Abstract:
The present invention relates to a novel α-catenin with a new, specific expression pattern in mainly heart and testis. The invention further relates to the use of this α-catenin in the prediction, diagnosis, and/or treatment of cadherin-catenin related diseases, in particular cardiomyopathies and male infertility.

Description:
CROSS-REFERENCE TO RELATED APPLICATION(S)  
       [0001]    This application is a continuation of PCT International Application Number PCT/EP01/07392, designating the United States of America (which itself claimed priority, inter alia, from U.S. Provisional Patent Application 60/218,309 filed Jul. 14, 2000), and published, in English, as PCT International Publication Number WO 02/04636 A1 on Jan. 17, 2002, the contents of the entirety of which is incorporated by this reference. 
     
    
     
       TECHNICAL FIELD  
         [0002]    The present invention relates generally to the field of biotechnology, and specifically to a novel α-catenin with a new, specific expression pattern in mainly heart and testis. The invention further relates to the use of the α-catenin in prediction, diagnosis or treatment of cadherin-catenin related diseases, such as cardiomyopathies and male infertility.  
         BACKGROUND  
         [0003]    The αE-catenin protein, a component of the epithelial cadherin-catenin adhesion complex, is a well-known invasion suppressor. To reach full functionality of the cadherin-catenin cell-cell adhesion complex, it is necessary to link the complex to the actin cytoskeleton. AlphaE-catenin provides this link by binding to β-catenin or plakoglobin through its amino terminal side, and by binding actin or the actin-binding molecule α-actinin through its carboxy-terminus (reviewed in Rudiger, 1998). It has been shown that loss of αE-catenin affects cell-cell adhesion and promotes tumorigenicity (Ewing et al., 1995). In many cases of invasive cells, αE-catenin defects are seen, and introduction of exogenous α-catenin can restore cell-cell aggregation and counteract invasiveness (Hirano et-al., 1992; van Hengel et al., 1997; Watabe et al., 1994).  
           [0004]    The family of α-catenins contains so far four known members. The αE-catenin protein is ubiquitously expressed, mainly in epithelial tissues. AlphaN-catenin protein has about 75% identity to αE-catenin, but is restricted in its expression to neural tissues (Hirano et al., 1992). In analogy with αE-catenin, it can also bind to β-catenin and plakoglobin and is supposed to bind α-actinin and actin. Although the vinculin protein shows much less identity (20%) to αE- and αN-catenin, it shares some similar characteristics. This protein is mainly found in focal adhesions where it forms the link to the actin cytoskeleton and binds the integrin-binding molecule talin. Vinculin is sometimes found in cell-cell contacts as well, and it may even be able to take over the function of αE-catenin, by binding to β-catenin (Hazan et al., 1997). On the other hand, vinculin has been reported to bind to a central region of αE-catenin and to be essential for apical junctional organization (Watabe-Uchida et al., 1998). Moreover, vinculin has a unique proline-rich hinge domain, which is absent in the other family members and which allows the vinculin tail to bind to the head, thus masking some “cryptic” binding sites (Johnson &amp; Craig, 1995). For the recently reported α-catulin (Janssens et al., 1999; Zhang et al., 1998), the identity to other family members is about 25% at the amino acid level, but no functional evidence for adhesive properties was found yet. In addition to their structural role, it is becoming clear that α-catenins and vinculin also have a regulatory function in the coordination of assembly and disassembly of junctions (Rudiger, 1998), and that αN-catenin can locate to the nucleus where it inhibits β-catenin/Tcf signaling (Giannini et al., 2000).  
         DISCLOSURE OF THE INVENTION  
         [0005]    The present invention relates to a novel α-catenin with about 55% identity and 70% similarity to other α-catenins. Surprisingly, the novel catenin shows a specific expression in mainly heart and testis. Moreover, it interacts more strongly with β-catenin than the known α-catenins.  
           [0006]    In one aspect, the invention provides an isolated novel α-catenin polypeptide comprising the sequence as presented in SEQ ID NO: 2 for the human molecule, or variants thereof, which variants have at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity with such sequences, as measured by a BLAST search (Altschul et al. 1997). A specific embodiment of a variant is the mouse molecule of which the sequence comprises the sequence presented in SEQ ID NO: 5. Preferably, the variant consists essentially of SEQ ID NO: 5.  
           [0007]    In another aspect, the invention provides a nucleic acid sequence encoding the polypeptide. A preferred embodiment is a nucleic acid sequence, comprising the sequence presented in SEQ ID NO: 1 for the human molecule and SEQ ID NO: 4 for the mouse molecule. Such novel α-catenin polypeptide is expressed selected tissues or organs such as heart and/or testis, possibly combined with expression in brain, kidney, liver, lung, ovary, tongue, uterus and skeletal muscle.  
           [0008]    It is another aspect of the invention to provide the genomic organization of the gene, encoding the novel human α-catenin polypeptide. Human exon-intron boundaries have been determined and the exons have been allocated to different bacterial and P1 derived artificial chromosomes (BACs and PACs). The determination of the intronic sequences is a routine technique known to the person skilled in the art.  
           [0009]    Still another aspect of the invention is a nucleic acid comprising the promoter region of a gene, encoding a polypeptide according to the invention. A preferred embodiment is the nucleic acid comprising the human sequence presented in SEQ ID NO: 3 and the mouse sequence presented in SEQ ID NO: 6. The sequences were found to contain several putative binding sites for muscle-specific transcription factors such as Nkx2.5, MEF-2 and MEF-3. By modifying this promoter sequence, or by modulating the correspondingly binding transcription factors the transcription of the novel α-catenin can be influenced.  
           [0010]    Another aspect of the invention is the use of the novel α-catenin, or variants thereof, to modulate the cadherin-catenin related pathway. Alpha-catenin-like molecules can interact with the cadherin-catenin adhesion complex and are influencing as such the cell-cell adhesion and the β-catenin mediated signaling that is influencing the transcription of, as a non-limiting example, genes as c-myc, cyclin-D1, matrilysin, and c-jun. As the novel α-catenin of the invention is more strongly interacting with β-catenin than any other of the known α-catenin like molecules, it can be useful to use this molecule, or its β-catenin binding domain, or a polypeptide comprising this β-catenin binding domain either to block the α-catenin/β-catenin interaction, or to restore a deficient interaction. In a preferred embodiment, the cadherin-catenin related pathway is specifically modulated in selected tissues or organs such as heart and/or testis, eventually combined with modulation of the pathway in brain, kidney, liver, skeletal muscle.  
           [0011]    Still another aspect of the invention is the use of the novel α-catenin, or variants thereof, to treat cadherin-catenin related diseases. Such diseases include, but are not limited to cancer, cardiomyopathies including dilated cardiomyopathy, and male infertility.  
           [0012]    Still another aspect of the invention is the use of the nucleic acid sequence encoding the novel α-catenin, or fragments thereof, or the gene encoding the novel α-catenin or fragments thereof for the diagnosis of cadherin-catenin related diseases, or for predicting the likelihood of developing the cadherin-catenin related diseases. A preferred embodiment is the use of the nucleic acid or the gene for predicting the likelihood and/or diagnosis of cancer. Another preferred embodiment is the use of the nucleic acid or the gene for predicting the likelihood and/or diagnosis of cardiomyopathies including dilated cardiomyopathy, and male infertility. The nucleic acid sequence, gene, or fragments thereof can be used for detecting mutations and/or for quantifying messenger RNA expression levels, by techniques know to the person skilled in the art, such as but not limited to PCR, DNA-DNA hybridization, DNA-RNA hybridization or fluorescent in situ hybridization (“FISH”).  
           [0013]    Still another aspect of the invention is the use of the nucleic acid sequence encoding the novel α-catenin, or functional fragments thereof, for the treatment of cadherin-catenin related diseases. Indeed, the nucleic acid sequence can be incorporated in a vector suitable for gene therapy. Such vectors are known to the person skilled in the art and do include, but are not limited to retroviral vectors, adenoviral vectors, adenovirus-associated viral vectors and lentiviral vectors.  
           [0014]    Another aspect of the invention is antibodies against a novel α-catenin according to the invention. Preferably, the antibodies are monoclonal antibodies. One embodiment is a monoclonal antibody deposited at BCCM under the number LMBP 5537CB. Another embodiment is a monoclonal antibody deposited at BCCM under the number LMBP 5728CB.  
           [0015]    Still another aspect of the invention is the use of the antibodies to treat cadherin-catenin related diseases. A preferred embodiment is the use of the antibodies whereby the disease is cancer. Another preferred embodiment is the use of the antibodies whereby the disease is a cardiomyopathy including dilated cardiomyopathy. Still another preferred embodiment is male infertility.  
           [0016]    Still another aspect of the invention is the use of the antibody for the diagnosis of cadherin-catenin related diseases, or for predicting the likelihood of developing the cadherin-catenin related diseases. A preferred embodiment is the use of the antibodies for predicting the likelihood and/or diagnosis of cancer. Another preferred embodiment is the use of the antibodies for predicting the likelihood and/or diagnosis of cardiomyopathies including dilated cardiomyopathy, and male infertility. Antibodies can be used in tests such as a Western blot or an ELISA test, known to the person skilled in the art, to compare expression levels of the protein.  
           [0017]    Definitions  
           [0018]    Fragment of a nucleic acid or gene, as used here means any fragment that can be used as specific probe in hybridization reaction or specific primer in PCR reaction.  
           [0019]    Functional fragment of the novel α-catenin means a fragment encoding a polypeptide that comprises a functional β-catenin binding domain.  
           [0020]    Promoter region of a gene as used here means a region that is sufficient to obtain transcription of the normally transcribed regions of the gene. Promoter region of a gene, encoding a polypeptide according to the invention means a promoter region that is in a natural, non-recombinant situation linked to a gene encoding a polypeptide comprising the sequence presented in SEQ ID NO: 2, or a variant thereof, which variant has at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity with the sequence.  
           [0021]    Cadherin-catenin related disease is a disease that is caused by overexpression, underexpression or dysfunction of one or more compounds of the cadherin-catenin cell adhesion complex and the related β-catenin mediated signaling pathway and includes, but is not limited to cancer, cardiomyopathies including dilated cardiomyopathy, and male infertility.  
           [0022]    β-catenin mediated signaling pathway as used here means the pathway that is influenced by protein complexes in which β-catenin takes part.  
           [0023]    Gene as used here, means the genomic structure of the gene, including the promoter, the terminator and the complete transcribed sequence, which includes both exonic and intronic sequences. 
       
    
    
     BRIEF DESCRIPTION OF THE TABLES AND FIGURES  
       [0024]    Table 1: List of genomic Genbank submissions containing CTNNA3-specific sequences, complemented with proprietary data on BAC 162A20 and PAC 320B7.  
         [0025]    Table 2: Sequences of CTNNA3 exon-intron and intron-exon boundaries. Coding sequences are shown in capital letters. The open reading frame is indicated, with translation to amino acids on top of the sequence. Position of exon boundaries in the cDNA sequence is annotated under the sequence. All introns start with gt and end with ag, conform to the rules for exon boundaries (Mount, 1982). Intron sizes are indicated as minimum size, based on the length of intronic sequences obtained so far.  
         [0026]    Table 3: Overview of CTNNA3 exon-specific primers. For each exon, primer sequences in flanking intronic sequences are given, with the length of the PCR product (in bp), the optimum MgCl 2  concentration and the recommended annealing temperature (A.T.) to be used for genomic PCR.  
         [0027]    [0027]FIG. 1: Isolation of the novel human αT-catenin cDNA by two consecutive 5′-RACE experiments. (A) On the basis of one single EST sequence (IMAGE clone #728263), primers for 5′-RACE were designed and used to obtain a product of 1,011 bp. As this fragment was still lacking a suitable start codon, a second 5′-RACE was performed, which yielded a product of 1,306 bp containing the start codon. By aligning sequences from these clones, a full-length cDNA sequence of 3,024 bp was obtained, containing an, open reading frame of 895 amino acid residues (boxed). Percentage identity (B) and similarity (C) by MegAlign sequence comparison (DNAStar, Madison, Wis.) after alignment of the protein sequences by the CLUSTALW method (Higgins &amp; Sharp, 1989) and distance calculation with GCG software (www.BEN.ac.be). GenBank accession numbers of the used human sequences are M33308 (vinculin), U97067 (α-catulin), AF091606 (αT-catenin), D14705 (αE-catenin) and M94151 (αN2-catenin).  
         [0028]    [0028]FIG. 2: Amino acid sequence alignment of the human α-catenins. MegAlign sequence comparison (DNAStar, Madison, Wis.) after alignment of the protein sequences by the CLUSTALW method (Higgins &amp; Sharp, 1989), shaded by the Boxshade server (http://ulrec3.unil.ch/softward/BOX_form.html). Partial 3-dimensional structure as, determined for αE-catenin (Pokutta &amp; Weis, 2000) is shown by the location of the respective α-helices. Two characteristic proline residues, inducing a kink in the α-helices, are marked with “P”. Annotated domains are: the β-catenin binding domain as determined by Pokutta &amp; Weis (2000) and by Huber et a/. (1997), the α-actinin binding domain (Nieset et al., 1997), the amphiphatic helices possibly responsible for actin binding (Rudiger, 1998) and the position of the alternatively spliced insert (Claverie et al., 1993), which is generally found for αN-catenin, but rarely for αE-catenin. GenBank accession numbers of the used sequences are AF091606 (αT-catenin), D14705 (αE-catenin) and M94151 (αN2-catenin).  
         [0029]    [0029]FIG. 3: Chromosomal localization of the CTNNA3 gene, encoding human αT-catenin. (A) Fluorescence in situ hybridization with a specific PAC clone; arrows point at the positive signal; (B) G-banding by DAPI staining of the same chromosomes as shown in (A); (C) Fine mapping by Genebridge4 PCR analysis reveals the CTNNA3 location between the markers as shown; the obtained PCR pattern was: 000000001000000000000010120000110010100011000001000000000000000010010001010000 001110000000100, and was used as such for analysis on Webpage http://www.hgmp.mrc.ac.uk/cgi-bin/contig/rhmapper.pl.  
         [0030]    [0030]FIG. 4: Positioning of exon-exon boundaries (boxed) in the protein sequences of human αT-catenin and αE-catenin. Three boundaries in the αT-catenin sequence that do not coincide with αE-catenin are shown in bold. Indicated numbers refer to exons of αT-catenin (see also Table 1, Table 2 and FIG. 5).  
         [0031]    [0031]FIG. 5: Overview of BAC and PAC clones covering the human CTNNA3 gene. Clones 162A20 and 320B7 are, respectively, a BAC and PAC clone isolated by us. All other sequences were obtained from GenBank and are annotated with their respective Accession Numbers. Lengths of contig sequences, in which a match is found with one or more CTNNA3 exons (indicated by “Ex” followed by the appropriate exon number), are mentioned in kilobase pairs (kb) or base pairs (bp). BAC or PAC clone lengths are mentioned between brackets and refer to the number of sequenced bp of the respective clones (GenBank including HTGS database, sequence versions as indicated).  
         [0032]    [0032]FIG. 6: (A) The human αT-catenin promoter sequence as determined from a human genomic BAC clone (clone 162A20). The sequence-listed comprises 1,237 bp of the upstream sequences, the sequence of exon-1 (bp 1238-1433) and part of intron-1 (bp 1434-1740). Several transcription factor binding sites as predicted by the Matinspector transcription factor binding site search program (Quandt et al., 1995), are indicated in bold and underlined. (B) Alignment of human and mouse αT-catenin promoter sequences, as determined from the human genomic BAC clone 162A20 and mouse genomic BAC clone 164N16. Transcription factor binding sites are indicated. The sequence conservation of the MEF2C site and two GATA-binding sites is striking. The arrow indicates the transcription initiation site.  
         [0033]    [0033]FIG. 7: Interaction between αT-catenin and β-catenin/plakoglobin in the yeast two-hybrid system. (A) Introduction in the two-hybrid system by cotransformation of the respective fusion constructs. Plasmids used for cotransformation were pGBT9-ATCTN(179-2860), pGBT9-αctl(50-2264), pGBT9-αECTN, pGBT9-αNCTN, pGAD424-ATβctn and pGAD424-Plakoglobin(227-2340), followed by XGAL staining of colonies on SD plates lacking leucine, tryptophan and histidine; pVA3 and pTD1 are control bait and prey plasmids (Clontech): they code for interacting fusion proteins derived of murine p53 and SV40 large T-antigen, respectively. (B) Quantification of interaction strength between β-catenin and either αE- or αT-catenin in the yeast two-hybrid system. β-Galactosidase activity was measured using CPRG as a substrate. Two different yeast strains and corresponding bait plasmids were used: the L40 yeast strain which was cotransformed with either pLexMG-αEctn (1) or pLexMG-αTctn (2) plus in each case pGAD424-βctn, and the Y190 yeast strain cotransformed with either pGBT9-αEctn (3) or pGBKT7-αTctn (4) plus in each case pGAD424-ATβctn. In both systems, the interaction between αT-catenin and β-catenin was found to be about 4 times stronger than the one between αE-catenin and β-catenin. (C) Confirmation of αT-catenin/β-catenin interactions by co-immunoprecipitation (IP) from HEK-293 cells transfected with plasmid pEF6MH-ATCTN(1-2860), encoding Myc-tagged αT-catenin. In the Western blots at the left, which serve as controls for efficient transfection, αT-catenin was detected by monoclonal antibody 892 — 24D2S and β-catenin by a polyclonal antibody (Sigma). The IP results at the right were obtained either with monoclonal anti-β-catenin antibody (Transduction) or with monoclonal anti-Myc antibody 9E10 (Oncogene, Cambridge, Mass.). SDS-PAGE was followed by Western blotting. A mixture of both antibodies was then used to probe this blot. In mock transfected cells, only β-catenin was detected as expected (data not shown). (D) Confirmation of αT-catenin/β-catenin interactions by co-immunoprecipitation (IP) from mouse tissues. IP from mouse testis and heart was performed with polyclonal antibody #952, specific for αT-catenin, and with a polyclonal antibody specific for β-catenin (Sigma). After Western blotting of total lysates (at the left) and coimmunoprecipitates (at the right), αT-catenin and β-catenin were detected by use of the same antibodies.  
         [0034]    [0034]FIG. 8: Tissue-specific expression patterns of αT-catenin. (A) Rapid-scan RT-PCR expression analysis of human αT-catenin and αE-catenin mRNAs. The specific 743-bp product of the first reaction was visible in heart, testis and skeletal muscle (not shown). After nested PCR, this first product of 743 bp is still visible, whereas the nested PCR product of 630 bp is detectable in the same three samples and a few more (brain, kidney, liver, fetal liver). PCR with αE-catenin-specific primers (yielding a 747-bp product) reveals expression in most tissues. PBL, peripheral blood lymphocytes. (B) RT-PCR analysis of αE-catenin, αT-catenin and αN-catenin mRNAs in mouse organs. GAPDH mRNA analysis served as a positive control. (C) Western blot analysis of αT-catenin, αE-catenin and β-catenin protein expression in various mouse organs. For detection of αT-catenin, polyclonal serum #952 was applied. In brain tissue, the 104-kDa band revealed by anti-αE-catenin corresponds to cross-reacting αN2-catenin protein.  
         [0035]    [0035]FIG. 9: Immunolocalization of αT-catenin in human tissues. (A) Frozen section of human heart stained with 892 — 24D2S monoclonal antibodies, showing localization of αT-catenin protein at intercalated discs of cardiac myocytes. (B) Enlarged detail of (A). (C) Frozen cross-section of human testis seminiferous tubules stained with 892 — 24D2S monoclonal antibodies, showing localization of αT-catenin protein in presumptive peritubular myoid cells at the basement membrane of seminiferous tubules. (D) Enlarged view of human testis staining with arrowhead pointing to a peritubular myoid cell.  
         [0036]    [0036]FIG. 10: Immunolocalization of αT-catenin in cryosections of human heart. (A) Double immunofluorescent staining of αT-catenin (monoclonal antibody 892 — 24D2S) and αE-catenin (polyclonal antibody) shows colocalization of the α-catenin proteins at intercalated discs of cardiomyocytes. (B) Double immunofluorescent staining of αT-catenin (polyclonal antibody #952) and N-cadherin (monoclonal antibody) shows colocalization at intercalated discs of cardiomyocytes. (C) Immunohistochemical staining for αT-catenin (monoclonal antibody 892 — 24D2) or desmin (monoclonal antibody 33) shows that αT-catenin is localized at the intercalated discs of cardiomyocytes, while desmin is present also at Z-discs. In the negative control (neg), only secondary antibody was used.  
         [0037]    [0037]FIG. 11: Immunolocalization of αT-catenin in cryosections of human testis. (A) Double immunofluorescent staining of αT-catenin (monoclonal antibody 892 — 24D2) and αE-catenin (polyclonal antibody) shows differential localization of these two related proteins. The αT-catenin is present in peritubular cells, clearly separated from αE-catenin, which is present in cells within the seminiferous tubules. (B) Immunohistochemical staining of consecutive sections for αT-catenin (monoclonal antibody 892 — 24D2) and desmin (monoclonal antibody 33) demonstrates that αT-catenin is localized in desmin-expressing peritubular myoid cells. In the negative control (neg), only secondary antibody was used.  
         [0038]    [0038]FIG. 12: Transient overexpression of αT-catenin in α-catenin-negative HCT-8/R1 colon carcinoma cells restores cadherin/catenin-mediated cell-cell adhesion. At 10 h after transfection with pE/L-GFP-ATCTN plasmid and simultaneous infection with ΔA36R vaccinia virus, opposing cells expressing GFP-αT-catenin show increased fluorescence at their common cell-cell contacts (A). This results in recruitment of β-catenin and E-cadherin to the same sites (B).  
         [0039]    [0039]FIG. 13: Relocalization of multiple cell-cell adhesion components in stably transfected colon cancer cells, expressing Myc-tagged αT-catenin. The α-catenin-negative parental HCT-8/R1 cells (left panels) were compared to the cloned transfectant HCT-8/R1/T31 (right panels). Cells were stained for the Myc tag (exogenous αT-catenin), for E-cadherin, desmoglein-2 or ZO-1 antigens.  
         [0040]    [0040]FIG. 14: Fast aggregation of α-catenin-negative HCT-8/R1 colon cancer cells is restored upon stable transfection with αT-catenin cDNA. After preparation of single-cell suspensions, cell aggregation was measured by determination of the volume % distribution in function of the particle diameter at the starting point (t0) and after 30 min (t30). HCT-8/R1, HCT-8/E11R1 and HCT-8/E8 cells were all obtained by subcloning HCT-8 cells, but only HCT-8/E8 cells express endogenous αE-catenin. HRpCαN2 is a cloned transfectant of HCT-8/E11R1 cells expressing exogenous αN-catenin (van Hengel et al., 1997); HCT-8/R1/T31 is a cloned transfectant of HCT-8/R1 cells expressing exogenous αT-catenin. MB2 is a monoclonal E-cadherin blocking antibody.  
         [0041]    [0041]FIG. 15: Slow aggregation and compaction of HCT-8 colon cancer cells on semi-solid agar. Images of two representative cultures were taken 24 h after seeding single-cell suspensions of the indicated cell lines. No aggregation is seen in cultures of either the untransfected α-catenin-negative HCT-8/R1 cells, or the control transfectant HCT-8/R1/1743 (also α-catenin-negative). Cells expressing either endogenous αE-catenin or exogenous αN-catenin (see, also, legend to FIG. 8) form small aggregates, whereas cells expressing exogenous αT-catenin form compacted large aggregates.  
         [0042]    [0042]FIG. 16: Isolation of the novel mouse αT-catenin cDNA by consecutive 5′-RACE and RT-PCR experiments. (A) On the basis of one single EST sequence (GenBank Accession No. AW556211), primers for 5′-RACE were designed and used to obtain a product of 1,177 bp. As this fragment was still lacking a suitable start codon, an RT-PCR was performed using primers MCB2335 and MCB2567. This yielded a product of 684 bp of which 668 bp were new sequences. A second 5′-RACE was performed to obtain a 696-bp fragment containing the start codon. By aligning sequences from these clones, a full-length cDNA sequence of 2,979 bp was obtained, containing an open reading frame of 895 amino acid residues (boxed). (B) Percentages of identity (and similarities) between mouse αT-catenin, human αT-catenin and other mouse α-catenins, after alignment of the protein sequences by the CLUSTALW method (Higgins &amp; Sharp, 1989) and distance calculation with GCG software (www.BEN.ac.be). GenBank accession numbers of the used sequences are AF091606 (human αT-catenin), AF344871 (mouse αT-catenin), NM — 009818 (mouse αE-catenin) and NM — 009819 (mouse αN-catenin).  
         [0043]    [0043]FIG. 17: The full-length mouse αT-catenin clone pGEMTeasy-maTctn(1-2979) was expressed in an in vitro coupled transcription and translation reaction, using the TNT® Coupled Reticulocyte Lysate System (Promega). The in vitro synthesized proteins were analyzed by Western blotting and compared with endogenous αT-catenin protein in mouse heart and testis. (A) For detection of αT-catenin, polyclonal serum #952 was applied. As a negative control we used the TNT product of human cDNA encoding p120 ctn  isoform 3B (Keirsebilck et al., 1998). (B) TNT products of human and mouse αT-catenin were compared with endogenous αT-catenin protein in lysates of human and mouse heart. The αT-catenin proteins were analyzed by Western blotting and detected with either the polyclonal serum #952 (at the left) or with the monoclonal antibody 893 — 32C6S (at the right).  
         [0044]    [0044]FIG. 18: Amino acid sequence alignment of the human and mouse αT-catenin. The sequences were aligned using the CLUSTALW program (Higgins &amp; Sharp, 1989). Alignments were shaded using the Boxshade server. GenBank accession numbers of the used sequences are AF091606 (human αT-catenin) and AF344871 (mouse αT-catenin). Arrows indicate the amino-terminal β-catenin binding domains (Pokutta and Weis, 2000).  
         [0045]    [0045]FIG. 19: Amino acid sequence alignment of the mouse α-catenins. The sequences were aligned using the CLUSTALW program (Higgins &amp; Sharp, 1989). Alignments were shaded using the Boxshade server. GenBank accession numbers of the used sequences are NM — 009818 (mouse αE-catenin), NM — 009819 (mouse αN-catenin) and AF344871 (mouse αT-catenin).  
         [0046]    [0046]FIG. 20: Northern blot analysis of mouse αT-catenin. RNAs of 10 different mouse tissues were separated by gel electrophoresis, blotted on a membrane and hybridized with a mouse αT-catenin-specific probe, consisting of 296 bp of the 3′ end of the mouse αT-catenin cDNA. The sizes of the mRNAs were determined by sequential hybridizations of the Northern blot with a mouse GAPDH probe, with a mouse αE-catenin probe and finally staining of ribosomal RNA with methylene blue.  
         [0047]    [0047]FIG. 21: Western blot analysis of αT-catenin protein expression in various mouse tissues. (A) Detection of αT-catenin with polyclonal serum #952; (B) Detection of αT-catenin with polyclonal serum #952 after pre-incubation with the specific peptide #893, which blocks detection not only of the 100-kDa band (full-length mouse αT-catenin) but also the 86-kDa band, the 66-kDa band and the 43-kDa band in heart and testis lysates.  
         [0048]    [0048]FIG. 22: Influence of MEF2C transcription factor on the αT-catenin promoter activity. AlphaT-catenin promoter luciferase constructs were cotransfected with a MEF2C encoding plasmid in P19 cells (mouse embryonal carcinoma cells). Cells were lysed 48 h after transfection and luciferase activity was measured. A β-galactosidase encoding plasmid was cotransfected to normalize for transfection efficiency. A luciferase construct without αT-catenin promoter serves as negative control and is insensitive to MEF2C. A construct with luciferase under the control of the αT-catenin promoter sequence shows background activity unless cotransfected with MEF2C. This leads to a 10-fold upregulation of the αT-catenin promoter activity. Deletion or mutation of the consensus MEF2C binding site abolishes this effect as illustrated. 
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
     EXAMPLES  
       [0049]    Materials and Methods to the Examples  
         [0050]    Cell Cultures  
         [0051]    Most of the cell lines used were purchased from the American Cell Type Culture Collection (ATCC, Rockville, Md.). HCT-8/E8, HCT-8/E11R1 and HCT-8/R1 cell lines were obtained by subcloning of the human ileocecal adenocarcinoma cell line HCT-8 (CCL-224), where E stands for epithelioid and R for round-cell variants lacking αE-catenin (VAN Hengel et al., 1997). PC-3 (CRL-1435) is a human prostate carcinoma cell line and HEK-293 (CRL-1 573) is a human embryonic kidney fibroblast cell line.  
         [0052]    Cloning and cDNA Isolation of Human and Mouse αT-catenin  
         [0053]    A human αT-catenin-specific EST clone (IMAGE #728263) was identified by BLAST analysis (Altschul et al., 1990) and requested from the IMAGE consortium UK-HGMP Resource Center (Hinxton, UK). Expression of the corresponding transcript was confirmed by RT-PCR on mRNA from the prostate cancer cell line PC3 with primers MCB967 (5′-TGAGGCAGAAAAAGAAAAGA-3′ (SEQ ID NO: 87)) and MCB968 (5′-AGTGTGGTTAGGCAGGATT-3′ (SEQ ID NO: 88)). In order to complete the cDNA sequence we performed two consecutive 5′ Marathon™ RACE experiments on a human testis Marathon cDNA library (Clontech, Palo Alto, Calif.). For the first 5′ RACE, the gene-specific primer was MCB1027 (5′-AATCTGCCGAGCAAGGACATCCA-3′ (SEQ ID NO: 90)) and the nested primer was MCB1028 (5′-TCAGGCAGTTGAGTCATCTTAGC-3′ (SEQ ID NO: 91)). RACE-PCR was performed on a Perkin Elmer 2400 thermal cycler (Perkin Elmer, Foster City, Calif.) following the supplied protocol (touchdown PCR). Obtained RACE fragments were purified from agarose gel on QIAquick™ columns (Qiagen, Chatsworth, Calif.) and cloned in the pGEMT® vector (Promega Corp., Madison, Wis.). The cloned fragment was called pGEMT-ATCTN-RACE1. As the obtained clone appeared to be incomplete, a second 5′ -RACE experiment was performed with gene-specific primer MCB1254 (5′-ACCCGTGACGATGTGAGCAACTC-3′ (SEQ ID NO: 92)) and nested primer MCB1255 (5′-GAGCTGTCTGCGAAGGTCTCTTG-3′ (SEQ ID NO: 93)). The obtained fragment was cloned in the pGEMT®-easy vector (Promega Corp), and called pGEMTeasy-ATCTN-RACE2.  
         [0054]    A mouse αT-catenin-specific expression tag (EST) clone sequence (GenBank Accession No. AW556211) of 624 bp was identified by BLAST analysis (Altschul et al., 1990). In order to complete the mouse cDNA sequence, we performed a 5′ RACE reaction (Life Technologies, Paisley, UK) on mouse cDNA. This cDNA was prepared with a gene-specific primer, MCB2461 (5′-CCCCAATGTTTTATGTTAT-3′ (SEQ ID NO: 114)) from RNA that was isolated with the RNeasy method (Qiagen, Chatsworth, Calif.) from mouse testis and heart tissues. For the 5′ RACE reaction, we used primer MCB2481 (5′-CTTGGTGGAGGCAATGTATGAC-3′ (SEQ ID NO: 118)) and nested primer MCB2482 (5′-TCTGCCGAGCAAGAACATCCAT-3′ (SEQ ID NO: 119)). The obtained RACE fragments were purified from agarose gel using the CONCERT™ Rapid Gel Extraction System (Life Technologies) and cloned into the pGEMT®-easy vector (Promega, Madison, Wis.). The resulting plasmid was called pGEMTeasy-maTctn(RACE 1 )cDNA.  
         [0055]    As the cloned cDNA was incomplete, we performed a PCR using human primer MCB2335 (5′-CCTCTTGCAACATGTGTC-3′ (SEQ ID NO: 110)) and mouse gene-specific primer MCB2567 (5′-GCGGAGGTCTCTTGTCTTCTT-3′ (SEQ ID NO: 120)), yielding a product of 684 bp. The obtained fragment was cloned into the pGEMT®-easy vector (Promega), and called pGEMTeasy-maTctn(PCR2567+2335).  
         [0056]    As the cloned cDNA was still incomplete we performed another 5′ RACE reaction using the GeneRacer™ Kit, version B (Invitrogen, San Diego, Calif.) on RNA isolated from mouse heart tissue. For this 5′ RACE reaction, primer MCB2569 (5′-CGCAGTCAGAGAGTTCTTGCTT-3′ (SEQ ID NO: 121)) was used to prepare gene-specific mouse cDNA. For the touchdown PCR we used primer MCB2711 (5′-CTTCCCGAGCTTCTGGTAGGTTCT-3′ (SEQ ID NO: 124)). The obtained RACE fragments were purified from agarose gel using the CONCERTT Rapid Gel Extraction system (Life Technologies) and cloned into the pGEMT®-easy vector (Promega). The resulting plasmid was called pGEMTeasy-maTctn(RACE3)cDNA.  
         [0057]    On the basis of the consensus mouse αT-catenin sequence, primers MCB2818 (5′-AACGCCTAGAAGCCATCATC-3′ (SEQ ID NO: 125)) and MCB2819 (5′-TGGCAAGAACAATGATGTCA-3′ (SEQ ID NO: 126)) were designed to amplify the full-length cDNA. The predicted 2979-bp product was obtained by PCR on heart cDNA, using the TaqPlus® Precision PCR System (Stratagene Cloning Systems, La Jolla, Calif.). The PCR-fragment was purified from agarose gel using the CONCERT™ Rapid Gel Extraction System (Life Technologies) and cloned into the pGEMT®-easy vector (Promega). The resulting plasmid was called pGEMTeasy-maTctn(1-2979). This full-length mouse αT-catenin clone was expressed in an in vitro coupled transcription and translation reaction, using the TNT® Coupled Reticulocyte Lysate System (Promega). The in vitro synthesized proteins were analyzed by Western blotting.  
         [0058]    All DNA sequences were obtained by the dideoxy chain termination method (Sanger et al., 1977), using fluorescent dye terminators in a 373ABI automated DNA sequencer (Applied Biosystems, Foster City, Calif.). Sequences were assembled and compared by the DNAstar (DNASTAR Inc, Madison, Wis.), and Staden gap4 software packages (Bonfield et al., 1995). Amino acid similarities alignments were obtained using the CLUSTALW program (Higgins &amp; Sharp, 1989; Thompson et al., 1994) and GCG software. Alignments were shaded using the WWW-BOXSHADE server (http://ulrec3.unil.ch/softward/BOX-form.html).  
         [0059]    PAC and BAC Human and Mouse Genomic Library Screening  
         [0060]    For the isolation of a human genomic clone of αT-catenin, the PAC genomic library RPCI1 constructed by Ioannou and de Jong (1996), and obtained from HGMP (UK), was screened by PCR with the 3′ located primers MCB1260 (5′-GAAAAAGAAAAGATTGCTGAG-3′ (SEQ. ID NO: 94)) and MCB1261 (5′-CCCTAGTGAAGTCTGTCATCT-3′ (SEQ ID NO: 95)), yielding a product of 157 bp. The PCR reaction was optimized on total human genomic DNA and performed with 2.5 mM MgCl 2  at an annealing temperature of 56° C. Clone 320B7 (#1487) was found to be specific by DNA sequencing using primers MCB1260 and MCB1261. However, this PAC clone was found to contain only the last exon of the CTNNA3 gene (exon 18 in FIGS. 4 and 5).  
         [0061]    A BAC (Bacterial Artificial Chromosome) human genomic library (Genome Systems, St. Louis, Mo.) was screened with primers located in the first protein encoding exon, i.e., primers MCB2099 (5′-TGTCATCTGCCTCTCAATTTG-3′ (SEQ ID NO: 104)) and MCB2100 (5′-ATGCTGCCTTTCTGTTTCTTC-3′ (SEQ ID NO: 105)), yielding a product of 149 bp at an annealing temperature of 52° C. and 2 mM MgCl 2 . Clone 162A20 was found to comprise this exon, as confirmed by direct sequencing and fragment subcloning. To clone the αT-catenin promoter region, the BAC162A20 clone was digested with the 6-bp cutters BamHI, BglII, EcoRI, HindIII, MluI, SstI, SalI, XbaI and XhoI. Fragments containing exon 1 of the αT-catenin gene were identified by Southern blot hybridization with a primer located in this first exon, i.e. MCB2099. An 8-kb BamHI fragment was cloned in the pGEM11 vector and positive clones were identified by colony hybridization with primer MCB2099. The promoter region in this clone was sequenced by primer walking using primers MCB2217 (5′-CAGATGACAGTGGGGCAGTC-3′ (SEQ ID NO: 106)), MCB2287 (5′-AACTTGTTACTGAAAATACT-3′ (SEQ ID NO: 109)), and MCB2350 (5′-CATTACCATTTTTCCGACTT-3′ (SEQ ID NO: 111)).  
         [0062]    A BAC mouse genomic library (Genome Systems) was screened by PCR with primers located in either exon 1 (primers MCB2820 and MCB2837), in exon 2 (primers MCB2840 and MCB2841) and in exon 3 of the mouse αT-catenin gene (primers MCB2838 and MCB2839). Sequences of these primers are:  
                               MCB2820:               5′-CCCCTTTCTCTCTTATCCTGAG-3′   (SEQ ID NO:127)               MCB2837:       5′-CTTTCTGATGCTTCCTACAAGTAAA-3′   (SEQ ID NO:128)               MCB2840:       5′-GTCGGCAGAAACGCCAATA-3′   (SEQ ID NO:131)               MCB2841:       5′-GAGGCTCCAGCAGTTTCTCC-3′   (SEQ ID NO:132)               MCB2838:       5′-CCGCAGAATCCTTCCAACA-3′   (SEQ ID NO:129)               MCB2839:       5′-GCTGCCAGCTCTTCCTTTAAA-3′.   (SEQ ID NO:130)          
 
         [0063]    Clone 164N16 was found to comprise these first three exons, as confirmed by Southern blot analysis.  
         [0064]    Fluorescence In Situ Hybridization (FISH)  
         [0065]    PAC clone 320B7 (#1487) was used for fluorescence in situ hybridization (Kievits et al., 1990). PAC DNA was prepared with Kb-Magnum purification columns (Genome Systems, St. Louis, Mo.) and nick-translated using a BioNick kit (Gibco-BRL). Denaturation of labeled probe and human chromosomes, hybridization and fluorescent detection were performed as described previously (van Hengel et al., 1995). The chromosomes were stained with DAPI to reproduce G-banding. The slides were observed using a Zeiss Axiophot fluorescent microscope (Zeiss, Jena, Germany) and images captured with a Photometrics Image Point CCD camera (Photometrics, Munich, Germany). Results were analyzed with the MacProbe software of PSI (Perceptive Scientific International, League City, Tex.). At least 20 metaphase spreads of normal human lymphocytes were analyzed.  
         [0066]    Confirmation of Human Chromosomal Localization by PCR-based Hybrid Mapping  
         [0067]    Genomic PCR was performed with primers  
                           MCB2056           (5′-GAAATGCCATGGAGCTCTAAC-3′ (SEQ ID NO:102)) and               MCB2057       (5′-ATGGGAAGGCAAACCAGTCAC-3′ (SEQ ID NO:103)).          
 
         [0068]    These primers correspond to intronic sequences and are flanking an exon, as deduced from the sequence of a genomic clone (GenBank N o  AQ163827) (Table 1; FIG. 5). A product of 274 bp was expected. As a positive control, genomic DNA from human placenta was used to optimize the annealing temperature of 52° C. and a concentration of 2 mM MgCl 2 . The PCR was performed on samples of the Genebridge 4 radiation hybrid mapping panel (HGMP-RC, UK), which allows construction of high-resolution contiguous maps of human chromosomes. Analysis was performed on the Web page http://www.hgmp.mrc.ac.uk/cgi-bin/contig/rhmapper.pl.  
         [0069]    PCR with the same primer set was also performed on a monochromosomal hybrid mapping panel (NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panel #2, Coriell Cell Repositories, Camden, N.J.). All cell hybrid templates were diluted to a final DNA concentration of 100 ng/μl, using 1 μl as PCR templates.  
         [0070]    Cloning of Two Hybrid Prey and Bait Plasmids  
         [0071]    After completion of plasmid constructs, as described below, all clones were checked by DNA sequencing. For all PCRs, Pfu polymerase (Stratagene, La Jolla, Calif.) with proofreading activity was used.  
         [0072]    αT-catenin  
         [0073]    For cloning of the full-length hαT-catenin cDNA in the pGBT9 vector (Clontech), in fusion with the GAL4 binding domain, four consecutive constructs were made. A PCR product of 1,134 bp was synthesized with primers MCB1607 (5′-AGAATTCTCAGCTGAAACACCAATCAC-3′ (SEQ ID NO: 96)) and MCB1609 (5′-AGGATCCTGCGAAGGTCTCTTGTCT-3′ (SEQ ID NO: 98)) using the pGEMTeasy-ATCTN-RACE2 clone as a template. This product was restricted with EcoRI plus BamHI and ligated to the EcoRI and BamHI sites of the pGBT9 vector, thus yielding pGBT9-ATCTN(179-949). This construct was opened with PstI, and the PstI insert of 1,111 bp from clone pGEMT-ATCTN-RACE2 was ligated to obtain pGBT9-ATCTN(179-1306). From this construct, the EcoRI-SspI insert of 1,082 bp was isolated and ligated together with fragment SspI-SalI from pGEMTeasy-ATCTN-RACE1 in the pGBT9 vector, restricted with EcoRI-SalI. In this way pGBT9-ATCTN(179-2176) was obtained. In order to have an overlapping 3′ clone, a PCR product of 890 bp was synthesized with primers MCB1610 (5′-GGATGATAATCAATTTGTGGACATCTC-3′ (SEQ ID NO: 99)) and MCB 1608 (5′-GGGATCCGTAGATTTGTCTTCCTCTAA-3′ (SEQ ID NO: 97)). For this PCR, template cDNA was synthesized from RNA prepared with the RNeasy kit (Qiagen) from the PC-3 prostate cancer cell line (American Type Culture Collection, Rockville, Md., U.S.A.). The PCR product was cut with BglII and SalI, and inserted in the BglII-SalI opened construct pGBT9-ATCTN(179-2176) to finally achieve the construct pGBT9-ATCTN(179-2860).  
         [0074]    From pGBT9-ATCTN(179-2860), the EcoRI-SacI insert was ligated together with the SacI-NotI fragment from the original EST clone pT3T7-EST728263, in the EcoRI-NotI digested pGBKT7 vector (Clontech). The obtained construct was designated pGBKT7-ATCTN(179-3024). Further, the EcoRI-SalI insert from pGBT9-ATCTN(179-2860) was cloned into the EcoRI-SalI sites of the LexA bait vector pLexMG (pGBT9 in which the GAL4 binding domain has been exchanged with the LexA binding domain by Mathias Gautel, EMBL, Heidelberg). In this way, pLexMG-ATCTN(179-2860) was obtained.  
         [0075]    α-catulin  
         [0076]    The full-length α-catulin cDNA sequence (GenBank Accession number U97067) was isolated by us in 3 steps (Janssens et al., 1999): The full-length sequence was compiled from 2 EST sequences (clones 36498 and 67201) and one 5′-RACE clone. In order to obtain a clone with the full-length sequence, these 3 clones were assembled in the pGEM11 vector (Clontech). First, the insert of EST clone 67201 was isolated by a SmaI-MunI digest and ligated to the SmaI-MunI opened vector pGEMT-αctlRACE. In that way the construct pGEMT-αctl(1-1369) was obtained. Part of the EST #36498 sequence (1,003 bp) was obtained by a BglII restriction digest, yielding a fragment comprising the complete 3′ part of the open reading frame (ORF) but only part of the 3′ untranslated region. This fragment was inserted in the plasmid pGEMT-αctl(1-1369) opened with BglII. This resulted in a clone containing the complete open reading frame of α-catulin, i.e. pGEMT-αctl(1-2264).  
         [0077]    This clone was used as a template to generate a PCR product with primer MCB725 (5′-TATTAGATATCGCCTCTCCCGGACCCGCC-3′ (SEQ ID NO: 86) comprising an EcoRV site) and primer MCB711 (5′-AGGGGGCAGTGGCTGAAGAAAGAAGTAATC-3′(SEQ ID NO: 85)). In a 3-point ligation this PCR product, cut with EcoRV +MunI, was ligated together with a MunI-SalI fragment of pGEMT-αctl(1-2264) into the BamHI(blunted)-SalI restricted pGBT9 two-hybrid vector (Clontech), in frame with the ORF encoding the GAL4 DNA binding domain. The obtained constructed was called pGBT9-αctl(50-2264).  
         [0078]    αE-catenin  
         [0079]    Screening of a human fetal kidney 5′ Stretch cDNA library in vector λDR2 (Clontech, Calif.) resulted in isolation of the pDR2αECTN plasmid, containing full-length αE-catenin cDNA. From this plasmid, Eco47III-SphI and SphI-SalI fragments were ligated in the SmaI-SalI digested pGBT9 vector, yielding pGBT9-αECTN, in which the full-length αE-catenin ORF is fused in frame with the OF encoding the GAL4 DNA binding domain.  
         [0080]    From pGBT9-αECTN, the EcoRI-SalI fragment was cloned into the pLexMG vector restricted with EcoRI and SalI. In this way pLexMG-αECTN was obtained in which the full-length αE-catenin ORF is fused in frame with the ORF encoding the LexA DNA binding domain.  
         [0081]    αN-catenin  
         [0082]    The cDNA for human αN-catenin was kindly provided as plasmid pPN-hANCTN by Dr. C. Petit (Claverie et al., 1993). Nearly full-length αN-catenin was amplified from pPN-hANCTN with primers MCB137 (5′-ACCCCCCGGGGGCAACTTCACCTATCATTC-3′ (SEQ ID NO: 83)) containing an XmaI site), and MCB138 (5′-GCCGCCGCCTTCCTTTTCATTTCCGCTCTT-3′(SEQ ID NO: 84)). The PCR fragment was digested with XmaI and BanI and ligated together with a BanI-HindIII fragment of pPN-hANCTN in the XmaI-HindIII digested pAS2 vector (Clontech). Thus the pAS2-αNCTN plasmid was obtained, in which codons 4 to 906 of αN-catenin are fused in frame with the ORF encoding the GAL4 DNA binding domain. From this construct, the XmaI-HindIII insert was transferred to the XmaI-HindIII opened pGBT9 vector, yielding pGBT9-αNCTN.  
         [0083]    β-catenin  
         [0084]    The full-length β-catenin cDNA was kindly provided as plasmid pBAT-βCAT (from Dr. J. Behrens, Berlin, Germany). The amino terminal fragment 239-717 was obtained as an NcoI-PstI restriction fragment, of which the NcoI cut end was filled in with Pfu polymerase. This fragment was cloned into the SmaI-PstI opened pGAD424 vector, by which the construct pGAD424-ATβctn was obtained.  
         [0085]    Plakoglobin  
         [0086]    Plasmid pHPGCa2.1 with the full-length human plakoglobin cDNA was kindly provided by Dr. W. Franke. PCR was performed on this plasmid, with primer MCB133 (5′-GGTGAATTCGTCAGCAGCAAGGGCATCAT-3′(SEQ ID NO: 81)), containing an EcoRI site) and primer MCB134 (5′-GGTTTGATGCAGGGTCCACAGGCAGTTCT-3′(SEQ ID NO: 82)). The obtained PCR product (encoding plakoglobin residues 227-1228) was digested with EcoRI and SacI (residues 227-559) and ligated together with the fragments SacI-BglII (residues 560-1856) and BglII-PstI (residues 1857-2340) from pHPGCa2.1, into the EcoRI-PstI opened pGAD424 vector (Clontech). Thus, the plasmid pGAD424-Plakoglobin(227-2340) was obtained.  
         [0087]    Yeast Two-Hybrid Transformation  
         [0088]    The yeast strain Y190 (Matchmaker, Clontech, CA), which contains GAL4 promoter driven His and β-galactosidase reporters, was used for cotransformation of pGBT9 bait and pGAD424 prey plasmids, comprising the cloned inserts of interest. The yeast strain L40, which contains LexA promoter driven His and β-galactosidase reporters, was used for cotransformation of pLexMG bait with pGAD424 prey plasmids, comprising the cloned inserts of interest.  
         [0089]    The yeast cells were grown in YPD medium until a log-phase culture with an O.D. 600  of about 0.8 was obtained, and transformed by the lithium acetate procedure (Gietz et al., 1992). Cotransformants were selected by plating the transformation mix on SD minimal medium plates lacking leucine and tryptophan. After three days, colonies were picked and grown overnight in SD without leucine and tryptophan, but containing 0.07 M potassium phosphate. Replica plates selecting for interaction were made on SD lacking leucine, tryptophan and histidine, but containing 0.07 M potassium phosphate, 40 mM 3-amino-triazol to suppress leaky His expression, and 80 mg/ml X-β-GAL (Duchefa, Haarlem, The Netherlands).  
         [0090]    CPRG Yeast Two-Hybrid Quantification of β-galactosidase  
         [0091]    To assay the strength of interaction between α-catenins and β-catenin, β-galactosidase activity was assayed using chlorophenol red-β-D-galactopyranoside (CPRG, Boehringer Mannheim, Del.) as a substrate, according to the provided protocol (Clontech Yeast Protocols Handbook). Briefly, transformed yeasts are grown until OD 600  of about 0.6, concentrated in three different dilutions (1.25, 2.5 and 5 times concentrated, respectively) and allowed to develop red color after addition of CPRG substrate (measured at that time point, at OD 578 ). One β-galactosidase unit is defined as the amount which hydrolyzes 1 μmol of CPRG to chlorophenol red and D-galactose per minute per cell (Miller et al., 1972). The amount of units is calculated as 10,000×OD 578 /(time×concentration factor×OD 600 ).  
         [0092]    Cloning of Expression Plasmids  
         [0093]    The full-length human αT-catenin cDNA was excised from the construct pGBT9-ATCTN(179-2860) with restriction enzymes EcoRI-SalI and inserted in the EcoRI-SalI digested pEGFPC2 vector (Clontech), in order to obtain an in-frame amino-terminal fusion with the GFP protein. The resulting plasmid was called pEGFPC2-ATCTN(179-2860). The EcoRI-NotI insert from pGBT9-ATCTN(179-2860) was ligated into the EcoRI-NotI digested vector pEF6MycHisA (Invitrogen), providing a C-terminal fusion between αT-catenin and the Myc and His epitopes in the construct pEF6MH-ATCTN(179-2860). In this construct, no in-frame start codon is present at the very 5′ side, but there is a start codon present at position 596. The full-length fusion construct, named pEF6MH-ATCTN(1-2860), was obtained by introducing the 5′ part of the αT-catenin cDNA from clone pGEMT-RACE2, cut with EcoRI-BstEII, into the EcoRI-BstEII opened vector pEF6MH-ATCTN(179-2860).  
         [0094]    Transfection Methods  
         [0095]    For vaccinia virus-mediated transient overexpression, α-catenins were cloned in the pE/L-GFP vector (Frischknecht et al., 1999). Cells were transfected with Lipofectin (Life Technologies) and simultaneously co-infected with vaccinia virus strain ΔA36R, which does not make actin tails (Parkinson and Smith, 1994). At 4 to 30 h after transfection, high levels of expression under control of the vaccinia virus early/late promoter (E/L) (Chakrabarti et al., 1997) were obtained of the cloned cDNA, amino-terminally fused to GFP. Human αT-catenin was amplified with Taq+Precision polymerase (Stratagene) using primers containing a 5′ NotI site and a 3′ EcoRI site (MCB2386, 5′-GGGGGCGGCCGCGGAGGGTCAGCTGAAACACCAATCACATTG-3′ (SEQ ID NO: 112) and MCB2387, 5 ′-CCCCGAATTCGCCGTGTGGTTAGGCAGGATTTTGTCATATAG-3′ (SEQ ID NO: 113)) and cloned into the NotI-EcoRI sites of the pE/L-GFP vector.  
         [0096]    For stable transfection of HCT-8/R1 carcinoma cells, 4×10 6  cells were electroporated (Easyject; Eurogentec, Seraing, Belgium) with 10 μg of plasmid pEF6MH-ATCTN(179-2860). Cells were plated and cultured in the presence of 6 μg/ml blasticidin (Invitrogen) to select for stable transfectants. Colonies of blasticidin-resistant cells were isolated and tested by immunofluorescence and Western blotting for expression of αT-catenin. One stable clone was isolated and called HCT-8/R1/T31. As a negative control we transfected HCT-8/R1 cells with the empty pEF6MH vector, resulting in stable clones called HCT-8/R1/1743. A clone of HCT-8/E11R1 cells, stably transfected with αN-catenin cDNA and designated HRpCαN2 (van Hengel et al., 1997), was used in comparison. Likewise, HCT-8/E11R1 carcinoma cells were transfected with plasmid pEGFPC2-ATCTN(179-2860). After selection with G418 (800 μg/ml), one stable αT-catenin expressing clone was isolated and called HCT-8/E11R1/T14.  
         [0097]    Expression Analysis by RT-PCR  
         [0098]    Expression analysis using the human Rapid-Scan panel (OriGene Technologies Inc, Rockville, Md.) was performed on 100-times diluted template, followed by a nested PCR ({fraction (1/10)} of the end volume of the first reaction was used). The end-point determination method used does not allow a reliable determination of expression levels to be deduced from the amount of PCR product visualized on gel. Therefore, visual presence of a signal (even weak) was scored as positive, and complete absence was scored as negative. Primers MCB967 (5′-TGAGGCAGAAAAAGAAAAGA-3′ (SEQ ID NO: 87)) and MCB968 (5′-AGTGTGGTTAGGCAGGATT-3′(SEQ ID NO: 88)) were used for the first PCR, yielding a product of 743 bp. For nested PCR, primers MCB967 (5′-TGAGGCAGAAAAAGAAAAGA-3′(SEQ ID NO: 87)) and MCB1010 (5′-GCTGAGCCTCGTCTGAC-3′(SEQ ID NO: 89)) were combined, yielding a smaller product of 630 bp. Amplified products were checked for specificity by sequence analysis, showing that the double bands observed after nested RT-PCR of heart and testis samples in particular are indeed the larger primary product and the smaller nested product.  
         [0099]    As a control, an αE-catenin-specific product of 747 bp was amplified with primers MCB53 (5′-CTTCGGGCCTCTGGAATTTA-3′(SEQ ID NO: 79)) and MCB73 (5′-CGACATCAGGGTGCTGTAGG-3′(SEQ ID NO: 80)).  
         [0100]    For RT-PCR analysis of mouse tissues, RNA was prepared from different tissues with the RNAeasy method (Qiagen) and cDNA was prepared using a commercial kit (Life Technologies, Ghent, Belgium). For mouse αT-catenin, primers MCB2461 (5′-CCCCAATGTTTTATGTTAT-3′ (SEQ ID NO: 114)) and MCB2463 (5′-GGGGAGAACTCATCGTAT-3′ (SEQ ID NO: 115)) were designed on the sequence of an EST clone (GenBank Accession No. AW556211), resulting in amplification of a 442-bp product. For mouse αE-catenin (GenBank Accession No. NM — 009818), a 733-bp product was amplified with primers MCB2636 (5′-GAAGGCCCCTGAGAAGAA-3′ (SEQ ID NO: 122)) and MCB2637 (5′-CCCGAATAAAGCAACTCCAT-3′ (SEQ ID NO: 123)). For mouse αN-catenin (GenBank Accession No. NM — 009819), a 858-bp product was amplified with primers MCB2479 (5′-GCCCTGATTGAGTTTGATAA-3′ (SEQ ID NO: 116)) and MCB2480 (5′-CCCAGCTTCATAGTTCTCC-3′ (SEQ ID NO: 117)). As a control, a 452-bp fragment of mouse GAPDH was amplified with primers MCB2219 (5′-ACCACAGTCCATGCCATCAC-3′ (SEQ ID NO: 107)) and MCB2220 (5′-TCCACCACCCTGTTGCTG TA-3′ (SEQ ID NO: 108)).  
         [0101]    Expression Analysis by Northern Blotting  
         [0102]    RNA was prepared from different mouse tissues, using the RNeasy method (Qiagen). For each tissue sample, 25 μg RNA was separated on a 1% agarose gel. RNA was transferred by Northern blotting on a Hybond™-N +  membrane (Amersham Pharmacia Biotech, Rainham, UK).  
         [0103]    A mouse αT-catenin-specific probe of 296 bp was generated by PCR with primer MCB2043 (5′-TCGAGGATGAAGGCTCTG-3′ (SEQ ID NO: 100)) and primer MCB2044 (5′-TGTTTAACCCCAATGTTT-3′ (SEQ ID NO: 101)). The PCR product was labeled with α[ 32 P]-dCTP using the Radprime DNA labeling System (Life Technologies). After hybridization according to standard procedures, the blot was washed at high stringency. For detection, a Phosphor Imager cassette (Molecular Dynamics, Sunnyvale, Calif.) was exposed for 4 days and scanned with a Molecular Imager® FX using the Quantity One software (BioRad, Richmond, Calif.).  
         [0104]    Antibodies  
         [0105]    Peptides corresponding to, respectively, the amino-terminus (MSAETPITLNIDPQDLQ-C (SEQ ID NO: 133)) and the carboxy-terminus (C-KIHPLQVMSEFRGRQIY (SEQ ID NO: 134)) of the human αT-ctn protein were synthesized and coupled to keyhole-limpet hemocyanin via the additional cysteine residue at either the carboxyterminal or the amino terminal end of the peptides. 200 μg of peptide was injected in each of three rabbits using Titermax (Sigma, St Louis, Mo.) as adjuvant. Boosts were given with intervals of minimum two weeks. Sera were tested by ELISA on the peptide used for injection, using the non-relevant peptide as a negative control. The sera #952 (specific for the carboxy-terminal peptide) and #954 (specific for the amino-terminal peptide) were affinity purified on hydroxymercuribenzoate-agarose (Sigma, St Louis, Mo.), coupled to the respective immunizing peptides. Crude and purified sera were tested on lysates of HEK cells transfected with plasmids pEGFPC2-ATCTN(179-2860) and pEF6MH-ATCTN(1-2860), encoding respectively full-length Myc-tagged and GFP-tagged αT-catenin. For Western blotting, a dilution of 1:1,000 was used for the crude polyclonal sera and a dilution of 1:250 for the affinity purified sera. Recognition of αT-catenin was inhibited by incubation of the polyclonal antibody with the antigenic peptide for one hour prior to use. Serum #952, but not serum #954, turned out to cross-react with mouse αT-catenin.  
         [0106]    Monoclonal antibodies were generated by injection of the N-terminal peptide (MSAETPITLNIDPQDLQ-C (SEQ ID NO: 133)) or the C-terminal peptide (C-KIHPLQVMSEFRGRQIY (SEQ ID NO: 134)) in C57B1/6 mice. Boosts were given with intervals of 2 weeks, and sera were tested by ELISA until a titer of 1:10,000 without loss of reactivity was obtained after 6 weeks. Hybridomas were generated by fusion of spleen cells with Sp20_Ag14 myeloma cells. Supernatants of hybridoma cell lines were tested by ELISA.  
         [0107]    For the N-terminal peptide, up to 72 strongly reacting clones were tested on Western blots for recognition of αT-catenin, fused at its amino terminus to GFP. From the 17 positive hybridomas identified in this way, 4 were also able to recognize native αT-catenin protein in MCF-7 cells transfected with plasmid pEGFPC2-ATCTN(179-2860). A subclone of hybridoma 892 — 24D2, 892 — 24D2S (deposited at BCCM under the number LMBP 5537CB), was used for further analysis.  
         [0108]    For the C-terminal peptide, 30 out of 96 ELISA-positive clones recognized GFP-αT-catenin by Western Blotting, from which 3 were able to recognize native αT-catenin protein by immunofluorescence. A subclone, called hybridoma 893 — 32C6S, was deposited at BCCM under the number LMBP 5728CB.  
         [0109]    Neither monoclonal antibody was found to cross-react with mouse αT-catenin.  
         [0110]    SDS-PAGE and Western Blot Analysis  
         [0111]    Protein lysates from various mouse tissues were prepared by isolating the tissues from normal BALB/c mice and mixing them in Laemmli buffer (Laemmli, 1970). Debris was removed by centrifugation and protein concentration was measured by the BioRad DC kit (BioRad, Richmond, Calif.). Lysates from subconfluent cultures of cell lines were also prepared in Laemmli buffer, followed by sonication and centrifugation. Of each protein lysate, 40 μg was diluted with 6× sample buffer (0.35 M Tris-HCl, pH 6.8, 10.28% SDS, 36% glycerol, 5% β-mercaptoethanol, 0.012% bromophenol blue), boiled for 5 min and subjected to separation on 10% polyacrylamide gels. Proteins were transferred onto Immobilon-P membranes (Millipore, Bredford, Mass.) and blocked with 5% nonfat dry milk, 0.1% Tween-20 in Tris-buffered saline buffer (100 mM Tris-HCl, pH 7.4, 1.4 M NaCl) prior to incubation with the primary antibody. Detection was carried out by phosphatase-coupled secondary antibodies (Sigma) and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) as a substrate.  
         [0112]    Co-immunoprecipitation was performed on lysates of transfected HEK cells, prepared in PBS containing 1% NP-40 and a protease inhibitor cocktail (Boehringer). Lysate (800 μg) was incubated overnight with 4 μg of the respective antibody, after which 100 μl of 50% protein-G (Amersham Pharmacia Biotech, Rainham, UK) was added to monoclonal antibodies, whereas protein-A Sepharose (Amersham Pharmacia Biotech) was added to polyclonal antibodies. After 2 h of incubation, the Sepharose beads were washed three times with PBS containing 0.1% NP-40, followed by boiling for 5 min in Laemmli buffer, before being subjected to SDS-PAGE and Western blotting. On these Western blots, protein was detected by the ECL detection system using secondary antibodies coupled to horseradish peroxidase (Amersham Pharmacia Biotech).  
         [0113]    Tissue Staining  
         [0114]    Frozen sections of human heart and testis tissue were treated for 20 min with 0.3% H 2 O 2  diluted in methanol, then washed in water and PBS, and pretreated for 10 min with goat serum diluted 1:10. The sections were then incubated for 30 min with crude monoclonal 892 — 24D2S hybridoma supernatans, which was diluted 1:5 in PBS containing 1% bovine serum albumin. The secondary antibody used was biotin-labeled goat-anti-mouse Ig (DAKO, Denmark), which was subsequently linked to the streptavidin-ABC complex coupled to horseradish peroxidase. Detection was carried out by a 5 min incubation with the chromogenic peroxidase substrate diaminobenzidine (Sigma). Cell nuclei were counterstained for 5 min with haematoxylin (Sigma), after which the slides were dehydrated by washing in 70%, 90% and two times 100% ethanol. Finally the slides were cleared in toluol and mounted.  
         [0115]    For double immunofluorescent staining, frozen sections were air dried, fixed in acetone at 4° C. for 10 min, washed in PBS and preincubated with 10% goat serum for 10 min. The slides were then incubated for 45 min with mixtures of primary antibodies diluted in PBS: either 1:5 monoclonal antibody 892 — 24D2S plus 1:500 polyclonal anti-αE-catenin, or 1:500 polyclonal antibody #952 plus 1:500 monoclonal anti-N-cadherin. The secondary goat anti-mouse IgG and goat anti-rabbit IgG antibodies used were labeled with FITC or TRITC (Santa Cruz, Santa Cruz, Calif.), or with Alexa 488 or Alexa 594 (Molecular Probes, Eugene, Oreg.).  
         [0116]    Immunocytochemistry of Cell Cultures  
         [0117]    Cells were grown on glass coverslips until confluency, rinsed briefly with PBS and fixed with either ice-cold methanol for 1 min, or with 3% paraformaldehyde (Merck, Darmstadt, Germany) for 10 min at room temperature, followed by permeabilization in 0.2% Triton X-100 (Sigma) for 2 min. Cells were then incubated for 30 min with primary antibody diluted in blocking solution (20 mM Tris/HCl pH 7.5, 154 mM NaCl, 2 mM EDTA, 2 mM MgCl 2 , with 1% BSA and 1% goat serum), washed in PBS, and incubated for 30 min with secondary antibodies diluted in blocking solution. Secondary anti-mouse IgG or anti-rabbit IgG antibodies were coupled to either Alexa 594 or Alexa 488 (Molecular Probes) and used at dilution 1:300. Finally, cells were treated for 10 sec with a 4′-6-diamidine-2-phenylindole-dihydrochloride solution (DAPI; Roche Diagnostics, Mannheim, Germany) to mark nuclear DNA, followed by mounting in Vectashield (Vector Laboratories, Burlingame, Calif.) to prevent photobleaching. Samples were examined with a Zeiss Axiophot microscope and images were recorded with a high-performance charge-coupled digital camera (Cohu, San Diego, Calif.) and NIH image software (version 1.62), or with a MicroMAX camera (Princeton, Trenton, N.J.) and MetaMorph software (Image Universal Corporation, New York, N.Y.).  
         [0118]    Fast Aggregation Assay  
         [0119]    Cell-cell adhesion was numerically evaluated in an aggregation assay as described before (Bracke et al., 1993). In brief, cultures were dissociated into single-cell suspensions under E-cadherin-saving conditions using collagenase. They were incubated under Gyrotory shaking (New Brunswick Scientific, New Brunswick, N.J.) at 80 rpm for 30 min in an isotonic buffer containing either 1 mM EGTA or 1.25 mM Ca 2+ . E-cadherin could be functionally blocked by treatment with MB2 anti-cadherin monoclonal antibody, starting 30 min before aggregation at 4° C. and continued throughout aggregation at 37° C. The volume % distribution in function of the particle diameter was measured by an LS200 particle size analyzer (Coulter Electronics Ltd., Luton, UK), at the start of the incubation at 37° C. (t0) and after 30 min (t30).  
         [0120]    Slow Aggregation Assay  
         [0121]    Slow aggregation was performed as described (Boterberg et al., 2000). Briefly, single-cell suspensions were seeded onto a semi-solid agar medium. After 24 h, aggregate formation was evaluated subjectively by phase contrast microscopy at 40 times magnification.  
       Example 1  
     Isolation of Novel α-catenin cDNAs  
       [0122]    By performing BLAST analyses (Altschul et al., 1990) with αE-catenin sequences as a query, human EST sequences with GenBank Accession Nos. AA393647 and AA400832 (both originating from IMAGE clone-#728263) were found to be, similar but not identical to αE- or αN-catenin. By RT-PCR, we confirmed faint expression of this novel transcript in the PC3 prostate carcinoma cell line. Two consecutive 5′ RACE experiments provided us with a full-length cDNA sequence (FIG. 1A), which was deposited with GenBank under the Accession No AF091606. The 3024-bp sequence (SEQ ID NO: 1) contains a Kozak-consensus start codon (Kozak, 1991) at position 176, preceded by a stop codon at position 137. A stop codon terminating the long open reading frame (ORF) is located at position 2861, and a putative poly-adenylation signal is seen at the 3′ untranslated region at 38 bp before the end of the sequence. The ORF encodes a protein of 895 amino acid residues (SEQ ID NO: 2), with a predicted molecular weight of 100 kDa and an overall identity to αE-catenin (102 kDa) and αN-catenin (104 kD) of respectively 58 and 56% (FIG. 1B). The overall homology is higher, as similarities were calculated of 74 and 70% with respectively αE- and αN-catenin. This novel protein is therefore to be considered a true α-catenin family member, and was called αT-catenin because its transcript was discovered in testis-derived mRNA. When the three main homology domains, as proposed by Herrenknecht (Herrenknecht et al., 1991), are aligned separately, we noticed that sequence conservation is elevated up to 71.5 % identity in the carboxy-terminal domains. In the alignment of the three full-length α-catenin proteins, high sequence conservation in previously described functional domains was observed, but also in other regions (FIG. 2).  
         [0123]    RACE and RT-PCR experiments provided us with a full-length mouse αT-catenin cDNA sequence of 2979 bp (FIG. 16; SEQ ID NO: 4), which we cloned in the pGEMT®-easy vector. The obtained sequence is deposited with GenBank under the Accession No AF344871. This 2979-bp sequence contains a start codon at position 160, preceded by a stop codon at position 114. The stop codon terminating the ORF is located at position 2846. The ORF encodes a protein of 895 amino acid residues (SEQ ID NO: 5), with a predicted molecular weight of 100 kDa. Indeed, after in vitro transcription/translation of plasmid pGEMTeasy-maTctn(1-2979), encoding the full-length mouse αT-catenin cDNA, a protein of approximately 100 kDa was detected (FIG. 17A).  
         [0124]    The overall identity of the mouse αT-catenin to the human αT-catenin protein (FIGS. 16B and 18) is about 95%, whereas the overall identity to mouse αE- and αN-catenin is about 66% and 67%. Hence, we can conclude that the cloned sequence is the mouse orthologue of human αT-catenin.  
       Example 2  
     Analysis of Human and Mouse αT-catenin Genes  
       [0125]    By PCR screening, we isolated a human genomic PAC clone, called clone 320B7 (#1487). This clone was used to perform fluorescence in situ hybridization (FISH), which revealed the localization of the αT-catenin gene CTNNA3 on chromosome band 10q21 (FIGS. 3A and 3B). This localization was confirmed by monochromosomal hybrid mapping and by Genebridge4™ PCR screening. The obtained pattern of PCR products indeed pointed to localization on 10q21, close to marker D10S1461 (FIG. 3C). The region 10q21-23 has been identified as a candidate region for autosomal dominant dilated cardiomyopathy (Bowles et al., 1996). However, up to now, there was no indication of a candidate gene in that region. Partial sequencing of PAC clone #1487 revealed that the clone contains only the last exon of CTNNA3 (exon 18 in FIGS. 4 and 5) besides intronic sequences preceding this exon. Upon database mining by BLAST algorithms, it was found that several genomic sequences (listed in Table 1 and 2) comprise boundaries of different exons of the CTNNA3 gene (FIGS. 4 and 5).  
         [0126]    In order to obtain a human genomic clone containing the 5′ end of CTNNA3, a human BAC library was screened by PCR with 5′ located primers. Clone 162A20 indeed contains the upstream genomic region but comprises only exons 1 and 2 with flanking intronic sequences, besides the upstream gene-regulatory 5′ sequences of the CTNNA3 gene. Indeed, from this genomic BAC clone about 1.2 kb of αT-catenin promoter region was sequenced (FIG. 6A and SEQ ID NO: 3). This promoter sequence was found to bear several putative binding sites for muscle specific transcription factors as predicted by the “Matinspector-Transcription-Factor-binding-site search program” (Quandt et al., 1995). The functional relevance of such sites is suggested by the conservation across species (FIG. 6B) and indeed demonstrated for the MEF2C binding site (FIG. 22).  
         [0127]    All genomic data were deposited with GenBank under accession numbers AF282678 to AF282692 and AF391792 to AF391794. Primers were designed on intronic sequences flanking each exon in order to amplify each of the 18 CTNNA3 exons for applications such as analysis of mutations and polymorphisms by SSCP or denaturing HPLC (Table 3). At the amino acid level, most exon-exon boundaries (boxed in FIG. 4) coincide with the boundaries determined for the αE-catenin CTNNA1 gene (Furukawa et al., 1994) and the CTNNAL1 gene (Janssens et al., 1999), pointing towards a common ancestor for all α-catenin genes. Interestingly, divergence in the genomic structure is observed for the CTNNA3 region covering exons 13 to 15. This domain corresponds to a region where the a-catulin gene CTNNAL1 also shows a divergent genomic organization, besides a “gap” in the open reading frame.  
         [0128]    In order to obtain a mouse genomic clone containing the 5′ end of the Ctnna3 gene, a mouse BAC library was screened by PCR with primers located in the first three exons of the mouse Ctnna3 gene. Genomic clone 164N 16 was found to contain these three exons. Part of the mouse promoter sequence was determined (SEQ ID NO: 6).  
       Example 3  
     αT-catenin Interacts Stronger with β-catenin than Other α-catenins Do  
       [0129]    The colocalization of αT-catenin and β-catenin suggested interaction between these two proteins. We confirmed this interaction in the two-hybrid system, by cotransformation of full-length αT-catenin, αE-catenin, αN-catenin and α-catulin bait fusions with prey fusions containing an amino terminal part of β-catenin and nearly full-length plakoglobin (FIG. 7A). In this way, we confirmed the reported interaction between αE-catenin and β-catenin (Aberle et al., 1994; Funayama et al., 1995; Hulsken et al., 1994; Jou et al., 1995), and between αN-catenin and β-catenin (Sehgal et al., 1997). Interestingly, α-catulin does not interact with β-catenin. On the other hand, we demonstrated the presumptive interaction between αT-catenin and β-catenin, and noticed strong blue staining as compared to other positive interactions (FIG. 7A), suggesting that α-catenins bind to β-catenin with the following decreasing strength: αT-catenin &gt;αN-catenin &gt;αE-catenin.  
         [0130]    When the interaction with β-catenin was quantified in the two-hybrid system, by using CPRG as a substrate for β-galactosidase, the values found for interaction with αT-catenin were about four times higher than these found for interaction with αE-catenin (FIG. 7B). This confirms that αT-catenin is able to interact in a stronger way to β-catenin than other α-catenins do. The interaction between αT-catenin and β-catenin could be confirmed by coimmunoprecipitation from lysates of HEK-293 cells overexpressing Myc-tagged αT-catenin (FIG. 7C), and also by coimmunoprecipitation from lysates of mouse heart and testis tissues (FIG. 7D). Thus, the interaction between αT-catenin and β-catenin occurs also in vivo.  
       Example 4  
     The αT-catenin Protein is Preferentially Expressed in Heart and Testis Tissues  
       [0131]    A human cDNA Rapid Scan panel (OriGene Technologies, Rockville, Md.) was screened by PCR for αT-catenin expression. A first PCR reaction revealed expression in heart and testis tissues only, whereas a second, nested PCR amplified low amounts in some other tissues (brain, kidney, liver, skeletal muscle, fetal liver) (FIG. 8A). In comparison to the ubiquitously expressed αE-catenin (FIG. 8A), the novel αT-catenin shows a very restricted expression pattern. Besides the original testis-derived EST clone (Accession Nos. AA393647 and AA400832), one additional αT-catenin-specific EST sequence, derived from kidney, was identified recently (Accession No AW444927). Weak amplification of the αT-catenin transcript is indeed seen by us in kidney tissue (FIG. 8A).  
         [0132]    These findings were confirmed by RT-PCR analysis (FIG. 8B) of several mouse tissues. The brain-specific expression of mouse αN-catenin mRNA is in line with the literature (Hirano et al., 1992).  
         [0133]    We generated αT-catenin-specific polyclonal antibodies #952, specific for a carboxyterminal peptide of human αT-catenin with sequence C-KIHPLQVMSEFRGRQIY (SEQ ID NO: 134). Using serum #952 on several mouse tissue protein lysates, we observed strong expression of αT-catenin in heart, lower levels in testis but hardly any αT-catenin protein in kidney, ovary, spleen or colon tissue, whereas these same tissues contain αE-catenin and β-catenin protein (FIG. 8C). This confirms the observed tissue-specificity of the αT-catenin mRNA (FIG. 8B) at the protein level. Monoclonal antibody 893 — 32C6S, generated against the same antigenic peptide as serum #952, is specific for human αT-catenin but does not cross-react with mouse αT-catenin (FIG. 17B).  
         [0134]    Northern blot analysis confirmed the strong expression of mouse αT-catenin in heart and testis. Different strong signals, which appear smaller (±2000 nt and ±2500 nt) than the full-length mouse αT-catenin mRNA (2,979 nt) on agarose gel, suggest the expression of alternative transcripts of mouse αT-catenin in both organs (FIG. 20). Also in Western blot analysis, some bands with smaller apparent molecular weights (of about 43 kDa, 66kDa and 86 kDa) were detected in heart and testis lysates (FIG. 21A). Immunodetection of all these bands is competed out by addition of the αT-catenin-specific immunogenic peptide (FIG. 21B). Importantly, the smallest mRNA transcript is very abundant in testis. Correspondingly, the 66-kDa band on Western blot also appears to be stronger than the full-length mouse αT-catenin protein in lysates of testis. As the probes and antibody used are specific for respectively the 3′-end of the αT-catenin transcript or the C-terminal end of the αT-catenin protein, the alternative variants may be N-terminally truncated and possibly deficient for β-catenin binding.  
       Example 5  
     Human αT-catenin Can be Detected in Cardiomyocytes and Testis Tissue  
       [0135]    Frozen sections of human heart and testis tissue were stained with the monoclonal 892 — 24D2S antibodies, shown to be specific for αT-catenin. Human αT-catenin protein can be detected in high amounts at intercalated discs, which are the specific heart cell-cell junctions to which actin microfilaments anchor (FIG. 9A and 9B). In testis, weaker but specific staining can be seen in interstitial elongated cells nearby the basement membrane of seminiferous tubules, which are probably peritubular myoid cells (FIG. 11A and 11B). These results suggest that αT-catenin protein is expressed in specific contractile cells of heart and testis tissues.  
         [0136]    In double labeling experiments, it co-localizes with αE-catenin (FIG. 10A) as well as N-cadherin (FIG. 10B). The muscle marker desmin can be detected at both intercalated discs and sarcomeric Z-lines, whereas αT-catenin expression is confined to intercalated discs (FIG. 10C). In human testis, αT-catenin protein was detected mainly in spindle-shaped cells surrounding testicular tubuli (FIG. 11). Interestingly, αT-catenin did not co-localize here with αE-catenin, as the latter showed an abundant intratubular expression pattern (FIG. 11A). The αT-catenin expressing cells in testis correspond to desmin-positive cells, and therefore could be identified as peritubular myoid cells (FIG. 11B). These stainings strongly suggest that αT-catenin expression is confined to specific muscle cell types  
       Example 6  
     αT-catenin Functionally Restores Cell Aggregation in α-catenin-Negative Cancer Cells  
       [0137]    To assess whether αT-catenin binding to β-catenin has functional implications for the formation of cell-cell contacts, we carried out rescue experiments by overexpression of αT-catenin in round HCT-8/R1 cells lacking α-catenins (Vermeulen et al., 1995; Vermeulen et al., 1999). Vaccinia virus-mediated expression was used to obtain high transient transfection efficiencies (between 30 and 70%). Cell-cell adhesion was found to be restored if neighboring cells were expressing the ectopic protein that became enriched at the cell-cell contacts, whereas solitary expressing cells remained round with diffuse expression of the ectopic protein (FIG. 12A). Moreover, when GFP-tagged αT-catenin was overexpressed in neighboring cells, its enrichment in cell-cell contacts recruited both β-catenin and E-cadherin to these sites (FIG. 12B). However, when αT-catenin was overexpressed for longer time periods, it tended to form cytoplasmic rod-like aggregates.  
         [0138]    In order to quantify the restoration of cell-cell adhesion by αT-catenin expression in HCT-8/R1 cells, these cells were transfected with a plasmid encoding Myc-tagged αT-catenin. A stable transfectant was cloned and called HCT-8/R1/T31. Western blotting with polyclonal antiserum #952 showed high expression of αT-catenin protein in this cell line. Immunofluorescent analysis of HCT-8/R1/T31 cells with anti-Myc antibodies showed that the αT-catenin-Myc protein was localized at restored cell-cell contacts (FIG. 13). Components of the adherens junctions (E-cadherin, β-catenin and plakoglobin) were recruited to such αT-catenin positive sites (illustrated in FIG. 13), but also desmosomes (desmoglein-2) and tight junctions (ZO-1, occludin) showed reassembly (illustrated in FIG. 13).  
         [0139]    By using the fast aggregation assay, we were able to show that HCT-8/RI/T31 cells are strongly aggregating in contrast to nonaggregating parental HCT-8/R1 cells (FIG. 14). HCT-8/R1/T31 cells aggregated to similar extent as αE-catenin-positive HCT-8/E8 cells and αN-catenin-transfected HRpCαN2 cells, thus showing that αT-catenin is able to functionally restore aggregation. These findings were confirmed using another transfected cell line, HCT-8/E11R1/T14, stably expressing a GFP-αT-catenin fusion protein.  
         [0140]    In a slow aggregation assay, αT-catenin-transfected HCT-8/R1/T31 cells were compacting even better than αE-catenin-positive HCT-8/E8 cells or αN-catenin-transfected HRpCαN2 cells (FIG. 15).  
                                                     TABLE 1                           Corresponding                       Genomic Clone   BAC   Size (bp)       CTNNA   (Name or   size (bp)   of exon-   position of       3 exon   GenBank   as far as   containing   exon       #   Acc. No.)   sequenced   contig   in contig                                1   BAC 162A20   &gt;9,000   8,000   2,500-2,600       2   BAC 162A20   &gt;9,000   1,000           AC009037.6   30,769   30,769   7,602-7,499       3   AC026394.9   180,805   54,182   25,892-26,084           AC027668.2   32,000   9,167   91,612-91,804       4   AC026394.9   180,805   23,941   17,471-17,637       5   AC026394.9   180,805   11,415   541-660       6   AC022534.7   185,679   185,679   56,887-56621            AC022401.3   172,591   172,591   163,589-163,323       7   AC024602.5   175,504   175,504   48,365-48,559           AC022534.7   185,679   185,679   17,533-17,330           AQ163827.1   396   396    16-289           AC022401.3   172,591   172,591   124,236-124,033       8   n.a.   n.a.   n.a.   n.a.       9   n.a.   n.a.   n.a.   n.a.       10   AQ351427.1   476   476   151-243           AC020642.6   145,695   145,695   110,853-110,945           AQ355080.1   677   677   151-243       11   AC023847.2   80,449   8,532   7,255-7,411           AC027668.3   200,268   140,908   93,854-93,699       12   AC016819.4   136,685   17,473   4,170-4,370           AC027675.5   160,439   1,919   561-761       13   AC027675.5   160,439   2,474   208-359       14   AC021888.3   174,470   7,744   6,707-6799           AC022017.5   183,665   141,718   46,258-46,350           AC022024.2   369,279   2,919   2,863-2,919           AL592075   73,925   7,743   6,705-6,797       15   AC022017.5   183,665   141,718   80,018-80,400           AC022024.2   369,279   8,442   3,097-3,278           AP001355.2   186,290   15,133   1,474-1,655           AL513126.4   163,341   77,255   61,704-561,522       16   AC022024.2   369,279   2,571   549-654           AP001355.2   186,290   11,104   1,303-1,408           AC022017.5   183,665   21,147   1,709-1,818           AL513126.4   163,341   68,244   66,959-66,854       17   AP001355.2   186,290   6,741   5,376-5,510           AL513126.4   163,341   68,244   44,908-44,774       18   AP001355.2   186,290   46,144   31,409-31,852           PAC 320B7   &gt;1,000   1,000           AC018979.6   356,758   3,084   2,111-2,557                  
 
         [0141]    [0141]                                                                     TABLE 2                       EXON   exon size   slice donor   intron size   slice acceptor                                                &gt;0.5   kb   axxctgggtgaa  CAACGCTCA-                                                 1                                                              M       1   170   bp   AAC-AGA-A AG    gt aagaatcaag   &gt;23.2   kb   tttgtgcagc ag   -GC-AGC-ATG-                           170                          171                            I   I   Q                          V   T   T       2   104   bp   ATA-ATC-C AG    gt attaatacca   &gt;8.6   kb   ttccaatttt ag    GT T-ACC-ACA-                           274                         275                            K   E                          S   E   A       3   193   bp   AAA-GAA-A--   gt gagtactcca   67   kb   tgtatttttc ag   - GT -GAA-GCT-                           467                          468                            V   S   A                          F   Q   R       4   167   bp   GTG-TCA-GCT   gt aagtaaaga.   17.5   kb   tttcaatttc ag   TTT-CAA-AGG-                           634                         635                            R   Q   Q                          D   L   K       5   120   bp   CGT-CAG-C AG    gt aggagtcaga.   &gt;100   kb   ttaccttctc ag   GAC-TTA-AAA-                           754                         755                            E   L   E                          N   L   I       6   264   bp   GAG-CTG-G AG    gt aagtcgggag.   39.5   kb   ttcttctctt ag   AAT-TTA-ATT-                          1018                         1019                            M   N   N                           A   G   K       7   204   bp   ATG-AAC-AAC   gt aagtatagtt.   &gt;50   kb   tcttcctttgc ag   GCT-GGA-AAA-                          1222                          1223                            R   R   Q                          L   R   K       8   81   bp   CGC-AGA-C AG    gt gagggaagag.   9   kb   atttcttctc ag   CTC-CGC-AAG-                          1303                         1304                            L   V   E                          V   A   N       9   153   bp   CTT-GTA-G AG    gt aagcatgcta.   &gt;150   kb   attgtattta ag    GT G-GCA-AAT-                          1456                         1457                            C   P   Q                          I   I   N       10   93   bp   TGT-CCA-C AG    gt atgacaacta.   100   kb   ttatctttat ag   ATT-ATT-AAT-                          1549                         1550                            V   S                          E   S   H       11   157   bp   GTA-TCT-G--   gt atgtttttat.   &gt;2.5   kb   atttacttac ag   -AA-AGC-CAT-                        1706                          1707                            S   T                          V   I   P       12   201   bp   AGT-ACT-G--   gt aagtcagttg.   &gt;7.2   kb   ttattttaac ag   -TA-ATT-CCT-                        1907                          1908                            M   I   R                              T   P       13   151   bp   ATG-ATT-CG-   gt aagtttgctt.   &gt;6.7   kb   ttctttttat ag   --G-ACC-CCA-                         2058                           2059                            T   D   R                          A   K   M       14   94   bp   ACT-GAT-AGG   gt atgtcacttc.   34   kb   cacatgtttt ag   GCT-AAG-ATG-                          2152                         2153                            F   T   R                              G   K       15   182   bp   TTC-ACT- AG -   gt aattatgtgg   81   kb   atttttttcc ag   --G-GGC-AAA-                         2334                           2335                            A   N   Q                          C   P   D       16   106   bp   GCT-AAT-C AG    gt gagttactta.   22   kb   atgcatattt ag   TGC-CCA-GAT-                          2440                         2441                            M   S   A                          L   D   S       17   135   bp   ATG-TCA-GCT   gt gagtactgcc.   46   kb   ttttccctac ag   TTG-GAC-AGT-                          2575                         2576               18   445   bp   ATA-TAT-A--  tttgggatcatt   &gt;200   kb                        3019                    
         [0142]    [0142]                                                                               TABLE 3                           Overview of CTNNA3 exon-specific PCRs                            [MgC12]   Product   A.T.           Exon   Upper Primer   Lower Primer   (mM)   (bp)   (° C)                    1   FVR 2513 (SEQ ID NO:43)   FVR 2514 (SEQ ID NO:44)   2   395   61               5′ TTGCTTGTAACCTCCCCTTT 3′   5′ GCGTGAAAGCCTACGTTTCT 3′               2   FVR 2515 (SEQ ID NO:45)   FVR 2516 (SEQ ID NO:46)   2   407   55.8           5′ TAATTTGTTACAGGACCTAAGC 3′   5′ TCTTCATTATTCATTTTTCCCAC 3′               3   FVR 2517 (SEQ ID NO:47)   FVR 2518 (SEQ ID NO:48)   2   353   59.6           5′ TATCCCAGGACTGTGTTCTC 3′   5′ TGGAGCCAAAAACAAAACA 3′               4   FVR 2519 (SEQ ID NO:49)   FVR 2520 (SEQ ID NO:50)   2   252   56.7           5′ TGGGGTTGTATTTTTCAGGTG 3′   5′ GCCAGGTTCAGAGAATGAAAT 3′               5   FVR 2521 (SEQ ID NO:51)   FVR 2522 (SEQ ID NO:52)   2   406   59.3           5′ GGACTGAACAGGCTTCTCAT 3′   5′ GCAGGAAGCCTAAAGTGTTC 3′               6   FVR 2523 (SEQ ID NO:53)   FVR 2524 (SEQ ID NO:54)   2   404   59.3           5′ GTCTTTCTCCCATAACCCATT 3′   5′ CGCCAACATGTGGATCTTCT 3′               7   FVR 2525 (SEQ ID NO:55)   FVR 2526 (SEQ ID NO:56)   2   326   57.8           5′ TGAAATGCCATGGAGCTCTAA 3′   5′ ACGGAAAGTATCTCAGCCTAT 3′               8   FVR 2958 (SEQ ID NO:57)   FVR 2959 (SEQ ID NO:58)   2   156   57.8           5′ CCATTGCTTATGTCGTTTTTTC 3′   5′ TTAGCCCCTATGTTTCTGACT 3′               9   FVR 2960 (SEQ ID NO:59)   FVR 2961 (SEQ ID NO:60)   2   259   56.5           5′ AGAAAAGGAAACACAGTGAACT 3′   5′ TTCTCCTGGACTTTAGTGAGTT 3′               10   FVR 2527 (SEQ ID NO:61)   FVR 2528 (SEQ ID NO:62)   2   267   62.4           5′ TGTTGCTGCATTTCCTTGCTA 3′   5′ GCGAGACCTGGTCTCAAAAA 3′               11   FVR 2529 (SEQ ID NO:63)   FVR 2530 (SEQ ID NO:64)   2   300   62.4           5′ GTGCCCATCACCCAAATAGT 3′   5′ CCATGCCTGTCCCAGTATTA 3′               12   FVR 2531 (SEQ ID NO:65)   FVR 2532 (SEQ ID NO:66)   2   350   61.0           5′ CCATTTCCAATGTGCACTCTA 3′   5′ AATTGTGCAGCTGTTATTGGC 3′               13   FVR 2956 (SEQ ID NO:67)   FVR 2957 (SEQ ID NO:68)   2   217   60.8           5′ ACAAAGAGGACAATCTTCTCC 3′   5′ TCAATGGAAGGAAAAGCAAAC 3′               14   FVR 2533 (SEQ ID NO:69)   FVR 2534 (SEQ ID NO:70)   2   301   64.3           5′ TGGGAGTGAAATTGCTGGGT 3′   5′ TAGAGGCTGCCTAGATTGAC 3′               15   FVR 2535 (SEQ ID NO:71)   FVR 2536 (SEQ ID NO:72)   2   326   56.7           5′ TGCTTTTGACATAGTGGAATGA 3′   5′ TGGCACTTGACACTCAGAGA 3′               16   FVR 2537 (SEQ ID NO:73)   FVR 2538 (SEQ ID NO:74)   2   295   56.7           5′ CCGTTCTTTGGGATGCGAAT 3′   5′ GGCAAAGAGCAATTAGCATGA 3′               17   FVR 2539 (SEQ ID NO:75)   FVR 2540 (SEQ ID NO:76)   2   313   59.3           5′ AAGGTACCTGCCATGTGAATA 3′   5′AGATTTGGTCATGTAAACAAGG 3′               18   FVR 2541 (SEQ ID NO:77)   FVR 2542 (SEQ ID NO:78)   2   552   62.8           5′ CCACGCTTGGCAATAATTAAC 3′   5′TGCTGACCATACAGAAATGAC 3′                    
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         1 
         
           
             134  
           
           
             1  
             3024  
             DNA  
             Homo sapiens  
             
               CDS  
               (176)..(2863)  
             
           
            1 

caacgctcag cgaaattgac tgccccactg tcatctgcct ctcaatttgg tactctgtaa     60 

ctctgtgacc accaagaagc ctttttccgt cccccacaaa gctctttttg gaaaattccc    120 

tacgggagct gaattttaag cccatttact ttataggaag aaacagaaag gcagc atg     178 
                                                             Met 
                                                               1 

tca gct gaa aca cca atc aca ttg aat atc gat cct cag gat ctg cag      226 
Ser Ala Glu Thr Pro Ile Thr Leu Asn Ile Asp Pro Gln Asp Leu Gln 
              5                  10                  15 

gtc caa aca ttc acc gtg gag aag cta ctg gag cct ctc ata atc cag      274 
Val Gln Thr Phe Thr Val Glu Lys Leu Leu Glu Pro Leu Ile Ile Gln 
         20                  25                  30 

gtt acc aca ctt gta aac tgt ccc cag aac cct tcc agc agg aaa aaa      322 
Val Thr Thr Leu Val Asn Cys Pro Gln Asn Pro Ser Ser Arg Lys Lys 
     35                  40                  45 

gga cgt tcg aaa aga gcc agt gtc ctt cta gct tct gtg gag gaa gca      370 
Gly Arg Ser Lys Arg Ala Ser Val Leu Leu Ala Ser Val Glu Glu Ala 
 50                  55                  60                  65 

act tgg aat tta tta gac aag gga gag aag att gcc cag gaa gct aca      418 
Thr Trp Asn Leu Leu Asp Lys Gly Glu Lys Ile Ala Gln Glu Ala Thr 
                 70                  75                  80 

gtt tta aag gat gag ctt acg gct tca ctt gag gaa gtt cgc aaa gaa      466 
Val Leu Lys Asp Glu Leu Thr Ala Ser Leu Glu Glu Val Arg Lys Glu 
             85                  90                  95 

agt gaa gct ctg aaa gta tca gct gag aga ttt aca gat gac ccc tgt      514 
Ser Glu Ala Leu Lys Val Ser Ala Glu Arg Phe Thr Asp Asp Pro Cys 
        100                 105                 110 

ttt ctc cca aaa agg gag gct gtg gtt caa gct gcc cgt gcc ttg ctg      562 
Phe Leu Pro Lys Arg Glu Ala Val Val Gln Ala Ala Arg Ala Leu Leu 
    115                 120                 125 

gct gcg gtg acg aga ctc ctt atc ctt gcg gac atg att gat gtc atg      610 
Ala Ala Val Thr Arg Leu Leu Ile Leu Ala Asp Met Ile Asp Val Met 
130                 135                 140                 145 

tgc ctc ttg caa cat gtg tca gct ttt caa agg aca ttt gag act ctc      658 
Cys Leu Leu Gln His Val Ser Ala Phe Gln Arg Thr Phe Glu Thr Leu 
                150                 155                 160 

aaa aat gtt gcc aac aaa tct gac ctc cag aaa acc tac cag aag ctt      706 
Lys Asn Val Ala Asn Lys Ser Asp Leu Gln Lys Thr Tyr Gln Lys Leu 
            165                 170                 175 

gga aag gag ctg gaa aat ttg gat tat tta gcc ttc aaa cgt cag cag      754 
Gly Lys Glu Leu Glu Asn Leu Asp Tyr Leu Ala Phe Lys Arg Gln Gln 
        180                 185                 190 

gac tta aaa tct cca aat cag aga gat gaa att gca gga gcc cga gct      802 
Asp Leu Lys Ser Pro Asn Gln Arg Asp Glu Ile Ala Gly Ala Arg Ala 
    195                 200                 205 

tca ctg aag gag aac tct ccc ctc ttg cat tca att tgt tca gct tgt      850 
Ser Leu Lys Glu Asn Ser Pro Leu Leu His Ser Ile Cys Ser Ala Cys 
210                 215                 220                 225 

ttg gag cat tct gat gtt gct tcc ctc aaa gca agc aag gac aca gtt      898 
Leu Glu His Ser Asp Val Ala Ser Leu Lys Ala Ser Lys Asp Thr Val 
                230                 235                 240 

tgt gaa gaa att cag aat gct ctc aat gta att tca aat gct tca caa      946 
Cys Glu Glu Ile Gln Asn Ala Leu Asn Val Ile Ser Asn Ala Ser Gln 
            245                 250                 255 

ggg atc cag aat atg aca acc cca cca gaa cct cag gca gca acc ctg      994 
Gly Ile Gln Asn Met Thr Thr Pro Pro Glu Pro Gln Ala Ala Thr Leu 
        260                 265                 270 

gga agt gcc ctt gat gag ctg gag aat tta att gtc ctg aat cca ctc     1042 
Gly Ser Ala Leu Asp Glu Leu Glu Asn Leu Ile Val Leu Asn Pro Leu 
    275                 280                 285 

aca gta act gag gag gaa ata cga cca tca cta gag aaa cgc ctt gaa     1090 
Thr Val Thr Glu Glu Glu Ile Arg Pro Ser Leu Glu Lys Arg Leu Glu 
290                 295                 300                 305 

gcc att atc agt ggg gct gct ctg ctg gcg gat tct tca tgt acg agg     1138 
Ala Ile Ile Ser Gly Ala Ala Leu Leu Ala Asp Ser Ser Cys Thr Arg 
                310                 315                 320 

gac tta cac cga gag cgg att atc gca gaa tgc aac gcc att cgc cag     1186 
Asp Leu His Arg Glu Arg Ile Ile Ala Glu Cys Asn Ala Ile Arg Gln 
            325                 330                 335 

gct ctt cag gat ctg ctt tca gag tac atg aac aac gct gga aaa aaa     1234 
Ala Leu Gln Asp Leu Leu Ser Glu Tyr Met Asn Asn Ala Gly Lys Lys 
        340                 345                 350 

gaa agg agt aat acc ctg aat att gct tta gac aac atg tgt aag aag     1282 
Glu Arg Ser Asn Thr Leu Asn Ile Ala Leu Asp Asn Met Cys Lys Lys 
    355                 360                 365 

aca aga gac ctt cgc aga cag ctc cgc aag gct att ata gat cat gtg     1330 
Thr Arg Asp Leu Arg Arg Gln Leu Arg Lys Ala Ile Ile Asp His Val 
370                 375                 380                 385 

tca gac tct ttc ctg gat acg aca gtc cct ctt ttg gtt ctc att gaa     1378 
Ser Asp Ser Phe Leu Asp Thr Thr Val Pro Leu Leu Val Leu Ile Glu 
                390                 395                 400 

gct gct aag aat ggc cgg gaa aag gaa ata aaa gaa tat gct gcg ata     1426 
Ala Ala Lys Asn Gly Arg Glu Lys Glu Ile Lys Glu Tyr Ala Ala Ile 
            405                 410                 415 

ttt cat gaa cac acc agc agg ctt gta gag gtg gca aat ctt gct tgt     1474 
Phe His Glu His Thr Ser Arg Leu Val Glu Val Ala Asn Leu Ala Cys 
        420                 425                 430 

tcc atg tca aca aat gaa gat gga att aaa att gtc aaa att gca gcc     1522 
Ser Met Ser Thr Asn Glu Asp Gly Ile Lys Ile Val Lys Ile Ala Ala 
    435                 440                 445 

aat cat ttg gaa acc ttg tgt cca cag att att aat gct gca ctt gct     1570 
Asn His Leu Glu Thr Leu Cys Pro Gln Ile Ile Asn Ala Ala Leu Ala 
450                 455                 460                 465 

ttg gct gca aga ccc aaa agt caa gcg gtc aaa aac acc atg gaa atg     1618 
Leu Ala Ala Arg Pro Lys Ser Gln Ala Val Lys Asn Thr Met Glu Met 
                470                 475                 480 

tac aag cgt aca tgg gag aat cat ata cat gtc ctc act gaa gcc gta     1666 
Tyr Lys Arg Thr Trp Glu Asn His Ile His Val Leu Thr Glu Ala Val 
            485                 490                 495 

gat gac att aca agc att gat gac ttc ctt gct gta tct gaa agc cat     1714 
Asp Asp Ile Thr Ser Ile Asp Asp Phe Leu Ala Val Ser Glu Ser His 
        500                 505                 510 

atc ttg gaa gat gtc aac aag tgt atc ata gcc tta aga gac cag gat     1762 
Ile Leu Glu Asp Val Asn Lys Cys Ile Ile Ala Leu Arg Asp Gln Asp 
    515                 520                 525 

gct gat aat tta gac cgt gct gcg ggt gct atc aga ggc cgg gca gca     1810 
Ala Asp Asn Leu Asp Arg Ala Ala Gly Ala Ile Arg Gly Arg Ala Ala 
530                 535                 540                 545 

aga gtt gct cac atc gtc acg ggt gaa atg gac agt tac gag cca ggg     1858 
Arg Val Ala His Ile Val Thr Gly Glu Met Asp Ser Tyr Glu Pro Gly 
                550                 555                 560 

gct tac acg gaa ggt gta atg aga aat gtt aac ttc ctt aca agt act     1906 
Ala Tyr Thr Glu Gly Val Met Arg Asn Val Asn Phe Leu Thr Ser Thr 
            565                 570                 575 

gta att cct gaa ttt gta aca caa gtg aat gtt gcc ttg gaa gcc tta     1954 
Val Ile Pro Glu Phe Val Thr Gln Val Asn Val Ala Leu Glu Ala Leu 
        580                 585                 590 

agc aaa agc tca ttg aat gtg ttg gat gat aat caa ttt gtg gac atc     2002 
Ser Lys Ser Ser Leu Asn Val Leu Asp Asp Asn Gln Phe Val Asp Ile 
    595                 600                 605 

tca aag aag atc tat gat aca att cat gat atc aga tgt tca gtc atg     2050 
Ser Lys Lys Ile Tyr Asp Thr Ile His Asp Ile Arg Cys Ser Val Met 
610                 615                 620                 625 

atg att cgg acc cca gag gaa ctg gag gat gtt tct gac ctt gaa gag     2098 
Met Ile Arg Thr Pro Glu Glu Leu Glu Asp Val Ser Asp Leu Glu Glu 
                630                 635                 640 

gaa cac gag gtc cgc agt cac acc agc att cag acc gaa ggg aaa act     2146 
Glu His Glu Val Arg Ser His Thr Ser Ile Gln Thr Glu Gly Lys Thr 
            645                 650                 655 

gat agg gct aag atg act caa ctg cct gag gca gaa aaa gaa aag att     2194 
Asp Arg Ala Lys Met Thr Gln Leu Pro Glu Ala Glu Lys Glu Lys Ile 
        660                 665                 670 

gct gag caa gtt gct gat ttc aag aaa gta aag agt aag ctg gat gct     2242 
Ala Glu Gln Val Ala Asp Phe Lys Lys Val Lys Ser Lys Leu Asp Ala 
    675                 680                 685 

gag att gag ata tgg gat gat aca agc aac gac atc att gtt ctg gcc     2290 
Glu Ile Glu Ile Trp Asp Asp Thr Ser Asn Asp Ile Ile Val Leu Ala 
690                 695                 700                 705 

aag aac atg tgt atg atc atg atg gag atg aca gac ttc act agg ggc     2338 
Lys Asn Met Cys Met Ile Met Met Glu Met Thr Asp Phe Thr Arg Gly 
                710                 715                 720 

aaa gga cca cta aag cat aca act gat gtg atc tat gca gcg aaa atg     2386 
Lys Gly Pro Leu Lys His Thr Thr Asp Val Ile Tyr Ala Ala Lys Met 
            725                 730                 735 

ata tca gaa tca gga tca agg atg gat gtc ctt gct cgg cag att gct     2434 
Ile Ser Glu Ser Gly Ser Arg Met Asp Val Leu Ala Arg Gln Ile Ala 
        740                 745                 750 

aat cag tgc cca gat cca tct tgt aaa cag gac ttg ttg gcc tac ctg     2482 
Asn Gln Cys Pro Asp Pro Ser Cys Lys Gln Asp Leu Leu Ala Tyr Leu 
    755                 760                 765 

gaa cag att aag ttc tac tcc cac caa ctg aaa atc tgc agt caa gtt     2530 
Glu Gln Ile Lys Phe Tyr Ser His Gln Leu Lys Ile Cys Ser Gln Val 
770                 775                 780                 785 

aaa gct gag atc cag aac ctg gga gga gag ctc atc atg tca gct ttg     2578 
Lys Ala Glu Ile Gln Asn Leu Gly Gly Glu Leu Ile Met Ser Ala Leu 
                790                 795                 800 

gac agt gtc aca tcc ctg atc caa gca gcc aaa aat tta atg aat gct     2626 
Asp Ser Val Thr Ser Leu Ile Gln Ala Ala Lys Asn Leu Met Asn Ala 
            805                 810                 815 

gta gtg caa aca gtg aaa atg tct tac att gcc tca acc aag atc atc     2674 
Val Val Gln Thr Val Lys Met Ser Tyr Ile Ala Ser Thr Lys Ile Ile 
        820                 825                 830 

cga atc cag agt cct gct ggg ccc cgg cac cca gtt gtg atg tgg aga     2722 
Arg Ile Gln Ser Pro Ala Gly Pro Arg His Pro Val Val Met Trp Arg 
    835                 840                 845 

atg aag gct cct gca aaa aaa ccc ttg att aaa aga gag aag cca gag     2770 
Met Lys Ala Pro Ala Lys Lys Pro Leu Ile Lys Arg Glu Lys Pro Glu 
850                 855                 860                 865 

gaa acg tgt gca gct gtc aga cga ggc tca gca aag aaa aaa atc cat     2818 
Glu Thr Cys Ala Ala Val Arg Arg Gly Ser Ala Lys Lys Lys Ile His 
                870                 875                 880 

cca ttg caa gtc atg agt gaa ttt aga gga aga caa atc tac tga         2863 
Pro Leu Gln Val Met Ser Glu Phe Arg Gly Arg Gln Ile Tyr 
            885                 890                 895 

aaccactatt ctacatatag tgcctatatg acaaaatcct gcctaaccac actgctttat   2923 

tttacactta agaagttctg taatttcact aagttttggt gtttaactca caaataacat   2983 

aaaatattgg gcgctaaatc aacaaaagca atatataaaa a                       3024 

 
           
             2  
             895  
             PRT  
             Homo sapiens  
           
            2 

Met Ser Ala Glu Thr Pro Ile Thr Leu Asn Ile Asp Pro Gln Asp Leu 
  1               5                  10                  15 

Gln Val Gln Thr Phe Thr Val Glu Lys Leu Leu Glu Pro Leu Ile Ile 
             20                  25                  30 

Gln Val Thr Thr Leu Val Asn Cys Pro Gln Asn Pro Ser Ser Arg Lys 
         35                  40                  45 

Lys Gly Arg Ser Lys Arg Ala Ser Val Leu Leu Ala Ser Val Glu Glu 
     50                  55                  60 

Ala Thr Trp Asn Leu Leu Asp Lys Gly Glu Lys Ile Ala Gln Glu Ala 
 65                  70                  75                  80 

Thr Val Leu Lys Asp Glu Leu Thr Ala Ser Leu Glu Glu Val Arg Lys 
                 85                  90                  95 

Glu Ser Glu Ala Leu Lys Val Ser Ala Glu Arg Phe Thr Asp Asp Pro 
            100                 105                 110 

Cys Phe Leu Pro Lys Arg Glu Ala Val Val Gln Ala Ala Arg Ala Leu 
        115                 120                 125 

Leu Ala Ala Val Thr Arg Leu Leu Ile Leu Ala Asp Met Ile Asp Val 
    130                 135                 140 

Met Cys Leu Leu Gln His Val Ser Ala Phe Gln Arg Thr Phe Glu Thr 
145                 150                 155                 160 

Leu Lys Asn Val Ala Asn Lys Ser Asp Leu Gln Lys Thr Tyr Gln Lys 
                165                 170                 175 

Leu Gly Lys Glu Leu Glu Asn Leu Asp Tyr Leu Ala Phe Lys Arg Gln 
            180                 185                 190 

Gln Asp Leu Lys Ser Pro Asn Gln Arg Asp Glu Ile Ala Gly Ala Arg 
        195                 200                 205 

Ala Ser Leu Lys Glu Asn Ser Pro Leu Leu His Ser Ile Cys Ser Ala 
    210                 215                 220 

Cys Leu Glu His Ser Asp Val Ala Ser Leu Lys Ala Ser Lys Asp Thr 
225                 230                 235                 240 

Val Cys Glu Glu Ile Gln Asn Ala Leu Asn Val Ile Ser Asn Ala Ser 
                245                 250                 255 

Gln Gly Ile Gln Asn Met Thr Thr Pro Pro Glu Pro Gln Ala Ala Thr 
            260                 265                 270 

Leu Gly Ser Ala Leu Asp Glu Leu Glu Asn Leu Ile Val Leu Asn Pro 
        275                 280                 285 

Leu Thr Val Thr Glu Glu Glu Ile Arg Pro Ser Leu Glu Lys Arg Leu 
    290                 295                 300 

Glu Ala Ile Ile Ser Gly Ala Ala Leu Leu Ala Asp Ser Ser Cys Thr 
305                 310                 315                 320 

Arg Asp Leu His Arg Glu Arg Ile Ile Ala Glu Cys Asn Ala Ile Arg 
                325                 330                 335 

Gln Ala Leu Gln Asp Leu Leu Ser Glu Tyr Met Asn Asn Ala Gly Lys 
            340                 345                 350 

Lys Glu Arg Ser Asn Thr Leu Asn Ile Ala Leu Asp Asn Met Cys Lys 
        355                 360                 365 

Lys Thr Arg Asp Leu Arg Arg Gln Leu Arg Lys Ala Ile Ile Asp His 
    370                 375                 380 

Val Ser Asp Ser Phe Leu Asp Thr Thr Val Pro Leu Leu Val Leu Ile 
385                 390                 395                 400 

Glu Ala Ala Lys Asn Gly Arg Glu Lys Glu Ile Lys Glu Tyr Ala Ala 
                405                 410                 415 

Ile Phe His Glu His Thr Ser Arg Leu Val Glu Val Ala Asn Leu Ala 
            420                 425                 430 

Cys Ser Met Ser Thr Asn Glu Asp Gly Ile Lys Ile Val Lys Ile Ala 
        435                 440                 445 

Ala Asn His Leu Glu Thr Leu Cys Pro Gln Ile Ile Asn Ala Ala Leu 
    450                 455                 460 

Ala Leu Ala Ala Arg Pro Lys Ser Gln Ala Val Lys Asn Thr Met Glu 
465                 470                 475                 480 

Met Tyr Lys Arg Thr Trp Glu Asn His Ile His Val Leu Thr Glu Ala 
                485                 490                 495 

Val Asp Asp Ile Thr Ser Ile Asp Asp Phe Leu Ala Val Ser Glu Ser 
            500                 505                 510 

His Ile Leu Glu Asp Val Asn Lys Cys Ile Ile Ala Leu Arg Asp Gln 
        515                 520                 525 

Asp Ala Asp Asn Leu Asp Arg Ala Ala Gly Ala Ile Arg Gly Arg Ala 
    530                 535                 540 

Ala Arg Val Ala His Ile Val Thr Gly Glu Met Asp Ser Tyr Glu Pro 
545                 550                 555                 560 

Gly Ala Tyr Thr Glu Gly Val Met Arg Asn Val Asn Phe Leu Thr Ser 
                565                 570                 575 

Thr Val Ile Pro Glu Phe Val Thr Gln Val Asn Val Ala Leu Glu Ala 
            580                 585                 590 

Leu Ser Lys Ser Ser Leu Asn Val Leu Asp Asp Asn Gln Phe Val Asp 
        595                 600                 605 

Ile Ser Lys Lys Ile Tyr Asp Thr Ile His Asp Ile Arg Cys Ser Val 
    610                 615                 620 

Met Met Ile Arg Thr Pro Glu Glu Leu Glu Asp Val Ser Asp Leu Glu 
625                 630                 635                 640 

Glu Glu His Glu Val Arg Ser His Thr Ser Ile Gln Thr Glu Gly Lys 
                645                 650                 655 

Thr Asp Arg Ala Lys Met Thr Gln Leu Pro Glu Ala Glu Lys Glu Lys 
            660                 665                 670 

Ile Ala Glu Gln Val Ala Asp Phe Lys Lys Val Lys Ser Lys Leu Asp 
        675                 680                 685 

Ala Glu Ile Glu Ile Trp Asp Asp Thr Ser Asn Asp Ile Ile Val Leu 
    690                 695                 700 

Ala Lys Asn Met Cys Met Ile Met Met Glu Met Thr Asp Phe Thr Arg 
705                 710                 715                 720 

Gly Lys Gly Pro Leu Lys His Thr Thr Asp Val Ile Tyr Ala Ala Lys 
                725                 730                 735 

Met Ile Ser Glu Ser Gly Ser Arg Met Asp Val Leu Ala Arg Gln Ile 
            740                 745                 750 

Ala Asn Gln Cys Pro Asp Pro Ser Cys Lys Gln Asp Leu Leu Ala Tyr 
        755                 760                 765 

Leu Glu Gln Ile Lys Phe Tyr Ser His Gln Leu Lys Ile Cys Ser Gln 
    770                 775                 780 

Val Lys Ala Glu Ile Gln Asn Leu Gly Gly Glu Leu Ile Met Ser Ala 
785                 790                 795                 800 

Leu Asp Ser Val Thr Ser Leu Ile Gln Ala Ala Lys Asn Leu Met Asn 
                805                 810                 815 

Ala Val Val Gln Thr Val Lys Met Ser Tyr Ile Ala Ser Thr Lys Ile 
            820                 825                 830 

Ile Arg Ile Gln Ser Pro Ala Gly Pro Arg His Pro Val Val Met Trp 
        835                 840                 845 

Arg Met Lys Ala Pro Ala Lys Lys Pro Leu Ile Lys Arg Glu Lys Pro 
    850                 855                 860 

Glu Glu Thr Cys Ala Ala Val Arg Arg Gly Ser Ala Lys Lys Lys Ile 
865                 870                 875                 880 

His Pro Leu Gln Val Met Ser Glu Phe Arg Gly Arg Gln Ile Tyr 
                885                 890                 895 

 
           
             3  
             3412  
             DNA  
             Homo sapiens  
             
               promoter sequence alpha-Tcatenin  
             
           
            3 

ggatccagca cacaagagag attagccttt ggtaagagca agaggacttc ttgcatttta     60 

ataggaggga gggtagagaa gatgaggctc ggtgttagtg ggtttataga tttgatgatg    120 

gaaagatgtg aaacatcttc cagatggctt ctgttcttct tagtgaggca ggagagttgg    180 

ttagctgcta ataataagga tggttgggaa gggagagtga gaaacatttt ttttttttgt    240 

attaatagta cataagacct tcaaatcagg ggcagattca tttagattca cctaaaatac    300 

ctgacaaggt gccagaagta tttatcacat atttttgaaa aaaatggttt taattatcac    360 

aaacagggat tattcctagc tttaaatctc attttagaaa ggtattatgg ctgttcagaa    420 

aattacatat ttttaaagcc cgaacctgaa attgccagta ttttgagata ttatggacag    480 

atgaaatgga ctcatggttc atgcaaaagt gattttcatt ctataaggga ttttattgac    540 

aagaagaaac tactccattc tgactaaaga acatattgca ccctggagga tttattttcc    600 

aagagaggct actgacaggg atagaattat cttctttttg gaggcctccc ttctctcctg    660 

taatcttcta gctggcgcct aacacagggc tctgcatcaa ggggcttctc aatacatcca    720 

attgaatgac tttgtttggg gccggtcctg atgcccagaa cgaaaaaact tacttttgat    780 

tacctcggga gaattgctag taatggccct gatttattct ctctcttttt tctctctttt    840 

ttttttcccc agctcttcta ccctcaatgt ttgattgctt gcaagaggca tgtgagccaa    900 

aattttaaag tttgcaaagt ataaagaaag gaaacaagta aatagggaag gagaataaat    960 

actgaacaag actgggcaag gtgacaagaa agaactactg tgaaaatctg gaagagggcc   1020 

aggatgcaaa atgcaggggc tgtgtagttt acaggaacta tttagcctcc agcatgcctc   1080 

agcgatcaca cagagaaagg cagatttctc tgcttttgtc tcctacccta tagttagcta   1140 

tggaaatttg acataggtga tatatgtttt ctgtcagcaa actgatacaa atacagagga   1200 

ccatggggcc tgtgatcaac aaatccaacg tgatttgaat tgctaggagg ggacctcttg   1260 

tggctatagt gggaaatact tcagctaaat actaccttgg aggctgcaga tatttttatg   1320 

aaggaattaa aaaaaaaccc actataaaag ctctttaaaa ttacatttca gatatttata   1380 

atatttaatt gttttgataa caccaaattc tgtaagaagg ttttatctgt attgcatctc   1440 

tgaagagttc agtgtaattt aaagctcttc agcttttatc catttaattc acatatcctc   1500 

tgaaaagcaa atgaaaagaa taagagttct catctacatt ttgtaggtgg aaaggagagg   1560 

tacagggcag gtaagtaact tattgtggtt tcacagcagg acaagctagg cttatgatag   1620 

aatttggata tctcatcttg ggctcaattc aacagtcctg gtttggaaaa cattcattat   1680 

taaagctcta aaacaaaacc tgctattttg caagtgtcaa gtgatttctt tattcaagaa   1740 

aagatggatt gagagacata aaacttactc tttgtttcac tgtgaaaaga tgctattcct   1800 

agattctcca gggggaaaaa gctgctttaa aaaaatctgg ggaggatagc atgttagcaa   1860 

ctaagaatct ttagataaat atattgtcaa ttatgcccat tttaaaggta gctacataaa   1920 

aatacagttg ttttgaaggc tatcctgaaa atcatataaa atgaactcct ttcatagttg   1980 

attctctgac agttcccaga ccctgccttt cctcttggct ccctgaaatt tgtgctaaga   2040 

gtatctggag agccaataaa taaatgcttt ctttttattt tgaattcagc cttttaagaa   2100 

caggactgcc aaaactcaaa caagtagttc atattttagt tagcacctct tgttttagaa   2160 

gctattagaa gaaagtcgga aaaatggtaa tgtccaagga aatgccacag aagttcgagt   2220 

gggatgtcaa ggaattgatg aaatgataaa gattgtttca gtggatgtga agatattgag   2280 

ggagaagata tcaaaaaaaa gggaaaagga aatgtgaaaa agaatagtca tagagagaaa   2340 

aaataaattt tggtggagaa gacttttttt tttggtggct taaatttaat aatgggttaa   2400 

cctattgagt ttttggtaaa tcttcagttt agattcttta ctgataatga tgtggttcct   2460 

cataaatact ggaaggagag agtgtgatgc ttggtacaag ggatgagaca ggtaatattt   2520 

cagaagaaga aaaatacgat ctcagatgtg acacatggcc ttgatgccat catctctagg   2580 

gttctgaaga cattgaattt tacataattg atcttttgat gtgaggattt cctggactct   2640 

tgttttccct gctttatcat ttttcacttt caataattcc agcctttggc tttaattaga   2700 

tagaagaggt tcttcttttg gaaaggaact agagaaatgc aaatctaaac ttattcagag   2760 

ctatgtttgt aggtctctag gcaaagtatg tgtctggcct ttttcaacga agtattttca   2820 

gtaacaagtt gtcagtgagg tcagtgacta gcggttcagg attagatacc acccaccctg   2880 

gcttgtaacc tcccctttct ttcttatcct gggtgaacaa cgctcagcga aattgactgc   2940 

cccactgtca tctgcctctc aatttggtac tctgtaactc tgtgaccacc aagaagcctt   3000 

tttccgtccc ccacaaagct ctttttggaa aattccctac gggagctgaa ttttaagccc   3060 

atttacttta taggaagaaa cagaaaggta agaatcaagt ttgtaaagag aagagctgaa   3120 

cttcagcgaa ttctcatttc tgcattgaat tcctgtgtct tagttataat cataggttta   3180 

aaatttgggg ttttcttctg aactgaggaa gatcacatta ttgtatgaaa taggaatgtt   3240 

ttgactagtt atgagaaacg taggctttca cgctaatttt aaagttataa ataactttcg   3300 

aactattgcc aggggaagct ggtagccaag gtcgtgcttt gcattcagag agtttctggc   3360 

tataaaaagc cgattggata ctgtgcagga aaagataaga tatggcctgg at           3412 

 
           
             4  
             2979  
             DNA  
             Mus musculus  
             
               CDS  
               (160)..(2847)  
             
           
            4 

cccctttctc tcttatcctg agtgaatggt gctcagctaa agggactgcc ccaccagctc     60 

tgtctctttc ctcccaccct tcgagctctc tttggaaaaa ttccctccaa aagctgaacc    120 

caaagctcat ttacttgtag gaagcatcag aaaggcaac atg tcg gca gaa acg       174 
                                           Met Ser Ala Glu Thr 
                                             1               5 

cca ata acc ctg aat atg gac act cag gat ctg cag atc caa acg ttc      222 
Pro Ile Thr Leu Asn Met Asp Thr Gln Asp Leu Gln Ile Gln Thr Phe 
                 10                  15                  20 

act gtg gag aaa ctg ctg gag cct ctg ata atc cag gtt act acc ctg      270 
Thr Val Glu Lys Leu Leu Glu Pro Leu Ile Ile Gln Val Thr Thr Leu 
             25                  30                  35 

gtg aat tgt ccg cag aat cct tcc aac agg aag aaa gga cgt tca aag      318 
Val Asn Cys Pro Gln Asn Pro Ser Asn Arg Lys Lys Gly Arg Ser Lys 
         40                  45                  50 

aga gcc aga gtt ctt cta gct tcc gtg gag gaa gca act tgg aat ttg      366 
Arg Ala Arg Val Leu Leu Ala Ser Val Glu Glu Ala Thr Trp Asn Leu 
     55                  60                  65 

tta gac aag ggg gag atg att gct aag gaa gcc acg gtt tta aag gaa      414 
Leu Asp Lys Gly Glu Met Ile Ala Lys Glu Ala Thr Val Leu Lys Glu 
 70                  75                  80                  85 

gag ctg gca gct gca ctc cag gaa gtt cga aaa gag agc aaa gct ctg      462 
Glu Leu Ala Ala Ala Leu Gln Glu Val Arg Lys Glu Ser Lys Ala Leu 
                 90                  95                 100 

aag gta tca gct gag aga ttt aca gac gac ccc tgt tac ctc ccg aaa      510 
Lys Val Ser Ala Glu Arg Phe Thr Asp Asp Pro Cys Tyr Leu Pro Lys 
            105                 110                 115 

agg gag gcc gtg gtt caa gcc gcc cgc gcc ctg ttg gca gca gtt aca      558 
Arg Glu Ala Val Val Gln Ala Ala Arg Ala Leu Leu Ala Ala Val Thr 
        120                 125                 130 

aga ctc ctt gtt ctt gcc gac atg att gat gtc atg tgc ctc ttg cag      606 
Arg Leu Leu Val Leu Ala Asp Met Ile Asp Val Met Cys Leu Leu Gln 
    135                 140                 145 

cat gtg tca tct ttc caa aga aca ttc gag tct ctc aaa aat gtt tcc      654 
His Val Ser Ser Phe Gln Arg Thr Phe Glu Ser Leu Lys Asn Val Ser 
150                 155                 160                 165 

aac aag tcc gac ctc cag aga acc tac cag aag ctc ggg aag gag ctg      702 
Asn Lys Ser Asp Leu Gln Arg Thr Tyr Gln Lys Leu Gly Lys Glu Leu 
                170                 175                 180 

gaa agc ctg gat tat ttg gcc ttc aaa cgc cag cag gac cta aaa tct      750 
Glu Ser Leu Asp Tyr Leu Ala Phe Lys Arg Gln Gln Asp Leu Lys Ser 
            185                 190                 195 

cca agc cag agg gat gaa att gca ggg gcc cgg gcc acc ttg aag gag      798 
Pro Ser Gln Arg Asp Glu Ile Ala Gly Ala Arg Ala Thr Leu Lys Glu 
        200                 205                 210 

aac tcc cca ctc ctg cat tct att tgt tca gca tgc ttg gaa cat tcc      846 
Asn Ser Pro Leu Leu His Ser Ile Cys Ser Ala Cys Leu Glu His Ser 
    215                 220                 225 

gat gtt gct tcg ctc aaa gcc agt aag gac acc gtc tgt gaa gag atc      894 
Asp Val Ala Ser Leu Lys Ala Ser Lys Asp Thr Val Cys Glu Glu Ile 
230                 235                 240                 245 

cag aac gct ctt gat gta att tca aat gct tcc caa ggc atc cag aat      942 
Gln Asn Ala Leu Asp Val Ile Ser Asn Ala Ser Gln Gly Ile Gln Asn 
                250                 255                 260 

gcg cca gcg ccc cct gaa cct cag gca gca aca ctg gga agt gct ttt      990 
Ala Pro Ala Pro Pro Glu Pro Gln Ala Ala Thr Leu Gly Ser Ala Phe 
            265                 270                 275 

gat gag ctg gag aac tta att gtc ctg aac cca ctc aca gtg aca gag     1038 
Asp Glu Leu Glu Asn Leu Ile Val Leu Asn Pro Leu Thr Val Thr Glu 
        280                 285                 290 

gaa gat gta aga cca tca cta gag aaa cgc cta gaa gcc atc atc agt     1086 
Glu Asp Val Arg Pro Ser Leu Glu Lys Arg Leu Glu Ala Ile Ile Ser 
    295                 300                 305 

ggg gcc gca ctg ttg gcc gac tcg tcc tgc acc agg gac ctc cac cgg     1134 
Gly Ala Ala Leu Leu Ala Asp Ser Ser Cys Thr Arg Asp Leu His Arg 
310                 315                 320                 325 

gag cgg att atc gcc gag tgc aat gcc atc cgc cag gct ctc cag gac     1182 
Glu Arg Ile Ile Ala Glu Cys Asn Ala Ile Arg Gln Ala Leu Gln Asp 
                330                 335                 340 

ctg ctg acg gag tac atg agt aat act gga aaa aca gaa agg agt aat     1230 
Leu Leu Thr Glu Tyr Met Ser Asn Thr Gly Lys Thr Glu Arg Ser Asn 
            345                 350                 355 

acc ctg aat act gcc att gtc aac atg agc aag aag aca aga gac ctc     1278 
Thr Leu Asn Thr Ala Ile Val Asn Met Ser Lys Lys Thr Arg Asp Leu 
        360                 365                 370 

cgc aga cag ctc cgc aaa gct atc ata gat cac ata tca gat tct ttc     1326 
Arg Arg Gln Leu Arg Lys Ala Ile Ile Asp His Ile Ser Asp Ser Phe 
    375                 380                 385 

ttg gat aca aca gtt cca ctc ctg gtc ctc att gaa gct gcg aag aat     1374 
Leu Asp Thr Thr Val Pro Leu Leu Val Leu Ile Glu Ala Ala Lys Asn 
390                 395                 400                 405 

ggc cga gtc aag gaa atc aaa gac tat gct gcc ata ttt cat gag cac     1422 
Gly Arg Val Lys Glu Ile Lys Asp Tyr Ala Ala Ile Phe His Glu His 
                410                 415                 420 

act ggc agg ctc gtg gag gtg gca aat ctg gct tgt tcc atg tca acg     1470 
Thr Gly Arg Leu Val Glu Val Ala Asn Leu Ala Cys Ser Met Ser Thr 
            425                 430                 435 

aat gaa gat ggg att aaa atc gtc aga att gca gcc aat cac ctg gag     1518 
Asn Glu Asp Gly Ile Lys Ile Val Arg Ile Ala Ala Asn His Leu Glu 
        440                 445                 450 

acc ctg tgt cca cag atc ata aat gct gca ctt gct ttg gca tca aga     1566 
Thr Leu Cys Pro Gln Ile Ile Asn Ala Ala Leu Ala Leu Ala Ser Arg 
    455                 460                 465 

ccc aag agt caa gtg gtc aaa aac acc atg gaa atg tac aag cgc aca     1614 
Pro Lys Ser Gln Val Val Lys Asn Thr Met Glu Met Tyr Lys Arg Thr 
470                 475                 480                 485 

tgg gaa cac tac atc cac gtc ctc act gaa gct gta gat gac atc acc     1662 
Trp Glu His Tyr Ile His Val Leu Thr Glu Ala Val Asp Asp Ile Thr 
                490                 495                 500 

agc att gac gac ttt ctg gct gta tct gaa agc cac atc ctg gaa gat     1710 
Ser Ile Asp Asp Phe Leu Ala Val Ser Glu Ser His Ile Leu Glu Asp 
            505                 510                 515 

gtc aac aaa tgc att ata gcc ttg aga gat cag gac gct gat aat tta     1758 
Val Asn Lys Cys Ile Ile Ala Leu Arg Asp Gln Asp Ala Asp Asn Leu 
        520                 525                 530 

gac cga gct gcc ggt gcc atc aga gga cgg gcc gca aga gta gct cac     1806 
Asp Arg Ala Ala Gly Ala Ile Arg Gly Arg Ala Ala Arg Val Ala His 
    535                 540                 545 

atc gtt gcg ggt gaa atg gat agt tac gaa ccc ggc gct tac acc gaa     1854 
Ile Val Ala Gly Glu Met Asp Ser Tyr Glu Pro Gly Ala Tyr Thr Glu 
550                 555                 560                 565 

ggt gtg atg aga aat gtc aac ttc ctt aca agc act gtg atc ccg gag     1902 
Gly Val Met Arg Asn Val Asn Phe Leu Thr Ser Thr Val Ile Pro Glu 
                570                 575                 580 

ttc gtg aca caa gtg aat gtg gcc cta gat gct tta agc aag aac tct     1950 
Phe Val Thr Gln Val Asn Val Ala Leu Asp Ala Leu Ser Lys Asn Ser 
            585                 590                 595 

ctg act gcg ctt gat gat aat cag ttt gtg gac atc tcc aag aag atc     1998 
Leu Thr Ala Leu Asp Asp Asn Gln Phe Val Asp Ile Ser Lys Lys Ile 
        600                 605                 610 

tat gac aca atc cat gat atc agg tgt tcg gtc atg atg att cgg aca     2046 
Tyr Asp Thr Ile His Asp Ile Arg Cys Ser Val Met Met Ile Arg Thr 
    615                 620                 625 

cca gag gaa cta gag gat gtt tct gac ctt gaa gat gac cat gag gtc     2094 
Pro Glu Glu Leu Glu Asp Val Ser Asp Leu Glu Asp Asp His Glu Val 
630                 635                 640                 645 

cgt agc cac acc agc att cag aca gaa ggg aaa act gat cgg gcc aag     2142 
Arg Ser His Thr Ser Ile Gln Thr Glu Gly Lys Thr Asp Arg Ala Lys 
                650                 655                 660 

atg act caa ctg cct gag gca gaa aag gaa aag att gct gag caa gtc     2190 
Met Thr Gln Leu Pro Glu Ala Glu Lys Glu Lys Ile Ala Glu Gln Val 
            665                 670                 675 

gcc gac ttc aag aag gtg aag agc aag ctg gac gct gag att gag ata     2238 
Ala Asp Phe Lys Lys Val Lys Ser Lys Leu Asp Ala Glu Ile Glu Ile 
        680                 685                 690 

tgg gat gac aca agc aat gac atc att gtt ctt gcc aag aag atg tgc     2286 
Trp Asp Asp Thr Ser Asn Asp Ile Ile Val Leu Ala Lys Lys Met Cys 
    695                 700                 705 

atg atc atg atg gag atg acc gac ttc acg agg ggg aaa gga cca cta     2334 
Met Ile Met Met Glu Met Thr Asp Phe Thr Arg Gly Lys Gly Pro Leu 
710                 715                 720                 725 

aag cat acc act gat gta atc tat gca gct aaa atg ata tca gag tca     2382 
Lys His Thr Thr Asp Val Ile Tyr Ala Ala Lys Met Ile Ser Glu Ser 
                730                 735                 740 

gga tca agg atg gat gtt ctt gct cgg cag att gct aac cag tgt cca     2430 
Gly Ser Arg Met Asp Val Leu Ala Arg Gln Ile Ala Asn Gln Cys Pro 
            745                 750                 755 

gat cca ccg tgc aaa cag gac ttg ctg gct tac ctg gaa cag att aaa     2478 
Asp Pro Pro Cys Lys Gln Asp Leu Leu Ala Tyr Leu Glu Gln Ile Lys 
        760                 765                 770 

ttc tac tcc cac cag ctg aaa atc tgc agt caa gtt aaa gca gag atc     2526 
Phe Tyr Ser His Gln Leu Lys Ile Cys Ser Gln Val Lys Ala Glu Ile 
    775                 780                 785 

caa aat ctg ggg gga gaa ctc atc gta tca gct ttg gac agt gtc acc     2574 
Gln Asn Leu Gly Gly Glu Leu Ile Val Ser Ala Leu Asp Ser Val Thr 
790                 795                 800                 805 

tcc ctg atc cag gca gcc aag aat tta atg aat gct gta gtg caa aca     2622 
Ser Leu Ile Gln Ala Ala Lys Asn Leu Met Asn Ala Val Val Gln Thr 
                810                 815                 820 

gtg aaa atg tca tac att gcc tcc acc aag atc atc cgc atc cag agt     2670 
Val Lys Met Ser Tyr Ile Ala Ser Thr Lys Ile Ile Arg Ile Gln Ser 
            825                 830                 835 

tct gca gga ccc cgg cac cca gta gtc atg tgg agg atg aag gct ccg     2718 
Ser Ala Gly Pro Arg His Pro Val Val Met Trp Arg Met Lys Ala Pro 
        840                 845                 850 

gct aag aag ccc ttg att aaa aga gag aag cca gaa gaa aca tgg gca     2766 
Ala Lys Lys Pro Leu Ile Lys Arg Glu Lys Pro Glu Glu Thr Trp Ala 
    855                 860                 865 

gct gcc aga aga ggc tct gcc aag aaa aag atc cac cca gtt caa gtc     2814 
Ala Ala Arg Arg Gly Ser Ala Lys Lys Lys Ile His Pro Val Gln Val 
870                 875                 880                 885 

atg agt gaa ttc aga ggg aga caa gtc tac tga ataccctcat ccactctagt   2867 
Met Ser Glu Phe Arg Gly Arg Gln Val Tyr 
                890                 895 

gcccatttct acaccccagg ctaaccacac tgctttattt catggttcat tggttcttta   2927 

atttcaccaa gtttcagagt taagctcaca aataacataa aacattgggg tt           2979 

 
           
             5  
             895  
             PRT  
             Mus musculus  
           
            5 

Met Ser Ala Glu Thr Pro Ile Thr Leu Asn Met Asp Thr Gln Asp Leu 
  1               5                  10                  15 

Gln Ile Gln Thr Phe Thr Val Glu Lys Leu Leu Glu Pro Leu Ile Ile 
             20                  25                  30 

Gln Val Thr Thr Leu Val Asn Cys Pro Gln Asn Pro Ser Asn Arg Lys 
         35                  40                  45 

Lys Gly Arg Ser Lys Arg Ala Arg Val Leu Leu Ala Ser Val Glu Glu 
     50                  55                  60 

Ala Thr Trp Asn Leu Leu Asp Lys Gly Glu Met Ile Ala Lys Glu Ala 
 65                  70                  75                  80 

Thr Val Leu Lys Glu Glu Leu Ala Ala Ala Leu Gln Glu Val Arg Lys 
                 85                  90                  95 

Glu Ser Lys Ala Leu Lys Val Ser Ala Glu Arg Phe Thr Asp Asp Pro 
            100                 105                 110 

Cys Tyr Leu Pro Lys Arg Glu Ala Val Val Gln Ala Ala Arg Ala Leu 
        115                 120                 125 

Leu Ala Ala Val Thr Arg Leu Leu Val Leu Ala Asp Met Ile Asp Val 
    130                 135                 140 

Met Cys Leu Leu Gln His Val Ser Ser Phe Gln Arg Thr Phe Glu Ser 
145                 150                 155                 160 

Leu Lys Asn Val Ser Asn Lys Ser Asp Leu Gln Arg Thr Tyr Gln Lys 
                165                 170                 175 

Leu Gly Lys Glu Leu Glu Ser Leu Asp Tyr Leu Ala Phe Lys Arg Gln 
            180                 185                 190 

Gln Asp Leu Lys Ser Pro Ser Gln Arg Asp Glu Ile Ala Gly Ala Arg 
        195                 200                 205 

Ala Thr Leu Lys Glu Asn Ser Pro Leu Leu His Ser Ile Cys Ser Ala 
    210                 215                 220 

Cys Leu Glu His Ser Asp Val Ala Ser Leu Lys Ala Ser Lys Asp Thr 
225                 230                 235                 240 

Val Cys Glu Glu Ile Gln Asn Ala Leu Asp Val Ile Ser Asn Ala Ser 
                245                 250                 255 

Gln Gly Ile Gln Asn Ala Pro Ala Pro Pro Glu Pro Gln Ala Ala Thr 
            260                 265                 270 

Leu Gly Ser Ala Phe Asp Glu Leu Glu Asn Leu Ile Val Leu Asn Pro 
        275                 280                 285 

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

Glu Ala Ile Ile Ser Gly Ala Ala Leu Leu Ala Asp Ser Ser Cys Thr 
305                 310                 315                 320 

Arg Asp Leu His Arg Glu Arg Ile Ile Ala Glu Cys Asn Ala Ile Arg 
                325                 330                 335 

Gln Ala Leu Gln Asp Leu Leu Thr Glu Tyr Met Ser Asn Thr Gly Lys 
            340                 345                 350 

Thr Glu Arg Ser Asn Thr Leu Asn Thr Ala Ile Val Asn Met Ser Lys 
        355                 360                 365 

Lys Thr Arg Asp Leu Arg Arg Gln Leu Arg Lys Ala Ile Ile Asp His 
    370                 375                 380 

Ile Ser Asp Ser Phe Leu Asp Thr Thr Val Pro Leu Leu Val Leu Ile 
385                 390                 395                 400 

Glu Ala Ala Lys Asn Gly Arg Val Lys Glu Ile Lys Asp Tyr Ala Ala 
                405                 410                 415 

Ile Phe His Glu His Thr Gly Arg Leu Val Glu Val Ala Asn Leu Ala 
            420                 425                 430 

Cys Ser Met Ser Thr Asn Glu Asp Gly Ile Lys Ile Val Arg Ile Ala 
        435                 440                 445 

Ala Asn His Leu Glu Thr Leu Cys Pro Gln Ile Ile Asn Ala Ala Leu 
    450                 455                 460 

Ala Leu Ala Ser Arg Pro Lys Ser Gln Val Val Lys Asn Thr Met Glu 
465                 470                 475                 480 

Met Tyr Lys Arg Thr Trp Glu His Tyr Ile His Val Leu Thr Glu Ala 
                485                 490                 495 

Val Asp Asp Ile Thr Ser Ile Asp Asp Phe Leu Ala Val Ser Glu Ser 
            500                 505                 510 

His Ile Leu Glu Asp Val Asn Lys Cys Ile Ile Ala Leu Arg Asp Gln 
        515                 520                 525 

Asp Ala Asp Asn Leu Asp Arg Ala Ala Gly Ala Ile Arg Gly Arg Ala 
    530                 535                 540 

Ala Arg Val Ala His Ile Val Ala Gly Glu Met Asp Ser Tyr Glu Pro 
545                 550                 555                 560 

Gly Ala Tyr Thr Glu Gly Val Met Arg Asn Val Asn Phe Leu Thr Ser 
                565                 570                 575 

Thr Val Ile Pro Glu Phe Val Thr Gln Val Asn Val Ala Leu Asp Ala 
            580                 585                 590 

Leu Ser Lys Asn Ser Leu Thr Ala Leu Asp Asp Asn Gln Phe Val Asp 
        595                 600                 605 

Ile Ser Lys Lys Ile Tyr Asp Thr Ile His Asp Ile Arg Cys Ser Val 
    610                 615                 620 

Met Met Ile Arg Thr Pro Glu Glu Leu Glu Asp Val Ser Asp Leu Glu 
625                 630                 635                 640 

Asp Asp His Glu Val Arg Ser His Thr Ser Ile Gln Thr Glu Gly Lys 
                645                 650                 655 

Thr Asp Arg Ala Lys Met Thr Gln Leu Pro Glu Ala Glu Lys Glu Lys 
            660                 665                 670 

Ile Ala Glu Gln Val Ala Asp Phe Lys Lys Val Lys Ser Lys Leu Asp 
        675                 680                 685 

Ala Glu Ile Glu Ile Trp Asp Asp Thr Ser Asn Asp Ile Ile Val Leu 
    690                 695                 700 

Ala Lys Lys Met Cys Met Ile Met Met Glu Met Thr Asp Phe Thr Arg 
705                 710                 715                 720 

Gly Lys Gly Pro Leu Lys His Thr Thr Asp Val Ile Tyr Ala Ala Lys 
                725                 730                 735 

Met Ile Ser Glu Ser Gly Ser Arg Met Asp Val Leu Ala Arg Gln Ile 
            740                 745                 750 

Ala Asn Gln Cys Pro Asp Pro Pro Cys Lys Gln Asp Leu Leu Ala Tyr 
        755                 760                 765 

Leu Glu Gln Ile Lys Phe Tyr Ser His Gln Leu Lys Ile Cys Ser Gln 
    770                 775                 780 

Val Lys Ala Glu Ile Gln Asn Leu Gly Gly Glu Leu Ile Val Ser Ala 
785                 790                 795                 800 

Leu Asp Ser Val Thr Ser Leu Ile Gln Ala Ala Lys Asn Leu Met Asn 
                805                 810                 815 

Ala Val Val Gln Thr Val Lys Met Ser Tyr Ile Ala Ser Thr Lys Ile 
            820                 825                 830 

Ile Arg Ile Gln Ser Ser Ala Gly Pro Arg His Pro Val Val Met Trp 
        835                 840                 845 

Arg Met Lys Ala Pro Ala Lys Lys Pro Leu Ile Lys Arg Glu Lys Pro 
    850                 855                 860 

Glu Glu Thr Trp Ala Ala Ala Arg Arg Gly Ser Ala Lys Lys Lys Ile 
865                 870                 875                 880 

His Pro Val Gln Val Met Ser Glu Phe Arg Gly Arg Gln Val Tyr 
                885                 890                 895 

 
           
             6  
             566  
             DNA  
             Mus musculus  
             
               promoter  
               1...566  
               promoter sequence alpha-Tcatenin  
             
           
            6 

acacggcgat atgtatcatc gccctgtggt ggnaatctgg tagcgttgtg acagtgtgag     60 

acggtaagat ttcggcagaa aaaacgatct cagatgtgac ccatgactcc agagaccctg    120 

cattttagct agtaacgatg cagggatttc ctggactctt gtatctcccc gctttacagt    180 

ttctcacttc caggtaattg taaccttcgg ctttaattcg aaacggttcc ttgttttgga    240 

tggggatgac aaagttaaag ctgacttatt tagagcttcc gaagaagtat ggaggtctct    300 

agggaatgta tgtgtctggc ctttttcaac aaagtatttt cagcaacaag ttgtcagtga    360 

ggtcagcgcg gagcccagga ttagatacca cccacccctg gcttgtaacc tcccctttct    420 

ctcttatcct gagtgaatgg tgctcagcta aagggactgc cccaccagct ctgtctcttt    480 

cctcccaccc ttcgagctct ctttggaaaa attccctcca aaagctgaac ccaaagctca    540 

tttacttgta ggaagcatca gaaagg                                         566 

 
           
             7  
             21  
             DNA  
             Homo sapiens  
             
               misc_feature  
               1...21  
               splice acceptor 0  
             
           
            7 

annctgggtg aacaacgctc a                                               21 

 
           
             8  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 1  
             
           
            8 

aacagaaagg taagaatcaa g                                               21 

 
           
             9  
             20  
             DNA  
             Homo sapiens  
             
               splice acceptor 1  
             
           
            9 

tttgtgcagc aggcagcatg                                                 20 

 
           
             10  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 2  
             
           
            10 

ataatccagg tattaatacc a                                               21 

 
           
             11  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 2  
             
           
            11 

ttccaatttt aggttaccac a                                               21 

 
           
             12  
             19  
             DNA  
             Homo sapiens  
             
               splice donor 3  
             
           
            12 

aaagaaagtg agtactcca                                                  19 

 
           
             13  
             20  
             DNA  
             Homo sapiens  
             
               splice acceptor 3  
             
           
            13 

tgtatttttc aggtgaagct                                                 20 

 
           
             14  
             20  
             DNA  
             Homo sapiens  
             
               splice donor 4  
             
           
            14 

gtgtcagctg taagtaaaga                                                 20 

 
           
             15  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 4  
             
           
            15 

tttcaatttc agtttcaaag g                                               21 

 
           
             16  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 5  
             
           
            16 

cgtcagcagg taggagtcag a                                               21 

 
           
             17  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 5  
             
           
            17 

ttaccttctc aggacttaaa a                                               21 

 
           
             18  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 6  
             
           
            18 

gagctggagg taagtcggga g                                               21 

 
           
             19  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 6  
             
           
            19 

ttcttctctt agaatttaat t                                               21 

 
           
             20  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 7  
             
           
            20 

atgaacaacg taagtatagt t                                               21 

 
           
             21  
             22  
             DNA  
             Homo sapiens  
             
               splice acceptor 7  
             
           
            21 

tcttcctttg caggctggaa aa                                              22 

 
           
             22  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 8  
             
           
            22 

cgcagacagg tgagggaaga g                                               21 

 
           
             23  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 8  
             
           
            23 

atttcttctc agctccgcaa g                                               21 

 
           
             24  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 9  
             
           
            24 

cttgtagagg taagcatgct a                                               21 

 
           
             25  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 9  
             
           
            25 

attgtattta aggtggcaaa t                                               21 

 
           
             26  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 10  
             
           
            26 

tgtccacagg tatgacaact a                                               21 

 
           
             27  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 10  
             
           
            27 

ttatctttat agattattaa t                                               21 

 
           
             28  
             19  
             DNA  
             Homo sapiens  
             
               splice donor 11  
             
           
            28 

gtatctggta tgtttttat                                                  19 

 
           
             29  
             20  
             DNA  
             Homo sapiens  
             
               splice acceptor 11  
             
           
            29 

atttacttac agaaagccat                                                 20 

 
           
             30  
             19  
             DNA  
             Homo sapiens  
             
               splice donor 12  
             
           
            30 

agtactggta agtcagttg                                                  19 

 
           
             31  
             20  
             DNA  
             Homo sapiens  
             
               splice acceptor 12  
             
           
            31 

ttattttaac agtaattcct                                                 20 

 
           
             32  
             20  
             DNA  
             Homo sapiens  
             
               splice donor 13  
             
           
            32 

atgattcggt aagtttgctt                                                 20 

 
           
             33  
             19  
             DNA  
             Homo sapiens  
             
               splice acceptor 13  
             
           
            33 

ttctttttat aggacccca                                                  19 

 
           
             34  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 14  
             
           
            34 

actgataggg tatgtcactt c                                               21 

 
           
             35  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 14  
             
           
            35 

cacatgtttt aggctaagat g                                               21 

 
           
             36  
             20  
             DNA  
             Homo sapiens  
             
               splice donor 15  
             
           
            36 

ttcactaggt aattatgtgg                                                 20 

 
           
             37  
             19  
             DNA  
             Homo sapiens  
             
               splice acceptor 15  
             
           
            37 

atttttttcc aggggcaaa                                                  19 

 
           
             38  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 16  
             
           
            38 

gctaatcagg tgagttactt a                                               21 

 
           
             39  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 16  
             
           
            39 

atgcatattt agtgcccaga t                                               21 

 
           
             40  
             21  
             DNA  
             Homo sapiens  
             
               splice donor 17  
             
           
            40 

atgtcagctg tgagtactgc c                                               21 

 
           
             41  
             21  
             DNA  
             Homo sapiens  
             
               splice acceptor 17  
             
           
            41 

ttttccctac agttggacag t                                               21 

 
           
             42  
             19  
             DNA  
             Homo sapiens  
             
               splice donor 18  
             
           
            42 

atatatattt gggatcatt                                                  19 

 
           
             43  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2513  
             
           
            43 

ttgcttgtaa cctccccttt                                                 20 

 
           
             44  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2514  
             
           
            44 

gcgtgaaagc ctacgtttct                                                 20 

 
           
             45  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2515  
             
           
            45 

taatttgtta caggacctaa gc                                              22 

 
           
             46  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2516  
             
           
            46 

tcttcattat tcatttttcc cac                                             23 

 
           
             47  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2517  
             
           
            47 

tatcccagga ctgtgttctc                                                 20 

 
           
             48  
             19  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence loower 
      primer FVR2518  
             
           
            48 

tggagccaaa aacaaaaca                                                  19 

 
           
             49  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2519  
             
           
            49 

tggggttgta tttttcaggt g                                               21 

 
           
             50  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2520  
             
           
            50 

gccaggttca gagaatgaaa t                                               21 

 
           
             51  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2521  
             
           
            51 

ggactgaaca ggcttctcat                                                 20 

 
           
             52  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2522  
             
           
            52 

gcaggaagcc taaagtgttc                                                 20 

 
           
             53  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2523  
             
           
            53 

gtctttctcc cataacccat t                                               21 

 
           
             54  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2524  
             
           
            54 

cgccaacatg tggatcttct                                                 20 

 
           
             55  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2525  
             
           
            55 

tgaaatgcca tggagctcta a                                               21 

 
           
             56  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2526  
             
           
            56 

acggaaagta tctcagccta t                                               21 

 
           
             57  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2958  
             
           
            57 

ccattgctta tgtcgttttt tc                                              22 

 
           
             58  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2959  
             
           
            58 

ttagccccta tgtttctgac t                                               21 

 
           
             59  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2960  
             
           
            59 

agaaaaggaa acacagtgaa ct                                              22 

 
           
             60  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2961  
             
           
            60 

ttctcctgga ctttagtgag tt                                              22 

 
           
             61  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2527  
             
           
            61 

tgttgctgca tttccttgct a                                               21 

 
           
             62  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2528  
             
           
            62 

gcgagacctg gtctcaaaaa                                                 20 

 
           
             63  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2529  
             
           
            63 

gtgcccatca cccaaatagt                                                 20 

 
           
             64  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2530  
             
           
            64 

ccatgcctgt cccagtatta                                                 20 

 
           
             65  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2531  
             
           
            65 

ccatttccaa tgtgcactct a                                               21 

 
           
             66  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2532  
             
           
            66 

aattgtgcag ctgttattgg c                                               21 

 
           
             67  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2956  
             
           
            67 

acaaagagga caatcttctc c                                               21 

 
           
             68  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2957  
             
           
            68 

tcaatggaag gaaaagcaaa c                                               21 

 
           
             69  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2533  
             
           
            69 

tgggagtgaa attgctgggt                                                 20 

 
           
             70  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2534  
             
           
            70 

tagaggctgc ctagattgac                                                 20 

 
           
             71  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2535  
             
           
            71 

tgcttttgac atagtggaat ga                                              22 

 
           
             72  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2536  
             
           
            72 

tggcacttga cactcagaga                                                 20 

 
           
             73  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2537  
             
           
            73 

ccgttctttg ggatgcgaat                                                 20 

 
           
             74  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2538  
             
           
            74 

ggcaaagagc aattagcatg a                                               21 

 
           
             75  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2539  
             
           
            75 

aaggtacctg ccatgtgaat a                                               21 

 
           
             76  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2540  
             
           
            76 

agatttggtc atgtaaacaa gg                                              22 

 
           
             77  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence upper 
      primer FVR2541  
             
           
            77 

ccacgcttgg caataattaa c                                               21 

 
           
             78  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence lower 
      primer FVR2542  
             
           
            78 

tgctgaccat acagaaatga c                                               21 

 
           
             79  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB53  
             
           
            79 

cttcgggcct ctggaattta                                                 20 

 
           
             80  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB73  
             
           
            80 

cgacatcagg gtgctgtagg                                                 20 

 
           
             81  
             29  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB133  
             
           
            81 

ggtgaattcg tcagcagcaa gggcatcat                                       29 

 
           
             82  
             29  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB134  
             
           
            82 

ggtttgatgc agggtccaca ggcagttct                                       29 

 
           
             83  
             30  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB137  
             
           
            83 

accccccggg ggcaacttca cctatcattc                                      30 

 
           
             84  
             30  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB138  
             
           
            84 

gccgccgcct tccttttcat ttccgctctt                                      30 

 
           
             85  
             30  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB711  
             
           
            85 

agggggcagt ggctgaagaa agaagtaatc                                      30 

 
           
             86  
             29  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB725  
             
           
            86 

tattagatat cgcctctccc ggacccgcc                                       29 

 
           
             87  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB967  
             
           
            87 

tgaggcagaa aaagaaaaga                                                 20 

 
           
             88  
             19  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB968  
             
           
            88 

agtgtggtta ggcaggatt                                                  19 

 
           
             89  
             17  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1010  
             
           
            89 

gctgagcctc gtctgac                                                    17 

 
           
             90  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1027  
             
           
            90 

aatctgccga gcaaggacat cca                                             23 

 
           
             91  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1028  
             
           
            91 

tcaggcagtt gagtcatctt agc                                             23 

 
           
             92  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1254  
             
           
            92 

acccgtgacg atgtgagcaa ctc                                             23 

 
           
             93  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1255  
             
           
            93 

gagctgtctg cgaaggtctc ttg                                             23 

 
           
             94  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1260  
             
           
            94 

gaaaaagaaa agattgctga g                                               21 

 
           
             95  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1261  
             
           
            95 

ccctagtgaa gtctgtcatc t                                               21 

 
           
             96  
             27  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1607  
             
           
            96 

agaattctca gctgaaacac caatcac                                         27 

 
           
             97  
             27  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1608  
             
           
            97 

gggatccgta gatttgtctt cctctaa                                         27 

 
           
             98  
             25  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1609  
             
           
            98 

aggatcctgc gaaggtctct tgtct                                           25 

 
           
             99  
             27  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB1610  
             
           
            99 

ggatgataat caatttgtgg acatctc                                         27 

 
           
             100  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2043  
             
           
            100 

tcgaggatga aggctctg                                                   18 

 
           
             101  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2044  
             
           
            101 

tgtttaaccc caatgttt                                                   18 

 
           
             102  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2056  
             
           
            102 

gaaatgccat ggagctctaa c                                               21 

 
           
             103  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2057  
             
           
            103 

atgggaaggc aaaccagtca c                                               21 

 
           
             104  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2099  
             
           
            104 

tgtcatctgc ctctcaattt g                                               21 

 
           
             105  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2100  
             
           
            105 

atgctgcctt tctgtttctt c                                               21 

 
           
             106  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2217  
             
           
            106 

cagatgacag tggggcagtc                                                 20 

 
           
             107  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2219  
             
           
            107 

accacagtcc atgccatcac                                                 20 

 
           
             108  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2220  
             
           
            108 

tccaccaccc tgttgctg                                                   18 

 
           
             109  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2287  
             
           
            109 

aacttgttac tgaaaatact                                                 20 

 
           
             110  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2335  
             
           
            110 

cctcttgcaa catgtgtc                                                   18 

 
           
             111  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2350  
             
           
            111 

cattaccatt tttccgactt                                                 20 

 
           
             112  
             42  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2386  
             
           
            112 

gggggcggcc gcggagggtc agctgaaaca ccaatcacat tg                        42 

 
           
             113  
             42  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2387  
             
           
            113 

ccccgaattc gccgtgtggt taggcaggat tttgtcatat ag                        42 

 
           
             114  
             19  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2461  
             
           
            114 

ccccaatgtt ttatgttat                                                  19 

 
           
             115  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2463  
             
           
            115 

ggggagaact catcgtat                                                   18 

 
           
             116  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2479  
             
           
            116 

gccctgattg agtttgataa                                                 20 

 
           
             117  
             19  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2480  
             
           
            117 

cccagcttca tagttctcc                                                  19 

 
           
             118  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2481  
             
           
            118 

cttggtggag gcaatgtatg ac                                              22 

 
           
             119  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2482  
             
           
            119 

tctgccgagc aagaacatcc at                                              22 

 
           
             120  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2567  
             
           
            120 

gcggaggtct cttgtcttct t                                               21 

 
           
             121  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2569  
             
           
            121 

cgcagtcaga gagttcttgc tt                                              22 

 
           
             122  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2636  
             
           
            122 

gaaggcccct gagaagaa                                                   18 

 
           
             123  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2637  
             
           
            123 

cccgaataaa gcaactccat                                                 20 

 
           
             124  
             24  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2711  
             
           
            124 

cttcccgagc ttctggtagg ttct                                            24 

 
           
             125  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2818  
             
           
            125 

aacgcctaga agccatcatc                                                 20 

 
           
             126  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2819  
             
           
            126 

tggcaagaac aatgatgtca                                                 20 

 
           
             127  
             22  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2820  
             
           
            127 

cccctttctc tcttatcctg ag                                              22 

 
           
             128  
             25  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2837  
             
           
            128 

ctttctgatg cttcctacaa gtaaa                                           25 

 
           
             129  
             19  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2838  
             
           
            129 

ccgcagaatc cttccaaca                                                  19 

 
           
             130  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2839  
             
           
            130 

gctgccagct cttcctttaa a                                               21 

 
           
             131  
             19  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2840  
             
           
            131 

gtcggcagaa acgccaata                                                  19 

 
           
             132  
             20  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence primer 
      MCB2841  
             
           
            132 

gaggctccag cagtttctcc                                                 20 

 
           
             133  
             17  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence amino- 
      terminus of human alpha-Tcatenin  
             
           
            133 

Met Ser Ala Glu Thr Pro Ile Thr Leu Asn Ile Asp Pro Gln Asp Leu 
  1               5                  10                  15 

Gln 

 
           
             134  
             17  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence 
      carboxy-terminus of alpha-Tcatenin  
             
           
            134 

Tyr Ile Gln Arg Gly Arg Phe Glu Ser Met Val Gln Leu Pro His Ile 
  1               5                  10                  15 

Lys