WO1994008037A1 - Human homologs of the transducin-like enhancer of split gene and methods based thereon - Google Patents

Abstract

The present invention relates to nucleotide sequences of the human transducin-like Enhancer of split ('TLE') genes, and amino acid sequences of their encoded TLE proteins. The invention further relates to fragments and other derivatives, and analogs, of human TLE proteins, as well as antibodies thereto. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is provided. Binding partners of TLE proteins are also provided. In particular, the invention provides sequences of four distinct human homologs of the Drosophila TLE gene, and sequences of their unique encoded TLE proteins. In specific embodiments, the invention relates to derivatives and analogs of the human TLE proteins which are functionally active, or which comprise one or more domains of a human TLE protein, including but not limited to the 'Q domain', 'GP domain', 'CcN domain', 'SP domain', 'WD-40 domain', or a WD-40 repeat, casein kinase II (CK II) site, cdc2 kinase (cdc2) site, or nuclear localization sequence motif, consensus sequences for any of the foregoing, or any combination of the foregoing.

Description

HUMAN HOMOLOGS OF THE TRANSDUCIN-LIKE ENHANCER OF SPLIT GENE AND METHODS BASED THEREON

This application is a continuation-in-part of copending application Serial No. 07/955,011 filed September 30, 1992, which is incorporated by reference herein in its entirety.

This invention was made with government support under grant number NS26084 awarded by The National Institutes of Health. The government has certain rights in the invention.

1. INTRODUCTION The present invention relates to human transducin-like Enhancer of split (TLE) genes and their encoded protein products. The invention also relates to derivatives and analogs of the human TLE proteins. Production of human TLE proteins, derivatives and antibodies is also provided.

2. BACKGROUND OF THE INVENTION In Drosophila melanogaster, the so called "Notch group" of genes has been implicated in events crucial for the correct developmental choices of a ^w'de variety of precursor cells (for review, see Artavanis-Tsakonas and Simpson, 1991, Trends Genet. 7:403-408). The accumulated genetic and molecular studies suggest that these genes encode elements of a cell communication mechanism which includes cell surface, cytoplasmic, and nuclear components.

The central player of the Notch group is the Notch (N) locus which encodes a transmembrane protein containing EGF-like repeats in its extracellular domain (Wharton et al., 1985, Cell 43:567-581 ; Kidd et al., 1986, Mol. Cell. Biol. 6:3094-3108). This protein has been shown to interact molecularly and genetically with two other transmembrane, EGF-containing proteins of the Notch group: Serrate and Delta (Vaessin et al., 1985, J. Neurogenetics 2:291-308; Fehon et al., 1990, Cell 61:523-534; Fleming et al.. 1990, Genes Dev. 4:2188- 2201; Xu et al., 1990, Genes Dev. 4:464-475; Rebay et al., 1991, Cell 67:687- 699; Thomas et al., 1991, Development 111:749-761). The other members of the Notch group are deltex (Xu and Artavanis-Tsakonas, 1991, Genetics 126:665- 677), Enhancer of (split) \E(spl)] (Knust et al., 1987, EMBO J. 6:4113-4123; Hartley et al., 1988, Cell 55:785-795; Preiss et al., 1988, EMBO J. 7:3917-3927; Klambt et al., 1989, EMBO J. 8:203-210), and mastermind (mam) (Smoller et al., 1990, Genes Dev. 4:1688-1700). mastermind and Enhancer of (split) encode nuclear proteins (Smoller et al., 1990, Genes Dev. 4:1688-1700; Delidakis et al., 1991, Genetics 129:803-823).

Notch homologs have been isolated from a variety of vertebrate species and have been shown to be remarkably similar to their Drosophila counterpart in terms of structure, expression pattern and ligand binding properties (Rebay et al., 1991, Cell 67:687-699; Coffman et al., 1990, Science 249:1438- 1441; Ellisen et al, 1991, Cell 66:649-661; Weinmaster et al., 1991, Development 113:199-205). A human Notch (TAN-1) malfunction has been associated with a lymphatic cancer (Ellisen et al, 1991, Cell 66:649-661). E(spl) is a complex locus comprised of at least ten genetically related transcription units which have been separated into two distinct groups, both of which display genetic interactions with specific Notch mutations (Knust et al., 1987, EMBO J. 6:4113-4123; Hartley et al., 1988, Cell 55:785-795; Preiss et al., 1988, EMBO J. 7:3917-3927; Klambt et al., 1989, EMBO J. 8:203-210; Delidakis et al., 1991, Genetics 129:803-823). The first group codes for proteins containing the helix-loop-helix motif (Klambt et al., 1989, EMBO J. 8:203-210) while the second displays homology to the β subunit of transducin (Hartley et al., 1988, Cell 55:785-795). Knust et al. (1987, EMBO J. 6:4113-4123) have numbered the transcripts in the E(spl) region and, according to their numbering system, the transcripts coding for the transducin-homologous protein are termed m9/10. Several embryonic lethal alleles affecting this gene were isolated. Moreover, P element transformation analyses demonstrated that the mutation groucho, which affects bristle development in Drosophila, is allelic to the Enhancer of split m9/10 gene (Hartley et al., 1988, Cell 55:785-795; Preiss et al., 1988, EMBO J. 7:3917-3927). The 719 amino acid long product of the E(spl) m.9110 gene contains four tandemly arranged repeats spanning the carboxyl-terminal ~ 300 amino acid residues of the protein (Hartley et al., 1988, Cell 55:785-795). Each repeat is approximately 40 residues in length and is characterized by the presence of the conserved motif WDL. Such repeats are found similarly arranged in the β subunits of G proteins and have been referred to as the "WD-40 repeat" (for review, see Simon et al., 1991, Science 252:802-808). Several other proteins containing this structural motif include the products of the yeast cell cycle gene CDC 4 (Yochem and Byers, 1987, J. Mol. Biol. 195:233-245) and of the TUP1 gene, a mediator of glucose repression (Williams and Trumbly, 1990, Mol. Cell. Biol. 10:6500-6511.).

Very little is known about the mechanisms underlying cell fate choices in higher organisms such as vertebrates; a knowledge of such mechanisms could provide insights into pathologies associated with abnormal differentiation events. Thus, a need exists in the art to obtain and characterize the human members of the "Notch group" of genes, including Enhancer of split, since these genes appear to play crucial roles in the determination of cell fate.

Citation of a reference hereinabove shall not be construed as an admission that such reference is prior art to the present invention.

3. SUMMARY OF THE INVENTION The present invention relates to nucleotide sequences of the human transducin-like Enhancer of split ("TLE") genes, and amino acid sequences of their encoded TLE proteins. The invention further relates to fragments and other derivatives, and analogs, of human TLE proteins, as well as antibodies thereto. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is provided. Binding partners of TLE proteins, and multiprotein complexes containing TLE proteins are also provided. in particular, the invention provides sequences of four distinct human homologs of the Drosophila TLE gene, and sequences of their unique encoded TLE proteins. As described by way of example in Section 6, by use of sequence comparisons, we identified structural motifs which provided important insights into the function of these proteins. For example, the TLE proteins and their Drosophila homolog contain a motif implicated in nuclear/cytoplasmic protein transport, called the casein kinase II site/cdc2 kinase site/nuclear localization sequence motif (CcN motif). We show that the TLE proteins are found in the nucleus, consistent with a function as nuclear effector molecules, and that the TLE genes are broadly expressed in adult tissues, suggesting a widespread physiological role for their encoded proteins. In specific embodiments, the invention relates to human TLE protein derivatives and analogs of the invention which are functionally active, or which comprise one or more domains of a human TLE protein, including but not limited to the "Q domain," "GP domain," "CcN domain," "SP domain," "WD-40 domain," or a WD-40 repeat, casein kinase II (CK II) site, cdc2 kinase (cdc2) site, or nuclear localization sequence motif, or consensus sequences for any of the foregoing, or any combination of the foregoing.

4. DESCRIPTION OF THE FIGURES Figure 1. Nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO: 2) of TLE 1.

Figure 2. Nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO:4) of TLE 2.

Figure 3. Nucleotide sequence (SEQ ID NO: 5) and deduced amino acid sequence (SEQ ID NO:6) of TLE 3. Figure 4. Partial nucleotide sequence (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO: 8) of TLE 4.

Figure 5. Comparison of the amino acid sequence of Drosophila E(spl) m9/10 (SEQ ID NO: 10) and human TLE proteins. Amino acids are numbered on the left side. Identical residues in all compared sequences are boxed, while residues identical in either three out of four or four out of five sequences are indicated in boldface type. Alignments maximize continuity between all sequences. Underlined amino acid residues correspond to the CcN motif.

Figure 6. Comparison of the WD-40 domains of Drosophila E(spl) m9/10 and TLE proteins. Amino acids are numbered on the left side. Those residues that are identical in each of the five sequences are boxed, while residues identical in four out of five sequences are indicated in boldface type. Those amino acids that are present at a given position in at least 10 out of 20 repeats define the consensus residues (SEQ ID NO:9) indicated at the bottom of the figure. Figure 7. Expression of TLE mRNAs. Human poly (A)⁺ RNA

("MTN Blot", catalog #7760-1; 2 μg/lane) was obtained from Clontech. Northern blotting experiments were performed at 42° C in a buffer containing 50% formamide, 5X SSPE, 5X Denhardt's solution, 0.5% SDS, and 100 μg/ml of salmon sperm DNA. After hybridization for 16 hr in the presence of [³²P]-labeled probes, blots were washed in IX SCC, 0.1 % SDS once at room temperature and 3 times at 68°C, followed by three washes at 68°C in 0.2X SSC, 0.1 % SDS. Individual probes corresponded to the following amino acid regions: TLE 1 (a), residues 260 through 435; TLE 2 (b), 32 through 342; TLE 3 (c), 350 through 440; TLE 4 (d), the region corresponding to that covered by the TLE 3 probe. RNA size markers (in kb) are indicated at the left of each autoradiogram. The arrows on the right of each panel indicate the sizes of the major TLE-specific transcripts.

Figure 8. Immunocytochemical characterization of TLE proteins, (a) Western blotting analysis of TLE proteins. Protein extracts from human thymus (lane 1; 250 μg of protein/lane), spleen (lane 2; 250 μg of protein/lane), lung (lane 3; 200 μg of protein/lane), heart (lane 4; 180 μg of protein/lane), kidney (lane 5; 200 μg of protein/lane), SUP-T1 cells (Ellisen et al., 1991, Cell 66:649-661) (lane 6; 180 μg of protein/lane), and HeLa cells (lane 7; 150 μg of protein/lane) were prepared and subjected to SDS-polyacrylamide gel electrophoresis (PAGE) on a 6% gel as described in Section 6.3. Western blotting was performed in the presence of a 1: 10 dilution of the rat monoclonal antibody C597.4A. Bound antibodies were detected by incubation with goat anti- rat IgG conjugated to horseradish peroxidase (1:1000). Molecular size standards are also indicated, (b) Intracellular localization of TLE proteins. HeLa cells were grown on chamber slides, fixed with paraformaldehyde, and stained with monoclonal antibody C597.4A.

Figure 9. Western blot visualization of multiprotein complexes containing TLE proteins after non-denaturing polyacrylamide gel electrophoresis (PAGE). A high speed supernatant fraction from human HeLa cell lysates was subjected to non-denaturing PAGE, proteins were transferred to nitrocellulose filters and probed in a Western blotting procedure with monoclonal antibody C597.4A, which binds to all TLE proteins. Two major immunoreactive species were detected, with apparent molecular weights of greater than 670,000 daltons.

Figure 10. Gel filtration chromatography of multiprotein complexes containing TLE proteins. High speed supernatant fractions from HeLa cells were subjected to gel filtration chromatography using a Sephacryl S-300 matrix. The fractions collected from the column were analyzed for the presence of TLE proteins in Western blotting experiments with monoclonal antibody C597.4A. Panel A shows the results after SDS-PAGE under reducing conditions; Panel B shows the results after SDS-PAGE under nonreducing conditions. Positions of elution of Dextran Blue (D.B.) and of molecular weight standards of 116 kD and of 80 kD are shown at the top of Panel A.

Figure 11. Western blots of cross-linked protein complexes containing TLE proteins. Protein extracts from Drosophila embryos (lanes 1-3), HeLa ceils (lanes 4-6) or SUP-T1 cells (lanes 7-9) were incubated in the presence of increasing concentrations of the chemical cross-linker, DTSSP. Concentrations used of DTSSP were as follows (in mM): Lanes 1, 4, and 7: 0; Lane 2: 0.2; Lane 3: 0.5; Lane 5: 0.06; Lane 6: 0.18; Lane 8: 0.06; Lane 9: 0.18. The products of the cross-linking reactions were subjected to SDS-PAGE under non- reducing conditions, followed by transfer to nitrocellulose membranes, and Western blotting with either monoclonal antibody 3C, directed against Enhancer of split m9/10 (lanes 1-3), or monoclonal antibody C597.4A, directed against the TLE proteins (lanes 4-9). The positions of migration of molecular size markers are shown at right.

5. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to nucleotide sequences of the human transducin-like Enhancer of split [E(spl)] ("TLE") genes, and amino acid sequences of their encoded TLE proteins. As used herein, the term "TLE" with reference to genes or proteins, shall refer to the transducin-Hke E(spl)- homologous genes or their encoded proteins, as the case may be, without reference to any species. The invention further relates to fragments and other derivatives, and analogs, of human TLE proteins. Nucleic acids encoding such fragments or derivatives are also within the scope of the invention. Production of the foregoing proteins and derivatives, e.g., by recombinant methods, is provided. Binding partners of TLE proteins, and multiprotein complexes containing TLE proteins are also provided.

In particular, the invention provides sequences of four distinct human homologs of the Drosophila TLE gene, and sequences of their unique encoded TLE proteins. As described by way of example in Section 6, by use of sequence comparisons, we identified structural motifs which provided important insights into the ftinction of these proteins. For example, the TLE proteins and their Drosophila homolog contain a motif implicated in nuclear/cytoplasmic protein transport, called the casein kinase II site/cdc2 kinase site/nuclear localization sequence motif (CcN motif). We show that the TLE proteins are found in the nucleus, consistent with a function as nuclear effector molecules, and that the TLE genes are broadly expressed in adult tissues, suggesting a widespread physiological role for their encoded proteins.

The invention also relates to human TLE protein derivatives and analogs of the invention which are functionally active, i.e. , they are capable of displaying one or more known functional activities associated with a full-length (wild-type) TLE protein. Such functional activities include but are not limited to antigenicity [ability to bind (or compete with a TLE protein for binding) to an anti-TLE protein antibody], immunogenicity (ability to generate antibody which binds to a TLE protein), ability to bind (or compete with a TLE protein for binding) possibly to Notch or other toporythmic proteins or fragments thereof, ability to bind (or compete with a TLE protein for binding) to a receptor or ligand for a TLE protein. "Toporythmic proteins" as used herein, refers to the protein products of Notch, Delta, Serrate, Enhancer of split, and deltex, as well as other members of this interacting gene family which may be identified, e.g. , by virtue of the ability of their gene sequences to hybridize, or their homology to Delta, E(spl), Serrate, or Notch, or the ability of their genes to display phenotypic interactions.

The invention further relates to fragments (and derivatives and analogs thereof) of a human TLE protein which comprise one or more domains of a human TLE protein (see Section 6), including but not limited to the "Q domain," "GP domain," "CcN domain," "SP domain," "WD-40 domain," or a WD-40 repeat, casein kinase II (CK II) site, cdc2 kinase (cdc2) site, or nuclear localization sequence motif, or consensus sequences for any of the foregoing, or any combination of the foregoing.

Antibodies to TLE proteins, their derivatives and analogs, are additionally provided. E(spl) plays a critical role in development and other physiological processes. The nucleic acid and amino acid sequences and antibodies thereto of the invention can be used for the detection and quantitation of human TLE mRNA, to study expression thereof, to produce human TLE proteins, fragments and other derivatives, and analogs thereof, in the study and manipulation of differentiation and other physiological processes, and may be of therapeutic or diagnostic use, e.g. , for neoplastic and pre-neoplastic conditions such as the detection of cervical squamous metaplasias, dysplasias, and malignancies.

The invention is illustrated by way of examples infra which disclose, inter alia, the cloning and sequencing of four human homologs of D. melanogaster E(spl); the construction and recombinant expression of human TLE chimeric/fusion derivatives and production of antibodies thereto, and multiprotein complexes containing TLE proteins, and an about 17 kD component of such complexes.

For clarity of disclosure, and not by way of limitation, the detailed description of the invention will be divided into the following subsections: (i) Isolation of the Human Transducin-Like Enhancer of Split

(TLE) Genes; (ii) Expression of the Human TLE Genes; (iii) Identification and Purification of the Expressed Gene Products; (iv) Structure of the Human TLE Genes and Proteins;

(v) Generation of Antibodies to Human TLE Proteins and

Derivatives Thereof; (vi) Human TLE Protein Derivatives and Analogs; (vii) Assays of TLE Proteins, Derivatives, and Analogs; (viii) Proteins Which Form a Complex With TLE Proteins.

5.1. ISOLATION OF THE HUMAN TRANSDUCIN-LIKE ENHANCER OF SPLIT (TLE) GENES

The invention relates to the nucleotide sequences of human TLE nucleic acids. In specific embodiments, human TLE nucleic acids comprise the

TLE 1, TLE 2, TLE 3, or TLE 4 cDNAs. The invention provides nucleic acids consisting of at least 8 nucleotides (i.e. , a hybridizable portion) of a human TLE sequence; in other embodiments, the nucleic acids consist of at least 50 nucleotides, 100 nucleotides, 150 nucleotides, or 200 nucleotides of a human TLE sequence. In a preferred, but not limiting, aspect of the invention, a human TLE

DNA can be cloned and sequenced by the method described in Section 6, infra.

The invention also relates to nucleic acids hybridizable to or complementary to the foregoing sequences. In specific aspects, nucleic acids are provided which comprise a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a human TLE gene.

Nucleic acids encoding fragments and derivatives of human TLE proteins (see Section 5.6) are additionally provided. A preferred embodiment for the cloning of a human TLE gene, presented as a particular example but not by way of limitation, follows:

A human expression library is obtained or is constructed by methods known in the art. For example, human mRNA is isolated, cDNA is made and ligated into an expression vector (e.g. , a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced. Various screening assays can then be used to select for the expressed human TLE product. In one embodiment, anti-TLE protein antibodies can be used for selection. In another preferred aspect, PCR is used to amplify the desired sequence in the, library, prior to selection. Oligonucleotide primers representing known TLE protein sequences can be used as primers in PCR. In a preferred aspect, the oligonucleotide primers encode at least part of the conserved segments of strong homology between Drosophila and human TLE proteins (e.g. , in the Q domain, CcN domain, or WD-40 domain). The synthetic oligonucleotides may be utilized as primers to amplify by PCR sequences from a source (RNA or DNA), preferably a cDNA library, of potential interest. PCR can be carried out, e.g. , by use of a Perkin-Elmer Cetus thermal cycler and Taq polymerase (Gene Amp"). The DNA being amplified can include human mRNA or cDNA or genomic DNA. One can choose to synthesize several different degenerate primers, for use in the PCR reactions. It is also possible to vary the stringency of hybridization conditions used in priming the PCR reactions, to allow for greater or lesser degrees of nucleotide sequence similarity between the known TLE nucleotide sequence and the nucleic acid homolog being isolated. After successful amplification of a segment of a TLE gene homolog, that segment may be molecularly cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, will permit the determination of the gene's complete nucleotide sequence, the analysis of its expression, and the production of its protein product for functional analysis, as described infra. In this fashion, it is also possible that additional human genes encoding TLE proteins may be identified. The above-methods are not meant to limit the following general description of methods by which clones of human TLE genes may be obtained.

Any human cell potentially can serve as the nucleic acid source for the molecular cloning of the TLE gene. The DNA may be obtained by standard procedures known in the art from cloned DNA (e.g. , a DNA "library"), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell. (See, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II.) Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will lack introns and will contain only exon sequences. Whatever the source, the gene should be moleculariy cloned into a suitable vector for propagation of the gene. In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and poly acrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired gene may be accomplished in a number of ways. For example, if an amount of a portion of a TLE (of any species) gene or its specific RNA, or a fragment thereof, e.g. , a Q or WD-40 domain (see Section 5.6), is available and can be purified, or synthesized, and labeled, the generated DNA fragments may be screened by nucleic acid hybridization to the labeled probe (Benton and Davis, 1977, Science 196: 180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion(s) and comparison of fragment sizes with those expected according to a known restriction map, either available or deduced from a known nucleotide sequence. Further selection can be carried out on the basis of the properties of the gene. Alternatively, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, e.g. , has similar or identical electrophoretic migration, isolectric focusing behavior, proteolytic digestion maps, binding activity, or antigenic properties as known for a TLE protein. By use of an antibody to a TLE protein, the TLE protein may be identified by binding of labeled antibody to the putatively TLE protein synthesizing clones, in an ELISA (enzyme-linked immunosorbent assay)-type procedure.

The TLE gene can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified TLE DNA of human or of another species (e.g. , Drosophila). Immunoprecipitation analysis or functional assays (e.g. , binding to a receptor or ligand; see infra) of the in vitro translation products of the isolated products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences. In addition, specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against a TLE protein. A radiolabelled TLE cDNA can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template. The radiolabelled mRNA or cDNA may then be used as a probe to identify the TLE DNA fragments from among other genomic DNA fragments.

Alternatives to isolating the human TLE genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence or making cDNA to the mRNA which encodes a human TLE protein. For example, RNA for cDNA cloning of the human TLE gene can be isolated from cells which express a TLE protein (see Section 6.1.3). Other methods are possible and within the scope of the invention.

The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Such vectors include, but are not limited to, bacteriophages such as lambda derivatives, or plasmids such as PBR322 or pUC plasmid derivatives. The insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzy atically modified. Alternatively, any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences. In an alternative method, the cleaved vector and TLE gene may be modified by homopolymeric tailing. Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.

In an alternative method, the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector. in specific embodiments, transformation of host cells with recombinant DNA molecules that incorporate the isolated TLE gene, cDNA, or synthesized DNA sequence enables generation of multiple copies of the gene. Thus, the gene may be obtained in large quantities by growing transformants, isolating the recombinant DNA molecules from the transformants and, when necessary, retrieving the inserted gene from the isolated recombinant DNA. 5.2. EXPRESSION OF THE HUMAN TLE GENES The nucleotide sequence coding for a human TLE protein or a functionally active fragment or other derivative thereof (see Section 5.6), can be inserted into an appropriate expression vector, i.e. , a vector which contains the necessary elements for the transcription and translation of the inserted protein- coding sequence. The necessary transcriptional and translational signals can also be supplied by the native TLE gene and/or its flanking regions. A variety of host-vector systems may be utilized to express the protein-coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g. , vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g. , baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used. In a specific embodiment, a chimeric protein comprising the nuclear localization signal or other motif or domain of a human TLE protein is expressed. In other specific embodiments, a full-length human TLE cDNA is expressed, or a sequence encoding a functionally active portion of a human TLE protein. In yet another embodiment, a fragment of a human TLE protein comprising a domain of the protein, or other derivative, or analog of a human TLE protein is expressed.

Any of the methods previously described for the insertion of DNA fragments into a vector may be used to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinants (genetic recombination). Expression of a nucleic acid sequence encoding a human TLE protein or peptide fragment may be regulated by a second nucleic acid sequence so that the TLE protein or peptide is expressed in a host transformed with the recombinant DNA molecule. For example, expression of a TLE protein may be controlled by any promoter/enhancer element known in the art. Promoters which may be used to control TLE gene expression include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78: 1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al., 1982, Nature 296:39-42); prokaryotic expression vectors such as the /3-lactamase (Villa- Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), tac (DeBoer et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25), λP_L, or trc promoters; see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; plant expression vectors comprising the nopaline synthetase promoter region or the cauliflower mosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al., 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control region which is active in the liver (Kelsey et al., 1987, Genes and Devel. 1: 161-171), beta-globin gene control region which is active in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al., 1986, Science 234: 1372-1378).

Expression vectors containing human TLE gene inserts can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene functions, and (c) expression of inserted sequences. In the first approach, the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted TLE gene. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" gene functions (e.g. , thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector. For example, if the TLE gene is inserted within the marker gene sequence of the vector, recombinants containing the E(spl) insert can be identified by the absence of the marker gene function. In the third approach, recombinant expression vectors can be identified by assaying the foreign gene product expressed by the recombinant. Such assays can be based, for example, on the physical or functional properties of the TLE gene product in in vitro assay systems, e.g. , binding to a ligand or receptor, binding with antibody, possible aggregation (binding) with Notch.

Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g. , lambda), and plasmid and cosmid DNA vectors, to name but a few. In addition, a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of the genetically engineered TLE protein may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post- translational processing and modification (e.g. , phosphorylation) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.

Both cDNA and genomic sequences can be cloned and expressed.

5.3. IDENTIFICATION AND PURIFICATION OF THE EXPRESSED GENE PRODUCTS

Once a recombinant which expresses a human TLE gene sequence is identified, the gene product can be analyzed. This is achieved by assays based on the physical or functional properties of the product, including radioactive labelling of the product followed by analysis by gel electrophoresis, immunoassay, etc. (see Section 6, infra).

Once a human TLE protein is identified, it may be isolated and purified by standard methods including chromatography (e.g. , ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

The functional properties may be evaluated using any suitable assay (see Section

5.7).

Alternatively, the amino acid sequence of a human TLE protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant. Once the amino acid sequence is thus known, the protein can be synthesized by standard chemical methods known in the art (e.g. , see Hunkapiller et al., 1984, Nature 310:105-111).

By way of example, the deduced amino acid sequences (SEQ ID NOS:2, 4, 6, and 8) of four human TLE proteins or (with respect to TLE 4) a portion thereof are presented in Figures 1-4. In specific embodiments of the present invention, human TLE proteins, whether produced by recombinant DNA techniques or by chemical synthetic methods, include but are not limited to those containing, as a primary amino acid sequence, all or part of the amino acid sequences substantially as depicted in Figures 1-4 (SEQ ID NOS:2, 4, 6, and 8), as well as fragments and other derivatives, and analogs thereof.

5.4. STRUCTURE OF THE HUMAN TLE GENES AND PROTEINS The structure of the human TLE genes and proteins can be analyzed by various methods known in the art.

5.4.1. GENETIC ANALYSIS The cloned DNA or cDNA corresponding to the TLE gene can be analyzed by methods including but not limited to Southern hybridization (Southern, 1975, J. Mol. Biol. 98:503-517), Northern hybridization (see e.g. , Freeman et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:4094-4098, and Section 6.1.3, infra), restriction endonuclease mapping (Maniatis, 1982, Molecular Cloning, A Laboratory, Cold Spring Harbor, New York), and DNA sequence analysis (see Section 6.3.1 and Figs. 1-4). Polymerase chain reaction (PCR; U.S. Patent Nos. 4,683,202, 4,683,195 and 4,889,818; Gyllenstein et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7652-7656; Ochman et al., 1988, Genetics 120:621- 623; Loh et al., 1989, Science 243:217-220) followed by Southern hybridization with a TLE-specific probe can allow the detection of the human TLE genes in DNA from various cell types. In one embodiment, Southern hybridization can be used to determine the genetic linkage of TLE. Northern hybridization analysis can be used to determine the expression of the TLE genes. Various cell types, at various states of development or activity can be tested for TLE gene expression. Examples of some such techniques and their results are described in Section 6, infra. The stringency of the hybridization conditions for both Southern and Northern hybridization can be manipulated to ensure detection of nucleic acids with the desired degree of relatedness to the specific TLE probe used, whether it be human or Drosophila.

Restriction endonuclease mapping can be used to roughly determine the genetic structure of the human TLE gene. Restriction maps derived by restriction endonuclease cleavage can be confirmed by DNA sequence analysis. Alternatively, restriction maps can be deduced, once the nucleotide sequence is known.

DNA sequence analysis can be performed by any techniques known in the art, including but not limited to the method of Maxam and Gilbert (1980, Meth. Enzymol. 65:499-560), the Sanger dideoxy method (Sanger et al., 1977, Proc. Natl. Acad. Sci. U.S.A. 74:5463), the use of T7 DNA polymerase (Tabor and Richardson, U.S. Patent No. 4,795,699; Sequenase, U.S. Biochemical Corp.), or Taq polymerase, or use of an automated DNA sequenator (e.g. , Applied Biosystems, Foster City, CA). The cDNA sequence of three human TLE genes comprises the sequence substantially as depicted in Figures 1-3 (SEQ ID NOS:l, 3, and 5), and described in Section 6, infra. The cDNA sequence of a portion of a fourth human TLE gene is shown in Figure 4 (SEQ ID NO:7) and is described in Section 6, infra.

5.4.2. PROTEIN ANALYSIS The amino acid sequence of a human TLE protein can be derived by deduction from the DNA sequence, or alternatively, by direct sequencing of the protein, e.g. , with an automated amino acid sequencer. The amino acid sequence of a representative human TLE protein comprises one of the sequences substantially as depicted in Figures 1-4, and detailed in Section 6, infra.

The TLE protein sequence can be further characterized by a hydrophilicity analysis (Hopp and Woods, 1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824). A hydrophilicity profile can be used to identify the hydrophobic and hydrophilic regions of a TLE protein and the corresponding regions of the gene sequence which encode such regions.

Secondary, structural analysis (Chou and Fasman, 1974, Biochemistry 13:222) can also be done, to identify regions of a TLE protein that assume specific secondary structures.

Manipulation, translation, and secondary structure prediction, as well as open reading frame prediction and plotting, can also be accomplished using computer software programs available in the art.

Other methods of structural analysis can also be employed. These include but are not limited to X-ray crystallography (Engstom, 1974, Biochem. Exp. Biol. 11:7-13) and computer modeling (Fletterick and Zoller (eds.), 1986, Computer Graphics and Molecular Modeling, in Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York).

5.5. GENERATION OF ANTIBODIES TO HUMAN TLE PROTEINS AND DERIVATIVES THEREOF

According to the invention, a TLE protein, its fragments or other derivatives, or analogs thereof, may be used as an immunogen to generate antibodies which recognize such an immunogen. Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library. In a preferred embodiment, antibodies which specifically bind to human TLE proteins are produced. In one embodiment, such an antibody recognizes the human TLE proteins TLE 1, TLE 2, TLE 3, and TLE 4, or a portion thereof. In another embodiment, such an antibody specifically binds to one human TLE protein selected from among TLE 1, TLE 2, TLE 3, and TLE 4, but does not bind to a different human TLE protein. In another embodiment, antibodies to a particular domain of a TLE protein are produced. Various procedures known in the art may be used for the production of polyclonal antibodies to a human TLE protein or derivative or analog. In a particular embodiment, rabbit polyclonal antibodies to an epitope of one of the TLE proteins encoded by a sequence depicted in Figure 1, 2, 3 or 4, or a subsequence thereof, can be obtained. For the production of antibody, various host animals can be immunized by injection with a native TLE protein, or a synthetic version, or derivative (e.g. , fragment) thereof, including but not limited to rabbits, mice, rats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum.

In a preferred embodiment, polyclonal or monoclonal antibodies are produced by use of a hydrophilic portion of a TLE peptide (e.g. , identified by the procedure of Hopp and Woods (1981, Proc. Natl. Acad. Sci. U.S.A. 78:3824)). For preparation of monoclonal antibodies directed toward a TLE protein sequence or analog thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture may be used. For example, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) can be used. In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals (PCT Publication No. WO 89/12690 dated December 28, 1989). According to the invention, human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, pp. 77-96). or by other methods known in the art. In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison et al.. 1984, Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for a TLE protein together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention.

According to the invention, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce TLE protein-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for TLE proteins, derivatives, or analogs.

Antibody fragments which contain the idiotype (binding domain) of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab')₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')₂ fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent. In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g. ELISA (enzyme-linked immunosorbent assay). For example, to select antibodies which recognize a specific domain of a TLE protein, one may assay generated hybridomas for a product which binds to a TLE fragment containing such domain. For selection of an antibody specific to human TLE protein(s), one can select on the basis of positive binding to a human TLE protein and a lack of binding to Drosophila TLE protein. Antibodies which bind to only one TLE protein (e.g. , TLE 1 or TLE 2) selected from among TLE 1, 2, 3 and 4 can be selected by appropriate binding assays. in another aspect of the invention, antibodies to a non-TLE protein component of multiprotein complexes containing a TLE protein (see Section 7) are provided. Such antibodies can be obtained by a method comprising immunizing an animal with such multiprotein complexes.

The foregoing antibodies can be used in methods known in the art relating to the localization and activity of the protein sequences of the invention (e.g. , see Section 5.7, infra), e.g. , for imaging these proteins, measuring levels thereof in appropriate physiological samples, etc.

5.6. HUMAN TLE PROTEIN DERIVATIVES AND ANALOGS The invention further relates to derivatives (including but not limited to fragments) and analogs of human TLE proteins.

The production and use of derivatives and analogs related to human TLE proteins are within the scope of the present invention. In a specific embodiment, the derivative or analog is functionally active, i.e. , capable of exhibiting one or more functional activities associated with a full-length, wild-type human TLE protein. As one example, such derivatives or analogs which have the desired immunogenicity or antigenicity can be used, for example, in immunoassays, for immunization, for promotion or inhibition of TLE protein activity, etc. Such molecules which retain, or alternatively inhibit, a desired human TLE protein property, e.g. , binding to a receptor or ligand, such as possibly Notch protein, can be used as inducers, or inhibitors, respectively, of such property and its physiological correlates. Derivatives or analogs of TLE proteins can be tested for the desired activity by procedures known in the art, including but not limited to the assays described in Section 5.7.

In particular, TLE derivatives can be made by altering TLE sequences by substitutions, additions or deletions that provide for functionally equivalent molecules. Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode substantially the same amino acid sequence as a human TLE gene may be used in the practice of the present invention. These include but are not limited to nucleotide sequences comprising all or portions of human TLE genes which are altered by the substitution of different codons that encode a functionally equivalent amino acid residue within the sequence, thus producing a silent change. Likewise, the TLE derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a human TLE protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

In a specific embodiment of the invention, proteins consisting of or comprising a fragment of a human TLE protein consisting of at least fifty amino acids of the TLE protein is provided. In other embodiments, the fragment consists of at least 75 or 100 amino acids of the TLE protein. The human TLE protein derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, the cloned TLE gene sequence can be modified by any of numerous strategies known in the art (Maniatis, 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro. In the production of the gene encoding a derivative or analog of a human TLE protein, care should be taken to ensure that the modified gene remains within the same translational reading frame as the TLE gene, uninterrupted by translational stop signals, in the gene region where the desired TLE protein activity is encoded. Additionally, the TLE-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem 253:6551), use of TAB^® linkers (Pharmacia), etc.

Manipulations of the human TLE sequence may also be made at the protein level. Included within the scope of the invention are human TLE protein fragments or other derivatives or analogs which are differentially modified during or after translation, e.g. , by acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation, formylation, oxidation, reduction, etc.

In a preferred aspect, phosphorylation or, alternatively, dephosphorylation is carried out, which can be to various extents, on the purified human TLE protein or derivative thereof. The phosphorylation state of the molecule may determine the distribution of the TLE protein between the cellular compartments of the nucleus and the cytoplasm (see Section 6, infra). Thus, controlling the phosphorylation state may allow control of intracellular localization. Phosphorylation can be carried out by reaction with an appropriate kinase (e.g. , possibly cdc2 or CK II). Dephosphorylation can be carried out by reaction with an appropriate phosphatase.

In addition, analogs and derivatives of human TLE proteins can be chemically synthesized. For example, a peptide corresponding to a portion of a TLE protein which comprises the desired domain (see Section 5.6.1), or which mediates the desired activity in vitro, can be synthesized by use of a peptide synthesizer. Furthermore, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the human TLE protein sequence. Non-classical amino acids include but are not limited to the D- isomers of the common amino acids, α-amino isobutyric acid, 4-aminobutyric acid, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, /3-alanine, designer amino acids such as /3-methyl amino acids, Cα-methyl amino acids, and Nα-methyl amino acids.

In a specific embodiment, the human TLE derivative is a chimeric, or fusion, protein comprising a human TLE protein or fragment thereof (preferably consisting of at least a domain or motif of the TLE protein, or at least 50 amino acids of the TLE protein) joined at its amino or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. In one embodiment, such a chimeric protein is produced by recombinant expression of a nucleic acid encoding the protein (comprising a human TLE-coding sequence joined in-frame to a coding sequence for a different protein). Such a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product by methods commonly known in the art. Alternatively, such a chimeric product may be made by protein synthetic techniques, e.g. , by use of a peptide synthesizer. A specific embodiment relates to a chimeric protein comprising a fragment of a TLE protein which comprises a domain or motif of the TLE protein, e.g. , a Q domain, GP domain, CcN domain, SP domain, WD-40 domain, one or more WD-40 repeats or a consensus WD-40 repeat (Figure 6), NLS, CK II site, or cdc2 site. In a particular embodiment, a chimeric nucleic acid can be constructed, encoding a fusion protein consisting of a human TLE nuclear localization sequence (NLS) or CcN motif (see Table I, Section 6, infra) joined to a non-TLE protein. Such a chimeric protein may thus be localized intracellularly to the nucleus of a cell into which it is introduced, by virtue of its human TLE sequence. The invention thus provides a method for delivering any protein of interest to the nucleus of a cell, by linkage of such protein to a human TLE protein NLS or CcN motif (the CcN motif contains an NLS). As another example, and not by way of limitation, a recombinant molecule can be constructed according to the invention, comprising coding portions of both a human TLE gene and another toporythmic gene. Another specific embodiment relates to a chimeric protein comprising a fragment of a human TLE protein of at least six amino acids. A particular example of a 5 human TLE fusion protein, consisting of a human TLE fragment capable of generating anti-TLE antibody fused to the carboxyl-terminus of glutathione-S-transferase, is described in Section 7 hereof.

Other specific embodiments of derivatives and analogs are described in the subsections below and examples sections infra. 10

5.6.1. DERIVATIVES OF HUMAN TLE PROTEINS CONTAINING ONE OR MORE DOMAINS OF THE PROTEIN

In a specific embodiment, the invention relates to human TLE protein derivatives and analogs, in particular human TLE fragments and

- - derivatives of such fragments, that comprise one or more domains of a human TLE protein, including but not limited to a Q domain [amino acids (approximately) 1-131, 1-127, and 1-130 for TLE 1, TLE 2, and TLE 3, respectively], GP domain [amino acids (approximately) 132-199, 128-191, and 131-197 for TLE 1 , TLE 2, and TLE 3, respectively], CcN domain [amino acids 0 (approximately) 200-268, 192-254, and 198-267 for TLE 1, TLE 2, and TLE 3, respectively], SP domain [amino acids (approximately) 269-449, 255-422, and 268-450 for TLE 1, TLE 2, and TLE 3, respectively], WD-40 domain [amino acids (approximately) 450-770, 423-743, and 451-774, for TLE 1, TLE 2, and TLE 3, respectively, and the last —321 amino acids of TLE 4] (see Fig. 5 for 5 sequences of all of the foregoing), one or more WD-40 repeats (see Fig. 6), or a consensus WD-40 repeat (Fig. 6), NLS (Table I), CK II site (Table I), or cdc2 site (Table I). A consensus WD-40 repeat is shown in Figure 6, and consists of the following sequence (SEQ ID NO:9): PXXXX(D or E)XTXXXXXXXX(I or L)X(I or L)SPDG(T or S)XLX(T or S)GGXDGXVXXWDLX, where X is any amino acid. The CcN domains comprise the CcN motifs, which latter span approximately amino acids 225-269, 214-255, and 224-268, for TLE 1, TLE 2, and TLE 3, respectively.

5 5.6.2. DERIVATIVES OF HUMAN TLE PROTEINS THAT MEDIATE BINDING TO PROTEINS

The invention also provides for human TLE fragments, and analogs or derivatives of such fragments, which mediate binding to other proteins, and nucleic acid sequences encoding the foregoing. As shown in Section 7, infra,

TLE proteins associate in multiprotein complexes, and thus bind to other proteins.

In a specific embodiment, a non-TLE protein component of such multiprotein complexes is an — 17 kD protein.

5.7. ASSAYS OF TLE PROTEINS, DERIVATIVES AND ANALOGS

The functional activity of TLE proteins, derivatives and analogs can be assayed by various methods.

For example, in one embodiment, where one is assaying for the ability to bind or compete with a wild-type human TLE protein for binding to anti-TLE protein antibody, various immunoassays known in the art can be used, including but not limited to competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g. , gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labelled. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.

The ability to bind to another protein (be it a second TLE protein, a non-TLE component of the multiprotein complexes described in Sections 5.6 and 7, possibly a Notch protein, or otherwise) can be demonstrated by in vitro binding assays, noncompetitive or competitive, by methods known in the art. Thus, where a receptor or ligand for a TLE protein is identified, receptor or ligand binding can be assayed, e.g. , by means well known in the art. In another embodiment, physiological correlates of TLE introduction into cells can be assayed.

Other methods will be known to the skilled artisan and are within the scope of the invention.

5.8. PROTEINS WHICH FORM A COMPLEX

WITH TLE PROTEINS

The invention further provides proteins which associate in a multiprotein complex containing TLE proteins (see Section 7, infra). In one embodiment, such a protein binds to a TLE protein or a binding partner thereof. Such protein components of complexes containing TLE proteins may act as effector molecules in TLE protein signal transduction events and thus have potential uses in modulation of TLE protein activity. In a specific embodiment, a substantially purified multiprotein complex of about 670,000 daltons that contains a TLE protein or epitope thereof (as detected e.g. , by the ability to be bound by an anti-TLE protein antibody) is provided; in another specific embodiment, such a multiprotein complex has a molecular weight greater than about 670,000 daltons; in yet other aspects, such a complex has a molecular weight of about 110, 170, 190, or 230 kilodaltons. These complexes of smaller molecular weight may be components of the larger complexes. In another specific embodiment, the invention provides a substantially purified protein component of such a multiprotein complex, with a molecular weight in the range of about 15,000- 18,000 daltons, in particular, about 17,000 daltons, as detected by SDS- polyacrylamide gel electrophoresis. The invention also provides antibodies, in particular, monoclonal antibodies, which specifically bind to the non-TLE protein components of such multiprotein complexes. In one embodiment, such antibodies are obtained by using as immunogen the multiprotein TLE complexes and selecting for negative TLE protein reactivity and positive TLE complex reactivity. 6. THE HUMAN HOMOLOGS OF THE TRANSDUCIN-LIKE ENHANCER OF SPLIT GENE PRODUCT OF THE DROSOPHILA "NOTCH GROUP" DEFINE A NOVEL FAMILY OF NUCLEAR PROTEINS

The Drosophila m.9110 gene (groucho) of the Enhancer of split [E(spl)] complex is part of a genetic circuitry, the so-called Notch group of genes, which is required for a variety of cell fate choices in Drosophila including the segregation of neural and epidermal cell lineages. As described herein, we have characterized human cDNA clones encoding a family of proteins, designated TLE, that are homologous to the E(spl) m9/10 gene product. The TLE and E(spl) m9/10 proteins share two amino acid sequence motifs. The first is a tandem array of four so-called "WD-40" repeats at the carboxyl end of the molecule and the second, referred to as the "CcN motif", consists of a closely- spaced combination of a nuclear localization sequence and potential phosphorylation sites for both casein kinase II and cdc2 kinase. As described herein, the TLE proteins were shown to be predominantly nuclear in HeLa cells, and the Drosophila E(spl) m9/10 protein was shown to be phosphorylated. These results suggest a role for the E(spl) m9/10 and human TLE proteins as nuclear effector molecules.

6.1. RESULTS

6.1.1. ISOLATION AND CHARACTERIZATION OF HUMAN E(snl) m9/10 HOMOLOGS

We searched for human homologs of the E(spl) m9/10 gene using a fortuitously isolated 1.7-kb cDNA clone from a human testis cDNA library (see Materials and Methods). This clone contains a partial open reading frame (ORF) encoding a 282 residue long polypeptide chain exhibiting homology to the portion of the E(spi) m9/10 protein that includes the four WD-40 repeats.

This partial cDNA clone was used to screen a human fetal brain cDNA library, resulting in the isolation of four classes of clones, hereafter referred to as 7 E 7, 7 E 2, 7 E 3, and 7 E 4 (Transducin-Like Enhancer of split). cDNAs TLΕ 1 , TLΕ 2, and TLΕ 3 contain entire ORFs for three distinct proteins of 770 (M_r 83,000), 743 (M_r 80,000), and 774 (M_r 83,000) amino acids, respectively, while TLE 4 is a partial clone. All encoded proteins are homologous to E(spl) m.9110. The complete nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequence ((SEQ ID NO:2) for TLE 1 are shown in Figure 1. The complete nucleotide sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ ID NO:4) for TLE 2 are shown in Figure 2. The complete nucleotide sequence (SEQ ID NO:5) and deduced amino acid sequence (SEQ ID NO: 6) for TLE 3 are shown in Figure 3. The partial nucleotide sequence (SEQ ID NO:7) and deduced amino acid sequence (SEQ ID NO: 8) for TLE 4 are shown in Figure 4. As is the case with the E(spl) m9/10 protein (Hartley et al., 1988, Cell 55:785-795), analysis of hydropathy plots for TLE 1 , TLE 2, and TLE 3 indicated that the TLE proteins are quite hydrophilic and appear not to have a signal sequence (not shown).

6.1.2. DOMAIN STRUCTURE OF TLE PROTEINS A comparison of the Drosophila E(spl) m9/10 protein (SEQ ID

NO: 10) and human TLE proteins is shown in Figure 5. The first ~ 130 residues at the amino terminus of the proteins are highly conserved and have a high content of conserved glutamine residues. Thus, we refer to this region as the "Q domain". In this region, TLE 1 is 72%, TLE 2 is 68%, and TLE 3 is 71 % identical to E(spl) m9/10. Adams et al. (1991, Science 252:1651-1656) have recently described partial DNA sequences of more than 600 randomly selected cDNA clones from human brain. Sequencing of ~ 250 nucleotides of Adams et al. clone EST00256 identified a reading frame coding for a protein related to E(spl) m9/10; this short sequence maps within the first 100 residues of the amino terminus of m9/10. Comparing the corresponding region of the TLE 1, TLE 2, and 7L£ 3 cDNAs with the nucleotide and predicted amino acid sequence of cDNA EST00256, we failed to show identity among these cDNAs. This suggests that cDNA EST00256 is either pan of the sequence coding for TLE 4, the sequence of which remains to be fully determined, or part of yet another member of this family. A poorly conserved region of approximately 80 amino acid residues follows the Q domain. We refer to this portion of the molecules as the "GP domain" to indicate the presence of numerous glycine and proline residues. The lack of significant structural conservation in the GP domain ends approximately 200 residues from the amino terminus, in the "CcN domain" (Jans et al., 1991, J. Cell Biol. 115:1203-1212).

The CcN domain consists of a stretch of — 60 residues that harbors a sequence motif conforming to the definition of a casein kinase II (CK II) site/cdc2 kinase (cdc2) site/nuclear localization sequence (NLS) motif first reported for the SV40 T antigen (Jans et al., 1991, J. Cell Biol. 115: 1203-1212; Rihs et al., 1991, EMBO J. 10:633-639). NLS, a cluster of four positively charged amino acids preceded, at a distance of ten residues, by a block of two or three basic amino acids (Kalderon et al., 1984, Nature 311:499-509; Dingwall and Laskey, 1991, Trends Biochem. Sci. 16:478-481), is in proximity to possible phosphorylation sites for both casein kinase II (defined by the consensus sequence ^S/_TXX^D/_E) and cdc2 kinase (defined by the consensus sequence ^S/_TPXZ, with X being dispensable and Z being generally a basic residue). Table I shows a comparison of the CcN motif found in E(spl) m9/10, TLE 1, TLE 2, and TLE 3. E(spl) m9/10, TLE 1, and TLE 3 have conventional NLSs, while TLE 2 deviates from the general consensus. It is worth noting, however, that a certain degree of flexibility in the selection of the amino acids that form a NLS has been observed previously (Dingwall and Laskey, 1991 , Trends Biochem. Sci. 16:478-481).

Table I Comparison of the CcN motif between E(spl) m9/10 and human TLE proteins^*

Piotein NLS CK II site cdc2 site

E(spl) m9/IO 204 - YRTRS PLD i ENDSKRRK-DEKLOEDEGEKSDQD WDVANE-MESHS PRP

TLE 1 212 DKRRNGP-EFSNDIKKRKVDDKDSSH-YD-SDGDKSDDNLWDVSNED-PS-S PRASPAHSPR

TLE 2 203 EERPSGP — GGGGKQR- ADEKEPSGPYE-SDEDKSDYNLWD EDQPSE - P P - SPATTPC

TLE 3 211 EKHRGSA-DYSMEAKKRKVEEKDSLSRYD-SDGDKSDD-LWDVSNED-P-ATPRVSPAHSPP

The nuclear localization sequence (NLS) and the possible phosphorylation sites for casein kinase II (CK II) and cdc2 are indicated by large characters. Identical amino acids and conservative substitutions are underlined.

Table II shows the relationship between the NLS and putative phosphorylation sites in E(spl) m9/10, TLE 1, TLE 2, TLE 3, as well as other proteins bearing the CcN motif; these proteins were selected on the basis of demonstrated nuclear localization and susceptibility to phosphorylation. Most, if not all, of them play important roles in regulating nuclear functions such as transcription and mitosis, as well as other aspects of the cell cycle (for review, see Meisner and Czech, 1991, Curr. Op. Cell Biol. 3:474-483; Moreno and Nurse, 1990, Cell 61:549-551).

Table II^* Comparison of the CcN motif of E(spl) m9/10, TLE proteins, and proteins with demonstrated nuclear localization and susceptibility to phosphorylation by casein kinase II and/or cdc2

Standard one-letter amino acid symbols are used; see e.g. , Lehninger, 1975, Biochemistry, 2d. Ed., Worth Publishers, Inc., New York, p. 72.

Phosphoryla table Ser /Thr residues are numbered.

The Drosophila protein dorsal was included as one example of several other proteins bearing a putative CcN motif for which only translocation to the nucleus has been demonstrated.

Kalderon et al., 1984, Nature 311 :499-509

McVey et al., 1989, Nature 341:503-507

Grasser et al., 1988. Virology 165: 13-22

Dang and Lee, 1988, Mol. Cell. Biochem. 8:4048-4054

Luscher et al., 1989, EMBO J. 8: 1111-1119

Jenkins et al., 1984, Nature 312:651-654

Bischoff et al., 1990, Proc. Nad. Acad. Sci. USA 87:4766-4770

Meek et al., 1990, EMBO J. 9:3253-3260

Nomura et al., 1988, Nucleic Acids Res. 16: 11075-11089

Luscher et al., 1990, Nature 344:517-522

Steward, 1987, Science 238:692-694 A poorly conserved region of 150-180 amino acids rich in serine and proline residues, the "SP domain, " separates the CcN domain from the carboxyl-ter inal region which contains the WD-40 repeats and shows the most impressive similarity among the E(spl) m9/10 and the TLE proteins. Figure 6 shows the sequence conservation in the "WD-40 domain" among all five proteins: TLE 1 is 89%, TLE 2 is 83%, TLE 3 is 87%, and TLE 4 is 89% identical to the Drosophila m9/10 protein. Large blocks of sequence are highly conserved within each individual group of repeats and when all repeats are compared inter se, a consensus motif can be identified at the carboxyl-terminal end of each repeat, terminating in the conserved WDL sequence.

In conclusion, comparison of the deduced amino acid sequence of the Drosophila m9/10 and human TLE proteins reveals the presence of three conserved structural elements: the Q-, the CcN-, and the WD-40 domains (see Fig. 5). Interestingly, we noticed that a similar domain structure is exhibited by the product of the yeast TUP1 gene, isolated by Williams and Trumbly (1990, Mol. Cell. Biol. 10:6500-6511).

6.1.3. EXPRESSION OF TLE mRNAs The distribution of the various TLE mRNAs was determined by

Northern blotting analyses of poly A^<+) RNA. These studies revealed that the TLE 1 mRNA migrates as a major species of 4.5 kb detectable in all adult tissues examined, with the highest level of expression in brain, liver, and muscle (Fig. 7a). Minor species of 5.8 and 3.2 kb were also detectable. Two distinct TLE 2 mRNAs were detected (Fig. 7b). One transcript, of 2.8 kb, was expressed at different levels in all tissues examined and was noticeably abundant in heart, brain, and muscle; the second transcript, of 3.5 kb, appeared to be expressed only in brain. Three distinct TLE 3 transcripts were present, having sizes of 5.8, 4.8, and 3.7 kb. Placenta and lung are the only tissues where all these mRNAs were detected, while the remaining tissues only expressed either one or two of them (Fig. 7c). Finally, two major TLE 4 transcripts of 5.1 and 2.8 kb were observed. They were predominantly expressed in brain and muscle, but were also present in all other tissues investigated (Fig. 7d).

In aggregate, the TLE mRNAs are expressed in all tissues examined with individual transcripts showing specific patterns of expression.

5

6.1.4. IMMUNOCYTOCHEMICAL CHARACTERIZATION OF TLE PROTEINS

In order to determine the intracellular distribution of the TLE proteins, we raised monoclonal antibodies against a fusion protein containing the

10 carboxyl-terminal-most 282 amino acids of TLE 3 fused to glutathione S- transferase (Smith and Johnson, 1988, Gene 67:31-40). Figure 8a illustrates the results of Western blotting experiments performed with monoclonal antibody

C597.4A. A small number of closely spaced immunoreactive species with apparent molecular weights of — 85,000 was detected in all tissues examined.

I - Monoclonal antibody C597.4A was selected based on its ability to cross-react with Drosophila E(spl) m9/10, as well as with rat proteins also exhibiting apparent molecular weights of — 85,000 (not shown). Thus, this antibody seems to recognize an epitope that has been conserved across species boundaries. This is not surprising, since the TLE 3 fusion protein contained the highly conserved o WD-40 domain (as shown in Figure 6); in this domain TLE 3 is 87%, 94%, 85%, and 94% identical to E(spl) m9/10, TLE 1, TLE 2, and TLE 4, respectively. We therefore expect that, in addition to TLE 3, this antibody will recognize the remaining TLE proteins in humans and that the — 85-kDa bands observed in Figure 8a correspond to more than one TLE protein. As expected 5 from the transcript analysis described in Figure 7, these investigations revealed that the TLE proteins are expressed in a broad range of human tissues and cell lines, including the human SUP-Tl cell line which was established from an acute T lymphoblastic leukemia and was shown to contain a translocation interrupting the TAN-1 coding sequence (Ellisen et al., 1991, Cell 66:649-661). 0 We investigated the intracellular distribution of the TLE proteins in HeLa cells by indirect immunofluorescence microscopy using monoclonal antibody C597.4A. Figure 8b shows that the TLE proteins were predominantly

5 localized to the nucleus. However, a much weaker but nevertheless above background staining was consistently seen in the cytoplasm. Preliminary studies with monoclonal antibodies we have developed that are specific for TLE 1 and TLE 2 have revealed that both of these proteins are also expressed in HeLa cells. Identical results were obtained using the SUP-Tl cell line (not shown).

6.1.5. THE DROSOPHILA E(spl) m9/10 PROTEIN IS PHOSPHORYLATED

We have asked the question of whether or not the Drosophila E(spl) m9/10 protein is phosphorylated. Drosophila S2 cells were pulse-labeled with ³²P and lysed as described in Materials and Methods. A single phosphorylated protein was detected in the immunoprecipitates obtained with monoclonal antibody 3C, which is directed against Drosophila E(spl) m9/10 (Delidakis et al., 1991, Genetics 129:803-823). The electrophoretic mobility of this molecule corresponded to that expected for the m9/10 protein. Although we have not shown directly that m9/10 is phosphorylated on Ser/Thr residues, we do know that phosphorylation does not seem to occur at Tyr residues: when the immunoprecipitates were probed with a monoclonal antibody specific for phosphorylated Tyr residues (Glenney et al., 1988, J. Immunol. Meth. 109:277- 283), no detectable immunoreactivity was observed (not shown).

The expression profile of the Drosophila m9/10 protein during embryogenesis was revealed by Western blotting analysis. Two closely-spaced bands were detected with monoclonal antibody 3C. The lower band was predominant very early in development and became progressively less abundant at later stages, while the higher band showed exactly an opposite profile. Given that the E(spl) m9/10 protein was shown to be the product of a single gene (Hartley et al., 1988, Cell 55:785-795; Preiss et al., 1988, EMBO J. 7:3917-3927), it is possible that this electrophoretic profile reflects a developmental ly regulated post- translational modification such as phosphorylation. Overall, our results demonstrate two of the features suggested by the presence of a CcN motif, namely nuclear targeting and susceptibility to phosphorylation. 6.2. DISCUSSION In the present work we provide evidence that the m.9110 gene of E(spl), a member of the Notch group, has also been conserved during evolution to a surprising degree and that in humans there is a family of m9/10-homologous proteins. These gene products, named TLE, show a striking conservation at their carboxyl-terminal regions, where a tandemly duplicated organization of four — 40-residue long repeats defines an extraordinarily well conserved structural domain. Similarly organized arrangements of so-called WD-40 repeats have recently been observed in an expanding group of unrelated proteins including the β subunits of heterotrimeric G proteins (for review, see Simon et al., 1991,

Science 252:802-808), the products of the yeast genes CDC 4, which is involved in regulating the cell cycle (Yochem and Byers, 1987), TUP1, a mediator of glucose repression (Williams and Trumbly, 1990, Mol. Cell. Biol. 10:6500- 6511), PRP4, a stable component of the U4/U6 small nuclear ribonucleoprotein particle (Dalrymple et al., 1989, Cell 58:811-812), and MSI1, a negative regulator of the RAS-mediated induction of cAMP (Ruggieri et al., 1989, Proc. Natl. Acad. Sci. USA 86:8778-8782), as well as the product of the vertebrate 12.3 gene, initially identified by virtue of its physical linkage to the chicken major histocompatibility complex (Guillemat et al., 1989, Proc. Natl. Acad. Sci. USA 86:4594-4598).

Recent genetic (Goebl and Yanagida, 1991, Trends Biochem. Sci. 16:173-177) and molecular (Williams et al., 1991, Mol. Cell. Biol. 11:3307- 3316) investigations with several yeast mutants have provided evidence for the existence of interactions between some members of the WD-40 repeat family and some members of a novel group of proteins characterized by the presence of the so-called TPR snap helix repeat (for review, see Goebl and Yanagida, 1991, Trends Biochem. Sci. 16: 173-177). These genetic associations always involve a pair of genes, representing either the WD-40 repeat family or the TPR helix family, thus suggesting a possible mutual relation of these two motifs. Analysis of the cellular functions carried out by most of these proteins indicates that they are involved in nuclear activities ranging from regulation of mitosis to regulation of transcription.

A second noteworthy structural feature shared by E(spl) m9/10 and TLE proteins is the presence of the CcN motif (Fig. 5 and Table I). The CcN motif has been found in several nuclear proteins involved in regulating cell differentiation or proliferation (Jans et al., 1991 , J. Cell Biol. 115: 1203-1212). Studies with SV40 T antigen have demonstrated that absence of the Ser residue of the CK II site of the CcN motif, which can be phosphorylated, causes a reduction of the rate of nuclear transport of the protein (Rihs et al., 1991, EMBO J. 10:633-639). On the other hand, phosphorylation of Thr¹²⁴ of the cdc2 site within the CcN motif inhibits nuclear import of SV40 T antigen fusion proteins (Jans et al., 1991, J. Cell Biol. 115:1203-1212). These studies suggest that the phosphorylation state of the SV40 T antigen determined by these two kinases controls the cytoplasmic/nuclear distribution of the protein, thus providing a possible mechanistic explanation for the presence of CK II and cdc2 phosphorylation sites in many proteins with known nuclear functions (for review, see Meisner and Czech, 1991, Curr. Op. Cell Biol. 3:474-483; Moreno and Nurse, 1990, Cell 61:549-551). Such a mechanism could also be involved in mediating the predominantly, but not exclusively, nuclear localization of E(spl) m9/10 (Delidakis et al., 1991 , Genetics 129:803-823) and TLE proteins (Fig. 8b). Indeed, in agreement with the observation that most of the proteins carrying a CcN motif have been shown to be phosphorylated by CK II and/or cdc2 (Meisner and Czech, 1991 , Curr. Op. Cell Biol. 3:474-483), we have shown that the m9/10 protein is phosphorylated in cultured Drosophila S2 cells. We do not have direct evidence that m9/10 is phosphorylated in embryos, but it is possible that the developmental profile we observed reflects the presence of differentially phosphorylated forms of the protein. This possibility is particularly attractive since it would provide a possible mechanism for regulating the relative distribution of the protein between cytoplasm and nucleus. In turn, this would permit direct interactions with a membrane-bound molecule such as Notch. Such molecular interactions could explain the documented genetic interactions between specific intracellular Notch mutations and certain E(spl) m9H0 alleles (Xu et al., 1990, Genes Dev. 4:464-475). Moreover, phosphorylation/dephosphorylation reactions may be used to stabilize or prevent associations with other factors.

Thus, the demonstration of nuclear targeting and susceptibility to phosphorylation, two of the features associated with the presence of a CcN motif, lends support to the concept that this motif is an important functional element of these proteins. It is also worth noting that the nuclear/cytoplasmic distribution of the various TLE proteins may not be the same under varying physiological conditions. Subsets of these proteins may redistribute in specific ways between cellular compartments in response to intracellular as well as extracellular changes.

The extraordinary structural conservation among the Drosophila and human gene products described herein implies that the biochemical mechanisms involving E(spl) m9/10-like proteins may also be conserved across species boundaries, as part of a general and pleiotropic pathway involved in controlling many aspects of mammalian cell fate.

6.3. MATERIALS AND METHODS 6.3.1. GENERAL METHODS AND MATERIALS Standard molecular biology techniques were used (Sambrook et al., 1989, Molecular cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). SDS- polyacrylamide gel electrophoresis (PAGE), transfer to nitrocellulose, and Western blotting were as described by Stifani et al. (1988, Biochem. J. 250:467-475). Both strands of double-stranded cDNA clones were sequenced by the dideoxy chain termination method using the Sequenase kit (U.S. Biochemical Corp.) and a combination of synthetic oligonucleotide primers, SK, KS, universal, and reverse primers. Human genomic DNA and poly (A)⁺ RNA from bom fetal and adult brain were obtained from Clontech. Incubations were at 65 °C for 48 hr using double stranded [³²P]- labeled probes prepared by random oligonucleotide priming. 6.3.2. cDNA CLONING A human testis cDNA library (Clontech) was used for the isolation of the 1.7-kb TLE 3a cDNA. This cDNA was used to screen a human fetal brain cDNA library (Stratagene) resulting in isolation of the 7LE 1, TLE 2, TLE 3, and TLE 4 cDNAs. Recombinant phage were propagated in E. coli "XLl-Blue" cells. Plaques were screened using me TLE 3a cDNA labeled with [³²P]-dCTP by random oligonucleotide priming as a probe. Replicate filters were hybridized at 65 °C in buffer A [500 mM sodium phosphate (pH 7.2), 5% SDS, 1 mM ΕDTA., and 1 % bovine serum albumin (BSA)]. Filters were washed at 65 °C three times with a buffer containing 40 mM sodium phosphate (pH 7.2), 4% SDS, and 1 mM ΕDTA, and three times with the same buffer containing 1 % SDS. After two further rounds of plaque hybridization, 14 unique clones were isolated. Cloned inserts were recovered after in vivo excision and recircularization of the "pBluescript SK (-)" phagemids from XLl-Blue cells in the presence of the R408 helper phage. Infection of E. coli cells with the obtained phagemids yielded colonies harboring double-stranded phagemid DNA. The size of individual inserts was determined by agarose gel electrophoresis after digestion with ΕcoRI.

6.3.3. CELL CULTURE AND PREPARATION OF CELL AND TISSUE EXTRACTS

Human HeLa cells were obtained from the American Type Culture

Collection, Rockville, MD. Cells were grown at 37 °C in an atmosphere of 5%

CO₂, 95% air in the presence of MEM (Eagle) supplemented with non-essential amino acids, Earle's BSS, 10% fetal bovine serum (FBS), 2 mM L-glutamine,

100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml fungizone.

Cells from stock cells were dissociated by addition of phosphate-buffered saline

(PBS) containing 0.25% trypsin and 0.03% EDTA, and subcultured at a ratio of

1:3 to 1:5. For preparation of protein extracts, all operations were carried out at

4 °C. Cells were resuspended in buffer B (10 mM HEPES, pH 7.8, 150 M

NaCl, 2mM MgCl₂, 1 mM phenyl methylsuifonyl fluoride (PMSF), 2 μM leupeptin, 2.5 μg/ml aprotinin, and 2.5 μg/ml pepstatin A), homogenized using a

Dounce homogenizer (10 strokes; type- A pestle), and protein extracts were obtained in the presence of 1 % Triton X-100. Lysates were centrifuged at 12,000 x g for 15 min and the resulting supernatants were collected, calibrated for their protein content, and subjected to SDS-PAGE. Human tissue samples were processed essentially in the same way.

6.3.4. IMMUNOFLUORESCENCE MICROSCOPY HeLa cell monolayers were grown in tissue culture chamber slides (Nunc). Indirect immunofluorescence microscopy using the rat monoclonal antibody C597.4A (see infra) was performed essentially as described by Fehon et al. (1990, Cell 61:523-534). Cells were fixed with freshly made 2% (w/v) paraformaldehyde in 100 mM PIPES (pH 6.8), 2 mM EGTA, 1 mM MgSO₄, and incubated for 30 min in PBS containing 0.08% Triton X-100 and 3% normal goat serum (buffer C). After this, cells were incubated for 1 hr in buffer C containing a 1:10 dilution of the rat monoclonal antibody C597.4A, directed against a TLE 3/glutathione S-transferase fusion protein (see below). At the end of this incubation, cells were washed four times with buffer C and then incubated for 1 hr in buffer C containing a 1: 1000 dilution of Cy3-conjugated goat anti-rat IgG (Jackson Immunoresearch Laboratories). Cells were then rinsed four times with buffer C, incubated for 1 min in PBS containing 1 μg/ml of DAPI, rinsed extensively with PBS, and processed for immunoflourescence as described in Fehon et al. (1990, Cell 61:523-534).

6.3.5. METABOLIC LABELING OF DROSOPHILA S2 CELL, IMMUNOPRECIPITATION, ELECTROPHORETIC PROCEDURES, AND WESTERN BLOTTING

Drosophila S2 cells were cultured as described previously (Fehon et al., 1990, Cell 61:523-534). In a typical metabolic labeling reaction with [³ P]- orthophosphate, 10-15 ml of cell suspension ( — 2 x IO⁷ cells/ml) was used. Cells were washed twice with BSS, resuspended in 1 ml of phosphate-free M3 medium, and incubated at 24 °C for 45 min. After this time, cells were incubated for 3 hr at 24 °C in the presence of 750 μCi/ml of [³²P]-orthophosphate (Amersham; 370 MBq/ml). Incubations were performed in 6-well tissue culture plates with gentle rocking motion. At the end of the incubation time, cells were collected, washed twice with ice-cold buffer D (10 mM HEPES, pH 7.8, 60 mM KC1, 15 mM NaCl, 50 mM NaF, 10 mM sodium pyrophosphate, 5 mM MgCl,, 300 mM sucrose, 1 mM PMSF, 2 μM leupeptin, 2.5 μg/ml aprotinin, 2.5 μg/ml pepstatin A), resuspended, and lysed in buffer D containing 0.5% Triton X-100. For immunoprecipitation experiments, cell lysates were mixed with the appropriate antibodies (150 μg/ml) and incubated for 2 hr at 4°C in the presence of 3.5 mg/ml of BSA. Immunoprecipitates were collected by incubation with protein G- agarose beads, washed extensively with buffer E (25 mM Tris-HCl, pH 7.8, 200 mM NaCl, 2 mM EDTA, 2 mM EGTA, 20 mM NaF, 10 mM sodium pyrophosphate, and 0.5% Triton X-100), and subjected to SDS-polyacrylamide gel electrophoresis.

6.3.6. PREPARATION OF DROSOPHILA

EMBRYONIC EXTRACTS

Staged Drosophila Canton-S embryos were collected, dechorionated in 50% Clorox solution, and washed extensively with 0.7% NaCl,

0.002% Triton X-100. Dechorionated embryos were washed twice in PBS and then homogenized by 10 strokes of a Dounce homogenizer (type-A pestle) in buffer F (10 M HEPES, pH 7.8, 150 mM NaCl, 2 mM MgCl₂, 1 mM PMSF, 2 μM leupeptin, 2.5 μg/ml aprotinin, and 2.5 μg/ml pepstatin A). All operations were carried out at 4 °C. After incubation in the presence of 1 % Triton X-100, homogenates were centrifuged at 12,000 x g for 15 min and the supernatants were collected. Determination of the protein concentration of embryonic and cell extracts was obtained using the Biorad protein assay kit using BSA as standard.

6.3.7. PREPARATION OF FUSION PROTEINS AND IMMUNOLOGICAL PROCEDURES

Fusion proteins were obtained using the pGEX-3X expression vector system in E. coli BL21DE3 cells. The 1.7-kb testis cDNA subcloned in Bluescript SK II was excised with BamHI and EcoRV and ligated in frame into pGEX-3X. This fragment encodes the carboxyl-terminal 282 amino acid residues of the TLE 3 protein. This fusion construct produced an ~55-kDa chimeric protein containing the carboxyl terminus of glutathione S-transferase. Fusion proteins were produced and purified according to standard procedures (Smith and Johnson, 1988, Gene 67:31-40) and utilized for immunization of Long Evans rats according to the schedule described in Stifani et al. (1988, Biochem. J. 250:467- 475). The hybridoma cell line C597.4A was obtained from a rat immunized with the TLE 3 fusion protein as described above.

7. TLE PROTEINS ARE PART OF HIGH-MOLECULAR- WEIGHT MULTIPROTEIN COMPLEXES

Both in Drosophila and man the TLE proteins (the terminology "TLE" applies in general to both the fly and human homologs) are present as part of high-molecular-weight (high M_r) multiprotein complexes. This was demonstrated by using a combination of non-denaturing polyacrylamide gel electrophoresis (PAGE), gel filtration, and cross-linking experiments. The results of such investigations are described below, together with the details of the experimental procedures.

7.1. NON-DENATURING PAGE We subjected a high-speed supernatant fraction from HeLa cell lysates to electrophoresis through non-denaturing polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose filters and the replicas were used in Western blotting experiments with the monoclonal antibody C597.4A, which cross-reacts with all TLE proteins. As shown in Figure 9, two major immunoreactive species were observed, with apparent molecular weights of greater than 670,000. Since the expected molecular weight of the monomeric TLE proteins is roughly 85,000. these results suggest that in their native state the TLE proteins are associates with other proteins to form high-molecular-weight complexes. Corresponding immunoreactive components were also visualized when polyclonal antibodies specific for TLE 1 were used (not shown). Methods:

HeLA cells were grown at 37°C in an atmosphere of 5% CO₂, 95% air in the presence of MEM (Eagle) supplemented with non-essential amino acids, 10% FBS, 2 mM L-glutamine, 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml fungizone. Cells were collected by scraping, washed once with ice-cold PBS, resuspended in ice-cold buffer G (50 mM HEPES, pH 7.6, 10 mM iodoacetic acid, 10 mM KC1, 0.5 mM EGTA, 0.5 mM EDTA, 1 mM PMSF, 2 μM leupeptin, 2.5 μg/ml aprotinin, 2.5 μg/ml pepstatin A, and 2.5 μg/ml antipain), and homogenized by 10 strokes of a Dounce homogenizer (teflon pestle). The homogenate was centrifuged at 100,000 x g for 1 hr and the supernatant was recovered and immediately loaded onto 3-18% non- denaturing polyacrylamide gels. (This lysis procedure results in the recovery of more than 90% of the total cellular content of TLE proteins in the high-speed supernatant fraction.) Electrophoresis was performed at 150 V for 24 hr using a running buffer containing 90 mM Tris Base, 80 mM boric acid, and 3 mM EDTA (pH 8.4). After electrophoretic transfer of proteins to nitrocellulose, Western blotting experiments were performed as described in Section 6, supra, in the presence of a 1:20 dilution of monoclonal antibody C597.4A.

7.2. GEL FILTRATION CHROMATOGRAPHY

High-speed supernatant fractions from HeLa cells prepared as described above were subjected to gel filtration chromatography using a Sephacryl S-300 matrix. The fractions collected from the column were analyzed for the presence of TLE proteins in Western blotting experiments with monoclonal antibody C597.4A. As shown in panel A of Figure 10, TLE proteins were detected in fractions expected to contain molecules of size significantly larger man that predicted for the monomeric TLE proteins (see lanes 2-8). These results are therefore in agreement with the studies shown in Figure 9 in suggesting the presence of large protein complexes containing the TLE proteins. The heterogeneous nature of such complexes, suggested by the presence of multiple bands in Figure 9, is confirmed by the detection of TLE proteins in several column fractions spanning a large size interval (cfr. positions of elution of molecular size markers on top of Figure 10; D.B. Dextran Blue). When the same column fractions were subjected to electrophoresis under non-reducing conditions, additional bands were visualized with monoclonal antibody C597.4A (Figure 10, panel B). In particular, the presence of bands corresponding to species exhibiting slightly slower electrophoretic mobility than that of the TLE proteins suggests the existence of small proteins (M_r, 15,000-18,000) that can remain associated with the TLE proteins under these experimental conditions, but not under those used in panel A of Figure 10.

Methods:

High-speed supernatant from HeLa cells prepared as described above was loaded onto a Sephacryl S-300 HR column (110 x 1.5 cm), and elution was carried out in a buffer containing 50 mM HEPES, pH 7.4 and 100 mM NaCl at a flow rate of 10 ml/hr. 1-ml fractions were collected and 130-μl aliquots from each fraction were subjected to electrophoresis on a 4-18% SDS-polyacrylamide gel, followed by transfer to nitrocellulose and immunoblotting with monoclonal antibody C597.4A as described for Figure 9. In panel A, samples were treated with 50 mM DTT and were heated to 95 °C prior to electrophoresis (reducing conditions). Under non-reducing conditions (panel B) these two steps were omitted.

7.3. CROSS-LINKING STUDIES We incubated protein extracts from Drosophila embryos (Figure 11, lanes 1-3), HeLa cells (lanes 4-6), or SUP-Tl cells (lanes 7-9) in the presence of increasing concentrations of the chemical cross-linker, DTSSP [3,3'-dithiobis (sulfosuccinimidylpropionate)]. The products of the cross-linking reactions were then subjected to electrophoresis on SDS-polyacrylamide gels, followed by transfer to nitrocellulose and Western blotting. These experiments showed that the presence of the cross-linker in the reaction mixtures results in the appearance of new immunoreactive components of higher apparent M_r. In the case of Drosophila embryonic extracts, Western blotting revealed, in addition to the monomeric form of the Enhancer of split m9/10 protein (apparent M_r, 82,000), three bands at roughly 170-, 190-, and 230 kD, plus two additional components of very slow electrophoretic mobility (lanes 1-3). Similarly, cross- linking reactions with either HeLa or SUP-Tl cell lysates led to the identification of new immunoreactive species of roughly 170-, 190-, and 230 kD (lanes 4-9). These experiments also confirmed the observation shown in Figure 10, namely that 15-18-kD-proteins seem to be capable of interacting with the TLE proteins. As shown both in the case of HeLa and SUP-Tl cells, cross-linking reactions facilitated detection of - 110-kD species (see lanes 6 and 9), likely reflecting the association of TLE proteins with low M_r components. Although it is not possible from these experiments to further characterize the composition of the various products of the cross-linking reactions, the apparent molecular weights of some of them indicate that they do not simply represent oligomeric forms of the TLE proteins, but must involve other unrelated proteins. Even though no immunoreactive products could be detected in the 110-kD range when these experiments were performed with Drosophila embryonic extracts, it is worth noting that an approximately 17-kD protein was co-immunoprecipitated from the metabolically labelled Drosophila S2 cell lysates using a monoclonal antibody directed against Enhancer of split m9/10. Thus it seems that the molecular associations involving TLE proteins have been conserved from flies to humans.

Methods:

4-10-hr Drosophila Canton S embryos were collected, dechorionated in 50% Clorox solution, and washed extensively with 0.7% NaCl, 0.02% Triton X-100. Dechorionated embryos were washed twice in ice-cold PBS and then homogenized by 10 strokes of a Dounce homogenizer in buffer H (10 mM HEPES, pH 7.6, 150 mM NaCl, 0.5 mM EDTA, 0.5 mM EGTA, ImM PMSF, 2 μM leupeptin, 2.5 μg/ml aprotinin, 2.5 μg/ml pepstatin A, and 2.5 μg/ml antipain). The homogenate was centrifuged at 13,000 x g for 15 min and the supernatant was recovered and used in the cross-linking experiments. HeLa and SUP-Tl cell lysates were prepared as described in Section 7.1 except that the homogenates were centrifuged at 8,000 x g for 5 min. The resulting supernatants were collected and immediately incubated with the cross-linking agent, DTSSP. Cross-linking reactions were carried out for 30 min at room temperature in the presence of the amounts of DTSSP indicated in Figure 11. At the end of the incubations, glycine and Tris/HCl (pH 8.0) were added to a final concentration of 40 mM each, samples were further incubated for 5 min, and then subjected to SDS-PAGE under non-reducing conditions on 4-15% gradient gels. Following electrophoretic transfer to nitrocellulose, membranes were probed with either monoclonal antibody 3C, directed against Enhancer of split m9/10 (lanes 1-3), or monoclonal antibody C597.4A, directed against the TLE proteins (lanes 4-9).

In summary, these combined results demonstrate that the TLE proteins can interact with other proteins. One such component of these large complexes appears to be a — 17-kD protein(s) that can be detected both in Drosophila and man.

7.4. FURTHER CHARACTERIZATION OF MULTIPROTEIN COMPLEXES CONTAINING TLE PROTEINS In order to investigate more thoroughly the composition of these protein complexes, we have obtained a panel of unique monoclonal antibodies by immunizing animals with a crude preparation of the TLE complex isolated from HeLa cells. These reagents should allow us to identify additional candidate members of the complex, e.g., as follows: The TLE complex is isolated from HeLa cells by obtaining the appropriate gel filtration fractions (Fig. 10). The appropriate fractions containing the complex are then subjected to SDS-PAGE analysis (both under reducing and non-reducing conditions), followed by Western blotting with (a) one of the unique monoclonal antibodies described above; and (b) a monoclonal antibody against one or more of the TLE proteins. An individual protein that is a member of the multiprotein TLE complex is identified by its ability (a) after SDS-PAGE under reducing conditions, to be bound by the unique monoclonal antibody; and (b) after PAGE under nondenaturing conditions, to be bound by the unique monoclonal antibody while in a multiprotein complex, which complex is also able to be bound by the anti-TLE antibody. Cross-linking reagents and immunoprecipitation experiments can also be employed. Furthermore, we have raised antibodies specific for either TLE 1 or TLE 2; these also can be used to characterize the components of the TLE complexes.

8. CHROMOSOMAL MAPPING OF HUMAN TLE GENES

Screening of a human genomic DNA library with a mixture of probes corresponding to the entire sequence of both TLE 1 and TLE 2 resulted in the isolation of 49 clones. Eleven clones were characterized. One clone was shown to be derived from the TLE 1 gene by a combination of sequencing, Southern blotting, and polymerase chain reaction (PCR). Five clones were shown to be derived from the TLE 2 gene by Southern blotting and PCR, two clones were shown to be derived from the TLE 3 gene by Southern blotting and PCR, and two clones were tentatively identified as TLE 3 by Southern blotting and PCR, although it is possible that they are derived from either a pseudogene or a new TLE gene very related to TLE 3. One clone has not conclusively been identified.

These clones were used as probes to determine the chromosomal location of the TLE genes in Fluorescence in situ Hybridization (FISH) analyses. The results of these studies are summarized below in Table III.

Knowledge of the chromosomal location of the TLE genes allows them to find use as markers in the genetic mapping of diseases and disorders.

9. GENERATION OF ANTIBODIES SPECIFICALLY

REACTIVE WITH A HUMAN TLE PROTEIN

A rat monoclonal antibody against TLE 2, designated as C637.2, was obtained using standard procedures, against the synthetic peptide EEERPSGPGGGG (part of SEQ ID NO:4) at position 202-213 of the TLE 2 amino acid sequence. Monoclonal antibody C637.2 specifically recognizes

TLE 2, and does not cross-react with TLE 1 , 3, 4 or Drosophila TLE proteins.

A rat polyclonal antibody against TLE 1 , designated as α-TLE 1 , was obtained using standard procedures, against the synthetic peptide GTDKRRNGPEFS (part of SEQ ID NO:2) at position 210-221 of the TLE 1 amino acid sequence. Antibody α-TLE 1 specifically recognizes TLE 1 , and does not cross-react with TLE 2, 3, 4 or Drosophila TLE proteins.

Monoclonal antibody C597.4A (see Sections 6.1.4 and 6.3.7 supra) recognizes both human and Drosophila TLE proteins.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. Various publications are cited herein, the disclosures of which are incorporated by. reference in their entireties. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Artavanis-Tsakonas, Spyridon Stifani, Stefano Redhead, Nicola J. Hill, Robert E.

(ii) TITLE OF INVENTION: Human Homologs of the Transducin-Like Enhancer of Split Gene and Methods Based Thereon

(iii) NUMBER OF SEQUENCES: 25

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Pennie & Edmonds

(B) STREET: 1155 Avenue of the Americas

(C) CITY: New York

(D) STATE: New York

(E) COUNTRY: U.S.A.

(F) ZIP: 10036

(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 #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: Concurrently Herewith

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Misrock, S. Leslie

(B) REGISTRATION NUMBER: 18,872

(C) REFERENCE/DOCKET NUMBER: 7326-019

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (212) 790-9090

(B) TELEFAX: (212) 7908864/9741

(C) TELEX: 66141 PENNIE

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2352 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (cDNA)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 26..2335

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

ACAGAGCCCC GCCGCCGCCA GAGCG ATG TTC CCG CAG AGC CGG CAC CCG ACG 52

Met Phe Pro Gin Ser Arg His Pro Thr 1 5 CCG CAC CAG GCT GCA GGC CAG CCC TTC AAG TTC ACT ATC CCG GAG TCC 100 Pro His Gin Ala Ala Gly Gin Pro Phe Lys Phe Thr lie Pro Glu Ser 10 15 20 25

CTG GAC CGG ATT AAA GAG GAA TTC CAG TTC CTG CAG GCG CAG TAT CAC 148 Leu Asp Arg lie Lys Glu Glu Phe Gin Phe Leu Gin Ala Gin Tyr His 30 35 40

AGC CTT AAA TTG GAA TGT GAG AAA CTG GCA AGT GAA AAG ACA GAA ATG 196 Ser Leu Lys Leu Glu Cys Glu Lys Leu Ala Ser Glu Lys Thr Glu Met 45 50 55

CAG AGG CAC TAT GTG ATG TAT TAT GAA ATG TCA TAT GGA TTA AAC ATT 244 Gin Arg His Tyr Val Met Tyr Tyr Glu Met Ser Tyr Gly Leu Asn lie 60 65 70

GAA ATG CAC AAA CAG ACT GAA ATC GCC AAG AGA TTG AAT ACG ATT TGT 292 Glu Met His Lys Gin Thr Glu lie Ala Lys Arg Leu Asn Thr lie Cys 75 80 85

GCA CAA GTC ATC CCA TTT CTG TCT CAG GAA CAT CAA CAA CAG GTG GCC 340 Ala Gin Val He Pro Phe Leu Ser Gin Glu His Gin Gin Gin Val Ala 90 95 100 105

CAG GCT GTT GAA CGT GCC AAA CAG GTG ACC ATG GCA GAG TTG AAT GCC 388 Gin Ala Val Glu Arg Ala Lys Gin Val Thr Met Ala Glu Leu Asn Ala 110 115 120

ATC ATC GGG CAG CAG CAG TTG CAA GCT CAG CAT CTT TCT CAT GGC CAC 436 He He Gly Gin Gin Gin Leu Gin Ala Gin His Leu Ser His Gly His 125 130 135

GGA CCC CCA GTT CCC CTT ACG CCT CAC CCT TCG GGA CTT CAG CCT CCT 484 Gly Pro Pro Val Pro Leu Thr Pro His Pro Ser Gly Leu Gin Pro Pro 140 145 150

GGA ATC CCG CCC CTC GGG GGC AGT GCC GGC CTT CTT GCG CTG TCT AGT 532 Gly He Pro Pro Leu Gly Gly Ser Ala Gly Leu Leu Ala Leu Ser Ser 155 160 165

GCT CTG AGT GGG CAG TCT CAC TTG GCA ATA AAA GAT GAC AAG AAG CAC 580 Ala Leu Ser Gly Gin Ser His Leu Ala He Lys Asp Asp Lys Lys His 170 175 180 185

CAC GAT GCA GAG CAC CAC AGA GAC AGA GAG CCG GGC ACA AGT AAT TCC 628 His Asp Ala Glu His His Arg Asp Arg Glu Pro Gly Thr Ser Asn Ser 190 195 200

CTC CTG GTC CCA GAC AGT CTA AGA GGC ACA GAT AAA CGC AGA AAT GGA 676 Leu Leu Val Pro Asp Ser Leu Arg Gly Thr Asp Lys Arg Arg Asn Gly 205 210 215

CCT GAA TTT TCC AAT GAC ATC AAG AAA AGG AAG GTG GAT GAT AAG GAC 724 Pro Glu Phe Ser Asn Asp He Lys Lys Arg Lys Val Asp Asp Lys Asp 220 225 230

TCC AGC CAC TAT GAC AGT GAT GGT GAC AAA AGC GAT GAC AAC TTA GTT 772 Ser Ser His Tyr Asp Ser Asp Gly Asp Lys Ser Asp Asp Asn Leu Val 235 240 245

GTG GAT GTG TCT AAT GAG GAC CCT TCT TCT CCG CGA GCA AGC CCT GCC 820 Val Asp Val Ser Asn Glu Asp Pro Ser Ser Pro Arg Ala Ser Pro Ala 250 255 260 265

CAC TCG CCC CGG GAA AAT GGA ATC GAC AAA AAT CGC CTG CTA AAG AAG 868 His Ser Pro Arg Glu Asn Gly He Asp Lys Asn Arg Leu Leu Lys Lys 270 275 280 GAT GCT TCT AGC AGT CCA GCT TCC ACG GCC TCC TCG GCA AGT TCC ACT 916 Asp Ala Ser Ser Ser Pro Ala Ser Thr Ala Ser Ser Ala Ser Ser Thr 285 290 295

TCT TTG AAA TCC AAA GAA ATG AGC TTG CAT GAA AAA GCC AGC ACG CCT 964 Ser Leu Lys Ser Lys Glu Met Ser Leu His Glu Lys Ala Ser Thr Pro 300 305 310

GTT CTG AAA TCC AGC ACA CCA ACG CCT CGG AGC GAC ATG CCA ACG CCG 1012 Val Leu Lys Ser Ser Thr Pro Thr Pro Arg Ser Asp Met Pro Thr Pro 315 320 325

GGC ACC AGC GCC ACT CCA GGC CTC CGT CCA GGT CTC GGC AAG CCT CCA 1060 Gly Thr Ser Ala Thr Pro Gly Leu Arg Pro Gly Leu Gly Lys Pro Pro 330 335 340 345

GCC ATA GAC CCC CTC GTT AAC CAA GCG GCA GCT GGC TTG AGG ACA CCC 1108 Ala He Asp Pro Leu Val Asn Gin Ala Ala Ala Gly Leu Arg Thr Pro 350 355 360

CTG GCA GTG CCC GGC CCA TAT CCT GCT CCT TTT GGG ATG GTC CCC CAC 1156 Leu Ala Val Pro Gly Pro Tyr Pro Ala Pro Phe Gly Met Val Pro His 365 370 375

GCT GGC ATG AAC GGC GAG CTG ACC AGC CCA GGC GCT GCC TAC GCC AGT 1204 Ala Gly Met Asn Gly Glu Leu Thr Ser Pro Gly Ala Ala Tyr Ala Ser 380 385 390

TTA CAC AAC ATG TCG CCC CAG ATG AGC GCC GCA GCC GCC CGC GGC CGC 1252 Leu His Asn Met Ser Pro Gin Met Ser Ala Ala Ala Ala Arg Gly Arg 395 400 405

CGT GGT CGG TAC GGG CGC TCC CCC ATG GTG GGG TTT GAT CCT CCC CCT 1300 Arg Gly Arg Tyr Gly Arg Ser Pro Met Val Gly Phe Asp Pro Pro Pro 410 415 420 425

CAC ATG AGA GTA CCT ACC ATT CCT CCA AAC CTG GCA GGA ATC CCT GGG 1348 His Met Arg Val Pro Thr He Pro Pro Asn Leu Ala Gly He Pro Gly 430 435 440

GGG AAA CCT GCA TAC TCC TTC CAC GTT ACT GCA GAC GGT CAG ATG CAG 1396 Gly Lys Pro Ala Tyr Ser Phe His Val Thr Ala Asp Gly Gin Met Gin 445 450 455

CCT GTC CCT TTT CCC CCG ACG CCC CTC ATC GGA CCC GGA ATC CCC CGG 1444 Pro Val Pro Phe Pro Pro Thr Pro Leu He Gly Pro Gly He Pro Arg 460 465 470

CAT GCT CGC CAG ATC AAC ACC CTC AAC CAC GGG GAG GTG GTG TGC GCT 1492 His Ala Arg Gin He Asn Thr Leu Asn His Gly Glu Val Val Cys Ala 475 480 485

GTG ACC ATC AGC AAC CCC ACG AGA CAC GTG TAC ACA GGC GGG AAG GGC 1540 Val Thr He Ser Asn Pro Thr Arg His Val Tyr Thr Gly Gly Lys Gly 490 495 500 505

TGC GTC AAG GTC TGG GAC ATC AGC CAC CCT GGC AAT AAG AGC CCT GTC 1588 Cys Val Lys Val Trp Asp He Ser His Pro Gly Asn Lys Ser Pro Val 510 515 520

TCC CAG CTC GAC TGT CTG AAC AGA GAC AAT TAT ATC CGT TCC TGT AAA 1636 Ser Gin Leu Asp Cys Leu Asn Arg Asp Asn Tyr He Arg Ser Cys Lys 525 530 535

TTG CTA CCC GAT GGC TGC ACT CTC ATA GTG GGA GGG GAA GCC AGT ACT 1684 Leu Leu Pro Asp Gly Cys Thr Leu He Val Gly Gly Glu Ala Ser Thr 540 545 550 TTG TCC ATT TGG GAC CTG GCG GCT CCA ACC CCG CGC ATC AAG GCG GAG 1732 Leu Ser He Trp Asp Leu Ala Ala Pro Thr Pro Arg He Lys Ala Glu 555 560 565

CTG ACG TCC TCG GCC CCC GCC TGC TAC GCC CTG GCC ATC AGC CCC GAT 1780 Leu Thr Ser Ser Ala Pro Ala Cys Tyr Ala Leu Ala He Ser Pro Asp 570 575 580 585

TCC AAG GTC TGC TTC TCA TGC TGC AGC GAC GGC AAC ATC GCT GTG TGG 1828 Ser Lys Val Cys Phe Ser Cys Cys Ser Asp Gly Asn He Ala Val Trp 590 595 600

GAT CTG CAC AAC CAG ACA CTA GTG AGG CAA TTC CAG GGC CAC ACA GAC 1876 Asp Leu His Asn Gin Thr Leu Val Arg Gin Phe Gin Gly His Thr Asp 605 610 615

GGA GCC AGC TGT ATT GAC ATT TCT AAT GAT GGC ACC AAG CTC TGG ACG 1924 Gly Ala Ser Cys He Asp He Ser Asn Asp Gly Thr Lys Leu Trp Thr 620 625 630

GGT GGT TTG GAC AAC ACA GTC AGG TCC TGG GAC CTG CGC GAG GGG CGG 1972 Gly Gly Leu Asp Asn Thr Val Arg Ser Trp Asp Leu Arg Glu Gly Arg 635 640 645

CAG CTG CAG CAG CAC GAC TTC ACC TCC CAG ATC TTC TCC CTG GGG TAC 2020 Gin Leu Gin Gin His Asp Phe Thr Ser Gin He Phe Ser Leu Gly Tyr 650 655 660 665

TGC CCC ACC GGG GAG TGG CTG GCA GTG GGC ATG GAG AGC AGC AAT GTG 2068 Cys Pro Thr Gly Glu Trp Leu Ala Val Gly Met Glu Ser Ser Asn Val 670 675 680

GAG GTG CTG CAC GTG AAC AAG CCT GAC AAG TAC CAG CTG CAC CTG CAT 2116 Glu Val Leu His Val Asn Lys Pro Asp Lys Tyr Gin Leu His Leu His 685 690 695

GAG AGC TGC GTG CTG TCC CTG AAA TTT GCT TAC TGT GGT AAA TGG TTT 2164 Glu Ser Cys Val Leu Ser Leu Lys Phe Ala Tyr Cys Gly Lys Trp Phe 700 705 710

GTG AGT ACT GGA AAA GAT AAC CTC CTC AAT GCT TGG CGG ACC CCC TAT 2212 Val Ser Thr Gly Lys Asp Asn Leu Leu Asn Ala Trp Arg Thr Pro Tyr 715 720 725

GGA GCC AGC ATA TTC CAG TCC AAA GAG TCC TCG TCA GTG CTT AGC TGT 2260 Gly Ala Ser He Phe Gin Ser Lys Glu Ser Ser Ser Val Leu Ser Cys 730 735 740 745

GAC ATC TCT GTG GAT GAT AAG TAC ATA GTC ACT GGC TCG GGG GAC AAG 2308 Asp He Ser Val Asp Asp Lys Tyr He Val Thr Gly Ser Gly Asp Lys 750 755 760

AAG GCT ACA GTC TAT GAA GTC ATC TAC TGAAAACATT ATGTGGT 2352 Lys Ala Thr Val Tyr Glu Val He Tyr 765 770

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 770 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Phe Pro Gin Ser Arg His Pro Thr Pro His Gin Ala Ala Gly Gin 1 5 10 15

Pro Phe Lys Phe Thr He Pro Glu Ser Leu Asp Arg He Lys Glu Glu 20 25 30

Phe Gin Phe Leu Gin Ala Gin Tyr His Ser Leu Lys Leu Glu Cys Glu 35 40 45

Lys Leu Ala Ser Glu Lys Thr Glu Met Gin Arg His Tyr Val Met Tyr 50 55 60

Tyr Glu Met Ser Tyr Gly Leu Asn He Glu Met His Lys Gin Thr Glu 65 70 75 80

He Ala Lys Arg Leu Asn Thr He Cys Ala Gin Val He Pro Phe Leu 85 90 95

Ser Gin Glu His Gin Gin Gin Val Ala Gin Ala Val Glu Arg Ala Lys 100 105 110

Gin Val Thr Met Ala Glu Leu Asn Ala He He Gly Gin Gin Gin Leu 115 120 125

Gin Ala Gin His Leu Ser His Gly His Gly Pro Pro Val Pro Leu Thr 130 135 140

Pro His Pro Ser Gly Leu Gin Pro Pro Gly He Pro Pro Leu Gly Gly 145 150 155 160

Ser Ala Gly Leu Leu Ala Leu Ser Ser Ala Leu Ser Gly Gin Ser His 165 170 175

Leu Ala He Lys Asp Asp Lys Lys His His Asp Ala Glu His His Arg 180 185 190

Asp Arg Glu Pro Gly Thr Ser Asn Ser Leu Leu Val Pro Asp Ser Leu 195 200 205

Arg Gly Thr Asp Lys Arg Arg Asn Gly Pro Glu Phe Ser Asn Asp He 210 215 220

Lys Lys Arg Lys Val Asp Asp Lys Asp Ser Ser His Tyr Asp Ser Asp 225 230 235 240

Gly Asp Lys Ser Asp Asp Asn Leu Val Val Asp Val Ser Asn Glu Asp 245 250 255

Pro Ser Ser Pro Arg Ala Ser Pro Ala His Ser Pro Arg Glu Asn Gly 260 265 270

He Asp Lys Asn Arg Leu Leu Lys Lys Asp Ala Ser Ser Ser Pro Ala 275 280 285

Ser Thr Ala Ser Ser Ala Ser Ser Thr Ser Leu Lys Ser Lys Glu Met 290 295 300

Ser Leu His Glu Lys Ala Ser Thr Pro Val Leu Lys Ser Ser Thr Pro 305 310 315 320

Thr Pro Arg Ser Asp Met Pro Thr Pro Gly Thr Ser Ala Thr Pro Gly 325 330 335

Leu Arg Pro Gly Leu Gly Lys Pro Pro Ala He Asp Pro Leu Val Asn 340 345 350

Gin Ala Ala Ala Gly Leu Arg Thr Pro Leu Ala Val Pro Gly Pro Tyr 355 360 365

Pro Ala Pro Phe Gly Met Val Pro His Ala Gly Met Asn Gly Glu Leu 370 375 380

Thr Ser Pro Gly Ala Ala Tyr Ala Ser Leu His Asn Met Ser Pro Gin 385 390 395 400

Met Ser Ala Ala Ala Ala Arg Gly Arg Arg Gly Arg Tyr Gly Arg Ser 405 410 415

Pro Met Val Gly Phe Asp Pro Pro Pro His Met Arg Val Pro Thr He 420 425 430

Pro Pro Asn Leu Ala Gly He Pro Gly Gly Lys Pro Ala Tyr Ser Phe 435 440 445

His Val Thr Ala Asp Gly Gin Met Gin Pro Val Pro Phe Pro Pro Thr 450 455 460

Pro Leu He Gly Pro Gly He Pro Arg His Ala Arg Gin He Asn Thr 465 470 475 480

Leu Asn His Gly Glu Val Val Cys Ala Val Thr He Ser Asn Pro Thr 485 490 495

Arg His Val Tyr Thr Gly Gly Lys Gly Cys Val Lys Val Trp Asp He 500 505 510

Ser His Pro Gly Asn Lys Ser Pro Val Ser Gin Leu Asp Cys Leu Asn 515 520 525

Arg Asp Asn Tyr He Arg Ser Cys Lys Leu Leu Pro Asp Gly Cys Thr 530 535 540

Leu He Val Gly Gly Glu Ala Ser Thr Leu Ser He Trp Asp Leu Ala 545 550 555 560

Ala Pro Thr Pro Arg He Lys Ala Glu Leu Thr Ser Ser Ala Pro Ala 565 570 575

Cys Tyr Ala Leu Ala He Ser Pro Asp Ser Lys Val Cys Phe Ser Cys 580 585 590

Cys Ser Asp Gly Asn He Ala Val Trp Asp Leu His Asn Gin Thr Leu 595 600 605

Val Arg Gin Phe Gin Gly His Thr Asp Gly Ala Ser Cys He Asp He 610 615 620

Ser Asn Asp Gly Thr Lys Leu Trp Thr Gly Gly Leu Asp Asn Thr Val 625 630 635 640

Arg Ser Trp Asp Leu Arg Glu Gly Arg Gin Leu Gin Gin His Asp Phe 645 650 655

Thr Ser Gin He Phe Ser Leu Gly Tyr Cys Pro Thr Gly Glu Trp Leu 660 665 670

Ala Val Gly Met Glu Ser Ser Asn Val Glu Val Leu His Val Asn Lys 675 680 685

Pro Asp Lys Tyr Gin Leu His Leu His Glu Ser Cys Val Leu Ser Leu 690 695 700

Lys Phe Ala Tyr Cys Gly Lys Trp Phe Val Ser Thr Gly Lys Asp Asn 705 710 715 720 Leu Leu Asn Ala Trp Arg Thr Pro Tyr Gly Ala Ser He Phe Gin Ser 725 730 735

Lys Glu Ser Ser Ser Val Leu Ser Cys Asp He Ser Val Asp Asp Lys 740 745 750

Tyr He Val Thr Gly Ser Gly Asp Lys Lys Ala Thr Val Tyr Glu Val 755 760 765

He Tyr 770

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2271 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (cDNA)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 26..2254

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

CTGGGGGGCT TTTCGAATCG GCAGG ATG TAC CCC CAG GGA AGG CAC CCG ACC 52

Met Tyr Pro Gin Gly Arg His Pro Thr 1 5

CCG CTC CAG TCC GGC CAG CCC TTC AAG TTC TCG ATC TTG GAG ATC TGC 100 Pro Leu Gin Ser Gly Gin Pro Phe Lys Phe Ser He Leu Glu He Cys 10 15 20 25

GAC CGC ATC AAA GAA GAA TTC CAG TTT CTT CAG GCT CAA TAC CAC AGC 148 Asp Arg He Lys Glu Glu Phe Gin Phe Leu Gin Ala Gin Tyr His Ser 30 35 40

CTC AAG CTA GAA TGT GAG AAG CTG GCC AGC GAG AAG ACG GAA ATG CAG 196 Leu Lys Leu Glu Cys Glu Lys Leu Ala Ser Glu Lys Thr Glu Met Gin 45 50 55

CGA CAT TAT GTC ATG TAT TAT GAG ATG TCG TAC GGG CTC AAC ATT GAA 244 Arg His Tyr Val Met Tyr Tyr Glu Met Ser Tyr Gly Leu Asn He Glu 60 65 70

ATG CAT AAG CAG GCG GAG ATT GTG AAG CGT CTG AGC GGT ATC TGC GCT 292 Met His Lys Gin Ala Glu He Val Lys Arg Leu Ser Gly He Cys Ala 75 80 85

CAG ATT ATC CCC TTC CTG ACC CAG GAG CAT CAG CAG CAG GTG CTC CAG 340 Gin He He Pro Phe Leu Thr Gin Glu His Gin Gin Gin Val Leu Gin 90 95 100 105

GCC GTA GAA CGC GCC AAG CAG GTC ACC GTG GGG GAG CTG AAC AGC CTC 388 Ala Val Glu Arg Ala Lys Gin Val Thr Val Gly Glu Leu Asn Ser Leu 110 115 120

ATC GGG CAG CAG CTC CAG CCG CTG TCC CAC CAC GCA CCC CCT GTG CCC 436 He Gly Gin Gin Leu Gin Pro Leu Ser His His Ala Pro Pro Val Pro 125 130 135

CTC ACC CCC CGC CCA GCC GGG CTG GTG GGC GGC AGT GCT ACG GGG CTG 484 Leu Thr Pro Arg Pro Ala Gly Leu Val Gly Gly Ser Ala Thr Gly Leu 140 145 150

CTT GCT CTG TCT GGA GCC CTG GCT GCC CAG GCT CAG CTG GCG GCG GCT 532 Leu Ala Leu Ser Gly Ala Leu Ala Ala Gin Ala Gin Leu Ala Ala Ala 155 160 165

GTC AAG GAG GAC CGT GCG GGC GTG GAG GCC GAG GGG TCC AGA GTG GAG 580 Val Lys Glu Asp Arg Ala Gly Val Glu Ala Glu Gly Ser Arg Val Glu 170 175 180 185

AGA GCC CCG AGC AGG AGT GCA TCT CCC TCG CCC CCT GAG AGT CTC GTG 628 Arg Ala Pro Ser Arg Ser Ala Ser Pro Ser Pro Pro Glu Ser Leu Val 190 195 200

GAG GAG GAG CGA CCG AGT GGC CCT GGT GGT GGC GGG AAG CAG AGA GCA 676 Glu Glu Glu Arg Pro Ser Gly Pro Gly Gly Gly Gly Lys Gin Arg Ala 205 210 215

GAT GAG AAG GAG CCA TCA GGA CCT TAT GAA AGC GAC GAA GAC AAG AGT 724 Asp Glu Lys Glu Pro Ser Gly Pro Tyr Glu Ser Asp Glu Asp Lys Ser 220 225 230

GAT TAC AAT CTG GTG GTG GAC GAG GAC CAA CCC TCA GAG CCC CCC AGC 772 Asp Tyr Asn Leu Val Val Asp Glu Asp Gin Pro Ser Glu Pro Pro Ser 235 240 245

CCG GCT ACC ACC CCC TGC GGA AAG GTA CCC ATC TGC ATT CCT GCC CGT 820 Pro Ala Thr Thr Pro Cys Gly Lys Val Pro He Cys He Pro Ala Arg 250 255 260 265

CGG GAC CTG GTG GAC AGT CCA GCC TCC TTG GCC TCT AGC TTG CGG TCA 868 Arg Asp Leu Val Asp Ser Pro Ala Ser Leu Ala Ser Ser Leu Arg Ser 270 275 280

CCG CTG CCT AGA GCC AAG GAG CTC ATC CTG AAT GAC CTT CCC GCC AGC 916 Pro Leu Pro Arg Ala Lys Glu Leu He Leu Asn Asp Leu Pro Ala Ser 285 290 295

ACT CCT GCC TCC AAA TCC TGT GAC TCC TCC CCG CCC CAG GAC GCT TCC 964 Thr Pro Ala Ser Lys Ser Cys Asp Ser Ser Pro Pro Gin Asp Ala Ser 300 305 310

ACC CCC GGG CCC AGC TCG GCC AGT CAC CTC TGC CAG CTT GCG CTC AAG 1012 Thr Pro Gly Pro Ser Ser Ala Ser His Leu Cys Gin Leu Ala Leu Lys 315 320 325

CCA GCA CCT TCC ACG GAC AGC GTC GCC CTG AGG AGC CCC CTG ACT CTG 1060 Pro Ala Pro Ser Thr Asp Ser Val Ala Leu Arg Ser Pro Leu Thr Leu 330 335 340 345

TCC AGT CCC TTC ACC ACG TCC TTC AGC CTG GGC TCC CAC AGC ACT CTC 1108 Ser Ser Pro Phe Thr Thr Ser Phe Ser Leu Gly Ser His Ser Thr Leu 350 355 360

AAC GGA GAC CTC TCC GTG CCC AGC TCC TAC GTC AGC CTC CAC CTG TCC 1156 Asn Gly Asp Leu Ser Val Pro Ser Ser Tyr Val Ser Leu His Leu Ser 365 370 375

CCC CAG GTC AGC AGC TCT GTG GTG TAC GGA CGC TCC CCC GTG ATG GCA 1204 Pro Gin Val Ser Ser Ser Val Val Tyr Gly Arg Ser Pro Val Met Ala 380 385 390

TTT GAG TCT CAT CCC CAT CTC CGA GGG TCA TCC GTC TCT TCC TCC CTA 1252 Phe Glu Ser His Pro His Leu Arg Gly Ser Ser Val Ser Ser Ser Leu 395 400 405 CCC AGC ATC CCT GGG GGA AAG CCG GCC TAC TCC TTC CAC GTG TCT GCG 1300 Pro Ser He Pro Gly Gly Lys Pro Ala Tyr Ser Phe His Val Ser Ala 410 415 420 425

GAC GGG CAG ATG CAG CCG GTT CCC TTC CCC TCG GAT GCA CTG GTA GAC 1348 Aβp Gly Gin Met Gin Pro Val Pro Phe Pro Ser Asp Ala Leu Val Asp 430 435 440

GCG GGC ATC CCG CGG CAC GCC CGG CAG CTG CAC ACG CTG GCC CAT GGC 1396 Ala Gly He Pro Arg His Ala Arg Gin Leu His Thr Leu Ala His Gly 445 450 455

GAG GTG GTC TGC GCG GTC ACC ATC AGC GGC TCC ACA CAG CAT GTG TAC 1444 Glu Val Val Cys Ala Val Thr He Ser Gly Ser Thr Gin His Val Tyr 460 465 470

ACG GGC GGC AAG GGC TGT GTG AAG GTG TGG GAC GTG GGC CAG CCT GGG 1492 Thr Gly Gly Lys Gly Cys Val Lys Val Trp Asp Val Gly Gin Pro Gly' 475 480 485

GCC AAG ACG CCC GTG CGC CAG CTC GAC TGC CTG AAC CGA GAC AAC TAC 1540 Ala Lys Thr Pro Val Arg Gin Leu Asp Cys Leu Asn Arg Asp Asn Tyr 490 495 500 505

ATT CGT TCC TGC AAG TTG CTG CCG GAT GGC CGG AGT CTG ATC GTG GGC 1588 He Arg Ser Cys Lys Leu Leu Pro Asp Gly Arg Ser Leu He Val Gly 510 515 520

GGT GAG GCC AGC ACC TTG TCC ATT TGG GAC CTG GCG GCG CCC ACC CCC 1636 Gly Glu Ala Ser Thr Leu Ser He Trp Asp Leu Ala Ala Pro Thr Pro 525 530 535

CGT ATC AAG GCC GAG CTG ACT TCC TCA GCC CCA GCC TGC TAC GCC CTG 1684 Arg He Lys Ala Glu Leu Thr Ser Ser Ala Pro Ala Cys Tyr Ala Leu 540 545 550

GCC GTC AGC CCC GAC GCC AAG GTT TGC TTC TCC TGC TGC AGC GAT GGC 1732 Ala Val Ser Pro Asp Ala Lys Val Cys Phe Ser Cys Cys Ser Asp Gly 555 560 565

AAC ATT GTG GTC TGG GAC CTG CAG AAT CAG ACT ATG GTC AGG CAG TTC 1780 Asn He Val Val Trp Asp Leu Gin Asn Gin Thr Met Val Arg Gin Phe 570 575 580 585

CAG GGC CAC ACG GAC GGC GCC AGC TGC ATT GAT ATT TCC GAT TAC GGC 1828 Gin Gly His Thr Asp Gly Ala Ser Cys He Asp He Ser Asp Tyr Gly 590 595 600

ACT CGG CTC TGG ACA GGG GGC CTG GAC AAC ACG GTG CGC TGC TGG GAC 1876 Thr Arg Leu Trp Thr Gly Gly Leu Asp Asn Thr Val Arg Cys Trp Asp 605 610 615

CTG CGG GAG GGC CGC CAG CTG CAG CAG CAT GAC TTC AGC TCC CAG ATT 1924 Leu Arg Glu Gly Arg Gin Leu Gin Gin His Asp Phe Ser Ser Gin He 620 625 630

TTC TCC CCC TGC CAC TGC CCT AAC CAG GAC TGG CTG GCG GTC GGA ATG 1972 Phe Ser Pro Cys His Cys Pro Asn Gin Asp Trp Leu Ala Val Gly Met 635 640 645

GAG AGT AGC AAC GTG GAG ATC CTG CAC GTC GGC AAG CCG GAG AAA TAC 2020 Glu Ser Ser Asn Val Glu He Leu His Val Gly Lys Pro Glu Lys Tyr 650 655 660 665

CAG CTG CAC CTC CAC GAG AGC TGC GTG CTG TCC CTG AAG TTT GCC CCT 2068 Gin Leu His Leu His Glu Ser Cys Val Leu Ser Leu Lys Phe Ala Pro 670 675 680 TGC GGA CGG TGG TTT GTG AGC ACC GGG AAG GAC AAC CTG CTC AAC GCC 2116 Cys Gly Arg Trp Phe Val Ser Thr Gly Lys Asp Asn Leu Leu Asn Ala 685 690 695

TGG AGG ACG CCG TAC GGG GCC AGC ATT TTC CAG TCC AAG GAG TCG TCC 2164 Trp Arg Thr Pro Tyr Gly Ala Ser He Phe Gin Ser Lys Glu Ser Ser 700 705 710

TCA GTC CTG AGT TGT GAC ATC TCC AGA AAT AAC AAA TAC ATT GTG ACA 2212 Ser Val Leu Ser Cys Asp He Ser Arg Asn Asn Lys Tyr He Val Thr 715 720 725

GGC TCG GGG GAC AAG AAG GCC ACC GTG TAT GAG GTG GTC TAC 2254

Gly Ser Gly Asp Lys Lys Ala Thr Val Tyr Glu Val Val Tyr 730 735 740

TGAAGACATG ACCCCCC 2271

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 743 amino acids

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

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Tyr Pro Gin Gly Arg His Pro Thr Pro Leu Gin Ser Gly Gin Pro 1 5 10 15

Phe Lys Phe Ser He Leu Glu He Cys Asp Arg He Lys Glu Glu Phe 20 25 30

Gin Phe Leu Gin Ala Gin Tyr His Ser Leu Lys Leu Glu Cys Glu Lys 35 40 45

Leu Ala Ser Glu Lys Thr Glu Met Gin Arg His Tyr Val Met Tyr Tyr 50 55 60

Glu Met Ser Tyr Gly Leu Asn He Glu Met His Lys Gin Ala Glu He 65 70 75 80

Val Lys Arg Leu Ser Gly He Cys Ala Gin He He Pro Phe Leu Thr 85 90 95

Gin Glu His Gin Gin Gin Val Leu Gin Ala Val Glu Arg Ala Lys Gin 100 105 110

Val Thr Val Gly Glu Leu Asn Ser Leu He Gly Gin Gin Leu Gin Pro 115 120 125

Leu Ser His His Ala Pro Pro Val Pro Leu Thr Pro Arg Pro Ala Gly 130 135 140

Leu Val Gly Gly Ser Ala Thr Gly Leu Leu Ala Leu Ser Gly Ala Leu 145 150 155 160

Ala Ala Gin Ala Gin Leu Ala Ala Ala Val Lys Glu Asp Arg Ala Gly 165 170 175

Val Glu Ala Glu Gly Ser Arg Val Glu Arg Ala Pro Ser Arg Ser Ala 180 185 190

Ser Pro Ser Pro Pro Glu Ser Leu Val Glu Glu Glu Arg Pro Ser Gly 195 200 205

Pro Gly Gly Gly Gly Lys Gin Arg Ala Asp Glu Lys Glu Pro Ser Gly 210 215 220

Pro Tyr Glu Ser Asp Glu Asp Lys Ser Asp Tyr Asn Leu Val Val Asp 225 230 235 240

Glu Asp Gin Pro Ser Glu Pro Pro Ser Pro Ala Thr Thr Pro Cys Gly 245 250 255

Lys Val Pro He Cys He Pro Ala Arg Arg Asp Leu Val Asp Ser Pro 260 265 270

Ala Ser Leu Ala Ser Ser Leu Arg Ser Pro Leu Pro Arg Ala Lys Glu 275 280 285

Leu He Leu Asn Asp Leu Pro Ala Ser Thr Pro Ala Ser Lys Ser Cys. 290 295 300

Asp Ser Ser Pro Pro Gin Asp Ala Ser Thr Pro Gly Pro Ser Ser Ala 305 310 315 320

Ser His Leu Cys Gin Leu Ala Leu Lys Pro Ala Pro Ser Thr Asp Ser 325 330 335

Val Ala Leu Arg Ser Pro Leu Thr Leu Ser Ser Pro Phe Thr Thr Ser 340 345 350

Phe Ser Leu Gly Ser His Ser Thr Leu Asn Gly Asp Leu Ser Val Pro 355 360 365

Ser Ser Tyr Val Ser Leu His Leu Ser Pro Gin Val Ser Ser Ser Val 370 375 380

Val Tyr Gly Arg Ser Pro Val Met Ala Phe Glu Ser His Pro His Leu 385 390 395 400

Arg Gly Ser Ser Val Ser Ser Ser Leu Pro Ser He Pro Gly Gly Lys 405 410 415

Pro Ala Tyr Ser Phe His Val Ser Ala Asp Gly Gin Met Gin Pro Val 420 425 430

Pro Phe Pro Ser Asp Ala Leu Val Asp Ala Gly He Pro Arg His Ala 435 440 445

Arg Gin Leu His Thr Leu Ala His Gly Glu Val Val Cys Ala Val Thr 450 455 460

He Ser Gly Ser Thr Gin His Val Tyr Thr Gly Gly Lys Gly Cys Val 465 470 475 480

Lys Val Trp Asp Val Gly Gin Pro Gly Ala Lys Thr Pro Val Arg Gin 485 490 495

Leu Asp Cys Leu Asn Arg Asp Asn Tyr He Arg Ser Cys Lys Leu Leu 500 505 510

Pro Asp Gly Arg Ser Leu He Val Gly Gly Glu Ala Ser Thr Leu Ser 515 520 525

He Trp Asp Leu Ala Ala Pro Thr Pro Arg He Lys Ala Glu Leu Thr 530 535 540

Ser Ser Ala Pro Ala Cys Tyr Ala Leu Ala Val Ser Pro Asp Ala Lys 545 550 555 560 Val Cys Phe Ser Cys Cys Ser Asp Gly Asn He Val Val Trp Asp Leu 565 570 575

Gin Asn Gin Thr Met Val Arg Gin Phe Gin Gly His Thr Asp Gly Ala 580 585 590

Ser Cys He Asp He Ser Asp Tyr Gly Thr Arg Leu Trp Thr Gly Gly 595 600 605

Leu Asp Asn Thr Val Arg Cys Trp Asp Leu Arg Glu Gly Arg Gin Leu 610 615 620

Gin Gin His Asp Phe Ser Ser Gin He Phe Ser Pro Cys His Cys Pro 625 630 635 640

Asn Gin Asp Trp Leu Ala Val Gly Met Glu Ser Ser Asn Val Glu He 645 650 655

Leu His Val Gly Lys Pro Glu Lys Tyr Gin Leu His Leu His Glu Ser 660 665 670

Cys Val Leu Ser Leu Lys Phe Ala Pro Cys Gly Arg Trp Phe Val Ser 675 680 685

Thr Gly Lys Asp Asn Leu Leu Asn Ala Trp Arg Thr Pro Tyr Gly Ala 690 695 700

Ser He Phe Gin Ser Lys Glu Ser Ser Ser Val Leu Ser Cys Asp He 705 710 715 720

Ser Arg Asn Asn Lys Tyr He Val Thr Gly Ser Gly Asp Lys Lys Ala 725 730 735

Thr Val Tyr Glu Val Val Tyr 740

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2357 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (cDNA)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 22..2337

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

GAATCACGAC CCCTCCCTGC C ATG TAT CCG CAG GGC AGA CAT CCG GCT CCC 51

Met Tyr Pro Gin Gly Arg His Pro Ala Pro 1 5 10

CAT CAA CCC GGG CAG CCG GGA TTT AAA TTC ACG GTG GCT GAG TCT TGT 99 His Gin Pro Gly Gin Pro Gly Phe Lys Phe Thr Val Ala Glu Ser Cys 15 20 25

GAC AGG ATC AAA GAC GAA TTC CAG TTC CTG CAA GCT CAG TAT CAC AGC 147 Asp Arg He Lys Asp Glu Phe Gin Phe Leu Gin Ala Gin Tyr His Ser 30 35 40

CTC AAA GTG GAG TAC GAC AAG CTG GCA AAC GAG AAG ACG GAG ATG CAG 195 Leu Lys Val Glu Tyr Asp Lys Leu Ala Asn Glu Lys Thr Glu Met Gin 45 50 55

CGC CAT TAT GTG ATG TAC TAT GAG ATG TCC TAT GGC TTG AAC ATT GAA 243 Arg His Tyr Val Met Tyr Tyr Glu Met Ser Tyr Gly Leu Asn He Glu 60 65 70

ATG CAC AAG CAG ACA GAG ATT GCG AAG AGA CTG AAC ACA ATT TTA GCA 291 Met His Lys Gin Thr Glu He Ala Lys Arg Leu Asn Thr He Leu Ala 75 80 85 90

CAG ATC ATG CCT TTC CTG TCA CAA GAG CAC CAG CAG CAG GTG GCG CAG 339 Gin He Met Pro Phe Leu Ser Gin Glu His Gin Gin Gin Val Ala Gin 95 100 105

GCA GTG GAG CGC GCC AAG CAG GTC ACC ATG ACG GAG CTG AAC GCC ATC 387 Ala Val Glu Arg Ala Lys Gin Val Thr Met Thr Glu Leu Asn Ala He 110 115 120

ATC GGG CAG CAG CAG CTC CAG GCG CAG CAC CTC TCC CAT GCC ACA CAC 435 He Gly Gin Gin Gin Leu Gin Ala Gin His Leu Ser His Ala Thr His 125 130 135

GGC CCC CCG GTC CAG TTG CCA CCC CAC CCG TCA GGT CTC CAG CCT CCA 483 Gly Pro Pro Val Gin Leu Pro Pro His Pro Ser Gly Leu Gin Pro Pro 140 145 150

GGA ATC CCC CCA GTG ACA GGG AGC AGC TCC GGG CTG CTG GCA CTG GGC 531 Gly He Pro Pro Val Thr Gly Ser Ser Ser Gly Leu Leu Ala Leu Gly 155 160 165 170

GCC CTG GGC AGC CAG GCC CAT CTG ACG GTG AAG GAT GAG AAG AAC CAC 579 Ala Leu Gly Ser Gin Ala His Leu Thr Val Lys Asp Glu Lys Asn His 175 180 185

CAT GAA CTC GAT CAC AGA GAG AGA GAA TCC AGT GCG AAT AAC TCT GTG 627 His Glu Leu Asp His Arg Glu Arg Glu Ser Ser Ala Asn Asn Ser Val 190 195 200

TCA CCC TCG GAA AGC CTC CGG GCC AGT GAG AAG CAC CGG GGC TCT GCG 675 Ser Pro Ser Glu Ser Leu Arg Ala Ser Glu Lys His Arg Gly Ser Ala 205 210 215

GAC TAC AGC ATG GAA GCC AAG AAG CGG AAG GTG GAG GAG AAG GAC AGC 723 Asp Tyr Ser Met Glu Ala Lys Lys Arg Lys Val Glu Glu Lys Asp Ser 220 225 230

TTG AGC CGA TAC GAC AGT GAT GGA GAC AAG AGT GAT GAT CTG GTG GTG 771 Leu Ser Arg Tyr Asp Ser Asp Gly Asp Lys Ser Asp Asp Leu Val Val 235 240 245 250

GAT GTT TCC AAT GAG GAC CCC GCA ACG CCC CGG GTC AGC CCG GCA CAC 819 Asp Val Ser Asn Glu Asp Pro Ala Thr Pro Arg Val Ser Pro Ala His 255 260 265

TCC CCT CCT GAA AAT GGG CTG GAC AAG GCC CGT AGC CTG AAA AAA GAT 867 Ser Pro Pro Glu Asn Gly Leu Asp Lys Ala Arg Ser Leu Lys Lys Asp 270 275 280

GCC CCC ACC AGC CCT GCC TCG GTG GCC TCT TCC AGT AGC ACA CCT TCC 915 Ala Pro Thr Ser Pro Ala Ser Val Ala Ser Ser Ser Ser Thr Pro Ser 285 290 295

TCC AAG ACC AAA GAC CTT GGT CAT AAC GAC AAA TCC TCC ACC CCT GGG 963 Ser Lys Thr Lys Asp Leu Gly His Asn Asp Lys Ser Ser Thr Pro Gly 300 305 310 CTC AAG TCC AAC ACA CCA ACC CCA AGG AAC GAC GCC CCA ACT CCA GGC 1011 Leu Lys Ser Asn Thr Pro Thr Pro Arg Asn Asp Ala Pro Thr Pro Gly 315 320 325 330

ACC AGC ACG ACC CCA GGG CTC AGG TCG ATG CCG GGT AAA CCT CCG GGC 1059 Thr Ser Thr Thr Pro Gly Leu Arg Ser Met Pro Gly Lys Pro Pro Gly 335 340 345

ATG GAC CCG ATA GGT ATA ATG GCC TCG GCT CTG CGC ACG CCC ATC TCC 1107 Met Asp Pro He Gly He Met Ala Ser Ala Leu Arg Thr Pro He Ser 350 355 360

ATC ACC AGC TCC TAT GCG GCG CCC TTC GCC ATG ATG AGC CAC CAT GAG 1155 He Thr Ser Ser Tyr Ala Ala Pro Phe Ala Met Met Ser His His Glu 365 370 375

ATG AAC GGC TCC CTC ACC AGT CCT GGC GCC TAC GCC GGC CTC CAC AAC 1203 Met Asn Gly Ser Leu Thr Ser Pro Gly Ala Tyr Ala Gly Leu His Asn- 380 385 390

ATC CCA CCC CAG ATG AGC GCC GCC GCC GCT GCT GCA GCC GCT GCC TAT 1251 He Pro Pro Gin Met Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Tyr 395 400 405 410

GGC CGA TCG CCA ATG GTG AGC TTT GGA GCT GTT GGT TTT GAC CCT CAC 1299 Gly Arg Ser Pro Met Val Ser Phe Gly Ala Val Gly Phe Asp Pro His 415 420 425

CCC CCG ATG CGG GCC ACA GGC CTC CCC TCA AGC CTG GCC TCC ATT CCT 1347 Pro Pro Met Arg Ala Thr Gly Leu Pro Ser Ser Leu Ala Ser He Pro 430 435 440

GGA GGA AAA CCA GCG TAC TCA TTC CAT GTG AGT GCT GAT GGG CAG ATG 1395 Gly Gly Lys Pro Ala Tyr Ser Phe His Val Ser Ala Asp Gly Gin Met 445 450 455

CAG CCC GTG CCC TTC CCC CAC GAC GCC CTG GCA GGC CCC GGC ATC CCG 1443 Gin Pro Val Pro Phe Pro His Asp Ala Leu Ala Gly Pro Gly He Pro 460 465 470

AGG CAC GCC CGG CAG ATC AAC ACA CTC AGC CAC GGG GGG GTG GTG TGT 1491 Arg His Ala Arg Gin He Asn Thr Leu Ser His Gly Gly Val Val Cys 475 480 485 490

GCC GTG ACC ATC AGC AAC CCC AGC AGG CAC GTC TAC ACA GGT GGC AAG 1539 Ala Val Thr He Ser Asn Pro Ser Arg His Val Tyr Thr Gly Gly Lys 495 500 505

GGC TGC GTG AAG ATC TGG GAC ATC AGC CAG CCA GGC AGC AAG AGC CCC 1587 Gly Cys Val Lys He Trp Asp He Ser Gin Pro Gly Ser Lys Ser Pro 510 515 520

ATC TCC CAG CTG GAC TGC CTG AAC AGG GAC AAT TAC ATG CGC TCC TGC 1635 He Ser Gin Leu Asp Cys Leu Asn Arg Asp Asn Tyr Met Arg Ser Cys 525 530 535

AAG CTG CAC CCT GAT GGG CGC ACG CTC ATC GTG GGC GGC GAG GGC AGC 1683 Lys Leu His Pro Asp Gly Arg Thr Leu He Val Gly Gly Glu Gly Ser 540 545 550

ACG CTC ACC ATC TGG GAC CTG GCC TCG CCC ACG CCC CGC ATC AAG GCC 1731 Thr Leu Thr He Trp Asp Leu Ala Ser Pro Thr Pro Arg He Lys Ala 555 560 565 570

GAG CTG ACG TCC TCG GCT CCC GCC TGT TAT GCC CTG GCC ATT AGC CCT 1779 Glu Leu Thr Ser Ser Ala Pro Ala Cys Tyr Ala Leu Ala He Ser Pro 575 580 585 GAC GCC AAA GTC TGC TTC TCC TGC TGC AGC GAT GGG AAC ATT GCT GTC 1827 Asp Ala Lys Val Cys Phe Ser Cys Cys Ser Asp Gly Asn He Ala Val 590 595 600

TGG GAC CTG CAC AAC CAG ACC CTG GTC AGG CAG TTC CAG GGC CAC ACA 1875 Trp Asp Leu His Asn Gin Thr Leu Val Arg Gin Phe Gin Gly His Thr 605 610 615

GAT GGG GCC AGC TGC ATA GAC ATC TCC CAT GAT GGC ACC AAA CTG TGG 1923 Asp Gly Ala Ser Cys He Asp He Ser His Asp Gly Thr Lys Leu Trp 620 625 630

ACA GGG GGC CTG GAC AAC ACG GTG CGC TCC TGG GAC CTG CGG GAG GGC 1971 Thr Gly Gly Leu Asp Asn Thr Val Arg Ser Trp Asp Leu Arg Glu Gly 635 640 645 650

CGA CAG CTA CAG CAG CAT GAC TTC ACT TCC CAG ATC TTC TCG CTG GGC 2019 Arg Gin Leu Gin Gin His Asp Phe Thr Ser Gin He Phe Ser Leu Gly 655 660 665

TAC TGC CCC ACT GGG GAG TGG CTG GCT GTG GGC ATG GAG AGC AGC AAC 2067 Tyr Cys Pro Thr Gly Glu Trp Leu Ala Val Gly Met Glu Ser Ser Asn 670 675 680

GTG GAG GTG CTG CAC CAC ACC AAG CCT CAC AAG TAC CAG CTG CAC CTG 2115 Val Glu Val Leu His His Thr Lys Pro His Lys Tyr Gin Leu His Leu 685 690 695

CAC GAG AGC TGC GTG CTC TCC CTC AAG TTC GCC TAC TGC GGC AAG TGG 2163 His Glu Ser Cys Val Leu Ser Leu Lys Phe Ala Tyr Cys Gly Lys Trp 700 705 710

TTC GTG AGC ACT GGG AAA GAT AAC CTT CTC AAC GCC TGG AGG ACG CCT 2211 Phe Val Ser Thr Gly Lys Asp Asn Leu Leu Asn Ala Trp Arg Thr Pro 715 720 725 730

TAT GGA GCC AGC ATA TCC CAG TCT AAA GAA TCC TCG TCT GTC TTG AGT 2259 Tyr Gly Ala Ser He Ser Gin Ser Lys Glu Ser Ser Ser Val Leu Ser 735 740 745

TGT GAC ATT TCA GCG GAT GAC AAA TAC ATT GTA ACA GGC TCT GGT GAC 2307 Cys Asp He Ser Ala Asp Asp Lys Tyr He Val Thr Gly Ser Gly Asp 750 755 760

AAG AAG GCC ACA GTT TAT GAG GTC ATC TAC TAAACAAGAA CTCCAGCAGG 2357 Lys Lys Ala Thr Val Tyr Glu Val He Tyr 765 770

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 772 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Tyr Pro Gin Gly Arg His Pro Ala Pro His Gin Pro Gly Gin Pro 1 5 10 15

Gly Phe Lys Phe Thr Val Ala Glu Ser Cys Asp Arg He Lys Asp Glu 20 25 30

Phe Gin Phe Leu Gin Ala Gin Tyr His Ser Leu Lys Val Glu Tyr Asp 35 40 45

Lys Leu Ala Asn Glu Lys Thr Glu Met Gin Arg His Tyr Val Met Tyr 50 55 60

Tyr Glu Met Ser Tyr Gly Leu Asn He Glu Met His Lys Gin Thr Glu 65 70 75 80

He Ala Lys Arg Leu Asn Thr He Leu Ala Gin He Met Pro Phe Leu 85 90 95

Ser Gin Glu His Gin Gin Gin Val Ala Gin Ala Val Glu Arg Ala Lys 100 105 110

Gin Val Thr Met Thr Glu Leu Asn Ala He He Gly Gin Gin Gin Leu 115 120 125

Gin Ala Gin His Leu Ser His Ala Thr His Gly Pro Pro Val Gin Leu 130 135 140

Pro Pro His Pro Ser Gly Leu Gin Pro Pro Gly He Pro Pro Val Thr 145 150 155 160

Gly Ser Ser Ser Gly Leu Leu Ala Leu Gly Ala Leu Gly Ser Gin Ala 165 170 175

His Leu Thr Val Lys Asp Glu Lys Asn His His Glu Leu Asp His Arg 180 185 190

Glu Arg Glu Ser Ser Ala Asn Asn Ser Val Ser Pro Ser Glu Ser Leu 195 200 205

Arg Ala Ser Glu Lys His Arg Gly Ser Ala Asp Tyr Ser Met Glu Ala 210 215 220

Lys Lys Arg Lys Val Glu Glu Lys Asp Ser Leu Ser Arg Tyr Asp Ser 225 230 235 240

Asp Gly Asp Lys Ser Asp Asp Leu Val Val Asp Val Ser Asn Glu Asp 245 250 255

Pro Ala Thr Pro Arg Val Ser Pro Ala His Ser Pro Pro Glu Asn Gly 260 265 270

Leu Asp Lys Ala Arg Ser Leu Lys Lys Asp Ala Pro Thr Ser Pro Ala 275 280 285

Ser Val Ala Ser Ser Ser Ser Thr Pro Ser Ser Lys Thr Lys Asp Leu 290 295 300

Gly His Asn Asp Lys Ser Ser Thr Pro Gly Leu Lys Ser Asn Thr Pro 305 310 315 320

Thr Pro Arg Asn Asp Ala Pro Thr Pro Gly Thr Ser Thr Thr Pro Gly 325 330 335

Leu Arg Ser Met Pro Gly Lys Pro Pro Gly Met Asp Pro He Gly He 340 345 350

Met Ala Ser Ala Leu Arg Thr Pro He Ser He Thr Ser Ser Tyr Ala 355 360 365

Ala Pro Phe Ala Met Met Ser His His Glu Met Asn Gly Ser Leu Thr 370 375 380

Ser Pro Gly Ala Tyr Ala Gly Leu His Asn He Pro Pro Gin Met Ser 385 390 395 400 Ala Ala Ala Ala Ala Ala Ala Ala Ala Tyr Gly Arg Ser Pro Met Val 405 410 415

Ser Phe Gly Ala Val Gly Phe Asp Pro His Pro Pro Met Arg Ala Thr 420 425 430

Gly Leu Pro Ser Ser Leu Ala Ser He Pro Gly Gly Lys Pro Ala Tyr 435 440 445

Ser Phe His Val Ser Ala Asp Gly Gin Met Gin Pro Val Pro Phe Pro 450 455 460

His Asp Ala Leu Ala Gly Pro Gly He Pro Arg His Ala Arg Gin He 465 470 475 480

Asn Thr Leu Ser His Gly Gly Val Val Cys Ala Val Thr He Ser Asn 485 490 495

Pro Ser Arg His Val Tyr Thr Gly Gly Lys Gly Cys Val Lys He Trp 500 505 510

Asp He Ser Gin Pro Gly Ser Lys Ser Pro He Ser Gin Leu Asp Cys 515 520 525

Leu Asn Arg Asp Asn Tyr Met Arg Ser Cys Lys Leu His Pro Asp Gly 530 535 540

Arg Thr Leu He Val Gly Gly Glu Gly Ser Thr Leu Thr He Trp Asp 545 550 555 560

Leu Ala Ser Pro Thr Pro Arg He Lys Ala Glu Leu Thr Ser Ser Ala 565 570 575

Pro Ala Cys Tyr Ala Leu Ala He Ser Pro Asp Ala Lys Val Cys Phe 580 585 590

Ser Cys Cys Ser Asp Gly Asn He Ala Val Trp Asp Leu His Asn Gin 595 600 605

Thr Leu Val Arg Gin Phe Gin Gly His Thr Asp Gly Ala Ser Cys He 610 615 620

Asp He Ser His Asp Gly Thr Lys Leu Trp Thr Gly Gly Leu Asp Asn 625 630 635 640

Thr Val Arg Ser Trp Asp Leu Arg Glu Gly Arg Gin Leu Gin Gin His 645 650 655

Asp Phe Thr Ser Gin He Phe Ser Leu Gly Tyr Cys Pro Thr Gly Glu 660 665 670

Trp Leu Ala Val Gly Met Glu Ser Ser Asn Val Glu Val Leu His His 675 680 685

Thr Lys Pro His Lys Tyr Gin Leu His Leu His Glu Ser Cys Val Leu 690 695 700

Ser Leu Lys Phe Ala Tyr Cys Gly Lys Trp Phe Val Ser Thr Gly Lys 705 710 715 720 sp Asn Leu Leu Asn Ala Trp Arg Thr Pro Tyr Gly Ala Ser He Ser 725 730 735 in Ser Lys Glu Ser Ser Ser Val Leu Ser Cys Asp He Ser Ala Asp 740 745 750 sp Lys Tyr He Val Thr Gly Ser Gly Asp Lys Lys Ala Thr Val Tyr 755 760 765

Glu Val He Tyr 770

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1443 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: DNA (cDNA)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1344

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

CCT ACT CCA CGA ACT GAT GCG CCC ACC CCA GGC AGT AAC TCT ACT CCC 48 Pro Thr Pro Arg Thr Asp Ala Pro Thr Pro Gly Ser Asn Ser Thr Pro 1 5 10 15

GGA TTG AGG CCT GTA CCT GGA AAA CCA CCA GGA GTT GAC CCT TTG GCC 96 Gly Leu Arg Pro Val Pro Gly Lys Pro Pro Gly Val Asp Pro Leu Ala 20 25 30

TCA AGC CTA AGG ACC CCA ATG GCA GTA CCT TGT CCA TAT CCA ACT CCA 144 Ser Ser Leu Arg Thr Pro Met Ala Val Pro Cys Pro Tyr Pro Thr Pro 35 40 45

TTT GGG ATT GTG CCC CAT GCT GGA ATG AAC GGA GAG CTG ACC AGC CCC 192 Phe Gly He Val Pro His Ala Gly Met Asn Gly Glu Leu Thr Ser Pro 50 55 60

GGA GCG GCC TAC GCT GGG CTC CAC AAC ATC TCC CCT CAG ATG AGC GCA 240 Gly Ala Ala Tyr Ala Gly Leu His Asn He Ser Pro Gin Met Ser Ala 65 70 75 80

GCT GCT GCC GCC GCC GCT GCT GCT GCT GCC TAT GGG AGA TCA CCA GTG 288 Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Tyr Gly Arg Ser Pro Val 85 90 95

GTG GGA TTT GAT CCA CAC CAT CAC ATG CGT GTG CCA GCA ATA CCT CCA 336 Val Gly Phe Asp Pro His His His Met Arg Val Pro Ala He Pro Pro 100 105 110

AAC CTG ACA GGC ATT CCA GGA GGA AAA CCA GCA TAC TCC TTC CAT GTT 384 Asn Leu Thr Gly He Pro Gly Gly Lys Pro Ala Tyr Ser Phe His Val 115 120 125

AGC GCA GAT GGT CAG ATG CAG CCT GTC CCT TTT CCA CCC GAC CCC CTC 432 Ser Ala Asp Gly Gin Met Gin Pro Val Pro Phe Pro Pro Asp Pro Leu 130 135 140

ATC GGA CCT GGA ATC CCC CGG CAT GCT CGC CAG ATC AAC ACC CTC AAC 480 He Gly Pro Gly He Pro Arg His Ala Arg Gin He Asn Thr Leu Asn 145 150 155 160

CAC GGG GAG GTG GTG TGC GCG GTG ACC ATC AGC AAC CCC ACG AGA CAC 528 His Gly Glu Val Val Cys Ala Val Thr He Ser Asn Pro Thr Arg His 165 170 175 GTG TAC ACG GGT GGG AAG GGC GCG GTC AAG GTC TGG GAC ATC AGC CAC 576 Val Tyr Thr Gly Gly Lys Gly Ala Val Lys Val Trp Asp He Ser His 180 185 190

CCA GGC AAT AAG AGT CCT GTC TCC CAG CTC GAC TGT CTG AAC AGG GAT 624 Pro Gly Asn Lys Ser Pro Val Ser Gin Leu Asp Cys Leu Asn Arg Asp 195 200 205

AAC TAC ATC CGT TCC TGC AGA TTG CTC CCT GAT GGT CGC ACC CTA ATT 672 Asn Tyr He Arg Ser Cys Arg Leu Leu Pro Asp Gly Arg Thr Leu He 210 215 220

GTT GGA GGG GAA GCC AGT ACT TTG TCC ATT TGG GAC CTG GCG GCT CCA 720 Val Gly Gly Glu Ala Ser Thr Leu Ser He Trp Asp Leu Ala Ala Pro 225 230 235 240

ACC CCA CGC ATC AAG GCA GAG CTG ACA TCC TCG GCC CCC GCC TGC TAT 768 Thr Pro Arg He Lys Ala Glu Leu Thr Ser Ser Ala Pro Ala Cys Tyr 245 250 255

GCC CTG GCC ATC AGC CCC GAT TCC AAG GTC TGC TTC TCA TGC TGC AGC 816 Ala Leu Ala He Ser Pro Asp Ser Lys Val Cys Phe Ser Cys Cys Ser 260 265 270

GAC GGC AAC ATC GCT GTG TGG GAT CTG CAC AAC CAG ACC TTG GTG AGG 864 Asp Gly Asn He Ala Val Trp Asp Leu His Asn Gin Thr Leu Val Arg 275 280 285

CAA TTC CAG GGC CAC ACA GAT GGA GCC AGC TGT ATT GAC ATT TCT AAT 912 Gin Phe Gin Gly His Thr Asp Gly Ala Ser Cys He Asp He Ser Asn 290 295 300

GAT GGC ACC AAG CTC TGG ACA GGT GGT TTG GAC AAC ACG GTC AGG TCC 960 Asp Gly Thr Lys Leu Trp Thr Gly Gly Leu Asp Asn Thr Val Arg Ser 305 310 315 320

TGG GAC CTG CGG GAG GGG CGG CAG CTG CAG CAG CAC GAC TTC ACC TCC 1008 Trp Asp Leu Arg Glu Gly Arg Gin Leu Gin Gin His Asp Phe Thr Ser 325 330 335

CAG ATC TTT TCT CTG GGC TAC TGC CCA ACT GGA GAG TGG CTT GCA GTG 1056 Gin He Phe Ser Leu Gly Tyr Cys Pro Thr Gly Glu Trp Leu Ala Val 340 345 350

GGG ATG GAG AAC AGC AAT GTG GAA GTT TTG CAT GTC ACC AAG CCA GAC 1104 Gly Met Glu Asn Ser Asn Val Glu Val Leu His Val Thr Lys Pro Asp 355 360 365

AAA TAC CAA CTA CAT CTT CAT GAG AGC TGT GTG CTG TCG CTC AAG TTT 1152 Lys Tyr Gin Leu His Leu His Glu Ser Cys Val Leu Ser Leu Lys Phe 370 375 380

GCC CAT TGT GGC AAA TGG TTT GTA AGC ACT GGA AAG GAC AAC CTT CTG 1200 Ala His Cys Gly Lys Trp Phe Val Ser Thr Gly Lys Asp Asn Leu Leu 385 390 395 400

AAT GCC TGG AGA ACG CCT TAC GGG GCC AGT ATA TTC CAG TCC AAA GAA 1248 Asn Ala Trp Arg Thr Pro Tyr Gly Ala Ser He Phe Gin Ser Lys Glu 405 410 415

TCC TCA TCG GTG CTT AGC TGT GAC ATC TCC GTG GAC GAC AAA TAC ATT 1296 Ser Ser Ser Val Leu Ser Cys Asp He Ser Val Asp Asp Lys Tyr He 420 425 430

GTC ACT GGC TCT GGG GAT AAG AAG GCC ACA GTT TAT GAA GTT ATT TAT 1344 Val Thr Gly Ser Gly Asp Lys Lys Ala Thr Val Tyr Glu Val He Tyr 435 440 445 TAAAGACAAA TCTTCATGCA GACTGGACTT CTCCTCCTGG TAGCACTTTG CTCTGTCATC 1404 CTTTTTGTTC ACCCCCATCC CCGCATCTAA AACCAAGGA 1443

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 448 amino acids

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

(ii) MOLECULE TYPE: protein

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

Pro Thr Pro Arg Thr Asp Ala Pro Thr Pro Gly Ser Asn Ser Thr Pro 1 5 10 15

Gly Leu Arg Pro Val Pro Gly Lys Pro Pro Gly Val Asp Pro Leu Ala 20 25 30

Ser Ser Leu Arg Thr Pro Met Ala Val Pro Cys Pro Tyr Pro Thr Pro 35 40 45

Phe Gly He Val Pro His Ala Gly Met Asn Gly Glu Leu Thr Ser Pro 50 55 60

Gly Ala Ala Tyr Ala Gly Leu His Asn He Ser Pro Gin Met Ser Ala 65 70 75 80

Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Tyr Gly Arg Ser Pro Val 85 90 95

Val Gly Phe Asp Pro His His His Met Arg Val Pro Ala He Pro Pro 100 105 110

Asn Leu Thr Gly He Pro Gly Gly Lys Pro Ala Tyr Ser Phe His Val 115 120 125

Ser Ala Asp Gly Gin Met Gin Pro Val Pro Phe Pro Pro Asp Pro Leu 130 135 140

He Gly Pro Gly He Pro Arg His Ala Arg Gin He Asn Thr Leu Asn 145 150 155 160

His Gly Glu Val Val Cys Ala Val Thr He Ser Asn Pro Thr Arg His 165 170 175

Val Tyr Thr Gly Gly Lys Gly Ala Val Lys Val Trp Asp He Ser His 180 185 190

Pro Gly Asn Lys Ser Pro Val Ser Gin Leu Asp Cys Leu Asn Arg Asp 195 200 205

Asn Tyr He Arg Ser Cys Arg Leu Leu Pro Asp Gly Arg Thr Leu He 210 215 220

Val Gly Gly Glu Ala Ser Thr Leu Ser He Trp Asp Leu Ala Ala Pro 225 230 235 240

Thr Pro Arg He Lys Ala Glu Leu Thr Ser Ser Ala Pro Ala Cys Tyr

245 250 255

Ala Leu Ala He Ser Pro Asp Ser Lys Val Cys Phe Ser Cys Cys Ser 260 265 270 Asp Gly Asn He Ala Val Trp Asp Leu His Asn Gin Thr Leu Val Arg 275 280 285

Gin Phe Gin Gly His Thr Asp Gly Ala Ser Cys He Asp He Ser Asn 290 295 300

Asp Gly Thr Lys Leu Trp Thr Gly Gly Leu Asp Asn Thr Val Arg Ser 305 310 315 320

Trp Asp Leu Arg Glu Gly Arg Gin Leu Gin Gin His Asp Phe Thr Ser 325 330 335

Gin He Phe Ser Leu Gly Tyr Cys Pro Thr Gly Glu Trp Leu Ala Val 340 345 350

Gly Met Glu Asn Ser Asn Val Glu Val Leu His Val Thr Lys Pro Asp 355 360 365

Lys Tyr Gin Leu His Leu His Glu Ser Cys Val Leu Ser Leu Lys Phe 370 375 380

Ala His Cys Gly Lys Trp Phe Val Ser Thr Gly Lys Asp Asn Leu Leu 385 390 395 400

Asn Ala Trp Arg Thr Pro Tyr Gly Ala Ser He Phe Gin Ser Lys Glu 405 410 415

Ser Ser Ser Val Leu Ser Cys Asp He Ser Val Asp Asp Lys Tyr He 420 425 430

Val Thr Gly Ser Gly Asp Lys Lys Ala Thr Val Tyr Glu Val He Tyr 435 440 445

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 41 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 6

(D) OTHER INFORMATION: /label= X /note= ""X = Asp or Glu""

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 17

(D) OTHER INFORMATION: /label= X /note= ""X = He or Leu""

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 19

(D) OTHER INFORMATION: /label= X /note= ""X = He or Leu""

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 24

(D) OTHER INFORMATION: /label= X /note= ""X = Thr or Ser""

(ix) FEATURE:

(A) NAME/KEY: Modified-site

(B) LOCATION: 28

(D) OTHER INFORMATION: /label= X /note= ""X = Thr or Ser""

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Pro Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15

Xaa Xaa Xaa Ser Pro Asp Gly Xaa Xaa Leu Xaa Xaa Gly Gly Xaa Asp 20 25 30

Gly Xaa Val Xaa Xaa Trp Asp Leu Xaa 35 40

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 719 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: protein

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

Met Tyr Pro Ser Pro Val Arg His Pro Ala Ala Gly Gly Pro Pro Pro 1 5 10 15

Gin Gly Pro He Lys Phe Thr He Ala Asp Thr Leu Glu Arg He Lys 20 25 30

Glu Glu Phe Asn Phe Leu Gin Ala His Tyr His Ser He Lys Leu Glu 35 40 45

Cys Glu Lys Leu Ser Asn Glu Lys Thr Glu Met Gin Arg His Tyr Val 50 55 60

Met Tyr Tyr Glu Met Ser Tyr Gly Leu Asn Val Glu Met His Lys Gin 65 70 75 80

Thr Glu He Ala Lys Arg Leu Asn Thr Leu He Asn Gin Leu Leu Pro 85 90 95

Phe Leu Gin Ala Asp His Gin Gin Gin Val Leu Gin Ala Val Glu Arg 100 105 110

Ala Lys Gin Val Thr Met Gin Glu Leu Asn Leu He He Gly Gin Gin 115 120 125

He His Ala Gin Gin Val Pro Gly Gly Pro Pro Gin Pro Met Gly Ala 130 135 140

Leu Asn Pro Phe Gly Ala Leu Gly Ala Thr Met Gly Leu Pro His Gly 145 150 155 160

Pro Gin Gly Leu Leu Asn Lys Pro Pro Glu His His Arg Pro Asp He 165 170 175 Lys Pro Thr Gly Leu Glu Gly Pro Ala Ala Ala Glu Glu Arg Leu Arg 180 185 190

Asn Ser Val Ser Pro Ala Asp Arg Glu Lys Tyr Arg Thr Arg Ser Pro 195 200 205

Leu Asp He Glu Asn Asp Ser Lys Arg Arg Lys Asp Glu Lys Leu Gin 210 215 220

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

Asn Glu Met Glu Ser His Ser Pro Arg Pro Asn Gly Glu His Val Ser 245 250 255

Met Glu Val Arg Asp Arg Glu Ser Leu Asn Gly Glu Arg Leu Glu Lys 260 265 270

Pro Ser Ser Ser Gly He Lys Gin Glu Arg Pro Pro Ser Arg Ser Gly 275 280 285

Ser Ser Ser Ser Arg Ser Thr Pro Ser Leu Lys Thr Lys Asp Met Glu 290 295 300

Lys Pro Gly Thr Pro Gly Ala Lys Ala Arg Thr Pro Thr Pro Asn Ala 305 310 315 320

Ala Ala Pro Ala Pro Gly Val Asn Pro Lys Gin Met Met Pro Gin Gly 325 330 335

Pro Pro Pro Ala Gly Tyr Pro Gly Ala Pro Tyr Gin Arg Pro Ala Asp 340 345 350

Pro Tyr Gin Arg Pro Pro Ser Asp Pro Ala Tyr Gly Arg Pro Pro Pro 355 360 365

Met Pro Tyr Asp Pro His Ala His Val Arg Thr Asn Gly He Pro His 370 375 380

Pro Ser Ala Leu Thr Gly Gly Lys Pro Ala Tyr Ser Phe His Met Asn 385 390 395 400

Gly Glu Gly Ser Leu Gin Pro Val Pro Phe Pro Pro Asp Ala Leu Val 405 410 415

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

Gly Glu Val Val Cys Ala Val Thr He Ser Asn Pro Thr Lys Tyr Val 435 440 445

Tyr Thr Gly Gly Lys Gly Cys Val Lys Val Trp Asp He Ser Gin Pro 450 455 460

Gly Asn Lys Asn Pro Val Ser Gin Leu Asp Cys Leu Gin Arg Asp Asn 465 470 475 480

Tyr He Arg Ser Val Lys Leu Leu Pro Asp Gly Arg Thr Leu He Val 485 490 495

Gly Gly Glu Ala Ser Asn Leu Ser He Trp Asp Leu Ala Ser Pro Thr 500 505 510

Pro Arg He Lys Ala Glu Leu Thr Ser Ala Ala Pro Ala Cys Tyr Ala 515 520 525

Leu Ala He Ser Pro Asp Ser Lys Val Cys Phe Ser Cys Cys Ser Asp 530 535 540

Gly Asn He Ala Val Trp Asp Leu His Asn Glu He Leu Val Arg Gin 545 550 555 560

Phe Gin Gly His Thr Asp Gly Ala Ser Cys He Asp He Ser Pro Asp 565 570 575

Gly Ser Arg Leu Trp Thr Gly Gly Leu Asp Asn Thr Val Arg Ser Trp 580 585 590

Asp Leu Arg Glu Gly Arg Gin Leu Gin Gin His Asp Phe Ser Ser Gin 595 600 605

He Phe Ser Leu Gly Tyr Cys Pro Thr Gly Asp Trp Leu Ala Val Gly 610 615 620

Met Glu Asn Ser His Val Glu Val Leu His Ala Ser Lys Pro Asp Lys 625 630 635 640

Tyr Gin Leu His Leu His Glu Ser Cys Val Leu Ser Leu Arg Phe Ala 645 650 655

Ala Cys Gly Lys Trp Phe Val Ser Thr Gly Lys Asp Asn Leu Leu Asn 660 665 670

Ala Trp Arg Thr Pro Tyr Gly Ala Ser He Phe Gin Ser Lys Glu Thr 675 680 685

Ser Ser Val Leu Ser Cys Asp He Ser Thr Asp Asp Lys Tyr He Val 690 695 700

Thr Gly Ser Gly Asp Lys Lys Ala Thr Val Tyr Glu Val He Tyr 705 710 715

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

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

Lys Lys Arg Lys

1

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12s Ser Ser Asp Asp Glu 1 5

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

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

Ser Thr Pro Pro Lys 1 5

(2) INFORMATION FOR SEQ ID NO:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Arg Gin Arg Arg

1

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

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

Ser Ser Asp Thr Glu 1 5

(2) INFORMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Thr Ser Pro Arg Ser 1 5

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

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

Lys Lys Lys Pro 1

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

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

Thr Glu Glu Glu

1

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

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

Ser Ser Pro Gin Pro 1 5

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

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

1

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

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

Ser Leu Asn Asp 1

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

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

Thr Arg Leu Lys

1

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

Lys Arg Gin Lys

1

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 8 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

( i) SEQUENCE DESCRIPTION: SEQ ID NO:24: Ser Asp Gly Val Thr Ser Glu Ala 1 5

(2) INFORMATION FOR SEQ ID NO:25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

Thr Pro Arg Tyr

1

Claims

WHAT IS CLAIMED IS:

1. A substantially purified human transducin-like Enhancer of split protein.

2. The protein of claim 1 having the amino acid sequence depicted in Figure 1 (SEQ ID NO:2).

3. The protein of claim 1 having the amino acid sequence depicted in Figure 2 (SEQ ID NO:4).

4. The protein of claim 1 having the amino acid sequence depicted in Figure 3 (SEQ ID NO: 6).

5. The protein of claim 1 comprising the amino acid sequence depicted in Figure 4 (SEQ ID NO: 8).

6. A substantially purified protein comprising the amino acid sequence depicted in Figure 1 (SEQ ID NO:2).

7. A substantially purified protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO:4).

8. A substantially purified protein comprising the amino acid sequence depicted in Figure 3 (SEQ ID NO: 6).

9. A substantially purified protein comprising the amino acid sequence depicted in Figure 4 (SEQ ID NO: 8).

10. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein consisting of at least 50 amino acids of the transducin-like Enhancer of split protein.

11. A fragment of a human transducin-like Enhancer of split protein consisting of at least 50 amino acids, which displays one or more functional activities associated with a full-length human transducin-like Enhancer of split protein.

12. The protein of claim 10 in which the protein is able to be bound by an antibody to a human transducin-like Enhancer of split protein.

13. The fragment of claim 11 which is able to be bound by an antibody to a human transducin-like Enhancer of split protein.

14. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein, which fragment comprises a

Q domain of the transducin-like Enhancer of split protein.

15. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein, which fragment comprises a GP domain of the transducin-like Enhancer of split protein.

16. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein, which fragment comprises a CcN domain of the transducin-like Enhancer of split protein.

17. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein, which fragment comprises a SP domain of the transducin-like Enhancer of split protein.

18. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein, which fragment comprises a WD-40 domain of the transducin-like Enhancer of split protein.

19. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein, which fragment comprises a WD-40 repeat of the transducin-like Enhancer of split protein.

20. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein, which fragment comprises a nuclear localization sequence of the transducin-like Enhancer of split protein.

21. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein, which fragment comprises a potential phosphorylation site for casein kinase II of the transducin-like Enhancer of split protein.

22. A substantially purified protein comprising a fragment of a human transducin-like Enhancer of split protein, which fragment comprises a potential phosphorylation site for cdc2 kinase of the transducin-like Enhancer of split protein.

23. A substantially purified protein comprising the following amino acid sequence (SEQ ID NO:9): PXXXX(D or E)XTXXXXXXXX(I or L)X(I or L)SPDG(T or

S)XLX(T or S)GGXDGXVXXWDLX, where X is any amino acid.

24. The protein of claim 1 which is phosphorylated.

25. The protein of claim 2 having a molecular weight of about 83,000 daltons.

26. The protein of claim 3 having a molecular weight of about 80,000 daltons.

27. The protein of claim 4 having a molecular weight of about 83,000 daltons.

28. A substantially purified multiprotein complex characterized by the following properties:

(a) a molecular weight as determined by nondenaturing polyacrylamide gel electrophoresis of at least about 670,000 daltons; and (b) able to be bound by an antibody to a transducin-like

Enhancer of split protein.

29. The multiprotein complex of claim 28 which is further characterized as containing a protein having a molecular weight in the range of about 15,000 to 18,000 daltons, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

30. A substantially purified multiprotein complex characterized by the following properties: (a) a molecular weight as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under non-reducing conditions of about 150,000 daltons; and

(b) able to be bound by an antibody to a transducin-like Enhancer of split protein.

31. A substantially purified protein characterized by the following properties:

(a) having a molecular weight of about 17,000 daltons, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis; and

(b) able to associate in a multiprotein complex:

(i) having a molecular weight of at least 670,000 daltons as determined by nondenaturing polyacrylamide gel electrophoresis under non- reducing conditions, and (ii) able to be bound by an antibody to a transducin-like Enhancer of split protein.

32. A chimeric protein comprising a functionally active fragment of a human transducin-like Enhancer of split protein joined via a peptide bond to an amino acid sequence of a protein other than a transducin-like Enhancer of split protein.

33. The protein of claim 32 in which the fragment comprises a nuclear localization sequence.

34. A protein comprising a functionally active portion of a first human transducin-like Enhancer of split protein joined via a peptide bond to a functionally active portion of a second human transducin-like Enhancer of split protein, in which the first and second transducin-like Enhancer of split proteins are different.

35. A derivative or analog of the protein of claim 2, which is characterized by the ability to be bound by antibody to the protein of claim 2.

36. A derivative or analog of the protein of claim 3, which is characterized by the ability to be bound by antibody to the protein of claim 3.

37. A derivative or analog of the protein of claim 4, which is characterized by the ability to be bound by antibody to the protein of claim 4.

38. A derivative or analog of the protein of claim 5, which is characterized by the ability to be bound by antibody to the protein of claim 5.

39. A derivative or analog of the protein of claim 1, which is able to display one or more functional activities of the protein of claim 1.

40. An antibody which binds to a human transducin-like Enhancer of split protein and which does not bind to a Drosophila transducin-like Enhancer of split protein.

41. The antibody of claim 40 which is monoclonal.

42. The antibody of claim 41 which binds to the protein of claim 2, and which does not bind to the protein of claim 3.

43. The antibody of claim 41 which binds to the protein of claim 3 and which does not bind to the protein of claim 2.

44. A fragment or derivative of the antibody of claim 41 containing the idiotype of the antibody.

45. A substantially purified nucleic acid encoding a human transducin-like Enhancer of split protein.

46. The nucleic acid of claim 45 which lacks introns.

47. The nucleic acid of claim 45 which encodes the protein of claim 2.

48. The nucleic acid of claim 45 which encodes the protein of claim 3.

49. The nucleic acid of claim 45 which encodes the protein of claim 4.

50. The nucleic acid of claim 45 which encodes the protein of claim 5.

51. A substantially purified nucleic acid encoding a protein comprising the amino acid sequence depicted in Figure 1 (SEQ ID NO:2).

52. A substantially purified nucleic acid encoding a protein comprising the amino acid sequence depicted in Figure 2 (SEQ ID NO:4).

53. A substantially purified nucleic acid encoding a protein comprising the amino acid sequence depicted in Figure 3 (SEQ ID NO: 6).

54. A substantially purified nucleic acid encoding a protein comprising the amino acid sequence depicted in Figure 4 (SEQ ID NO: 8).

55. A substantially purified cDNA encoding a protein comprising a fragment of a human transducin-like Enhancer of split protein consisting of at least 50 amino acids of the transducin-like Enhancer of split protein.

56. A substantially purified nucleic acid encoding a protein comprising a Q domain of a human transducin-like Enhancer of split protein.

57. A substantially purified nucleic acid encoding a protein comprising a CcN domain of a human transducin-like Enhancer of split protein.

58. A substantially purified nucleic acid encoding a protein comprising a WD-40 domain of a human transducin-like Enhancer of split protein.

59. A substantially purified nucleic acid encoding a protein comprising the following amino acid sequence (SEQ ID NO:9): PXXXX(D or E)XTXXXXXXXX(I or L)X(I or L)SPDG(T or

S)XLX(T or S)GGXDGXVXXWDLX, where X is any amino acid.

60. A substantially purified nucleic acid comprising a nucleotide sequence complementary to at least ten nucleotides of the nucleic acid of claim 46.

61. A substantially purified cDNA molecule comprising the DNA sequence depicted in Figure 1 (SEQ ID NO: l).

62. A substantially purified cDNA molecule comprising the DNA sequence depicted in Figure 2 (SEQ ID NO:3).

63. A substantially purified cDNA molecule comprising the DNA sequence depicted in Figure 3 (SEQ ID NO:5).

64. A substantially purified cDNA molecule comprising the DNA sequence depicted in Figure 4 (SEQ ID NO: 7).

65. A nucleic acid encoding the chimeric protein of claim 32.

66. A nucleic acid encoding the chimeric protein of claim 34.

67. An antibody to the protein of claim 31.

68. A nucleic acid vector comprising the nucleic acid of claim

45.

69. A nucleic acid vector comprising the cDNA molecule of claim 46.

70. A nucleic acid vector comprising the nucleic acid of claim

60.

71. A recombinant cell containing the nucleic acid vector of claim 68.

72. A recombinant cell containing the nucleic acid vector of claim 69.

73. A recombinant cell containing the nucleic acid vector of claim 70.

74. A method for producing a human transducin-like Enhancer of split protein comprising growing the recombinant cell of claim 71, such that the human transducin-like Enhancer of split protein is expressed by the cell; and recovering the expressed human transducin-like Enhancer of split protein.

75. A method for producing a protein comprising growing a recombinant cell containing the cDNA of claim 55, such that the protein is expressed by the cell; and recovering the expressed protein.

76. A method for producing a protein comprising growing a recombinant cell containing the nucleic acid of claim 56, such that the protein is expressed by the cell; and recovering the expressed protein.

77. A substantially purified protein which is the product of the method of claim 74.

78. The antibody of claim 41 which binds to the protein of claim 2, and which does not bind to the protein of claim 3, 4, or 5.

79. The antibody of claim 41 which binds to the protein of claim 3, and which does not bind to the protein of claim 2, 4, or 5.