WO1999051742A2

WO1999051742A2 - Dadd, death activator death domain protein

Info

Publication number: WO1999051742A2
Application number: PCT/US1999/007517
Authority: WO
Inventors: Jean-Baptiste Telliez; Lih Ling Lin
Original assignee: Genetics Institute, Inc.
Priority date: 1998-04-07
Filing date: 1999-04-06
Publication date: 1999-10-14
Also published as: AU3473699A; CA2325339A1; AU768824B2; US20060189525A1; NZ507864A; US6893838B1; US7576178B2; EP1070129A2; WO1999051742A3; JP2002510491A

Abstract

Polynucleotides encoding DADD protein are also disclosed, along with vectors, host cells, and methods of making DADD protein. Methods of identifying inhibitors of DADD death domain binding and inhibitors identified by such methods are also disclosed.

Description

DADD, Death Activator Death Domain Protein

BACKGROUND OF THE INVENTION Tumor necrosis factor (herein "TNF") is a cytokine which produces a wide range of cellular activities. TNF causes an inflammatory response, which can be beneficial, such as in mounting an immune response to a pathogen, or when overexpressed can lead to other detrimental effects of inflammation.

The cellular effects of TNF are initiated by the binding of TNF to its receptors (TNF-Rs) on the surface of target cells. The isolation of polynucleotides encoding

TNF-Rs and variant forms of such receptors has been described in European patent publication Nos. EP 308,378, EP 393,438, EP 433,900, EP 526,905 and EP 568,925; in PCT patent publication Nos. WO91/03553 and WO93/19777; and by Schall et al, Cell 67:361-370 (1990) (disclosing the P55 type TNF receptor). Processes for purification of TNF-Rs have also been disclosed in U.S. Patent No. 5,296,592.

Native TNF-Rs are characterized by distinct extracellular, transmembrane and intracellular domains. The primary purpose of the extracellular domain is to present a binding site for TNF on the outside of the cell. When TNF is bound to the binding site, a "signal" is transmitted to the inside of the cell through the transmembrane and intracellular domains, indicating that binding has occurred.

Transmission or "transduction" of the signal to the inside of the cell occurs by a change in conformation of the transmembrane and/or intracellular domains of the receptor. This signal is "received" by the binding of proteins and other molecules to the intracellular domain ofthe receptor, resulting in the effects seen upon TNF stimulation. Two distinct TNF receptors of -55 kd ("TNF-R1 ") and -75 kd ("TNF-R2") have been identified. Numerous studies with anti-TNF receptor antibodies have demonstrated that

TNF-R 1 is the receptor which signals the majority of the pleiotropic activities of TNF.

The domain required for signaling cytotoxicity and other TNF-mediated responses has been mapped to the -80 amino acid near the C-terminus of TNF-R 1. This domain is therefore termed the "death domain" (hereinafter referred to as "TNF-R death domain") (see, Tartaglia et al, Cell 74:845-853 (1993)). Other proteins have been identified which also have regions homologous to the TNF-R death domain. These regions are also referred to genetically as "death domains." Examples of proteins having such a death domain include Fas (Tartaglia et al., Cell 74: 845-853 (1993)), FADD (Chinnaiyan et al., Cell 81: 505-512 (1995)), RIP (Stanger et al., Cell 81: 513- 523 (1995)), TRADD (Hsu et al., Cell 81: 495-504 (1995)), DR3 (Chinnaiyan et al., Science 274: 990-992 (1996)), and DR4 (Pan et al., Science, 276: 111-113 (1997)).

One activity produced by the interaction of TNF with TNF-R is cell death or apoptosis. It has been determined that the cell death process is mediated by the interaction ofthe death domains of TNF-R and other death doma -contøining proteins. After binding of TNF to TNF-R, such proteins associate, forming homodimer and heterodimers, resulting in the instigation of the apoptotic process. As a result, inhibiting the interaction of death domain proteins will inhibit the induction of apoptosis.

It would, therefore, be desireable to identify new death domain-containing proteins which may be involved in the apoptotic process in order to in turn identify inhibitors of death domain associations and the apoptotic process resulting therefrom.

SUMMARY OF THE INVENTION

Applicants have for the first time identified novel DADD proteins and have isolated polynucleotides encoding such proteins. In one embodiment, the present invention provides a composition comprising an isolated polynucleotide encoding a protein having DADD protein activity. In preferred embodiments, the polynucleotide is selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID

NO: 12 from nucleotide 794 to nucleotide 3052;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12 from nucleotide 1295 to nucleotide 3052;

(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12 from nucleotide 2726 to nucleotide 2929; (e) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO: 12;

(f) a polynucleotide encoding a DADD protein comprising the amino acid sequence of SEQ ID NO: 13; (g) a polynucleotide encoding a DADD protein comprising the amino acid sequence of SEQ ID NO: 13 from amino acid 167 to amino acid 753;

(h) a polynucleotide encoding a DADD protein comprising the amino acid sequence of SEQ ID NO: 13 from amino acid 645 to amino acid 712;

(i) a polynucleotide encoding a DADD protein comprising a fragment of the amino acid sequence of SEQ ID NO: 13; and

(j) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). In certain preferred embodiments, the polynucleotide is operably linked to an expression control sequence. The invention also provides a host cell, including bacterial, yeast, insect and mammalian cells, transformed with such polynucleotide compositions.

Processes are also provided for producing a DADD protein, which comprises:

(a) growing a culture of the host cell transformed with such polynucleotide compositions in a suitable culture medium; and

(b) purifying the DADD protein from the culture. The protein produced according to such methods is also provided by the present invention.

Compositions comprising a protein having DADD protein activity are also disclosed. In preferred embodiments the protein comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO: 13 ;

(b) the amino acid sequence of SEQ ID NO: 13 from amino acid 167 to amino acid 753;

(c) the amino acid sequence of SEQ ID NO : 13 from amino acid 645 to amino acid 712; and (d) fragments of the amino acid sequence of SEQ ID NO: 13; the protein being substantially free from other mammalian proteins. Such compositions may further comprise a pharmaceutically acceptable carrier.

Compositions comprising an antibody which specifically reacts with such DADD protein are also provided by the present invention. Methods are also provided for identifying an inhibitor of binding of a DADD protein to a second protein having a death domain which comprise:

(a) combining said DADD protein with said second protein, said combination forming a first binding mixture;

(b) measuring the amount of binding between the DADD protein and the second protein in the first binding mixture;

(c) combining a compound with the DADD protein and the second protein to form a second binding mixture;

(d) measuring the amount of binding between the DADD protein and the second protein in the second binding mixture; and (e) comparing the amount of binding in the first binding mixture with the amount of binding in the second binding mixture; wherein the compound is capable of inhibiting binding when a decrease in the amount of binding occurs in the second mixture as compared to the first mixture. In certain preferred embodiments the second protein is either a protein comprising the death domain of TNF-R or a DADD protein. In other preferred embodiments, the DADD protein used in such method comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO: 13;

(c) the amino acid sequence of SEQ ID NO: 13 from amino acid 645 to amino acid 712; and

(d) fragments of the amino acid sequence of SEQ ID NO: 13 ; Compositions comprising inhibitors identified according to such method are also provided. Such compositions may include pharmaceutically acceptable carriers. Methods are also provided for preventing or ameliorating an inflammatory condition which comprises administering a therapeuticaUy effective amount of a composition comprising a protein having DADD protein activity and a pharmaceutically acceptable carrier. Other embodiments provide methods of inhibiting TNF-R death domain binding comprising administering a therapeuticaUy effective amount of a composition comprising a protein having DADD protein activity and a pharmaceutically acceptable carrier.

Methods of preventing or ameliorating an inflammatory condition or of inhibiting DADD death domain binding are provided, which comprise administering to a mammalian subject a therapeuticaUy effective amount of inhibitors of DADD death domain binding, are also provided.

Methods of identifying an inhibitor of DADD death domain binding are also provided by the present invention which comprise: (a) transforming a ceU with a first polynucleotide encoding a DADD protein, a second polynucleotide encoding a second protein having a death domain, and at least one reporter gene, wherein the expression of the reporter gene is regulated by the binding of the DADD protein encoded by the first polynucleotide to the second protein encoded by the second polynucleotide; (b) growing the cell in the presence of and in the absence of a compound; and

(c) comparing the degree of expression of the reporter gene in the presence of and in the absence of the compound; wherein the compound is capable of inhibiting DADD death domain binding when a decrease in the degree of expression of the reporter gene occurs. In preferred embodiments, the second protein is a DADD protein or a protein containing the TNF-R death domain. In other preferred embodiments, the cell is a yeast cell and the first polynucleotide is selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12; (b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12 from nucleotide 794 to nucleotide 3052;

(c) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12 from nucleotide 1295 to nucleotide 3052; (d) a polynucleotide comprising the nucleotide sequence of SEQ ID

NO: 12 from nucleotide 2726 to nucleotide 2929;

(e) a polynucleotide comprising a fragment of the nucleotide sequence of SEQ ID NO: 12;

(f) a polynucleotide encoding a DADD protein comprising the amino acid sequence of SEQ ID NO: 13;

(g) a polynucleotide encoding a DADD protein comprising the amino acid sequence of SEQ ID NO: 13 from amino acid 167 to amino acid 753;

(h) a polynucleotide encoding a DADD protein comprising the amino acid sequence of SEQ ID NO: 13 from amino acid 645 to amino acid 712; (i) a polynucleotide encoding a DADD protein comprising a fragment of the amino acid sequence of SEQ ID NO: 13; and

(j) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i). In other preferred embodiments, the second polynucleotides is also selected from the preceding list.

DETAILED DESCRIPTION OF THE INVENTION

The EST database (GenBank) was screened using the following sequence from the death domain sequence of human RTP (tblastn):

IRENLGKHWKNCARKLGFTQSQIDEIDHDYERDGLKEKVΥQMLQK VMREGIKGAT VGKLAQAHQCSRIDLLSSLT (SEQ ID NO:l)

A homo sapiens cDNA clone (Length = 433) (gb N55392) was identified in the screen. The clone had 39% identity (15/38) and 50% homology (19/38) with RTP as shown below: RIP RrjGLKEKWQMLQKWVMREGIKGATVGKIAQALHQCSR ( 32- 69 SEQ ID NO : l )

RD E++ ML W R+ + G L QAL Q R clone RDDLDEQIRHMLFSWAERQAGQPGAVGLLVQALEQSDR ( 668-705 SEQ ID NO : 13 )

The sequence of the clone N55392 follows:

TGGACTGGCC AGCCGTGGCC AAAACCTGGG GGTGTCCTAC CGGGAGTGCA

GCGATCCGGC ACGAGTTCCG GGATGATCTG GATGAGCAGA TCCGTCACAT GCTCTTCTCC TGGGCTGAGC GCCAGGCTGG GCAGCCAGGG NTGTNGGGGC

TCCTGGTGCA GGCCCTGGAG CAGAGTGACC GGCAGACCGT GGCTGAAGAG

GTGCGCGCAG TCTTGGAGCT CGGCCGCCGC AAGTACCAGG ACAGCATCCG

ACGCATGGGC TTGGCCCCAA GGACCCCGCT CTGCCTGGCT CCTCGGGCTC

CACAGCCCCC AGGAGCCTGC CCCAGGCCTT AGGGCCCCAA CAGAACTTTT TAGGCTGGGC CCAGAATATT CCCCAGGTGG AATGGGCAGA ACCCCAACCN

TTCAAAGTCT CTCCAAGTGTG TGGGGGACG NTT (SEQ ID NO:2)

This clone also showed 93% identity with another Homo sapiens cDNA clone (gb N39432) over a 159bp overlap. This clone is 423bp. The sequence of clone N39432 (3'-->5') follows:

GAAAGAAACA GTGCAGTTTT GTTGCTCACA GGGACCCGTC CCCACACACT

GGAGAGACTT GAAGGTGGGG GCTCTGCATN CCACTGGGGA ATATCTGGGC

CAGCCTAAAA GTCTGTGGGG CCTAGGCTGG GCAGGCTCTG GGGGCTGTGG AGCCGAGGAG CCAGGCAGAG CGGGGTCCTT GGGGGCCAAG CCATGCGTCG

GATGCTGTCC TGGTACTTGC GGCGGCCGAG CTCCAAGACT GCGCGCACCT

CTTCAGCCAC GTCCTGCCGG TCACTCTGCT CCAGGGCCTG CACCAGGAAG

CCCCACAGCC CCTGGCTGCC CAGCCTNGGC GCTCAGCCAG GAGAAGAGCA

TGTGACGGAT CTGCTCATCC AGATCATCCC GGAACTCGTG CCGGATGCGC TGCACCTCCC GGTAGACACC CCN (SEQ ID NO:3)

Two sets of PCR primers were designed to amplify 423bp and 187bp from human cDNA. An HL60 cDNA library was used as template. The first set was designed to cover the entire sequence ofthe clone N55392 to amplify 423bp. 5'primer: GGG GGT GTC CTA CCGGGA GTG CA (23mer) (SEQ ID NO:4)

3' primer: GAA AGA AAC AGT GCA GTT TTG TTG CTC (27 mer) (SEQ ID NO:5)

The second set was design to amplify 187bp, using the sequence of both clones identified in the search.

5' primer: CCG CCG CCA GTA CCA GGA CAG CAT (24 mer) (SEQ ID NO: 6) 3' primer: ccc ACA CAC TGG AGA GAC TTG AAG (24 mer) (SEQ ID NO:7)

The second set of primers generated a 187bp fragment with an HL60 library as template. This DNA fragment was used as a probe to screen an HL60 library. The HL60 cDNA library used for this first screening was cloned into the pMT vector for screening in bacteria (colony lifting method).

Two clones (clonel (SEQ ID NO:8) & clone2 (SEQ ID NO:9)) were isolated from this screen. The sequences are 2844 bp and 2195 bp respectively. Clone 2 contains two potential open reading frames of 642 and 655 amino acids depending of which ATG is considered. The region containing these two ATGs is not present in clone 1. The predicted proteins encoded by clone 2 contain a potential death domain at the C-terminus. A second library was screened using a random primed U937 cDNA library in lambda phage. The probe used for this screening was a 779bp fragment from clone 2 from the first screen. This fragment was at the 3 'end of clone 2 and was generated by AccI-EcoRI digestion.

Three positive clones were isolated. The sequence of clone32 thus isolated is reported as SEQ ID NO: 10. The sequence of these clones gave more information on the 5'end but didn't give the full length cDNA.

To clone the 5'end of the cDNA RACE PCR and primer extended methods were used. In both cases, a primer at the 5'end of clone 32 was used to extend the cDNA toward the 5'end. A human heart and human brain libraries were used for the RACE PCR and a human brain library was used for the primer extended library. Multiple clones were obtained by both techniques. The sequence of clone PE6 thus isolated is reported as SEQ ID NO: 11.

Compilation of the sequences for clone 2, clone 32 and clone PE6 give a complete cDNA of 3205bp for DADD (SEQ ID NO: 12). The predicted amino acid sequence encoded thereby is reported as SEQ ID NO: 13. There are two potential predicted open reading frames in the same frame, encoding 753 amino acids (for the most upstream ATG at amino acid 1 of SEQ ID NO: 13) and 586 amino acids starting at the downstream ATG (amino acid 167 of SEQ ID NO: 13). The predicted proteins have a death domain at the C-terminus of the protein (amino acids 645-712 of SEQ ID NO: 13).

Rabbit antibodies were raised against the death domain portion of DADD. The antigen was in the form of a GST-DD fusion protein or a MBP-DD fusion protein.

Western blot analysis showed a specific band around 55kDa in Cos cells with the serum

7008 as well as with 7006 to a lesser extent. Multiple human tissue analysis clearly showed a band around the same size (55kDa) in kidney and liver and to a lesser extent in heart. There was no detection of this protein in brain, lung and skeletal muscle.

Two expression vectors with the two potential open reading frames of 753 and

586 amino acids vere expressed with a Flag tag at the N-terminus in Cos cells. Western blot using the serum raised against the DADD death domain showed, in both cases, a band around 55kDa (as the endogenous protein) plus a band at the expected size of the full length protein around 83kDa and 65kDa respectively. On the other hand, a Western blot with the Flag antibody shows a band at the full length size in both cases plus a band at around 30kDa and 15kDa respectively.

These results show that the two proteins of 753aa and 586aa are cleaved at the same site giving a 55kDa C-terminal product which comigrate with the endogenous protein originally detected, and an N-terminal product around 30kDa and 15kDa respectively.

Co-immunoprecipitation experiments using the serum 7008 raised against the

DD of DADD shows that the 30kDa N-terminus portion of the 753aa protein is co- immunoprecipitated with the C-terminal part containing the DD. Preliminary experiments also show that DADD co-immunoprecipitate FADD and MADD (Chinnaiyan et al., Cell 81:505-512 (1995); Schierellaet al., J. Biol. Chem. 272: 12063-

12075 (1997)).

The DADD protein sequence contains five full and two half leucine rich repeats.

There are five tandems of 23 amino acid residues plus two half repeats of 12 and 9 residues. This leucine rich region is believed to be a protein-protein interaction domain.

The region between this Leu-rich domain and the death domain shows some homology with ankyrin proteins. The homology between DADD and ankyrins correspond to the spectrin domain of the ankyrins, which is know to be a protein-protein interaction domain. DADD was analysed in assays to investigate different pathways which are known to be activated by TNF (apoptosis, Jnk activation and NFkB activation). DADD activated apoptosis in the SEAP assay (secreted alkaline phosphatase).

The DADD cDNA was deposited with the American Type Culture Collection on April 1, 1998, as accession number ATCC XXXXX. The deposited cDNA encodes the protein of SEQ ID NO: 13 with an additional Flag tag as described above.

Polynucleotides hybridizing to the polynucleotides of the present invention under stringent conditions and highly stringent conditions are also part of the present invention. As used herein, "highly stringent conditions" include, for example, 0.2xSSC at 65°C; and "stringent conditions" include, for example, 4xSSC at 65°C or 50% formamide and 4xSSC at 42°C.

For the purposes ofthe present application, "DADD protein" includes proteins which exhibit DADD protein activity. For the purposes of the present application, a protein is defined as having "DADD protein activity" when it binds to a protein having a death domain, including without limitation the TNF-R death domain or the DADD death domain. Activity can be measured by using any assay which will detect binding to a death domain protein. Examples of such assays include without limitation the interaction trap assays and assays in which TNF-R death domain protein which is affixed to a surface in a manner conducive to observing binding. Fragments of the DADD protein which are capable of interacting with death domains or which are capable of inhibiting death domain binding (i.e., exhibit DADD protein activity) are also encompassed by the present invention. Fragments of the DADD protein may be in linear form or they may be cyclized using known methods, for example, as described in H.U. Saragovi, et al. , Bio/Technology JO, 773-778 (1992) and in R.S. McDowell, et al, J. Amer. Chem. Soc. U4, 9245-9253 (1992), both of which are incorporated herein by reference. Such fragments may be fused to carrier molecules such as immunoglobulins for many purposes, including increasing the valency of DADD protein binding sites. For example, fragments of the DADD protein may be fused through "linker" sequences to the Fc portion of an immunoglobulin. For a bivalent form of the DADD protein, such a fusion could be to the Fc portion of an IgG molecule. Other immunoglobulin isotypes may also be used to generate such fusions. For example, a DADD protein - IgM fusion would generate a decavalent form of the DADD protein of the invention. The isolated polynucleotide of the invention may be operably linked to an expression control sequence such as the pMT2 or pED expression vectors disclosed in Kaufman et al, Nucleic Acids Res. 19, 4485-4490 (1991), in order to produce the DADD protein recombinantly. Many suitable expression control sequences are known in the art. General methods of expressing recombinant proteins are also known and are exemplified in R. Kaufman, Methods in Enzymology 185. 537-566 (1990). As defined herein "operably linked" means that the isolated polynucleotide ofthe invention and the expression control sequence are situated within a vector or cell in such a way that the DADD protein is expressed by a host cell which has been transformed (transfected) with the ligated polynucleotide/expression control sequence. A number of types of cells may act as suitable host cells for expression of the

DADD protein. Host cells include, for example, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK or Jurkat cells. The DADD protein may also be produced by operably linking the isolated polynucleotide of the invention to suitable control sequences in one or more insect expression vectors, and employing an insect expression system. Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, e.g., Invitrogen, San Diego, California, U.S.A. (the MaxBac® kit), and such methods are well known in the art, as described in Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). incorporated herein by reference.

Alternatively, it may be possible to produce the DADD protein in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Potentially suitable yeast strains include Saccharomyces cerevisiae, Schizosaccharomycespom.be, Kluyveromyces strains, Candida, or any yeast strain capable of expressing heterologous proteins. Potentially suitable bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium, or any bacterial strain capable of expressing heterologous proteins. If the DADD protein is made in yeast or bacteria, it may be necessary to modify the protein produced therein, for example by phosphorylation or glycosylation ofthe appropriate sites, in order to obtain the functional DADD protein. Such covalent attachments may be accomplished using known chemical or enzymatic methods.

The DADD protein of the invention may also be expressed as a product of transgenic animals, e.g., as a component ofthe milk of transgenic cows, goats, pigs, or sheep which are characterized by somatic or germ cells containing a nucleotide sequence encoding the DADD protein.

The DADD protein ofthe invention may be prepared by culturing transformed host cells under culture conditions suitable to express the recombinant protein. The resulting expressed protein may then be purified from such culture (i.e., from culture medium or cell extracts) using known purification processes, such as gel filtration and ion exchange chromatography. The purification of the DADD protein may also include an affinity column containing the TNF-R death domain, the DADD death domain or other death domain protein; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography.

Alternatively, the DADD protein of the invention may also be expressed in a form which will facilitate purification. For example, it may be expressed as a fusion protein, such as those of maltose binding protein (MBP) or glutathione-S-transferase

(GST). Kits for expression and purification of such fusion proteins are commercially avaUable from New England BioLab (Beverly, MA) and Pharmacia (Piscataway, NJ), respectively. The TNF-R ligand protein can also be tagged with an epitope and subsequen y purified by using a specific antibody directed to such epitope. One such epitope ("Flag") is commercially available from Kodak (New Haven, CT).

Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the DADD protein. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous isolated recombinant protein.

The DADD protein thus purified is substantially free of other mammalian proteins and is defined in accordance with the present invention as an "isolated DADD protein."

DADD proteins may also be produced by known conventional chemical synthesis. Methods for constructing the proteins of the present invention by synthetic means are known to those skilled in the art. The synthetically-constructed protein sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with DADD proteins may possess biological properties in common therewith, including DADD protein activity. Thus, they may be employed as biologically active or immunological substitutes for natural, purified DADD proteins in screening of therapeutic compounds and in immunological processes for the development of antibodies.

The DADD proteins provided herein also include proteins characterized by amino acid sequences similar to those of purified DADD proteins but into which modification are naturally provided or deliberately engineered. For example, modifications in the peptide or DNA sequences can be made by those skilled in the art using known techniques. Modifications of interest in the DADD protein sequences may include the replacement, insertion or deletion of a selected amino acid residue in the coding sequence. For example, one or more ofthe cysteine residues may be deleted or replaced with another amino acid to alter the conformation of the molecule. Mutagenic techniques for such replacement, insertion or deletion are well known to those skilled in the art (see, e.g., U.S. Patent No. 4,518,584).

Other fragments and derivatives of the sequences of DADD proteins which would be expected to retain DADD protein activity in whole or in part and may thus be useful for screening or other immunological methodologies may also be easUy made by those skilled in the art given the disclosures herein. Such modifications are believed to be encompassed by the present invention.

DADD protein of the invention may also be used to screen for agents which are capable of inhibiting or blocking binding of a DADD protein to the death domain of TNF-R, DADD or other protein, and thus may act as inhibitors of death domain binding and/or the biological activity normally brought on by such binding (e.g., apoptosis). Binding assays using a desired binding protein, immobilized or not, are well known in the art and may be used for this purpose using the DADD protein of the invention. Appropriate screening assays may be cell-based or cell-free. Alternatively, purified protein based screening assays may be used to identify such agents. For example, DADD protein may be immobilized in purified form on a carrier and binding to purified death domain proteins may be measured in the presence and in the absence of potential inhibiting agents. A suitable binding assay may alternatively employ purified death domain protein immobilized on a carrier, with a soluble form of a DADD protein ofthe invention. Any DADD protein may be used in the screening assays described above.

In such a screening assay, a first binding mixture is formed by combining a death domain-containing protein and DADD protein, and the amount of binding in the first binding mixture (B₀) is measured. A second binding mixture is also formed by combining the death domain-containing protein, DADD protein, and the compound or agent to be screened, and the amount of binding in the second binding mixture (B) is measured. The amounts of binding in the first and second binding mixtures are compared, for example, by performing a B/B₀ calculation. A compound or agent is considered to be capable of inhibiting binding if a decrease in binding in the second binding mixture as compared to the first binding mixture is observed. The formulation and optimization of binding mixtures is within the level of skiU in the art. Such binding mixtures may also contain buffers and salts necessary to enhance or to optimize binding, and additional control assays may be included in the screening assay of the invention.

Alternatively, appropriate screening assays may be cell based. For example, the binding or interaction between a DADDprotein and death domain protein can be measured in yeast.

Compounds found to reduce, preferably by at least about 10%, more preferably greater than about 50% or more, the binding activity of DADD protein to a death domain may thus be identified and then secondarily screened in other binding assays, including in vivo assays. By these means compounds having inhibitory activity for DADD death domain binding which may be suitable as anti-inflammatory agents may be identified. Isolated DADD protein may be useful in treating, preventing or ameliorating inflammatory conditions and other conditions, such as cachexia, autoimmune disease, graft versus host reaction, osteoporosis, colitis, myelogenous leukemia, diabetes, wasting, and atherosclerosis. Isolated DADD protein may be used itself as an inhibitor of TNF-R death domain binding or to design inhibitors of TNF-R death domain binding. Inhibitors of binding of DADD protein to the TNF-R death domain ("TNF-R intracellular binding inhibitors") are also useful for treating such conditions.

The present invention encompasses both pharmaceutical compositions and therapeutic methods of treatment or use which employ isolated DADD protein and/or binding inhibitors of TNF-R intracellular binding. Isolated DADD protein or binding inhibitors (from whatever source derived, including without limitation from recombinant and non-recombinant cell lines) may be used in a pharmaceutical composition when combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to DADD protein or binding inhibitor and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration. The pharmaceutical composition of the invention may also contain cytokines, lymphokines, or other hematopoietic factors such as M-CSF, GM-CSF, TNF, IL-2, IL-3, IL-4, IL-5, E -6, IL-7, IL-8, IL-9, G-CSF, Meg- CSF, stem cell factor, and erythropoietin. The pharmaceutical composition may further contain other anti-inflammatory agents. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with isolated DADD protein or binding inhibitor, or to minimize side effects caused by the isolated DADD protein or binding inhibitor. Conversely, isolated DADD protein or binding inhibitor may be included in formulations of the particular cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent to minimize side effects of the cytokine, lymphokine, other hematopoietic factor, thrombolytic or anti-thrombotic factor, or anti-inflammatory agent.

The pharmaceutical composition of the invention may be in the form of a liposome in which isolated DADD protein or binding inhibitor is combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lameUar layers in aqueous solution. Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art, as disclosed, for example, in U.S. Patent No. 4,235,871; U.S. Patent No. 4,501,728; U.S. Patent No.4,837,028; and U.S. Patent No. 4,737,323, all of which are incorporated herein by reference.

As used herein, the term "therapeuticaUy effective amount" means the total amount of each active component of the pharmaceutical composition or method that is sufficient to show a meaningful patient benefit, i.e., treatment, healing, prevention or amelioration of an inflammatory response or condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

In practicing the method of treatment or use of the present invention, a therapeuticaUy effective amount of isolated DADD protein or binding inhibitor is administered to a mammal having a condition to be treated. Isolated DADD protein or binding inhibitor may be administered in accordance with the method of the invention either alone or in combination with other therapies such as treatments employing cytokines, lymphokines or other hematopoietic factors. When co-administered with one or more cytokines, lymphokines or other hematopoietic factors, isolated DADD protein or binding inhibitor may be administered either simultaneously with the cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors, or sequentially. If administered sequentially, the attending physician will decide on the appropriate sequence of administering isolated DADD protein or binding inhibitor in combination with cytokine(s), lymphokine(s), other hematopoietic factor(s), thrombolytic or anti-thrombotic factors.

Administration of isolated DADD protein or binding inhibitor used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as oral ingestion, inhalation, or cutaneous, subcutaneous, or intravenous injection. Intravenous administration to the patient is preferred.

When a therapeuticaUy effective amount of isolated DADD protein or binding inhibitor is administered orally, isolated DADD protein or binding inhibitor will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition ofthe invention may additionaUy contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% isolated DADD protein or binding inhibitor, and preferably from about 25 to 90% isolated DADD protein or binding inhibitor. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of isolated DADD protein or binding inhibitor, and preferably from about 1 to 50% isolated DADD protein or binding inhibitor. When a therapeuticaUy effective amount of isolated DADD protein or binding inhibitor is administered by intravenous, cutaneous or subcutaneous injection, isolated DADD protein or binding inhibitor will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to isolated DADD protein or binding inhibitor, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art. The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.

The amount of isolated DADD protein or binding inhibitor in the pharmaceutical composition ofthe present invention will depend upon the nature and severity of the condition being treated, and on the nature of prior treatments which the patient has undergone. Ultimately, the attending physician will decide the amount of isolated DADD protein or binding inhibitor with which to treat each individual patient. Initially, the attending physician will administer low doses of isolated DADD protein or binding inhibitor and observe the patient's response. Larger doses of isolated DADD protein or binding inhibitor may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further. It is contemplated that the various pharmaceutical compositions used to practice the method ofthe present invention should contain about 0.1 μg to about 100 mg of isolated DADD protein or binding inhibitor per kg body weight.

The duration of intravenous therapy using the pharmaceutical composition of the present invention will vary, depending on the severity of the disease being treated and the condition and potential idiosyncratic response of each individual patient. It is contemplated that the duration of each application of the isolated DADD protein or binding inhibitor will be in the range of 12 to 24 hours of continuous intravenous administration. Ultimately the attending physician will decide on the appropriate duration of intravenous therapy using the pharmaceutical composition of the present invention.

Isolated DADD protein ofthe invention may also be used to immunize animals to obtain polyclonal and monoclonal antibodies which specifically react with the DADD protein and which may inhibit TNF-R death domain binding. Such antibodies may be obtained using either the entire DADD protein or fragments of DADD protein as an immunogen. The peptide immunogens additionally may contain a cysteine residue at the carboxyl terminus, and are conjugated to a hapten such as keyhole limpet hemocyanin (KLH). Methods for synthesizing such peptides are known in the art, for example, as in R.P. Merrifield, J. Amer.Chem.Soc. 85, 2149-2154 (1963); J.L. Krstenansky, et al, FEBS Lett. 2H, 10 (1987). Monoclonal antibodies binding to DADD protein or to complex carbohydrate moieties characteristic of the DADD glycoprotein may be useful diagnostic agents for the immunodetection of TNF-R ligand protein.

Neutralizing monoclonal antibodies binding to DADD protein or to complex carbohydrates characteristic of DADD glycoprotein may also be useful therapeutics for both inflammatory conditions and also in the treatment of some forms of cancer where abnormal expression of DADD protein is involved. These neutralizing monoclonal antibodies are capable of blocking the signaling function of the DADD protein. By blocking the binding of DADD protein, certain biological responses to TNF are either abolished or markedly reduced. In the case of cancerous cells or leukemic cells, neutralizing monoclonal antibodies against DADD protein may be useful in detecting and preventing the metastatic spread ofthe cancerous cells, which may be mediated by the DADD protein.The present invention also provides genes corresponding to the polynucleotide sequences disclosed herein. "Corresponding genes" are the regions of the genome that are transcribed to produce the mRNAs from which cDNA polynucleotide sequences are derived and may include contiguous regions of the genome necessary for the regulated expression of such genes. Corresponding genes may therefore include but are not limited to coding sequences, 5' and 3' untranslated regions, alternatively spUced exons, introns, promoters, enhancers, and silencer or suppressor elements. The corresponding genes can be isolated in accordance with known methods using the sequence information disclosed herein. Such methods include the preparation of probes or primers from the disclosed sequence information for identification and/or amplification of genes in appropriate genomic libraries or other sources of genomic materials. An "isolated gene" is a gene that has been separated from the adjacent coding sequences, if any, present in the genome of the organism from which the gene was isolated.

Organisms that have enhanced, reduced, or modified expression of the gene(s) corresponding to the polynucleotide sequences disclosed herein are provided. The desired change in gene expression can be achieved through the use of antisense polynucleotides or ribozymes that bind and/or cleave the mRNA transcribed from the gene (Albert and Morris, 1994, Trends Pharmacol. Sci. 15(7): 250-254; Lavarosky et al., 1997, Biochem. Mol. Med. 62(1): 11-22; and Hampel, 1998, Prog. Nucleic Acid Res. Mol. Biol. 58: 1-39; aU of which are incorporated by reference herein). Transgenic animals that have multiple copies of the gene(s) corresponding to the polynucleotide sequences disclosed herein, preferably produced by transformation of cells with genetic constructs that are stably maintained within the transformed ceUs and their progeny, are provided. Transgenic animals that have modified genetic control regions that increase or reduce gene expression levels, or that change temporal or spatial patterns of gene expression, are also provided (see European Patent No. 0 649 464 Bl, incorporated by reference herein). In addition, organisms are provided in which the gene(s) corresponding to the polynucleotide sequences disclosed herein have been partiaUy or completely inactivated, through insertion of extraneous sequences into the corresponding gene(s) or through deletion of all or part of the corresponding gene(s).

Partial or complete gene inactivation can be accomplished through insertion, preferably foUowed by imprecise excision, of transposable elements (Plasterk, 1992, Bioessays 14(9): 629-633; Zwaal et al, 1993, Proc. Natl. Acad. Sci. USA 90(16): 7431-7435; Clark et al, 1994, Proc. Natl. Acad. Sci. USA 91(2): 719-722; all of which are incorporated by reference herein), or through homologous recombination, preferably detected by positive/negative genetic selection strategies (Mansour et al, 1988, Nature 336: 348-352; U.S. Patent Nos. 5,464,764; 5,487,992; 5,627,059; 5,631,153; 5,614, 396; 5,616,491; and 5,679,523; all of which are incorporated by reference herein). These organisms with altered gene expression are preferably eukaryotes and more preferably are mammals. Such organisms are useful for the development of non-human models for the study of disorders involving the corresponding gene(s), and for the development of assay systems for the identification of molecules that interact with the protein product(s) of the corresponding gene(s).

AU references cited herein are incorporated as if fullyset forht herein.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: GENETICS INSTITUTE, INC

(ii) TITLE OF INVENTION: DADD (Death Activator Death Domain Protein)

(iii) NUMBER OF SEQUENCES: 13

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Genetics Institute, Inc.

(B) STREET: 87 CambridgePark Drive

(C) CITY: Cambridge

(D) STATE: MA

(E) COUNTRY: USA

(F) ZIP: 02140

(V) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 09/056,383

(B) FILING DATE: 07 April 1998

(C) CLASSIFICATION:

(viii) ATTORNE /AGENT INFORMATION:

(A) NAME: Brown, Scott A.

(B) REGISTRATION NUMBER: 32,724

(C) REFERENCE/DOCKET NUMBER: GI5317

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617-498-8224

(B) TELEFAX: 617-876-5851

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 77 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS :

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:l:

lie Arg Glu Asn Leu Gly Lys His Trp Lys Asn Cys Ala Arg Lys Leu 1 5 10 15

Gly Phe Thr Gin Ser Gin lie Asp Glu lie Asp His Asp Tyr Glu Arg 20 25 30

Asp Gly Leu Lys Glu Lys Val Tyr Gin Met Leu Gin Lys Trp Val Met 35 40 45

Arg Glu Gly lie Lys Gly Ala Thr Val Gly Lys Leu Ala Gin Ala Leu 50 55 60

His Gin Cys Ser Arg He Asp Leu Leu Ser Ser Leu Thr 65 70 75

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 433 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

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

TGGACTGGCC AGCCGTGGCC AAAACCTGGG GGTGTCCTAC CGGGAGTGCA GCGATCCGGC 60

ACGAGTTCCG GGATGATCTG GATGAGCAGA TCCGTCACAT GCTCTTCTCC TGGGCTGAGC 120

GCCAGGCTGG GCAGCCAGGG NTGTNGGGGC TCCTGGTGCA GGCCCTGGAG CAGAGTGACC 180

GGCAGACCGT GGCTGAAGAG GTGCGCGCAG TCTTGGAGCT CGGCCGCCGC AAGTACCAGG 240

ACAGCATCCG ACGCATGGGC TTGGCCCCAA GGACCCCGCT CTGCCTGGCT CCTCGGGCTC 300 CACAGCCCCC AGGAGCCTGC CCCAGGCCTT AGGGCCCCAA CAGAACTTTT TAGGCTGGGC 360

CCAGAATATT CCCCAGGTGG AATGGGCAGA ACCCCAACCN TTCAAAGTCT CTCCAAGTGT 420

GTGGGGGACG NTT 433

(2) INFORMATION FOR SEQ ID Nθ:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 423 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

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

GAAAGAAACA GTGCAGTTTT GTTGCTCACA GGGACCCGTC CCCACACACT GGAGAGACTT 60

GAAGGTGGGG GCTCTGCATN CCACTGGGGA ATATCTGGGC CAGCCTAAAA GTCTGTGGGG 120

CCTAGGCTGG GCAGGCTCTG GGGGCTGTGG AGCCGAGGAG CCAGGCAGAG CGGGGTCCTT 180

GGGGGCCAAG CCATGCGTCG GATGCTGTCC TGGTACTTGC GGCGGCCGAG CTCCAAGACT 240

GCGCGCACCT CTTCAGCCAC GTCCTGCCGG TCACTCTGCT CCAGGGCCTG CACCAGGAAG 300

CCCCACAGCC CCTGGCTGCC CAGCCTNGGC GCTCAGCCAG GAGAAGAGCA TGTGACGGAT 360

CTGCTCATCC AGATCATCCC GGAACTCOTG CCGGATGCGC TGCACCTCCC GGTAGACACC 420

CCN 423

(2) INFORMATION FOR SEQ ID NO: 4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: :

GGGGGTGTCC TACCGGGAGT GCA 23

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 27 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

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

GAAAGAAACA GTGCAGTTTT GTTGCTC 27

(2) INFORMATION FOR SEQ ID NO: 6:

<i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

CCGCCGCCAG TACCAGGACA GCAT 24

(2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid (A) DESCRIPTION: /desc = "primer"

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:7:

CCCACACACT GGAGAGACTT GAAG 24

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2884 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

GAATTCCGTC GACTCTAGAG GGCTCTGGGG GCCCTCCCCG CCCTCACCTT CCTCATAGTG 60

ACACACAACC GCCTGCAGAC GCTGCCCCCA GCACTGGGGG CCCTATCCAC CCTGCAGCGC 120

CTCGATCTCT CTCAGAATCT GCTGGACACG CTACCTCCTG AGATTGGAGG CCTGGGCAGC 180

CTCCTGGAGC TCAACCTGGC CTCCCAACCG GCTGCAGAGC CTCCCAGCCT CTCTGGGTGA 240

GTAGCCCCTG CGCCCCGACA CACTGGCCCC ACGGGAGGGT CCCTGAAGCC TGCCTGTCTT 300

CTGCAGGGGC CTCTGCACCC ACAGGCTTGG TCCACAGCTG CCTCTTGGTT GTCCCTCCAC 360

CTCCCTGGCC TTTGAGACTC CCTCAGTGGC TTCGTCAGAG TTCTCTGAGC CCAGCTGTGG 420

AGGAGAGTCT GAAACAGCTG CTCTGGGAGG CGGCAGCAGG AGTGTCCCAG CGCCGTGGGC 480

TGGGCTGGTG CCAAGCCTAA GCCAGCACCT GCCCGCAGCG GGACTTCGGT CCTTGCGGCT 540 CCTTGTCCTG CACAGCAACC TCCTGGCCTC TGTGCCAGCT GACTTGGCCC GCCTTCCACT 600

CCTCACCCGG CTCGACCTGA GGGACAACCA GCTCCGGGAC CTGCCCCCTG AGCTGCTAGA 660

CGCCCCCTTT GTGCGCCTGC AGGGGAACCC CCTGGGTGAG GCCTCGCCAG ACGCCCCGAG 720

TTCACCAGTG GCAGCCCTCA TTCCAGAAAT GCCCAGACTG TTCCTGACCT CAGATTTGGA 780

CAGCTTTCCT GTGACCCCTC GAGGCTGCTC AGTGACCCTG GCCTGTGGCG TCCGCCTGCA 840

GTTCCCAGCG GGAGCCACCG CCACCCCCAT CACCATCCGC TATCGGCTGC TGCTGCCGGA 900

GCCAGGCCTC GTCCCCCTGG GTCCTCATGA CGCCCTGCTC AGCCATGTGC TGGAGCTGCA 960

GCCCCATGGG GTGGCCTTCC AGCAGGATGT GGGGCTGTGG CTGCTCTTCA CCCCACCGCA 1020

GGCCCGGCGC TGCCGTGAAG TGGTGGTCAG GACCCGGAAT GACAACAGCT GGGGTGACCT 1080

GGAGACCTAC CTGGAGGAAG AGGCACCCCA GCGGCTCTGG GCTCACTGCC AGGTGCCCCA 1140

CTTCTCCTGG TTCCTTGTGG TTTCCCGCCC TGTGTCCAAT GCCTGCCTGG TGCCACCGGA 1200

GGGGACACTG CTGTGCTCCT CGGGTCATCC TGGGGTCAAA GTCATCTTCC CCCCTGGGGC 1260

CACTGAGGAG CCTCGTCGAG TCTCCATGCA GGTGGTGCGC ATGGCTGGCC GAGAGCTGCA 1320

GGCCCTCCTG GGAGAACCAG AGGCTGCAGT GAGCCCCCTG CTGTGCCTGT CACAGAGCGG 1380

TCCCCCCAGC TTCCTCCAAC CGGTCACCGT GCAGCTGCCT CTGCCCTCTG GCATCACAGG 1440

CCTCAGTCTG GACCGCTCCC GCCTGCACCT GTTGTACTGG GCCCCTCCTG CAGCCACCTG 1500

GGATGACATC ACAGCTCAGG TGGTCCTGGA GCTCACCCAC CTGTACGCAC GCTTCCAGGT 1560

CACACACTTC TCCTGGTCAG TGCCCCCCAG CTTTCTCAGC CCCCCTCCCC CAGTCTGTAC 1620

AGCCCTCCTC ACCCCCAGCT CTCCCAGGTA CTGGCTCTGG TACACCACCA AGAACTGTGT 1680

GGGAGGCCTG GCTCGGAAGG CCTGGGAGCG GCTGCGGCTG CACCGTGTGA ACCTCATCGC 1740

TCTGCAGCGG CGCCGGGACC CTGAGCAGGT CCTGCTGCAG TGCCTGCCCC GAAACAAGGT 1800

GGACGCCACC CTTCGGCGGC TGCTGGAGCG GTACCGGGGC CCCGAGCCCT CTGACACGGT 1860

GGAGATGTTC GAGGGCGAAG AGTTCTTTGC GGCCTTCGAG CGCGGCATCG ACGTGGATGC 1920 TGACCGCCCT GACTGTGTGG AGGGCAGAAT CTGCTTTGTC TTCTACTCGC ACCTGAAGAA 1980

TGTGAAGGAG GTATACGTGA CCACCACTCT GGACCGGGAG GCTCAGGCTG TGCGGGGCCA 2040

GGTGTCCTTC TACCGTGGCG CGGTGCCTGT GCGGGTGCCC GAGGAGGCTG AGGCTGCCCG 2100

GCAGAGGAAG GGCGCAGACG CCCTGTGGAT GGCCACTCTG CCCATCAAGC TGCCGGTGGG 2160

ACTGAGGGAC AGCAGAGGGG CGGGGCAGGA CCGAGGCCCA GGGGTGACCA GGGTGACATG 2220

GTGGAGTTGG GGGTGGAGCC CAGGGCTTAA TGCACTTTTT CCTTCCAACA GAGACTTCGA 2280

GGGTCCGAGG GGCCACGGCG GGGGGCTGGC CTCTCCTTGG CACCCTTGAA TCTGGGAGAT 2340

GCCGAGACCG GCTTTCTGAC GCAGAGCAAC CTGCTGAGTG TGGCTGGGCG TCTGGGTCTG 2400

GACTGGCCAG CCGTGGCCCT GCACCTGGGG GTGTCCTACC GGGAGGTGCA GCGCATCCGG 2460

CACGAGTTCC GGGATGATCT GGATGAGCAG ATCCGTCACA TGCTCTTCTC CTGGGCTGAG 2520

CGCCAGGCTG GGCAGCCAGG GGCTGTGGGG CTCCTGGTGC AGGCCCTGGA GCAGAGTGAC 2580

CGGCAGGACG TGGCTGAAGA GGTGCGCGCA GTCTTGGAGC TCGGCCGCCG CAAGTACCAG 2640

GACAGCATCC GACGCATGGG CTTGGCCCCC AAGGACCCCG CTCTGCCTGG CTCCTCGGCT 2700

CCACAGCCCC CAGAGCCTGC CCAGGCCTAG GCCCCACAGA CTTTTAGGCT GGCCCAGATA 2760

TTCCCCAGTG GATGGGCAGA GCCCCCACCT TCAAGTCTCT CCAGTGTGTG GGGACGGGTC 2820

CCTGTGAGCA ACAAAACTGC ACTGTTTCTT TCAAAAAAAA AAAACTCTAG AGTCGACGGA 2880

ATTC 2884

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2209 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

TCTAGAGGTC CCCCTGGGTC CTCATGACGC CCTGCTCAGC CATGTGCTGG AGCTGCAGCC 60

CCATGGGGTG GCCTTCCAGC AGGCATGGAC AGGGCGTGGC GGGGGCAGTG TGTGGGCCAG 120

GGCATGGCAG GGGCAGTGTG CGGGCCCGGG CGTGGCTCCA GCGCTCACAC CATCCTTGTC 180

TGGCAGGATG TGGGGCTGTG GCTGCTCTTC ACCCCACCGC AGGCCCGGCG CTGCCGTGAA 240

GTGGTGGTCA GGACCCGGAA TGACAACAGC TGGGGTGACC TGGAGACCTA CCTGGAGGAA 300

GAGGCACCCC AGGTGAGGGC CACCCAGGCC TGCCGGTGGC GAGTGGAGAG CTGCTGCCCT 360

GAGCCGTGCA CCTCTGCCCA GAGCCTCACC CTGGCACCTT CCACCCTGCC CCGTCCCTCC 420

TGGATCCTGC TTCCCTATGT CCCTGGCACC TTCCACCCCA CCCCGTCCCT CCTGGATCCT 480

GCTTCCCTGT GTCCCTGGCA CCTTCCACCC CACCCCGTCC CTCCTGGATC CTGCTCCCTG 540

TGTCCCTGGC ACCTNTCCAC CCCGCCCCGT CTCTCCTGGA TNCTGCTCCC TGTGTCCCTG 600

ACTGGCTGTG CCCTGACCCA GGCTCCTGTG ACCTCCTCTC TCCCCCCATC CCAGCGGCTC 660

TGGGCTCACT GCCAGGTGCC CCACTTCTCC TGGTTCCTTG TGGTTTCCCG CCCTGTGTCC 720

AATGCCTGCC TGGTGCCACC GGAGGGGACA CTGCTGTGCT CCTCGGGTCA TCCTGGGGTC 780

AAAGTCATCT TCCCCCCTGG GGCCACTGAG GAGCCTCGTC GAGTCTCCAT GCAGGTGGTG 840

CGCATGGCTG GCCGAGAGCT GCAGGCCCTC CTGGGAGAAC CAGAGGCTGC AGTGAGCCCC 900

CTGCTGTGCC TGTCACAGAG CGGTCCCCCC AGCTTCCTCC AACCGGTCAC CGTGCAGCTG 960

CCTCTGCCCT CTGGCATCAC AGGCCTCAGT CTGGACCGCT CCCGCCTGCA CCTGTTGTAC 1020

TGGGCCCCTC CTGCAGCCAC CTGGGATGAC ATCACAGCTC AGGTGGTCCT GGAGCTCACC 1080

CACCTGTACT GGCTCTGGTA CACCACCAAG AACTGTGTGG GAGGCCTGGC TCGGAAGGCC 1140

TGGGAGCGGC TGCGGCTGCA CCGTGTGAAC CTCATCGCTC TGCAGCGGCG CCGGGACCCT 1200

GAGCAGGTCC TGCTGCAGTG CCTGCCCCGA AACAAGGTGG ACGCCACCCT TCGGCGGCTG 1260

CTGGAGCGGT ACCGGGGCCC CGAGCCCTCT GACACGGTGG AGATGTTCGA GGGCGAAGAG 1320 TTCTTTGCGG CCTTCGAGCG CGGCATCGAC GTGGATGCTG ACCGCCCTGA CTGTGTGGAG 1380

GGCAGAATCT GCTTTGTCTT CTACTCGCAC CTGAAGAATG TGAAGGAGGT ATACGTGACC 1440

ACCACTCTGG ACCGGGAGGC TCAGGCTGTG CGGGGCCAGG TGTCCTTCTA CCGTGGCGCG 1500

GTGCCTGTGC GGGTGCCCGA GGAGGCTGAG GCTGCCCGGC AGAGGAAGGG CGCAGACGCC 1560

CTGTGGATGG CCACTCTGCC CATCAAGCTG CCGAGACTTC GAGGGTCCGA GGGGCCACGG 1620

CGGGGGGCTG GCCTCTCCTT GGCACCCTTG AATCTGGGAG ATGCCGAGAC CGGCTTTCTG 1680

ACGCAGAGCA ACCTGCTGAG TGTGGCTGGG CGTCTGGGTC TGGACTGGCC AGCCGTGGCC 1740

CTGCACCTGG GGGTGTCCTA CCGGGAGGTG CAGCGCATCC GGCACGAGTT CCGGGATGAT 1800

CTGGATGAGC AGATCCGTCA CATGCTCTTC TCCTGGGCTG AGCGCCAGGC TGGGCAGCCA 1860

GGGGCTGTGG GGCTCCTGGT GCAGGCCCTG GAGCAGAGTG ACCGGCAGGA CGTGGCTGAA 1920

GAGGTGCGCG CAGTCTTGGA GCTCGGCCGC CGCAAGTACC AGGACAGCAT CCGACGNATG 1980

GGCTTGGCCC CCAAGGACCC CGTTCTGCCT GGCTCCTCGG CTCCACAGCC CCCAGAGCCT 2040

GCCCAGGCAT AGGCCCCACA GAATTTTAGG CTGGCCCAGA TATTCCCCAG TGGATGGGCA 2100

GAGCCCCCAC CTTCAAGTCT CTCCAGTGTG TGGGGACGGG TCCCTGTGAG CAACAAAACT 2160

GCACTGTTTC TTTCACCTCG AAAAAAAAAA AAAAAAAAAA AACTCTAGA 2209

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1903 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GGCCTGGGCA GCCTCCTGGA GCTCAACCTG GCCTCCAACC GGCTGCAGAG CCTCCCAGCC 60 TCTCTGGCGG GACTTCGGTC CTTGCGGCTC CTTGTCCTGC ACAGCAACCT CCTGGCCTCT 120

GTGCCAGCTG ACTTGGCCCG CCTTCCACTC CTCACCCGGC TCGACCTGAG GGACAACCAG 180

CTCCGGGACC TGCCCCCTGA GCTGCTAGAC GCCCCCTTTG TGCGCCTGCA GGGGAACCCC 240

CTGGGTGAGG CCTCGCCAGA CGCCCCGAGT TCACCAGTGG CAGCCCTCAT TCCAGAAATG 300

CCCAGACTGT TCCTGACCTC AGATTTGGAC AGCTTTCCTG TGACCCCTCG AGGCTGCTCA 360

GTGACCCTGG CCTGTGGCGT CCGCCTGCAG TTCCCAGCGG GAGCCACCGC CACCCCCATC 420

ACCATCCGCT ATCGGCTGCT GCTGCCGGAG CCAGGCCTCG TCCCCCTGGG TCCTCATGAC 480

GCCCTGCTCA GCCATGTGCT GGAGCTGCAG CCCCATGGGG TGGCCTTCCA GCAGGATGTG 540

GGGCTGTGGC TGCTCTTCAC CCCACCGCAG GCCCGGCGCT GCCGTGAAGT GGTGGTCAGG 600

ACCCGGAATG ACAACAGCTG GGGTGACCTG GAGACCTACC TGGAGGAAGA GGCACCCCAG 660

CGGCTCTGGG CTCACTGCCA GGTGCCCCAC TTCTCCTGGT TCCTTGTGGT TTCCCGCCCT 720

GTGTCCAATG CCTGCCTGGT GCCACCGGAG GGGACACTGC TGTGCTCCTC GGGTCATCCT 780

GGGGTCAAAG TCATCTTCCC CCCTGGGGCC ACTGAGGAGC CTCGTCGAGT CTCCATGCAG 840

GTGGTGCGCA TGGCTGGCCG AGAGCTGCAG GCCCTCCTGG GAGAACCAGA GGCTGCAGTG 900

AGCCCCCTGC TGTGCCTGTC ACAGAGCGGT CCCCCCAGCT TCCTCCAACC GGTCACCGTG 960

CAGCTGCCTC TGCCCTCTGG CATCACAGGC CTCAGTCTGG ACCGCTCCCG CCTGCACCTG 1020

TTGTACTGGG CCCCTCCTGC AGCCACCTGG GATGACATCA CAGCTCAGGT GGTCCTGGAG 1080

CTCACCCACC TGTACTGGCT CTGGTACACC ACCAAGAACT GTGTGGGAGG CCTGGCTCGG 1140

AAGGCCTGGG AGCGGCTGCG GCTGCACCGT GTGAACCTCA TCGCTCTGCA GCGGCGCCGG 1200

GACCCTGAGC AGGTCCTGCT GCAGTGCCTG CCCCGAAACA AGGTGGACGC CACCCTTCGG 1260

CGGCTGCTGG AGCGGTACCG GGGCCCCGAG CCCTCTGACA CGGTGGAGAT GTTCGAGGGC 1320

GAAGAGTTCT TTGCGGCCTT CGAGCGCGGC ATCGACGTGG ATGCTGACCG CCCTGACTGT 1380

GTGGAGGGCA GAATCTGCTT TGTCTTCTAC TCGCACCTGA AGAATGTGAA GGAGGTGTCC 1440 TTCTACCGTG GCGCGGTGCC TGTGCGGGTG CCCGAGGAGG CTGAGGCTGC CCGGCAGAGG 1500

AAGGGCGCAG ACGCCCTGTG GATGGCCACT CTGCCCATCA AGCTGCCGAG ACTTCGAGGG 1560

TCCGAGGGGC CACGGCGGGG GGCTGGCCTC TCCTTGGCAC CCTTGAATCT GGGAGATGCC 1620

GAGACCGGCT TTCTGACGCA GAGCAACCTG CTGAGTGTGG CTGGGCGTCT GGGTCTGGAC 1680

TGGCCAGCCG TGGCCCTGCA CCTGGGGGTG TCCTACCGGG AGGTGCAGCG CATCCGGCAC 1740

GAGTTCCGGG ATGATCTGGA TGAGCAGATC CGTCACATGC TCTTCTCCTG GGCTGAGCGC 1800

CAGGCTGGGC AGCCAGGGGC TGTGGGGCTC CTGGTGCAGG CCCTGGAGCA GAGTGACCGG 1860

CAGGACGTGG CTGAAGAGGT GCGCGCAGTC TTGGAGCTCG GCC 1903

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1030 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GTCGACTCTC CTGCGTAGCC ATGGCGGTCC CATTCCCCAA CCCTCTTTCC AGTGGTGACC 60

CAAGCCTCCG GGGTTCAGGG TGACCACNAT GTCTCCCCCA CTCTGCATCC CAGGCCCATA 120

TGGCCTGGCT CTANAGCTCC CCACTCCATC CANAGTCCCT GTTTCCCCAA AGAGAANGGC 180

CCACCCCGGC TCCCGCTCAC TCCTCCTCCT GCCTCTGCAT CTTCCCCGGG CGCTGCCTGG 240

ACAGGCCTGC CTGCGTGCTG GGACATGTCT GGCCTCCAAG GACCGTCGGT GGGCGATGGC 300

TGCAACGGTG GAGGGGCCAG AGCTGGAGGC AGCTGCTGCC GCAGGAGATG CTTCAGAGGA 360

TTCGGACGCA GGGTCCAGGG CGCTTCCTTT CCTGGGCGGC AACCGGCTGA GCTTGGACCT 420

GTACCCCGGG GGCTGCCAGC AGCTGCTGCA CCTGTGTGTC CAGCAGCCTC TTCAGCTGCT 480 GCAGGTGGAA TTCTTGCGTC TGAGCACTCA CGAGGACCCT CAGCTGCTGG AGGCCNCCCT 540

GGCCCAGCTG CCTCAGAGCC TGTCCTGCCT CCGCTCCGTG GTCCTCAAAG GGTCGATCTG 600

GGACCTCGGA CCCTGGCTCT GAGGGCCACA TCCGCCTCCC CCCTTCCCAG GAGGGCAACG 660

CCGGGACACA CTGGGTGCCT GTCTCCGGGG TGCCCTGACC AACCTGCCCG CTGGTCTGAG 720

TGGCCTGGCC CATCTGGCCC ACCTGGACCT GAGCTTCAAC AGCCTGGAGA CACTGCCGGC 780

CTGTGTCCTG CAGATGCGAG GTCTGGGTGC GCTCTTGCTG TCTCACAACT GCCTCTTTGA 840

GCTGCCTGAG GCTCTGGGGG CCCTCCCCGC CCTCACCTTC CTCATAGTGA CACACAACCG 900

CCTGCAGACG CTGCCCCCAG CACTGGGGGC CCTATCCACC CTGCAGCGCC TCGATCTCTC 960

TCAGAATCTG CTGGACACGC TACCTCCTGA GATTGGAGGC CTGGGCAGCC TCCTGGAGCT 1020

CAACCTGGCC 1030

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3205 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GTCGACTCTC CTGCGTAGCC ATGGCGGTCC CATTCCCCAA CCCTCTTTCC AGTGGTGACC 60

CAAGCCTCCG GGGTTCAGGG TGACCACNAT GTCTCCCCCA CTCTGCATCC CAGGCCCATA 120

TGGCCTGGCT CTANAGCTCC CCACTCCATC CANAGTCCCT GTTTCCCCAA AGAGAANGGC 180

CCACCCCGGC TCCCGCTCAC TCCTCCTCCT GCCTCTGCAT CTTCCCCGGG CGCTGCCTGG 240

ACAGGCCTGC CTGCGTGCTG GGACATGTCT GGCCTCCAAG GACCGTCGGT GGGCGATGGC 300

TGCAACGGTG GAGGGGCCAG AGCTGGAGGC AGCTGCTGCC GCAGGAGATG CTTCAGAGGA 360 TTCGGACGCA GGGTCCAGGG CGCTTCCTTT CCTGGGCGGC AACCGGCTGA GCTTGGACCT 420

GTACCCCGGG GGCTGCCAGC AGCTGCTGCA CCTGTGTGTC CAGCAGCCTC TTCAGCTGCT 480

GCAGGTGGAA TTCTTGCGTC TGAGCACTCA CGAGGACCCT CAGCTGCTGG AGGCCNCCCT 540

GGCCCAGCTG CCTCAGAGCC TGTCCTGCCT CCGCTCCGTG GTCCTCAAAG GGTCGATCTG 600

GGACCTCGGA CCCTGGCTCT GAGGGCCACA TCCGCCTCCC CCCTTCCCAG GAGGGCAACG 660

CCGGGACACA CTGGGTGCCT GTCTCCGGGG TGCCCTGACC AACCTGCCCG CTGGTCTGAG 720

TGGCCTGGCC CATCTGGCCC ACCTGGACCT GAGCTTCAAC AGCCTGGAGA CACTGCCGGC 780

CTGTGTCCTG CAGATGCGAG GTCTGGGTGC GCTCTTGCTG TCTCACAACT GCCTCTTTGA 840

GCTGCCTGAG GCTCTGGGGG CCCTCCCCGC CCTCACCTTC CTCATAGTGA CACACAACCG 900

CCTGCAGACG CTGCCCCCAG CACTGGGGGC CCTATCCACC CTGCAGCGCC TCGATCTCTC 960

TCAGAATCTG CTGGACACGC TACCTCCTGA GATTGGAGGC CTGGGCAGCC TCCTGGAGCT 1020

CAACCTGGCC TCCAACCGGC TGCAGAGCCT CCCAGCCTCT CTGGCGGGAC TTCGGTCCTT 1080

GCGGCTCCTT GTCCTGCACA GCAACCTCCT GGCCTCTGTG CCAGCTGACT TGGCCCGCCT 1140

TCCACTCCTC ACCCGGCTCG ACCTGAGGGA CAACCAGCTC CGGGACCTGC CCCCTGAGCT 1200

GCTAGACGCC CCCTTTGTGC GCCTGCAGGG GAACCCCCTG GGTGAGGCCT CGCCAGACGC 1260

CCCGAGTTCA CCAGTGGCAG CCCTCATTCC AGAAATGCCC AGACTGTTCC TGACCTCAGA 1320

TTTGGACAGC TTTCCTGTGA CCCCTCGAGG CTGCTCAGTG ACCCTGGCCT GTGGCGTCCG 1380

CCTGCAGTTC CCAGCGGGAG CCACCGCCAC CCCCATCACC ATCCGCTATC GGCTGCTGCT 1440

GCCGGAGCCA GGCCTCGTCC CCCTGGGTCC TCATGACGCC CTGCTCAGCC ATGTGCTGGA 1500

GCTGCAGCCC CATGGGGTGG CCTTCCAGCA GGATGTGGGG CTGTGGCTGC TCTTCACCCC 1560

ACCGCAGGCC CGGCGCTGCC GTGAAGTGGT GGTCAGGACC CGGAATGACA ACAGCTGGGG 1620

TGACCTGGAG ACCTACCTGG AGGAAGAGGC ACCCCAGCGG CTCTGGGCTC ACTGCCAGGT 1680

GCCCCACTTC TCCTGGTTCC TTGTGGTTTC CCGCCCTGTG TCCAATGCCT GCCTGGTGCC 1740 ACCGGAGGGG ACACTGCTGT GCTCCTCGGG TCATCCTGGG GTCAAAGTCA TCTTCCCCCC 1800

TGGGGCCACT GAGGAGCCTC GTCGAGTCTC CATGCAGGTG GTGCGCATGG CTGGCCGAGA 1860

GCTGCAGGCC CTCCTGGGAG AACCAGAGGC TGCAGTGAGC CCCCTGCTGT GCCTGTCACA 1920

GAGCGGTCCC CCCAGCTTCC TCCAACCGGT CACCGTGCAG CTGCCTCTGC CCTCTGGCAT 1980

CACAGGCCTC AGTCTGGACC GCTCCCGCCT GCACCTGTTG TACTGGGCCC CTCCTGCAGC 2040

CACCTGGGAT GACATCACAG CTCAGGTGGT CCTGGAGCTC ACCCACCTGT ACTGGCTCTG 2100

GTACACCACC AAGAACTGTG TGGGAGGCCT GGCTCGGAAG GCCTGGGAGC GGCTGCGGCT 2160

GCACCGTGTG AACCTCATCG CTCTGCAGCG GCGCCGGGAC CCTGAGCAGG TCCTGCTGCA 2220

GTGCCTGCCC CGAAACAAGG TGGACGCCAC CCTTCGGCGG CTGCTGGAGC GGTACCGGGG 2280

CCCCGAGCCC TCTGACACGG TGGAGATGTT CGAGGGCGAA GAGTTCTTTG CGGCCTTCGA 2340

GCGCGGCATC GACGTGGATG CTGACCGCCC TGACTGTGTG GAGGGCAGAA TCTGCTTTGT 2400

CTTCTACTCG CACCTGAAGA ATGTGAAGGA GGTATACGTG ACCACCACTC TGGACCGGGA 2460

GGCTCAGGCT GTGCGGGGCC AGGTGTCCTT CTACCGTGGC GCGGTGCCTG TGCGGGTGCC 2520

CGAGGAGGCT GAGGCTGCCC GGCAGAGGAA GGGCGCAGAC GCCCTGTGGA TGGCCACTCT 2580

GCCCATCAAG CTGCCGAGAC TTCGAGGGTC CGAGGGGCCA CGGCGGGGGG CTGGCCTCTC 2640

CTTGGCACCC TTGAATCTGG GAGATGCCGA GACCGGCTTT CTGACGCAGA GCAACCTGCT 2700

GAGTGTGGCT GGGCGTCTGG GTCTGGACTG GCCAGCCGTG GCCCTGCACC TGGGGGTGTC 2760

CTACCGGGAG GTGCAGCGCA TCCGGCACGA GTTCCGGGAT GATCTGGATG AGCAGATCCG 2820

TCACATGCTC TTCTCCTGGG CTGAGCGCCA GGCTGGGCAG CCAGGGGCTG TGGGGCTCCT 2880

GGTGCAGGCC CTGGAGCAGA GTGACCGGCA GGACGTGGCT GAAGAGGTGC GCGCAGTCTT 2940

GGAGCTCGGC CGCCGCAAGT ACCAGGACAG CATCCGACGN ATGGGCTTGG CCCCCAAGGA 3000

CCCCGTTCTG CCTGGCTCCT CGGCTCCACA GCCCCCAGAG CCTGCCCAGG CATAGGCCCC 3060

ACAGAATTTT AGGCTGGCCC AGATATTCCC CAGTGGATGG GCAGAGCCCC CACCTTCAAG 3120 TCTCTCCAGT GTGTGGGGAC GGGTCCCTGT GAGCAACAAA ACTGCACTGT TTCTTTCACC 3180

TCGAAAAAAA AAAAAAAAAA AAAAA 3205

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 753 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

Met Arg Gly Leu Gly Ala Leu Leu Leu Ser His Asn Cys Leu Phe Glu 1 5 10 15

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

Thr His Asn Arg Leu Gin Thr Leu Pro Pro Ala Leu Gly Ala Leu Ser 35 40 45

Thr Leu Gin Arg Leu Asp Leu Ser Gin Asn Leu Leu Asp Thr Leu Pro 50 55 60

Pro Glu He Gly Gly Leu Gly Ser Leu Leu Glu Leu Asn Leu Ala Ser 65 70 75 80

Asn Arg Leu Gin Ser Leu Pro Ala Ser Leu Ala Gly Leu Arg Ser Leu 85 90 95

Arg Leu Leu Val Leu His Ser Asn Leu Leu Ala Ser Val Pro Ala Asp 100 105 110

Leu Ala Arg Leu Pro Leu Leu Thr Arg Leu Asp Leu Arg Asp Asn Gin 115 120 125

Leu Arg Asp Leu Pro Pro Glu Leu Leu Asp Ala Pro Phe Val Arg Leu 130 135 140 Gin Gly Asn Pro Leu Gly Glu Ala Ser Pro Asp Ala Pro Ser Ser Pro 145 150 155 160

Val Ala Ala Leu He Pro Glu Met Pro Arg Leu Phe Leu Thr Ser Asp 165 170 175

Leu Asp Ser Phe Pro Val Thr Pro Arg Gly Cys Ser Val Thr Leu Ala 180 185 190

Cys Gly Val Arg Leu Gin Phe Pro Ala Gly Ala Thr Ala Thr Pro He 195 200 205

Thr He Arg Tyr Arg Leu Leu Leu Pro Glu Pro Gly Leu Val Pro Leu 210 215 220

Gly Pro His Asp Ala Leu Leu Ser His Val Leu Glu Leu Gin Pro His 225 230 235 240

Gly Val Ala Phe Gin Gin Asp Val Gly Leu Trp Leu Leu Phe Thr Pro 245 250 255

Pro Gin Ala Arg Arg Cys Arg Glu Val Val Val Arg Thr Arg Asn Asp 260 265 270

Asn Ser Trp Gly Asp Leu Glu Thr Tyr Leu Glu Glu Glu Ala Pro Gin 275 280 285

Arg Leu Trp Ala His Cys Gin Val Pro His Phe Ser Trp Phe Leu Val 290 295 300

Val Ser Arg Pro Val Ser Asn Ala Cys Leu Val Pro Pro Glu Gly Thr 305 310 315 320

Leu Leu Cys Ser Ser Gly His Pro Gly Val Lys Val He Phe Pro Pro 325 330 335

Gly Ala Thr Glu Glu Pro Arg Arg Val Ser Met Gin Val Val Arg Met 340 345 350

Ala Gly Arg Glu Leu Gin Ala Leu Leu Gly Glu Pro Glu Ala Ala Val 355 360 365

Ser Pro Leu Leu Cys Leu Ser Gin Ser Gly Pro Pro Ser Phe Leu Gin 370 375 380

Pro Val Thr Val Gin Leu Pro Leu Pro Ser Gly He Thr Gly Leu Ser 385 390 395 400

Leu Asp Arg Ser Arg Leu His Leu Leu Tyr Trp Ala Pro Pro Ala Ala 405 410 415

Thr Trp Asp Asp He Thr Ala Gin Val Val Leu Glu Leu Thr His Leu 420 425 430

Tyr Trp Leu Trp Tyr Thr Thr Lys Asn Cys Val Gly Gly Leu Ala Arg 435 440 445

Lys Ala Trp Glu Arg Leu Arg Leu His Arg Val Asn Leu He Ala Leu 450 455 460

Gin Arg Arg Arg Asp Pro Glu Gin Val Leu Leu Gin Cys Leu Pro Arg 465 470 475 480

Asn Lys Val Asp Ala Thr Leu Arg Arg Leu Leu Glu Arg Tyr Arg Gly 485 490 495

Pro Glu Pro Ser Asp Thr Val Glu Met Phe Glu Gly Glu Glu Phe Phe 500 505 510

Ala Ala Phe Glu Arg Gly He Asp Val Asp Ala Asp Arg Pro Asp Cys 515 520 525

Val Glu Gly Arg He Cys Phe Val Phe Tyr Ser His Leu Lys Asn Val 530 535 540

Lys Glu Val Tyr Val Thr Thr Thr Leu Asp Arg Glu Ala Gin Ala Val 545 550 555 560

Arg Gly Gin Val Ser Phe Tyr Arg Gly Ala Val Pro Val Arg Val Pro 565 570 575

Glu Glu Ala Glu Ala Ala Arg Gin Arg Lys Gly Ala Asp Ala Leu Trp 580 585 590

Met Ala Thr Leu Pro He Lys Leu Pro Arg Leu Arg Gly Ser Glu Gly 595 600 605

Pro Arg Arg Gly Ala Gly Leu Ser Leu Ala Pro Leu Asn Leu Gly Asp 610 615 620

Ala Glu Thr Gly Phe Leu Thr Gin Ser Asn Leu Leu Ser Val Ala Gly 625 630 635 640 Arg Leu Gly Leu Asp Trp Pro Ala Val Ala Leu His Leu Gly Val Ser 645 650 655

Tyr Arg Glu Val Gin Arg He Arg His Glu Phe Arg Asp Asp Leu Asp 660 665 670

Glu Gin He Arg His Met Leu Phe Ser Trp Ala Glu Arg Gin Ala Gly 675 680 685

Gin Pro Gly Ala Val Gly Leu Leu Val Gin Ala Leu Glu Gin Ser Asp 690 695 700

Arg Gin Asp Val Ala Glu Glu Val Arg Ala Val Leu Glu Leu Gly Arg 705 710 715 720

Arg Lys Tyr Gin Asp Ser He Arg Arg Met Gly Leu Ala Pro Lys Asp 725 730 735

Pro Val Leu Pro Gly Ser Ser Ala Pro Gin Pro Pro Glu Pro Ala Gin 740 745 750

Ala

Claims

CLAIMSWhat is claimed is:

1. An isolated polynucleotide encoding a protein having DADD protein activity selected from the group consisting of:

(a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12;

(b) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12 from nucleotide 794 to nucleotide 3052;

(d) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12 from nucleotide 2726 to nucleotide 2929;

(j) a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a)-(i).

2. A composition of claim 1 wherein said polynucleotide is operably Unked o an expression control sequence.

3. A host cell transformed with a composition of claim 2.

4. The host cell of claim 3, wherein said cell is a mammalian cell.

5. A process for producing a DADD protein, which comprises:

(a) growing a culture of the host cell of claim 4 in a suitable culture medium; and

(b) purifying the DADD protein from the culture.

6. A composition comprising a protein having DADD protein activity which comprises an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO: 13;

(d) fragments of the amino acid sequence of SEQ ID NO: 13; said protein being substantially free from other mammalian proteins.

7. The composition of claim 6, further comprising a pharmaceutically acceptable carrier.

8. A composition comprising an antibody which specifically reacts with the DADD protein of claim 6.

9. A method of identifying an inhibitor of binding of a DADD protein to a second protein having a death domain which comprise:

(c) combining a compound with the DADD protein and the second protein to form a second binding mixture; (d) measuring the amount of binding between the DADD protein and the second protein in the second binding mixture; and

(e) comparing the amount of binding in the first binding mixture with the amount of binding in the second binding mixture; wherein the compound is capable of inhibiting binding when a decrease in the amount of binding occurs in the second mixture as compared to the first mixture.

10. The method of claim 9 wherein said second protein is a protein comprising the death domain of TNF-R.

11. The method of claim 9 wherein said second protein is a protein comprising an amino acid sequence selected from the group consisting of:

(a) the amino acid sequence of SEQ ID NO: 13;

(d) fragments of the amino acid sequence of SEQ ED NO: 13.

12. A method of preventing or ameliorating an inflammatory condition which comprises administering a therapeuticaUy effective amount of a composition of claim 7.

13. DADD protein produced according to the method of claim 5.

14. A method of inhibiting DADD death domain binding comprising administering a therapeuticaUy effective amount of a composition of claim 7.

15. A composition comprising an inhibitor identified according to the method of claim 9.

16. The composition of claim 15 further comprising a pharmaceutically acceptable carrier.

17. A method of preventing or ameliorating an inflammatory condition comprising administering to a mammalian subject a therapeuticaUy effective amount of the composition of claim 6.

18. A method of inhibiting DADD death domain binding comprising administering to a mammalian subject a therapeuticaUy effective amount of the composition of claim 16.

19. A method of identifying an inhibitor of DADD death domain binding are also provided by the present invention which comprise:

(a) transforming a cell with a first polynucleotide encoding a DADD protein, a second polynucleotide encoding a second protein having a death domain, and at least one reporter gene, wherein the expression of the reporter gene is regulated by the binding of the DADD protein encoded by the first polynucleotide to the second protein encoded by the second polynucleotide;

(b) growing the cell in the presence of and in the absence of a compound; and

(c) comparing the degree of expression of the reporter gene in the presence of and in the absence of the compound; wherein the compound is capable of inhibiting DADD death domain binding when a decrease in the degree of expression of the reporter gene occurs.

20. The method of claim 19 wherein the second protein is selected from the group consisting of a DADD protein or a protein containing the TNF-R death domain.

21. The method of claim 20 wherein the first polynucleotide is selected from the group consisting of: (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 12;

22. The method of claim 19 wherein the cell is a yeast cell.