ser Leu Ser Pro Gin Ser Leu Val Asp Ala He Val Gin Leu Gin 210 215 220

Gly Leu Thr Tyr Thr Ala Ser Gly He Gin Lys Val Val Lys Glu Leu 225 230 235 240

Phe His Ser Lys Asn Gly Ala Arg Lys Ser Ala Lys Lys He Leu He 245 250 255

Val He Thr Asp Gly Gin Lys Phe Arg Asp Pro Leu Glu Tyr Arg His 260 265 270

Val He Pro Glu Ala Glu Lys Ala Gly He He Arg Tyr Ala He Gly 275 280 285

Val Gly Asp Ala Phe Arg Glu Pro Thr Ala Leu Gin Glu Leu Asn Thr 290 295 300

He Gly Ser Ala Pro Ser Gin Asp His Val Phe Lys Val Gly Asn Phe 305 310 315 320

Val Ala Leu Arg Ser He Gin Arg Gin He Gin Glu Lys He Phe Ala 325 330 335

He Glu Gly Thr Glu Ser Arg Ser Ser Ser Ser Phe Gin His Glu Met 340 345 350

Ser Gin Glu Gly Phe Ser Ser Ala Leu Ser Met Asp Gly Pro Val Leu 355 360 365

Gly Ala Val Gly Gly Phe Ser Trp Ser Gly Gly Ala Phe Leu Tyr Pro 370 375 380

Ser Asn Met Arg Ser Thr Phe He Asn Met Ser Gin Glu Asn Glu Asp 385 390 395 400

Met Arg Asp Ala Tyr Leu Gly Tyr Ser Thr Ala Leu Ala Phe Trp Lys 405 410 415

Gly Val His Ser Leu He Leu Gly Ala Pro Arg His Gin His Thr Gly 420 425 430

Lys Val Val He Phe Thr Gin Glu Ser Arg His Trp Arg Pro Lys Ser 435 440 445

Glu Val Arg Gly Thr Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys 450 455 460

Ser Val Asp Met Asp Arg Asp Gly Ser Thr Asp Leu Val Leu He Gly 465 470 475 480

Val Pro His Tyr Tyr Glu His Thr Arg Gly Gly Gin Val Ser Val Cys 485 490 495

Pro Met Pro Gly Val Arg Ser Arg Trp His Cys Gly Thr Thr Leu His 500 505 510

Gly Glu Gin Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val 515 520 525

Leu Gly Asp Val Asn Gly Asp Ser Leu Ala Asp Val Ala He Gly Ala 530 535 540

Pro Gly Glu Glu Glu Asn Arg Gly Ala Val Tyr He Phe His Gly Ala 545 550 555 560

Ser Arg Gin Asp He Ala Pro Ser Pro Ser Gin Arg Val Thr Gly Ser 565 570 575

Gin Leu Phe Leu Arg Leu Gin Tyr Phe Gly Gin Ser Leu Ser Gly Gly 580 585 590

Gin Asp Leu Thr Gin Asp Gly Leu Val Asp Leu Ala Val Gly Ala Gin 595 600 605

Gly His Val Leu Leu Leu Arg Ser Leu Pro Leu Leu Lys Val Gly He 610 615 620

Ser He Arg Phe Ala Pro Ser Glu Val Ala Lys Thr Val Tyr Gin Cys 625 630 635 640

Trp Gly Arg Thr Pro Thr Val Leu Glu Ala Gly Glu Ala Thr Val Cys 645 650 655

Leu Thr Val Arg Lys Gly Ser Pro Asp Leu Leu Gly Asp Val Gin Ser 660 665 670

Ser Val Arg Tyr Asp Leu Ala Leu Asp Pro Gly Arg Leu He Ser Arg 675 680 685

Ala He Phe Asp Glu Thr Lys Asn Cys Thr Leu Thr Arg Arg Lys Thr 690 695 700

Leu Gly Leu Gly Asp His Cys Glu Thr Met Lys Leu Leu Leu Pro Asp 705 710 715 720

Cys Val Glu Asp Ala Val Thr Pro He He Leu Arg Leu Asn Leu Ser 725 730 735

Leu Ala Gly Asp Ser Ala Pro Ser Arg Asn Leu Arg Pro Val Leu Ala 740 745 750

Val Gly Ser Gin Asp His Val Thr Ala Ser Phe Pro Phe Glu Lys Asn 755 760 765

Cys Glu Gly Asn Leu Gly Val Ser Phe Asn Phe Ser Gly Leu Gin Val 770 775 780

Leu Glu Val Gly Ser Ser Pro Glu Leu Thr Val Thr Val Thr Val Trp 785 790 795 800

Asn Glu Gly Glu Asp Ser Tyr Gly Thr Leu He Lys Phe Tyr Tyr Pro 805 810 815

Ala Glu Leu Ser Tyr Arg Arg Val Thr Arg Ala Gin Gin Pro His Pro 820 825 830

Tyr Pro Leu Arg Leu Ala Cys Glu Ala Glu Pro Thr Gly Gin Glu Ser 835 840 845

Leu Arg Ser Ser Ser Cys Ser He Asn His Pro He Phe Arg Glu Gly 850 855 860

Ala Lys Ala Thr Phe Met He Thr Phe Asp Val Ser Tyr Lys Ala Phe 865 870 875 880

Leu Gly Asp Arg Leu Leu Leu Arg Ala Ser Ala Ser Ser Glu Asn Asn 885 890 895

Lys Pro Glu Thr Ser Lys Thr Ala Phe Gin Leu Glu Leu Pro Val Lys 900 905 910

Tyr Thr Val Tyr Thr Val He Ser Arg Gin Glu Asp Ser Thr Lys His 915 920 925

Phe Asn Phe Ser Ser Ser His Gly Glu Arg Gin Lys Glu Ala Glu His 930 935 940

Arg Tyr Arg Val Asn Asn Leu Ser Pro Leu Thr Leu Ala He Ser Val 945 950 955 960

Asn Phe Trp Val Pro He Leu Leu Asn Gly Val Ala Val Trp Asp Val 965 970 975

Thr Leu Arg Ser Pro Ala Gin Gly Val Ser Cys Val Ser Gin Arg Glu 980 985 990

Pro Pro Gin His Ser Asp Leu Leu Thr Gin He Gin Gly Arg Ser Val 995 1000 1005

Leu Asp Cys Ala He Ala Asp Cys Leu His Leu Arg Cys Asp He Pro 1010 1015 1020

Ser Leu Gly Thr Leu Asp Glu Leu Asp Phe He Leu Lys Gly Asn Leu 1025 1030 1035 1040

Ser Phe Gly Trp He Ser Gin Thr Leu Gin Lys Lys Val Leu Leu Leu 1045 1050 1055

Ser Glu Ala Glu He Thr Phe Asn Thr Ser Val Tyr Ser Gin Leu Pro 1060 1065 1070

Gly Gin Glu Ala Phe Leu Arg Ala Gin Val Ser Thr Met Leu Glu Glu 1075 1080 1085

Tyr Val Val Tyr Glu Pro Val Phe Leu Met Val Phe Ser Ser Val Gly 1090 1095 1100

Gly Leu Leu Leu Leu Ala Leu He Thr Val Ala Leu Tyr Lys Leu Gly 1105 1110 1115 1120

Phe Phe Lys Arg Gin Tyr Lys Glu Met Leu Asp Leu Pro Ser Ala Asp 1125 1130 1135

Pro Asp Pro Ala Gly Gin Ala Asp Ser Asn His Glu Thr Pro Pro His 1140 1145 1150

Leu Thr Ser 1155

(2) INFORMATION FOR SEQ ID NO:47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 49 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: AGTTACGGAT CCGGCACCAT GACCTTCGGC ACTGTGATCC TCCTGTGTG 49

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: GCTGGACGAT GGCATCCAC 19

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GTAGAGTTAC GGATCCGGCA CCAT 24

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: GCAGCCAGCT TCGGACAGAC 20

(2) INFORMATION FOR SEQ ID NO:51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51: CCATGTCCAC AGAACAGAGA G 21

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3803 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..3486

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

ATG GTC CGT GGA GTT GTG ATC CTC CTG TGT GGC TGG GCC CTG GCT TCC 48 Met Val Arg Gly Val Val He Leu Leu Cys Gly Trp Ala Leu Ala Ser 1 5 10 15

TGT CAT GGG TCT AAC CTG GAT GTG GAG AAG CCC GTC GTG TTC AAA GAG 96 Cys His Gly Ser Asn Leu Asp Val Glu Lys Pro Val Val Phe Lys Glu 20 25 30

GAT GCA GCC AGC TTC GGA CAG ACT GTG GTG CAG TTT GGT GGA TCT CGA 144 Asp Ala Ala Ser Phe Gly Gin Thr Val Val Gin Phe Gly Gly Ser Arg 35 40 45

CTC GTG GTG GGA GCC CCT CTG GAG GCG GTG GCA GTC AAC CAA ACA GGA 192 Leu Val Val Gly Ala Pro Leu Glu Ala Val Ala Val Asn Gin Thr Gly 50 55 60

CAG TCG TCT GAC TGT CCG CCT GCC ACT GGC GTG TGC CAG CCC ATC TTA 240 Gin Ser Ser Asp Cys Pro Pro Ala Thr Gly Val Cys Gin Pro He Leu 65 70 75 80

CTG CAC ATT CCC CTA GAG GCA GTG AAC ATG TCC CTG GGC CTG TCT CTG 288 Leu His He Pro Leu Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu 85 90 95

GTG GCT GAC ACC AAT AAC TCC CAG TTG CTG GCT TGT GGT CCA ACT GCA 336 Val Ala Asp Thr Asn Asn Ser Gin Leu Leu Ala Cys Gly Pro Thr Ala 100 105 110

CAG AGA GCT TGT GCA AAG AAC ATG TAT GCA AAA GGT TCC TGC CTC CTT 384 Gin Arg Ala Cys Ala Lys Asn Met Tyr Ala Lys Gly Ser Cys Leu Leu 115 120 125

CTG GGC TCC AGC TTG CAG TTC ATC CAG GCA ATC CCT GCT ACC ATG CCA 432 Leu Gly Ser Ser Leu Gin Phe He Gin Ala He Pro Ala Thr Met Pro 130 135 140

GAG TGT CCA GGA CAA GAG ATG GAC ATT GCT TTC CTG ATT GAT GGC TCC 480 Glu Cys Pro Gly Gin Glu Met Asp He Ala Phe Leu He Asp Gly Ser 145 150 155 160

GGC AGC ATT GAT CAA AGT GAC TTT ACC CAG ATG AAG GAC TTC GTC AAA 528 Gly Ser He Asp Gin Ser Asp Phe Thr Gin Met Lys Asp Phe Val Lys 165 170 175

GCT TTG ATG GGC CAG TTG GCG AGC ACC AGC ACC TCG TTC TCC CTG ATG 576 Ala Leu Met Gly Gin Leu Ala Ser Thr Ser Thr Ser Phe Ser Leu Met 180 185 190

CAA TAC TCA AAC ATC CTG AAG ACT CAT TTT ACC TTC ACG GAA TTC AAG 624 Gin Tyr Ser Asn He Leu Lys Thr His Phe Thr Phe Thr Glu Phe Lys 195 200 205

AGC AGC CTG AGC CCT CAG AGC CTG GTG GAT GCC ATC GTC CAG CTC CAA 672 Ser Ser Leu Ser Pro Gin Ser Leu Val Asp Ala He Val Gin Leu Gin 210 215 220

GGC CTG ACG TAC ACA GCC TCG GGC ATC CAG AAA GTG GTG AAA GAG CTA 720 Gly Leu Thr Tyr Thr Ala Ser Gly He Gin Lys Val Val Lys Glu Leu 225 230 235 240

TTT CAT AGC AAG AAT GGG GCC CGA AAA AGT GCC AAG AAG ATA CTA ATT 768 Phe His Ser Lys Asn Gly Ala Arg Lys Ser Ala Lys Lys He Leu He 245 250 255

GTC ATC ACA GAT GGG CAG AAA TTC AGA GAC CCC CTG GAG TAT AGA CAT 816 Val He Thr Asp Gly Gin Lys Phe Arg Asp Pro Leu Glu Tyr Arg His 260 265 270

GTC ATC CCT GAA GCA GAG AAA GCT GGG ATC ATT CGC TAT GCT ATA GGG 864 Val He Pro Glu Ala Glu Lys Ala Gly He He Arg Tyr Ala He Gly 275 280 285

GTG GGA GAT GCC TTC CGG GAA CCC ACT GCC CTA CAG GAG CTG AAC ACC 912 Val Gly Asp Ala Phe Arg Glu Pro Thr Ala Leu Gin Glu Leu Asn Thr 290 295 300

ATT GGC TCA GCT CCC TCG CAG GAC CAC GTG TTC AAG GTG GGC AAT TTT 960 He Gly Ser Ala Pro Ser Gin Asp His Val Phe Lys Val Gly Asn Phe 305 310 315 320

GTA GCA CTT CGC AGC ATC CAG CGG CAA ATT CAG GAG AAA ATC TTT GCC 1008 Val Ala Leu Arg Ser He Gin Arg Gin He Gin Glu Lys He Phe Ala 325 330 335

ATT GAA GGA ACC GAA TCA AGG TCA AGT AGT TCC TTT CAG CAC GAG ATG 1056 He Glu Gly Thr Glu Ser Arg Ser Ser Ser Ser Phe Gin His Glu Met 340 345 350

TCA CAA GAA GGT TTC AGC TCA GCT CTC TCA ATG GAT GGA CCA GTT CTG 1104 Ser Gin Glu Gly Phe Ser Ser Ala Leu Ser Met Asp Gly Pro Val Leu 355 360 365

GGG GCT GTG GGA GGC TTC AGC TGG TCT GGA GGT GCC TTC TTG TAC CCC 1152 Gly Ala Val Gly Gly Phe Ser Trp Ser Gly Gly Ala Phe Leu Tyr Pro 370 375 380

TCA AAT ATG AGA TCC ACC TTC ATC AAC ATG TCT CAG GAG AAC GAG GAT 1200 Ser Asn Met Arg Ser Thr Phe He Asn Met Ser Gin Glu Asn Glu Asp 385 390 395 400

ATG AGG GAC GCT TAC CTG GGT TAC TCC ACC GCA CTG GCC TTT TGG AAG 1248 Met Arg Asp Ala Tyr Leu Gly Tyr Ser Thr Ala Leu Ala Phe Trp Lys 405 410 415

GGG GTC CAC AGC CTG ATC CTG GGG GCC CCT CGC CAC CAG CAC ACG GGG 1296 Gly Val His Ser Leu He Leu Gly Ala Pro Arg His Gin His Thr Gly 420 425 430

AAG GTT GTC ATC TTT ACC CAG GAA TCC AGG CAC TGG AGG CCC AAG TCT 1344 Lys Val Val He Phe Thr Gin Glu Ser Arg His Trp Arg Pro Lys Ser 435 440 445

GAA GTC AGA GGG ACA CAG ATC GGC TCC TAC TTT GGG GCA TCT CTC TGT 1392 Glu Val Arg Gly Thr Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys 450 455 460

TCT GTG GAC ATG GAT AGA GAT GGC AGC ACT GAC CTG GTC CTG ATT GGA 1440 Ser Val Asp Met Asp Arg Asp Gly Ser Thr Asp Leu Val Leu He Gly 465 470 475 480

GTC CCC CAT TAC TAT GAG CAC ACC CGA GGG GGG CAG GTG TCG GTG TGC 1488 Val Pro His Tyr Tyr Glu His Thr Arg Gly Gly Gin Val Ser Val Cys 485 490 495

CCC ATG CCT GGT GTG AGG AGC AGG TGG CAT TGT GGG ACC ACC CTC CAT 1536 Pro Met Pro Gly Val Arg Ser Arg Trp His Cys Gly Thr Thr Leu His 500 505 510

GGG GAG CAG GGC CAT CCT TGG GGC CGC TTT GGG GCG GCT CTG ACA GTG 1584 Gly Glu Gin Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val 515 520 525

CTA GGG GAC GTG AAT GGG GAC AGT CTG GCG GAT GTG GCT ATT GGT GCA 1632 Leu Gly Asp Val Asn Gly Asp Ser Leu Ala Asp Val Ala He Gly Ala 530 535 540

CCC GGA GAG GAG GAG AAC AGA GGT GCT GTC TAC ATA TTT CAT GGA GCC 1680 Pro Gly Glu Glu Glu Asn Arg Gly Ala Val Tyr He Phe His Gly Ala 545 550 555 560

TCG AGA CAG GAC ATC GCT CCC TCG CCT AGC CAG CGG GTC ACT GGC TCC 1728 Ser Arg Gin Asp He Ala Pro Ser Pro Ser Gin Arg Val Thr Gly Ser 565 570 575

CAG CTC TTC CTG AGG CTC CAA TAT TTT GGG CAG TCA TTA AGT GGG GGT 1776 Gin Leu Phe Leu Arg Leu Gin Tyr Phe Gly Gin Ser Leu Ser Gly Gly 580 585 590

CAG GAC CTT ACA CAG GAT GGC CTG GTG GAC CTG GCC GTG GGA GCC CAG 1824 Gin Asp Leu Thr Gin Asp Gly Leu Val Asp Leu Ala Val Gly Ala Gin 595 600 605

GGG CAC GTG CTG CTG CTT AGG AGT CTG CCT TTG CTG AAA GTG GGG ATC 1872 Gly His Val Leu Leu Leu Arg Ser Leu Pro Leu Leu Lys Val Gly He 610 615 620

TCC ATT AGA TTT GCC CCC TCA GAG GTG GCA AAG ACT GTG TAC CAG TGC 1920 Ser He Arg Phe Ala Pro Ser Glu Val Ala Lys Thr Val Tyr Gin Cys 625 630 635 640

TGG GGA AGG ACT CCC ACT GTC CTC GAA GCT GGA GAG GCC ACC GTC TGT 1968 Trp Gly Arg Thr Pro Thr Val Leu Glu Ala Gly Glu Ala Thr Val Cys 645 650 655

CTC ACT GTC CGC AAA GGT TCA CCT GAC CTG TTA GGT GAT GTC CAA AGC 2016 Leu Thr Val Arg Lys Gly Ser Pro Asp Leu Leu Gly Asp Val Gin Ser 660 665 670

TCT GTC AGG TAT GAT CTG GCG TTG GAT CCG GGC CGT CTG ATT TCT CGT 2064 Ser Val Arg Tyr Asp Leu Ala Leu Asp Pro Gly Arg Leu He Ser Arg 675 680 685

GCC ATT TTT GAT GAG ACG AAG AAC TGC ACT TTG ACC CGA AGG AAG ACT 2112 Ala He Phe Asp Glu Thr Lys Asn Cys Thr Leu Thr Arg Arg Lys Thr 690 695 700

CTG GGG CTT GGT GAT CAC TGC GAA ACA ATG AAG CTG CTT TTG CCA GAC 2160 Leu Gly Leu Gly Asp His Cys Glu Thr Met Lys Leu Leu Leu Pro Asp 705 710 715 720

TGT GTG GAG GAT GCA GTG ACC CCT ATC ATC CTG CGC CTT AAC TTA TCC 2208 Cys Val Glu Asp Ala Val Thr Pro He He Leu Arg Leu Asn Leu Ser 725 730 735

CTG GCA GGG GAC TCT GCT CCA TCC AGG AAC CTT CGT CCT GTG CTG GCT 2256 Leu Ala Gly Asp Ser Ala Pro Ser Arg Asn Leu Arg Pro Val Leu Ala 740 745 750

GTG GGC TCA CAA GAC CAT GTA ACA GCT TCT TTC CCG TTT GAG AAG AAC 2304 Val Gly Ser Gin Asp His Val Thr Ala Ser Phe Pro Phe Glu Lys Asn 755 760 765

TGT AAG CAG GAG CTC CTG TGT GAG GGG AAC CTG GGC GTC AGC TTC AAC 2352 Cys Lys Gin Glu Leu Leu Cys Glu Gly Asn Leu Gly Val Ser Phe Asn 770 775 780

TTC TCA GGC CTG CAG GTC TTG GAG GTA GGA AGC TCC CCA GAG CTC ACT 2400 Phe Ser Gly Leu Gin Val Leu Glu Val Gly Ser Ser Pro Glu Leu Thr 785 790 795 800

GTG ACA GTA ACA GTT TGG AAT GAG GGT GAG GAC AGC TAT GGA ACC TTA 2448 Val Thr Val Thr Val Trp Asn Glu Gly Glu Asp Ser Tyr Gly Thr Leu 805 810 815

ATC AAG TTC TAC TAC CCA GCA GAG CTA TCT TAC CGA CGG GTG ACA AGA 2496 lie Lys Phe Tyr Tyr Pro Ala Glu Leu Ser Tyr Arg Arg Val Thr Arg 820 825 830

GCC CAG CAA CCT CAT CCG TAC CCA CTA CGC CTG GCA TGT GAG GCT GAG 2544 Ala Gin Gin Pro His Pro Tyr Pro Leu Arg Leu Ala Cys Glu Ala Glu 835 840 845

ZCC ACG GGC CAG GAG AGC CTG AGG AGC AGC AGC TGT AGC ATC AAT CAC 2592 Pro Thr Gly Gin Glu Ser Leu Arg Ser Ser Ser Cys Ser He Asn His 850 855 860

CCC ATC TTC CGA GAA GGT GCC AAG GCC ACC TTC ATG ATC ACA TTT GAT 2640 Pro He Phe Arg Glu Gly Ala Lys Ala Thr Phe Met He Thr Phe Asp 865 870 875 880

GTC TCC TAC AAG GCC TTC CTG GGA GAC AGG TTG CTT CTG AGG GCC AGC 2688 Val Ser Tyr Lys Ala Phe Leu Gly Asp Arg Leu Leu Leu Arg Ala Ser 885 890 895

GCA AGC AGT GAG AAT AAT AAG CCT GAA ACC AGC AAG ACT GCC TTC CAG 2736 Ala Ser Ser Glu Asn Asn Lys Pro Glu Thr Ser Lys Thr Ala Phe Gin 900 905 910

CTG GAG CTT CCG GTG AAG TAC ACG GTC TAT ACC GTG ATC AGT AGG CAG 2784 _eu Glu Leu Pro Val Lys Tyr Thr Val Tyr Thr Val He Ser Arg Gin 915 920 925 AA GAT TCT ACC AAG CAT TTC AAC TTC TCA TCT TCC CAC GGG GAG AGA 2832 Glu Asp Ser Thr Lys His Phe Asn Phe Ser Ser Ser His Gly Glu Arg 930 935 940

.TTG AAA GAG GCC GAA CAT CGA TAT CGT GTG AAT AAC CTG AGT CCA TTG 2880 Gin Lys Glu Ala Glu His Arg Tyr Arg Val Asn Asn Leu Ser Pro Leu S 5 950 955 960

TCG CTG GCC ATC AGC GTT AAC TTC TGG GTC CCC ATC CTT CTG AAT GGT 2928 -T.r Leu Ala He Ser Val Asn Phe Trp Val Pro He Leu Leu Asn Gly 965 970 975

^" TG GCC GTG TGG GAT GTG ACT CTG AGG AGC CCA GCA CAG GGT GTC TCC 2976 . -1 Ala Val Trp Asp Val Thr Leu Arg Ser Pro Ala Gin Gly Val Ser 980 985 990

TGT GTG TCA CAG AGG GAA CCT CCT CAA CAT TCC GAC CTT CTG ACC CAG 3024 Cys Val Ser Gin Arg Glu Pro Pro Gin His Ser Asp Leu Leu Thr Gin 995 1000 1005

ATC CAA GGA CGC TCT GTG CTG GAC TGC GCC ATC GCC GAC TGC CTG CAC 3072 He Gin Gly Arg Ser Val Leu Asp Cys Ala He Ala Asp Cys Leu His 1010 1015 1020

CTC CGC TGT GAC ATC CCC TCC TTG GGC ACC CTG GAT GAG CTT GAC TTC 3120 Leu Arg Cys Asp He Pro Ser Leu Gly Thr Leu Asp Glu Leu Asp Phe 1025 1030 1035 1040

ATT CTG AAG GGC AAC CTC AGC TTC GGC TGG ATC AGT CAG ACA TTG CAG 3168 He Leu Lys Gly Asn Leu Ser Phe Gly Trp He Ser Gin Thr Leu Gin 1045 1050 1055

AAA AAG GTG TTG CTC CTG AGT GAG GCT GAA ATC ACA TTC AAC ACA TCT 3216 Lys Lys Val Leu Leu Leu Ser Glu Ala Glu He Thr Phe Asn Thr Ser 1060 1065 1070

GTG TAT TCC CAG CTG CCG GGA CAG GAG GCA TTT CTG AGA GCC CAG GTG 3264 Val Tyr Ser Gin Leu Pro Gly Gin Glu Ala Phe Leu Arg Ala Gin Val 1075 1080 1085

TCA ACG ATG CTA GAA GAA TAC GTG GTC TAT GAG CCC GTC TTC CTC ATG 3312 Ser Thr Met Leu Glu Glu Tyr Val Val Tyr Glu Pro Val Phe Leu Met 1090 1095 1100

GTG TTC AGC TCA GTG GGA GGT CTG CTG TTA CTG GCT CTC ATC ACT GTG 3360 Val Phe Ser Ser Val Gly Gly Leu Leu Leu Leu Ala Leu He Thr Val 1105 1110 1115 1120

GCG CTG TAC AAG CTT GGC TTC TTC AAA CGT CAG TAT AAA GAG ATG CTG 3408 Ala Leu Tyr Lys Leu Gly Phe Phe Lys Arg Gin Tyr Lys Glu Met Leu 1125 1130 1135

GAT CTA CCA TCT GCA GAT CCT GAC CCA GCC GGC CAG GCA GAT TCC AAC 3456 Asp Leu Pro Ser Ala Asp Pro Asp Pro Ala Gly Gin Ala Asp Ser Asn 1140 1145 1150

CAT GAG ACT CCT CCA CAT CTC ACG TCC TAGGAATCTA CTTTCCTGTA 3503

His Glu Thr Pro Pro His Leu Thr Ser 1155 1160

TATCTCCACA ATTACGAGAT TGGTTTTGCT TTTGCCTATG AATCTACTGG CATGGGAACA 3563

AGTTCTCTTC AGCTCTGGGC TAGCCTGGGA AACTTCCCAG AAATGATGCC CTACCTCCTG 3623

AGCTGGGAGA TTTTTATGGT TTGCCCATGT GTCAGATTTC AGTGCTGATC CACTTTTTTT 3683

GCAAGAGCAG GAATGGGGTC AGCATAAATT TACATATGGA TAAGAACTAA CACAAGACTG 3743

AGTAATATGC TCAATATTCA ATGTATTGCT TGTATAAATT TTTAAAAAAT AAAATGAAAN 3803

(2) INFORMATION FOR SEQ ID NO:53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1161 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

Met Val Arg Gly Val Val He Leu Leu Cys Gly Trp Ala Leu Ala Ser 1 5 10 15

Cys His Gly Ser Asn Leu Asp Val Glu Lys Pro Val Val Phe Lys Glu 20 25 30

Asp Ala Ala Ser Phe Gly Gin Thr Val Val Gin Phe Gly Gly Ser Arg 35 40 45

Leu Val Val Gly Ala Pro Leu Glu Ala Val Ala Val Asn Gin Thr Gly 50 55 60

Gin Ser Ser Asp Cys Pro Pro Ala Thr Gly Val Cys Gin Pro He Leu 65 70 75 80

Leu His He Pro Leu Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu 85 90 95

Val Ala Asp Thr Asn Asn Ser Gin Leu Leu Ala Cys Gly Pro Thr Ala 100 105 110

Gin Arg Ala Cys Ala Lys Asn Met Tyr Ala Lys Gly Ser Cys Leu Leu 115 120 125

Leu Gly Ser Ser Leu Gin Phe He Gin Ala He Pro Ala Thr Met Pro 130 135 140

Glu Cys Pro Gly Gin Glu Met Asp He Ala Phe Leu He Asp Gly Ser 145 150 155 160

Gly Ser He Asp Gin Ser Asp Phe Thr Gin Met Lys Asp Phe Val Lys 165 170 175

Ala Leu Met Gly Gin Leu Ala Ser Thr Ser Thr Ser Phe Ser Leu Met 180 185 190

Gin Tyr Ser Asn He Leu Lys Thr His Phe Thr Phe Thr Glu Phe Lys 195 200 205

Ser Ser Leu Ser Pro Gin Ser Leu Val Asp Ala He Val Gin Leu Gin 210 215 220

Gly Leu Thr Tyr Thr Ala Ser Gly He Gin Lys Val Val Lys Glu Leu 225 230 235 240

Phe His Ser Lys Asn Gly Ala Arg Lys Ser Ala Lys Lys He Leu He 245 250 255

Val He Thr Asp Gly Gin Lys Phe Arg Asp Pro Leu Glu Tyr Arg His 260 265 270

Val He Pro Glu Ala Glu Lys Ala Gly He He Arg Tyr Ala He Gly 275 280 285

Val Gly Asp Ala Phe Arg Glu Pro Thr Ala Leu Gin Glu Leu Asn Thr 290 295 300

He Gly Ser Ala Pro Ser Gin Asp His Val Phe Lys Val Gly Asn Phe 305 310 315 320

Val Ala Leu Arg Ser He Gin Arg Gin He Gin Glu Lys He Phe Ala 325 330 335

He Glu Gly Thr Glu Ser Arg Ser Ser Ser Ser Phe Gin His Glu Met 340 345 350

Ser Gin Glu Gly Phe Ser Ser Ala Leu Ser Met Asp Gly Pro Val Leu 355 360 365

Gly Ala Val Gly Gly Phe Ser Trp Ser Gly Gly Ala Phe Leu Tyr Pro 370 375 380

Ser Asn Met Arg Ser Thr Phe He Asn Met Ser Gin Glu Asn Glu Asp 385 390 395 400

Met Arg Asp Ala Tyr Leu Gly Tyr Ser Thr Ala Leu Ala Phe Trp Lys 405 410 415

Gly Val His Ser Leu He Leu Gly Ala Pro Arg His Gin His Thr Gly 420 425 430

Lys Val Val He Phe Thr Gin Glu Ser Arg His Trp Arg Pro Lys Ser 435 440 445

Glu Val Arg Gly Thr Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys 450 455 460

Ser Val Asp Met Asp Arg Asp Gly Ser Thr Asp Leu Val Leu He Gly 465 470 475 480

Val Pro His Tyr Tyr Glu His Thr Arg Gly Gly Gin Val Ser Val Cys 485 490 495

Pro Met Pro Gly Val Arg Ser Arg Trp His Cys Gly Thr Thr Leu His 500 505 510

Gly Glu Gin Gly His Pro Trp Gly Arg Phe Gly Ala Ala Leu Thr Val 515 520 525

Leu Gly Asp Val Asn Gly Asp Ser Leu Ala Asp Val Ala He Gly Ala 530 535 540

Pro Gly Glu Glu Glu Asn Arg Gly Ala Val Tyr He Phe His Gly Ala 545 550 555 560

Ser Arg Gin Asp He Ala Pro Ser Pro Ser Gin Arg Val Thr Gly Ser 565 570 575

Gin Leu Phe Leu Arg Leu Gin Tyr Phe Gly Gin Ser Leu Ser Gly Gly 580 585 590

Gin Asp Leu Thr Gin Asp Gly Leu Val Asp Leu Ala Val Gly Ala Gin 595 600 605

Gly His Val Leu Leu Leu Arg Ser Leu Pro Leu Leu Lys Val Gly He 610 615 620

Ser He Arg Phe Ala Pro Ser Glu Val Ala Lys Thr Val Tyr Gin Cys 625 630 635 640

Trp Gly Arg Thr Pro Thr Val Leu Glu Ala Gly Glu Ala Thr Val Cys 645 650 655

Leu Thr Val Arg Lys Gly Ser Pro Asp Leu Leu Gly Asp Val Gin Ser 660 665 670

Ser Val Arg Tyr Asp Leu Ala Leu Asp Pro Gly Arg Leu He Ser Arg 675 680 685

Ala He Phe Asp Glu Thr Lys Asn Cys Thr Leu Thr Arg Arg Lys Thr 690 695 700

Leu Gly Leu Gly Asp His Cys Glu Thr Met Lys Leu Leu Leu Pro Asp 705 710 715 720

Cys Val Glu Asp Ala Val Thr Pro He He Leu Arg Leu Asn Leu Ser 725 730 735

Leu Ala Gly Asp Ser Ala Pro Ser Arg Asn Leu Arg Pro Val Leu Ala 740 745 750

Val Gly Ser Gin Asp His Val Thr Ala Ser Phe Pro Phe Glu Lys Asn 755 760 765

Cys Lys Gin Glu Leu Leu Cys Glu Gly Asn Leu Gly Val Ser Phe Asn 770 775 780

Phe Ser Gly Leu Gin Val Leu Glu Val Gly Ser Ser Pro Glu Leu Thr 785 790 795 800

Val Thr Val Thr Val Trp Asn Glu Gly Glu Asp Ser Tyr Gly Thr Leu 805 810 815

He Lys Phe Tyr Tyr Pro Ala Glu Leu Ser Tyr Arg Arg Val Thr Arg 820 825 830

Ala Gin Gin Pro His Pro Tyr Pro Leu Arg Leu Ala Cys Glu Ala Glu 835 840 845

Pro Thr Gly Gin Glu Ser Leu Arg Ser Ser Ser Cys Ser He Asn His 850 855 860

Pro He Phe Arg Glu Gly Ala Lys Ala Thr Phe Met He Thr Phe Asp 865 870 875 880

Val Ser Tyr Lys Ala Phe Leu Gly Asp Arg Leu Leu Leu Arg Ala Ser 885 890 895

Ala Ser Ser Glu Asn Asn Lys Pro Glu Thr Ser Lys Thr Ala Phe Gin 900 905 910

Leu Glu Leu Pro Val Lys Tyr Thr Val Tyr Thr Val He Ser Arg Gin 915 920 925

Glu Asp Ser Thr Lys His Phe Asn Phe Ser Ser Ser His Gly Glu Arg 930 935 940

Gin Lys Glu Ala Glu His Arg Tyr Arg Val Asn Asn Leu Ser Pro Leu 945 950 955 960

Thr Leu Ala He Ser Val Asn Phe Trp Val Pro He Leu Leu Asn Gly 965 970 975

Val Ala Val Trp Asp Val Thr Leu Arg Ser Pro Ala Gin Gly Val Ser 980 985 990

Cys Val Ser Gin Arg Glu Pro Pro Gin His Ser Asp Leu Leu Thr Gin 995 1000 1005

He Gin Gly Arg Ser Val Leu Asp Cys Ala He Ala Asp Cys Leu His 1010 1015 1020

Leu Arg Cys Asp He Pro Ser Leu Gly Thr Leu Asp Glu Leu Asp Phe 1025 1030 1035 1040

He Leu Lys Gly Asn Leu Ser Phe Gly Trp He Ser Gin Thr Leu Gin 1045 1050 1055

Lys Lys Val Leu Leu Leu Ser Glu Ala Glu He Thr Phe Asn Thr Ser 1060 1065 1070

Val Tyr Ser Gin Leu Pro Gly Gin Glu Ala Phe Leu Arg Ala Gin Val 1075 1080 1085

Ser Thr Met Leu Glu Glu Tyr Val Val Tyr Glu Pro Val Phe Leu Met 1090 1095 1100

Val Phe Ser Ser Val Gly Gly Leu Leu Leu Leu Ala Leu He Thr Val 1105 1110 1115 1120

Ala Leu Tyr Lys Leu Gly Phe Phe Lys Arg Gin Tyr Lys Glu Met Leu 1125 1130 1135

Asp Leu Pro Ser Ala Asp Pro Asp Pro Ala Gly Gin Ala Asp Ser Asn 1140 1145 1150

His Glu Thr Pro Pro His Leu Thr Ser 1155 1160

(2) INFORMATION FOR SEQ ID NO:54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3597 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 40..3525

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

AGCTTTACAG CTCTCTACTT CTCAGTGCAC TGCTCAGTG ATG GCC GGT GGA GTT 54

Met Ala Gly Gly Val 1 5

GTG ATC CTC CTG TGT GGC TGG GTC CTG GCT TCC TGT CAT GGG TCT AAC 102 Val He Leu Leu Cys Gly Trp Val Leu Ala Ser Cys His Gly Ser Asn 10 15 20

CTG GAT GTG GAG GAA CCC ATC GTG TTC AGA GAG GAT GCA GCC AGC TTT 150 Leu Asp Val Glu Glu Pro He Val Phe Arg Glu Asp Ala Ala Ser Phe 25 30 35

GGA CAG ACT GTG GTG CAG TTT GGT GGA TCT CGA CTC GTG GTG GGA GCC 198 Gly Gin Thr Val Val Gin Phe Gly Gly Ser Arg Leu Val Val Gly Ala 40 45 50

CCT CTG GAG GCG GTG GCA GTC AAC CAA ACA GGA CGG TTG TAT GAC TGT 246 Pro Leu Glu Ala Val Ala Val Asn Gin Thr Gly Arg Leu Tyr Asp Cys 55 60 65

GCA CCT GCC ACT GGC ATG TGC CAG CCC ATC GTA CTG CGC AGT CCC CTA 294 Ala Pro Ala Thr Gly Met Cys Gin Pro He Val Leu Arg Ser Pro Leu 70 75 80 85

GAG GCA GTG AAC ATG TCC CTG GGC CTG TCT CTG GTG ACT GCC ACC AAT 342 Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu Val Thr Ala Thr Asn 90 95 100

AAC GCC CAG TTG CTG GCT TGT GGT CCA ACT GCA CAG AGA GCT TGT GTG 390 Asn Ala Gin Leu Leu Ala Cys Gly Pro Thr Ala Gin Arg Ala Cys Val 105 110 115

AAG AAC ATG TAT GCG AAA GGT TCC TGC CTC CTT CTC GGC TCC AGC TTG 438 Lys Asn Met Tyr Ala Lys Gly Ser Cys Leu Leu Leu Gly Ser Ser Leu 120 125 130

CAG TTC ATC CAG GCA GTC CCT GCC TCC ATG CCA GAG TGT CCA AGA CAA 486 Gin Phe He Gin Ala Val Pro Ala Ser Met Pro Glu Cys Pro Arg Gin 135 140 145

GAG ATG GAC ATT GCT TTC CTG ATT GAT GGT TCT GGC AGC ATT AAC CAA 534 Glu Met Asp He Ala Phe Leu He Asp Gly Ser Gly Ser He Asn Gin 150 155 160 165

AGG GAC TTT GCC CAG ATG AAG GAC TTT GTC AAA GCT TTG ATG GGA GAG 582 Arg Asp Phe Ala Gin Met Lys Asp Phe Val Lys Ala Leu Met Gly Glu 170 175 180

TTT GCG AGC ACC AGC ACC TTG TTC TCC CTG ATG CAA TAC TCG AAC ATC 630 Phe Ala Ser Thr Ser Thr Leu Phe Ser Leu Met Gin Tyr Ser Asn He 185 190 195

CTG AAG ACC CAT TTT ACC TTC ACT GAA TTC AAG AAC ATC CTG GAC CCT 678 Leu Lys Thr His Phe Thr Phe Thr Glu Phe Lys Asn He Leu Asp Pro 200 205 210

CAG AGC CTG GTG GAT CCC ATT GTC CAG CTG CAA GGC CTG ACC TAC ACA 726 Gin Ser Leu Val Asp Pro He Val Gin Leu Gin Gly Leu Thr Tyr Thr 215 220 225

GCC ACA GGC ATC CGG ACA GTG ATG GAA GAG CTA TTT CAT AGC AAG AAT 774 Ala Thr Gly He Arg Thr Val Met Glu Glu Leu Phe His Ser Lys Asn 230 235 240 245

GGG TCC CGT AAA AGT GCC AAG AAG ATC CTC CTT GTC ATC ACA GAT GGG 822 Gly Ser Arg Lys Ser Ala Lys Lys He Leu Leu Val He Thr Asp Gly 250 255 260

CAG AAA TAC AGA GAC CCC CTG GAG TAT AGT GAT GTC ATT CCC GCC GCA 870 Gin Lys Tyr Arg Asp Pro Leu Glu Tyr Ser Asp Val He Pro Ala Ala 265 270 275

GAC AAA GCT GGC ATC ATT CGT TAT GCT ATT GGG GTG GGA GAT GCC TTC 918 Asp Lys Ala Gly He He Arg Tyr Ala He Gly Val Gly Asp Ala Phe 280 285 290

CAG GAG CCC ACT GCC CTG AAG GAG CTG AAC ACC ATT GGC TCA GCT CCC 966 Gin Glu Pro Thr Ala Leu Lys Glu Leu Asn Thr He Gly Ser Ala Pro 295 300 305

CCA CAG GAC CAC GTG TTC AAG GTA GGC AAC TTT GCA GCA CTT CGC AGC 1014 Pro Gin Asp His Val Phe Lys Val Gly Asn Phe Ala Ala Leu Arg Ser 310 315 320 325

ATC CAG AGG CAA CTT CAG GAG AAA ATC TTC GCC ATT GAG GGA ACT CAA 1062 He Gin Arg Gin Leu Gin Glu Lys He Phe Ala He Glu Gly Thr Gin 330 335 340

TCA AGG TCA AGT AGT TCC TTT CAG CAC GAG ATG TCA CAA GAA GGT TTC 1110 Ser Arg Ser Ser Ser Ser Phe Gin His Glu Met Ser Gin Glu Gly Phe 345 350 355

AGT TCA GCT CTC ACA TCG GAT GGA CCC GTT CTG GGG GCC GTG GGA AGC 1158 Ser Ser Ala Leu Thr Ser Asp Gly Pro Val Leu Gly Ala Val Gly Ser 360 365 370

TTC AGC TGG TCC GGA GGT GCC TTC TTA TAT CCC CCA AAT ACG AGA CCC 1206 Phe Ser Trp Ser Gly Gly Ala Phe Leu Tyr Pro Pro Asn Thr Arg Pro 375 380 385

ACC TTT ATC AAC ATG TCT CAG GAG AAT GTG GAC ATG AGA GAC TCC TAC 1254 Thr Phe He Asn Met Ser Gin Glu Asn Val Asp Met Arg Asp Ser Tyr 390 395 400 405

CTG GGT TAC TCC ACC GCA GTG GCC TTT TGG AAG GGG GTT CAC AGC CTG 1302 Leu Gly Tyr Ser Thr Ala Val Ala Phe Trp Lys Gly Val His Ser Leu 410 415 420

ATC CTG GGG GCC CCG CGT CAC CAG CAC ACG GGG AAG GTT GTC ATC TTT 1350 He Leu Gly Ala Pro Arg His Gin His Thr Gly Lys Val Val He Phe 425 430 435

ACC CAG GAA GCC AGG CAT TGG AGG CCC AAG TCT GAA GTC AGA GGG ACA 1398 Thr Gin Glu Ala Arg His Trp Arg Pro Lys Ser Glu Val Arg Gly Thr 440 445 450

CAG ATC GGC TCC TAC TTC GGG GCC TCT CTC TGT TCT GTG GAC GTG GAT 1446 Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys Ser Val Asp Val Asp 455 460 465

AGA GAT GGC AGC ACY GAC CTG GTC CTG ATC GGA GCC CCC CAT TAC TAT 1494 Arg Asp Gly Ser Xaa Asp Leu Val Leu He Gly Ala Pro His Tyr Tyr 470 475 480 485

GAG CAG ACC CGA GGG GGG CAG GTC TCA GTG TTC CCC GTG CCC GGT GTG 1542

Glu Gin Thr Arg Gly Gly Gin Val Ser Val Phe Pro Val Pro Gly Val 490 495 500

AGG GGC AGG TGG CAG TGT GAG GCC ACC CTC CAC GGG GAG CAG GGC CAT 1590 Arg Gly Arg Trp Gin Cys Glu Ala Thr Leu His Gly Glu Gin Gly His 505 510 515

CCT TGG GGC CGC TTT GGG GTG GCT CTG ACA GTG CTG GGG GAC GTA AAC 1638 Pro Trp Gly Arg Phe Gly Val Ala Leu Thr Val Leu Gly Asp Val Asn

520 525 530

GGG GAC AAT CTG GCA GAC GTG GCT ATT GGT GCC CCT GGA GAG GAG GAG 1686 Gly Asp Asn Leu Ala Asp Val Ala He Gly Ala Pro Gly Glu Glu Glu 535 540 545

AGC AGA GGT GCT GTC TAC ATA TTT CAT GGA GCC TCG AGA CTG GAG ATC 1734 Ser Arg Gly Ala Val Tyr He Phe His Gly Ala Ser Arg Leu Glu He 550 555 560 565

ATG CCC TCA CCC AGC CAG CGG GTC ACT GGC TCC CAG CTC TCC CTG AGA 1782 Met Pro Ser Pro Ser Gin Arg Val Thr Gly Ser Gin Leu Ser Leu Arg 570 575 580

CTG CAG TAT TTT GGG CAG TCA TTG AGT GGG GGT CAG GAC CTT ACA CAG 1830 Leu Gin Tyr Phe Gly Gin Ser Leu Ser Gly Gly Gin Asp Leu Thr Gin 585 590 595

GAT GGC CTG GTG GAC CTG GCC GTG GGA GCC CAG GGG CAC GTA CTG CTG 1878 Asp Gly Leu Val Asp Leu Ala Val Gly Ala Gin Gly His Val Leu Leu 600 605 610

CTC AGG AGT CTG CCT CTG CTG AAA GTG GAG CTC TCC ATA AGA TTC GCC 1926 Leu Arg Ser Leu Pro Leu Leu Lys Val Glu Leu Ser He Arg Phe Ala

615 620 625

CCC ATG GAG GTG GCA AAG GCT GTG TAC CAG TGC TGG GAA AGG ACT CCC 1974

Pro Met Glu Val Ala Lys Ala Val Tyr Gin Cys Trp Glu Arg Thr Pro 630 635 640 645

ACT GTC CTC GAA GCT GGA GAG GCC ACT GTC TGT CTC ACT GTC CAC AAA 2022 Thr Val Leu Glu Ala Gly Glu Ala Thr Val Cys Leu Thr Val His Lys 650 655 660

GGC TCA CCT GAC CTG TTA GGT AAT GTC CAA GGC TCT GTC AGG TAT GAT 2070 Gly Ser Pro Asp Leu Leu Gly Asn Val Gin Gly Ser Val Arg Tyr Asp 665 670 675

CTG GCG TTA GAT CCG GGC CGC CTG ATT TCT CGT GCC ATT TTT GAT GAG 2118 Leu Ala Leu Asp Pro Gly Arg Leu He Ser Arg Ala He Phe Asp Glu 680 685 690

ACT AAG AAC TGC ACT TTG ACG GGA AGG AAG ACT CTG GGG CTT GGT GAT 2166 Thr Lys Asn Cys Thr Leu Thr Gly Arg Lys Thr Leu Gly Leu Gly Asp 695 700 705

CAC TGC GAA ACA GTG AAG CTG CTT TTG CCG GAC TGT GTG GAA GAT GCA 2214 His Cys Glu Thr Val Lys Leu Leu Leu Pro Asp Cys Val Glu Asp Ala 710 715 720 725

GTG AGC CCT ATC ATC CTG CGC CTC AAC TTT TCC CTG GTG AGA GAC TCT 2262 Val Ser Pro He He Leu Arg Leu Asn Phe Ser Leu Val Arg Asp Ser 730 735 740

GCT TCA CCC AGG AAC CTG CAT CCT GTG CTG GCT GTG GGC TCA CAA GAC 2310 Ala Ser Pro Arg Asn Leu His Pro Val Leu Ala Val Gly Ser Gin Asp 745 750 755

CAC ATA ACT GCT TCT CTG CCG TTT GAG AAG AAC TGT AAG CAA GAA CTC 2358 His He Thr Ala Ser Leu Pro Phe Glu Lys Asn Cys Lys Gin Glu Leu 760 765 770

CTG TGT GAG GGG GAC CTG GGC ATC AGC TTT AAC TTC TCA GGC CTG CAG 2406 Leu Cys Glu Gly Asp Leu Gly He Ser Phe Asn Phe Ser Gly Leu Gin 775 780 785

GTC TTG GTG GTG GGA GGC TCC CCA GAG CTC ACT GTG ACA GTC ACT GTG 2454 Val Leu Val Val Gly Gly Ser Pro Glu Leu Thr Val Thr Val Thr Val 790 795 800 805

TGG AAT GAG GGT GAG GAC AGC TAT GGA ACT TTA GTC AAG TTC TAC TAC 2502 Trp Asn Glu Gly Glu Asp Ser Tyr Gly Thr Leu Val Lys Phe Tyr Tyr 810 815 820

CCA GCA GGG CTA TCT TAC CGA CGG GTA ACA GGG ACT CAG CAA CCT CAT 2550 Pro Ala Gly Leu Ser Tyr Arg Arg Val Thr Gly Thr Gin Gin Pro His 825 830 835

CAG TAC CCA CTA CGC TTG GCC TGT GAG GCT GAG CCC GCT GCC CAG GAG 2598 Gin Tyr Pro Leu Arg Leu Ala Cys Glu Ala Glu Pro Ala Ala Gin Glu 840 845 850

GAC CTG AGG AGC AGC AGC TGT AGC ATT AAT CAC CCC ATC TTC CGA GAA 2646 Asp Leu Arg Ser Ser Ser Cys Ser He Asn His Pro He Phe Arg Glu 855 860 865

GGT GCA AAG ACC ACC TTC ATG ATC ACA TTC GAT GTC TCC TAC AAG GCC 2694 Gly Ala Lys Thr Thr Phe Met He Thr Phe Asp Val Ser Tyr Lys Ala 870 875 880 885

TTC CTA GGA GAC AGG TTG CTT CTG AGG GCC AAA GCC AGC AGT GAG AAT 2742 Phe Leu Gly Asp Arg Leu Leu Leu Arg Ala Lys Ala Ser Ser Glu Asn 890 895 900

AAT AAG CCT GAT ACC AAC AAG ACT GCC TTC CAG CTG GAG CTC CCA GTG 2790 Asn Lys Pro Asp Thr Asn Lys Thr Ala Phe Gin Leu Glu Leu Pro Val 905 910 915

AAG TAC ACC GTC TAT ACC CTG ATC AGT AGG CAA GAA GAT TCC ACC AAC 2838 Lys Tyr Thr Val Tyr Thr Leu He Ser Arg Gin Glu Asp Ser Thr Asn 920 925 930

CAT GTC AAC TTT TCA TCT TCC CAC GGG GGG AGA AGG CAA GAA GCC GCA 2886 His Val Asn Phe Ser Ser Ser His Gly Gly Arg Arg Gin Glu Ala Ala 935 940 945

CAT CGC TAT CGT GTG AAT AAC CTG AGT CCA CTG AAG CTG GCC GTC AGA 2934 His Arg Tyr Arg Val Asn Asn Leu Ser Pro Leu Lys Leu Ala Val Arg 950 955 960 965

GTT AAC TTC TGG GTC CCT GTC CTT CTG AAC GGT GTG GCT GTG TGG GAC 2982 Val Asn Phe Trp Val Pro Val Leu Leu Asn Gly Val Ala Val Trp Asp 970 975 980

GTG ACT CTG AGC AGC CCA GCA CAG GGT GTC TCC TGC GTG TCC CAG ATG 3030 Val Thr Leu Ser Ser Pro Ala Gin Gly Val Ser Cys Val Ser Gin Met 985 990 995

AAA CCT CCT CAG AAT CCC GAC TTT CTG ACC CAG ATT CAG AGA CGT TCT 3078 Lys Pro Pro Gin Asn Pro Asp Phe Leu Thr Gin He Gin Arg Arg Ser 1000 1005 1010

GTG CTG GAC TGC TCC ATT GCT GAC TGC CTG CAC TTC CGC TGT GAC ATC 3126 Val Leu Asp Cys Ser He Ala Asp Cys Leu His Phe Arg Cys Asp He 1015 1020 1025

CCC TCC TTG GAC ATC CAG GAT GAA CTT GAC TTC ATT CTG AGG GGC AAC 3174 Pro Ser Leu Asp He Gin Asp Glu Leu Asp Phe He Leu Arg Gly Asn 1030 1035 1040 1045

CTC AGC TTC GGC TGG GTC AGT CAG ACA TTG CAG GAA AAG GTG TTG CTT 3222 Leu Ser Phe Gly Trp Val Ser Gin Thr Leu Gin Glu Lys Val Leu Leu 1050 1055 1060

GTG AGT GAG GCT GAA ATC ACT TTC GAC ACA TCT GTG TAC TCC CAG CTG 3270 Val Ser Glu Ala Glu He Thr Phe Asp Thr Ser Val Tyr Ser Gin Leu 1065 1070 1075

CCA GGA CAG GAG GCA TTT CTG AGA GCC CAG GTG GAG ACA ACG TTA GAA 3318 Pro Gly Gin Glu Ala Phe Leu Arg Ala Gin Val Glu Thr Thr Leu Glu 1080 1085 1090

GAA TAC GTG GTC TAT GAG CCC ATC TTC CTC GTG GCG GGC AGC TCG GTG 3366 Glu Tyr Val Val Tyr Glu Pro He Phe Leu Val Ala Gly Ser Ser Val 1095 1100 1105

GGA GGT CTG CTG TTA CTG GCT CTC ATC ACA GTG GTA CTG TAC AAG CTT 3414 Gly Gly Leu Leu Leu Leu Ala Leu He Thr Val Val Leu Tyr Lys Leu 1110 1115 1120 1125

GGC TTC TYC AAA CGT CAG TAC AAA GAA ATG CTG GAC GGC AAG GCT GCA 3462 Gly Phe Xaa Lys Arg Gin Tyr Lys Glu Met Leu Asp Gly Lys Ala Ala 1130 1135 1140

GAT CCT GTC ACA GCC GGC CAG GCA GAT TTC GGC TGT GAG ACT CCT CCA 3510 Asp Pro Val Thr Ala Gly Gin Ala Asp Phe Gly Cys Glu Thr Pro Pro 1145 1150 1155

TAT CTC GTG AGC TAGGAATCCA CTCTCCTGCC TATCTCTGCA ATGAAGATTG 3562 Tyr Leu Val Ser 1160

GTCCTGCCTA TGAGTCTACT GGCATGGGAA CGAGT 3597

(2) INFORMATION FOR SEQ ID NO:55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1161 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:

Met Ala Gly Gly Val Val He Leu Leu Cys Gly Trp Val Leu Ala Ser 1 5 10 15

Cys His Gly Ser Asn Leu Asp Val Glu Glu Pro He Val Phe Arg Glu 20 25 30

Asp Ala Ala Ser Phe Gly Gin Thr Val Val Gin Phe Gly Gly Ser Arg 35 40 45

Leu Val Val Gly Ala Pro Leu Glu Ala Val Ala Val Asn Gin Thr Gly 50 55 60

Arg Leu Tyr Asp Cys Ala Pro Ala Thr Gly Met Cys Gin Pro He Val 65 70 75 80

Leu Arg Ser Pro Leu Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu 85 90 95

Val Thr Ala Thr Asn Asn Ala Gin Leu Leu Ala Cys Gly Pro Thr Ala 100 105 110

Gin Arg Ala Cys Val Lys Asn Met Tyr Ala Lys Gly Ser Cys Leu Leu 115 120 125

Leu Gly Ser Ser Leu Gin Phe He Gin Ala Val Pro Ala Ser Met Pro 130 135 140

Glu Cys Pro Arg Gin Glu Met Asp He Ala Phe Leu He Asp Gly Ser 145 150 155 160

Gly Ser He Asn Gin Arg Asp Phe Ala Gin Met Lys Asp Phe Val Lys 165 170 175

Ala Leu Met Gly Glu Phe Ala Ser Thr Ser Thr Leu Phe Ser Leu Met 180 185 190

Gin Tyr Ser Asn He Leu Lys Thr His Phe Thr Phe Thr Glu Phe Lys 195 200 205

Asn He Leu Asp Pro Gin Ser Leu Val Asp Pro He Val Gin Leu Gin 210 215 220

Gly Leu Thr Tyr Thr Ala Thr Gly He Arg Thr Val Met Glu Glu Leu 225 230 235 240

Phe His Ser Lys Asn Gly Ser Arg Lys Ser Ala Lys Lys He Leu Leu 245 250 255

Val He Thr Asp Gly Gin Lys Tyr Arg Asp Pro Leu Glu Tyr Ser Asp 260 265 270

Val He Pro Ala Ala Asp Lys Ala Gly He He Arg Tyr Ala He Gly 275 280 285

Val Gly Asp Ala Phe Gin Glu Pro Thr Ala Leu Lys Glu Leu Asn Thr 290 295 300

He Gly Ser Ala Pro Pro Gin Asp His Val Phe Lys Val Gly Asn Phe 305 310 315 320

Ala Ala Leu Arg Ser He Gin Arg Gin Leu Gin Glu Lys He Phe Ala 325 330 335

He Glu Gly Thr Gin Ser Arg Ser Ser Ser Ser Phe Gin His Glu Met 340 345 350

Ser Gin Glu Gly Phe Ser Ser Ala Leu Thr Ser Asp Gly Pro Val Leu 355 360 365

Gly Ala Val Gly Ser Phe Ser Trp Ser Gly Gly Ala Phe Leu Tyr Pro 370 375 380

Pro Asn Thr Arg Pro Thr Phe He Asn Met Ser Gin Glu Asn Val Asp 385 390 395 400

Met Arg Asp Ser Tyr Leu Gly Tyr Ser Thr Ala Val Ala Phe Trp Lys 405 410 415

Gly Val His Ser Leu He Leu Gly Ala Pro Arg His Gin His Thr Gly 420 425 430

Lys Val Val He Phe Thr Gin Glu Ala Arg His Trp Arg Pro Lys Ser 435 440 445

Glu Val Arg Gly Thr Gin He Gly Ser Tyr Phe Gly Ala Ser Leu Cys 450 455 460

Ser Val Asp Val Asp Arg Asp Gly Ser Xaa Asp Leu Val Leu He Gly 465 470 475 480

Ala Pro His Tyr Tyr Glu Gin Thr Arg Gly Gly Gin Val Ser Val Phe 485 490 495

Pro Val Pro Gly Val Arg Gly Arg Trp Gin Cys Glu Ala Thr Leu His 500 505 510

Gly Glu Gin Gly His Pro Trp Gly Arg Phe Gly Val Ala Leu Thr Val 515 520 525

Leu Gly Asp Val Asn Gly Asp Asn Leu Ala Asp Val Ala He Gly Ala 530 535 540

Pro Gly Glu Glu Glu Ser Arg Gly Ala Val Tyr He Phe His Gly Ala 545 550 555 560

Ser Arg Leu Glu He Met Pro Ser Pro Ser Gin Arg Val Thr Gly Ser 565 570 575

Gin Leu Ser Leu Arg Leu Gin Tyr Phe Gly Gin Ser Leu Ser Gly Gly 580 585 590

Gin Asp Leu Thr Gin Asp Gly Leu Val Asp Leu Ala Val Gly Ala Gin 595 600 605

Gly His Val Leu Leu Leu Arg Ser Leu Pro Leu Leu Lys Val Glu Leu 610 615 620

Ser He Arg Phe Ala Pro Met Glu Val Ala Lys Ala Val Tyr Gin Cys 625 630 635 640

Trp Glu Arg Thr Pro Thr Val Leu Glu Ala Gly Glu Ala Thr Val Cys 645 650 655

Leu Thr Val His Lys Gly Ser Pro Asp Leu Leu Gly Asn Val Gin Gly 660 665 670

Ser Val Arg Tyr Asp Leu Ala Leu Asp Pro Gly Arg Leu He Ser Arg 675 680 685

Ala He Phe Asp Glu Thr Lys Asn Cys Thr Leu Thr Gly Arg Lys Thr 690 695 700

Leu Gly Leu Gly Asp His Cys Glu Thr Val Lys Leu Leu Leu Pro Asp 705 710 715 720

Cys Val Glu Asp Ala Val Ser Pro He He Leu Arg Leu Asn Phe Ser 725 730 735

Leu Val Arg Asp Ser Ala Ser Pro Arg Asn Leu His Pro Val Leu Ala 740 745 750

Val Gly Ser Gin Asp His He Thr Ala Ser Leu Pro Phe Glu Lys Asn 755 760 765

Cys Lys Gin Glu Leu Leu Cys Glu Gly Asp Leu Gly He Ser Phe Asn 770 775 780

Phe Ser Gly Leu Gin Val Leu Val Val Gly Gly Ser Pro Glu Leu Thr 785 790 795 800

Val Thr Val Thr Val Trp Asn Glu Gly Glu Asp Ser Tyr Gly Thr Leu 805 810 815

Val Lys Phe Tyr Tyr Pro Ala Gly Leu Ser Tyr Arg Arg Val Thr Gly 820 825 830

Thr Gin Gin Pro His Gin Tyr Pro Leu Arg Leu Ala Cys Glu Ala Glu 835 840 845

Pro Ala Ala Gin Glu Asp Leu Arg Ser Ser Ser Cys Ser He Asn His 850 855 860

Pro He Phe Arg Glu Gly Ala Lys Thr Thr Phe Met He Thr Phe Asp 865 870 875 880

Val Ser Tyr Lys Ala Phe Leu Gly Asp Arg Leu Leu Leu Arg Ala Lys 885 890 895

Ala Ser Ser Glu Asn Asn Lys Pro Asp Thr Asn Lys Thr Ala Phe Gin 900 905 910

Leu Glu Leu Pro Val Lys Tyr Thr Val Tyr Thr Leu He Ser Arg Gin 915 920 925

Glu Asp Ser Thr Asn His Val Asn Phe Ser Ser Ser His Gly Gly Arg 930 935 940

Arg Gin Glu Ala Ala His Arg Tyr Arg Val Asn Asn Leu Ser Pro Leu 945 950 955 960

Lys Leu Ala Val Arg Val Asn Phe Trp Val Pro Val Leu Leu Asn Gly 965 970 975

Val Ala Val Trp Asp Val Thr Leu Ser Ser Pro Ala Gin Gly Val Ser 980 985 990

Cys Val Ser Gin Met Lys Pro Pro Gin Asn Pro Asp Phe Leu Thr Gin 995 1000 1005

He Gin Arg Arg Ser Val Leu Asp Cys Ser He Ala Asp Cys Leu His 1010 1015 1020

Phe Arg Cys Asp He Pro Ser Leu Asp He Gin Asp Glu Leu Asp Phe 1025 1030 1035 1040

He Leu Arg Gly Asn Leu Ser Phe Gly Trp Val Ser Gin Thr Leu Gin 1045 1050 1055

Glu Lys Val Leu Leu Val Ser Glu Ala Glu He Thr Phe Asp Thr Ser 1060 1065 1070

Val Tyr Ser Gin Leu Pro Gly Gin Glu Ala Phe Leu Arg Ala Gin Val 1075 1080 1085

Glu Thr Thr Leu Glu Glu Tyr Val Val Tyr Glu Pro He Phe Leu Val 1090 1095 1100

Ala Gly Ser Ser Val Gly Gly Leu Leu Leu Leu Ala Leu He Thr Val 1105 1110 1115 1120

Val Leu Tyr Lys Leu Gly Xaa Xaa Lys Arg Gin Tyr Lys Glu Met Leu 1125 1130 1135

Asp Gly Lys Ala Ala Asp Pro Val Thr Xaa Gly Gin Ala Asp Phe Gly 1140 1145 1150

Cys Glu Thr Pro Pro Tyr Leu Val Ser 1155 1160

(2) INFORMATION FOR SEQ ID NO:56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: CCTGTCATGG GTCTAACCTG 20

(2) INFORMATION FOR SEQ ID NO:57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: AGGTTAGACC CATGACAGG 19

(2) INFORMATION FOR SEQ ID NO:58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58: GGCCTTGCAG CTGGACAATG 20

(2) INFORMATION FOR SEQ ID NO:59:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: CCAAAGCTGG CTGCATCCTC TC 22

(2) INFORMATION FOR SEQ ID NO:60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60: CCGCCTGCCA CTGGCGTGTG C 21 (2) INFORMATION FOR SEQ ID NO:61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: CCCAGATGAA GGACTTCGTC AA 22 (2) INFORMATION FOR SEQ ID NO:62:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: GCTGGGATCA TTCGCTATGC 20 (2) INFORMATION FOR SEQ ID NO:63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: CAATGGATGG ACCAGTTCTG G 21 (2) INFORMATION FOR SEQ ID NO:64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: CAGATCGGCT CCTACTTTGG 20

(2) INFORMATION FOR SEQ ID NO:65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65: CATGGAGCCT CGAGACAGG 19

(2) INFORMATION FOR SEQ ID NO:66:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: CCACTGTCCT CGAAGCTGGA G 21

(2) INFORMATION FOR SEQ ID NO:67:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 26 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CTTCGTCCTG TGCTGGCTGT GGGCTC 26

(2) INFORMATION FOR SEQ ID NO:68:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68: CGCCTGGCAT GTGAGGCTGA G 21 (2) INFORMATION FOR SEQ ID NO:69:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69: CCGTGATCAG TAGGCAGGAA G 21 (2) INFORMATION FOR SEQ ID NO:70:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70: GTCACAGAGG GAACCTCC 18 (2) INFORMATION FOR SEQ ID NO:71:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: GCTCCTGAGT GAGGCTGAAA TCA 23 (2) INFORMATION FOR SEQ ID NO:72:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72: GAGATGCTGG ATCTACCATC TGC 23

(2) INFORMATION FOR SEQ ID NO:73:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73: CTGAGCTGGG AGATTTTTAT GG 22

(2) INFORMATION FOR SEQ ID NO:74:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74: GTGGATCAGC ACTGAAATCT G 21

(2) INFORMATION FOR SEQ ID NO:75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75: CGTTTGAAGA AGCCAAGCTT G 21

(2) INFORMATION FOR SEQ ID NO:76:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76: CACAGCGGAG GTGCAGGCAG 20 (2) INFORMATION FOR SEQ ID NO:77:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77: CTCACTGCTT GCGCTGGC 18 (2) INFORMATION FOR SEQ ID NO:78:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78: CGGTAAGATA GCTCTGCTGG 20 (2) INFORMATION FOR SEQ ID NO:79:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:

GAGCCCACAG CCAGCACAGG 20

(2) INFORMATION FOR SEQ ID NO:80:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80: GATCCAACGC CAGATCATAC C 21

(2) INFORMATION FOR SEQ ID NO:81:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81: CACGGCCAGG TCCACCAGGC 20

(2) INFORMATION FOR SEQ ID NO:82:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82: CACGTCCCCT AGCACTGTCA G 21

(2) INFORMATION FOR SEQ ID NO:83:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83: TTGACGAAGT CCTTCATCTG GG 22 (2) INFORMATION FOR SEQ ID NO:84:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84: GAACTGCAAG CTGGAGCCCA G 21 (2) INFORMATION FOR SEQ ID NO:85:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85: CTGGATGCTG CGAAGTGCTA C 21 (2) INFORMATION FOR SEQ ID NO:86:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86: GCCTTGGAGC TGGACGATGG C 21

(2) INFORMATION FOR SEQ ID NO:87:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87: GTAAGATCTC CAGAGTGTCC AAGACAAGAG ATG 33

(2) INFORMATION FOR SEQ ID NO:88:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88: CTTCTCGAGT GTGAGAGCTG AACTGAAACC TTC 33

(2) INFORMATION FOR SEQ ID NO:89:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89: CGCTGTGACG TCAGAGTTGA GTCCAAATAT GG 32

(2) INFORMATION FOR SEQ ID NO:90:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90: GGTGACACTA TAGAATAGGG C 21

(2) INFORMATION FOR SEQ ID NO:91:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91: AAGCAGGAGCTCCTGTGT 18

(2) INFORMATION FOR SEQ ID NO:92:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 852 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 61..852

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:

TGATCTCCCT CCAGGCCACT GTTCCCTCTC CACTTCCCCT CACCGCTGCA CTGCTCAGAG 60

ATG GCC CTT GGG GCT GTG GTC CTC CTT GGG GTC CTG GCT TCT TAC CAC 108 Met Ala Leu Gly Ala Val Val Leu Leu Gly Val Leu Ala Ser Tyr His 1 5 10 15

GGA TTC AAC TTG GAC GTG ATG AGC GGT GAT CTT CCA GGA AGA CGC AGC 156 Gly Phe Asn Leu Asp Val Met Ser Gly Asp Leu Pro Gly Arg Arg Ser 20 25 30

GGG CTT CGG GCA GAG CGT GAT GCA GTT TGG GGA TCT CGA CTC GTG GTG 204 Gly Leu Arg Ala Glu Arg Asp Ala Val Trp Gly Ser Arg Leu Val Val 35 40 45

GGA GCC CCC CTG GCG GTG GTG TCG GCC AAC CAC ACA GGA CGG CTG TAC 252 Gly Ala Pro Leu Ala Val Val Ser Ala Asn His Thr Gly Arg Leu Tyr 50 55 60

GAG TGT GCG CCT GCC TCC GGC ACC TGC ACG CCC ATT TTC CCA TTC ATG 300 Glu Cys Ala Pro Ala Ser Gly Thr Cys Thr Pro lie Phe Pro Phe Met 65 70 75 80

CCC CCC GAA GCC GTG AAC ATG TCC CTG GGC CTG TCC CTG GCA GCC TCC 348 Pro Pro Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu .Ala Ala Ser 85 90 95

CCC AAC CAT TCC CAG CTG CTG GCT TGT GGC CCG ACC GTG CAT AGA GCC 396 Pro Asn His Ser Gin Leu Leu Ala Cys Gly Pro Thr Val His Arg Ala 100 105 110

TGC GGG GAG GAC GTG TAC GCC CAG GGT TTC TGT GTG CTG CTG GAT GCC 444 Cys Gly Glu Asp Val Tyr Ala Gin Gly Phe Cys Val Leu Leu Asp Ala 115 120 125

CAC GCA CAG CCC ATC GGG ACT GTG CCA GCT GCC CTG CCC GAG TGC CCA 492 His Ala Gin Pro lie Gly Thr Val Pro Ala Ala Leu Pro Glu Cys Pro 130 135 140

GAT CAA GAG ATG GAC ATT GTC TTC CTG ATT GAC GGC TCT GGC AGC ATT 540 Asp Gin Glu Met Asp lie Val Phe Leu lie Asp Gly Ser Gly Ser lie 145 150 155 160

AGC TCA AAT GAC TTC CGC AAG ATG AAG GAC TTT GTC AGA GCT GTG ATG 588 Ser Ser Asn Asp Phe Arg Lys Met Lys Asp Phe Val Arg Ala Val Met 165 170 175

GAC CAG TTC AAG GAC ACC AAC ACC CAG TTC TCG CTG ATG CAG TAC TCC 636 Asp Gin Phe Lys Asp Thr Asn Thr Gin Phe Ser Leu Met Gin Tyr Ser 180 185 190

AAT GTG CTG GTG ACA CAT TTC ACC TTC AGC AGC TTC CGG AAC AGC TCC 684 Asn Val Leu Val Thr His Phe Thr Phe Ser Ser Phe Arg Asn Ser Ser 195 200 205

AAT CCT CAG GGC CTA GTG GAG CCC ATT GTG CAG CTG ACA GGC CTC ACG 732 Asn Pro Gin Gly Leu Val Glu Pro lie Val Gin Leu Thr Gly Leu Thr 210 215 220

TTC ACG GCC ACA GGG ATC CTG AAA GTG GTG ACA GAG CTG TTT CAA ACC 780 Phe Thr Ala Thr Gly lie Leu Lys Val Val Thr Glu Leu Phe Gin Thr 225 230 235 240

AAG AAC GGG GCC CGC GAA AGT GCC AAG AAG ATC CTC ATC GTC ATC ACA 828 Lys Asn Gly Ala Arg Glu Ser Ala Lys Lys lie Leu lie Val lie Thr 245 250 255

GAT GGG CAG AAG TAC AAA GCG GCA 852

Asp Gly Gin Lys Tyr Lys Ala Ala 260

(2) INFORMATION FOR SEQ ID NO:93:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 264 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:

Met Ala Leu Gly Ala Val Val Leu Leu Gly Val Leu Ala Ser Tyr His 1 5 10 15

Gly Phe Asn Leu Asp Val Met Ser Gly Asp Leu Pro Gly Arg Arg Ser 20 25 30

Gly Leu Arg Ala Glu Arg Asp Ala Val Trp Gly Ser Arg Leu Val Val 35 40 45

Gly Ala Pro Leu Ala Val Val Ser Ala Asn His Thr Gly Arg Leu Tyr 50 55 60

Glu Cys Ala Pro Ala Ser Gly Thr Cys Thr Pro lie Phe Pro Phe Met 65 70 75 80

Pro Pro Glu Ala Val Asn Met Ser Leu Gly Leu Ser Leu Ala Ala Ser 85 90 95

Pro Asn His Ser Gin Leu Leu Ala Cys Gly Pro Thr Val His Arg Ala 100 105 110

Cys Gly Glu Asp Val Tyr Ala Gin Gly Phe Cys Val Leu Leu Asp Ala 115 120 125

His Ala Gin Pro lie Gly Thr Val Pro Ala Ala Leu Pro Glu Cys Pro 130 135 140

Asp Gin Glu Met Asp lie Val Phe Leu lie Asp Gly Ser Gly Ser lie 145 150 155 160

Ser Ser Asn Asp Phe Arg Lys Met Lys Asp Phe Val Arg Ala Val Met 165 170 175

Asp Gin Phe Lys Asp Thr Asn Thr Gin Phe Ser Leu Met Gin Tyr Ser 180 185 190

Asn Val Leu Val Thr His Phe Thr Phe Ser Ser Phe Arg Asn Ser Ser 195 200 205

Asn Pro Gin Gly Leu Val Glu Pro lie Val Gin Leu Thr Gly Leu Thr 210 215 220

Phe Thr Ala Thr Gly lie Leu Lys Val Val Thr Glu Leu Phe Gin Thr 225 230 235 240

Lys Asn Gly Ala Arg Glu Ser Ala Lys Lys lie Leu lie Val lie Thr 245 250 255

Asp Gly Gin Lys Tyr Lys Ala Ala 260