EB1 GENE PRODUCT BINDS TO APC

Abstract: Inactivation of the APC tumor suppressor gene plays an important role in the development of both sporadic and familial forms of colorectal cancers. The majority of these mutations result in the loss of the carboxyl terminus of the APC protein. A cellular protein, EB1, that associates with the carboxyl terminus of APC both in vitro and in vivo has been identified. The EB1 gene is predicted to encode a 268 amino acid protein without significant homology to any protein with known function.

Description: EBl GENE PRODUCT BINDS TO APC This invention was made with support from the National Institutes of Health, Grant No. CA57345. The U.S. government therefore retains certain rights in the invention. BACKGROUND OF THE INVENTION The APC gene (adenomatous polyposis coli) was originally isolated by virtue of its alteration in familial and sporadic forms of colorectal cancer (1-4). Germline mutations of the APC gene account for most cases of familial adenomatous polyposis (FAP), an autosomal, dominantly inherited disease that predisposes patients to multiple colorectal polyps and cancer (reviewed in 5). APC mutations have also been found in cancers of the central nervous system. While FAP patients with germline mutations of APC account for less than 1 % of colorectal cancers in the United States, somatic mutations of APC occur in the majority of colorectal adenomas and cancers (6-9). These alterations appear to occur early as they can be identified in the smallest identifiable lesions including dysplastic aberrant crypt foci (6,10,11). The vast majority of both germline and somatic APC mutations are predicted to result in truncation of the APC protein due to either nonsense or frame-shifting mutations (5,6,7,8,9). Likewise, mice carrying homologous germline truncating mutations of APC are also predisposed to intestinal tumors (8, 9, 10). Altogether, these results strongly suggest that APC mutations are an early if not initiating event in the development of both sporadic an inherited forms of colorectal cancer. While disruption of normal APC function clearly plays a role in colorectal tumorigenesis, what this function might be remains unclear. The APC gene is predictesd to encode a protein of 2843 amino acids with limited functional homology to known proteins. The primary structure contains several Armadillo repeats that are sh,ared by proteins with apparently diverse functions (3, 15) as well as several regions of heptad repeats of the type that mediate oligomerization via coiled-coil structures (3). Indeed, the amino terminus of APC, which has a very strong potential for forming coiled-coil structures, has been shown to mediate the homo-oligomerization of APC protein (16, 17). Three additional repeats located between amino acids 1000 and 1200 of APC mediate an associate with a and β - catenins, critical cytoplasmic components of cadherin cell adhesion (18, 19). In addition, wild-type but not mutant forms of APC have been shown to associate with microtubule cytoskeleton (20, 21). While the aforementioned biochemical characteristics of APC provide important clues to its function, other functions remain undefined. Because mutant APC proteins almost uniformly lack their carboxyl terminus, we hypothesized that the carboxyl terminus of APC interacts with proteins that are essential for its normal function. To test this hypothesis we attempted to identify a protein that associates with the carboxyl terminus of APC. SUMMARY OF THE INVENTION It is an object of the invention to provide a nucleic acid molecule encoding a protein which binds to APC. It is an object of the invention to provide a protein molecule which binds to APC. It is another object of the invention to provide nucleic acid molecules which can be used to detect genes involved in neoplasia in a sample. It is yet another object of the invention to provide methods for determining a predisposition to colorectal and other neopl∑tsms. It is still another object of the invention to provide antibodies useful for detecting proteins which bind to APC. It is an object of the invention to provide methods for assessing susceptibility to colorectal and other cancer. It is an object of the invention to provide methods for diagnosing cancer. It is still another object of the invention to provide methods to assess treatment options for a cancer. It is yet another object of the invention to provide methods to assess the status of APC alleles in a cell. These and other objects of the invention are provided by one or more of the embodiments described below. In one embodiment of the invention a nucleic acid molecule is provided which comprises an EBl DNA according to SEQ ID NO: l. Also provided is a molecule which may contain at least 12, 18, or 20 contiguous nucleotides of EBl coding sequence. Also provided is a molecule which encodes at least about 6, 8, 10, or 20 contiguous EBl amino acids. In another embodiment of the invention an isolated and purified EBl protein is provided. The protein has an amino acid sequence according to SEQ ID NO:2. Polypeptides having at least 6, 8, 10, or 20 contiguous amino acids of said sequence are also provided. In still another embodiment of the invention a method for determining a predisposition to or a diagnosis of colorectal and other neoplasms is provided. The method comprises the step of: determining one or more mutations in one or more EBl alleles of a human tissue, wherein wild-type EBl is as shown in SEQ ID NO: l. In one embodiment of the invention a method for determining a predisposition to or diagnosis of colorectal and other neoplasms is provided. The method comprises the step of: assaying protein complexes in a cell, wherein said protein complexes comprise APC and EBl, wherein absence of said complexes or reduction in level of said complexes indicates a predisposition to neoplasms. In another embodiment of the invention an antibody preparation is provided. The antibody is specifically immunoreactive with an EBl protein according to SEQ ID NO:2. According to still another aspect of the invention a method for determining a diagnosis or predisposition to cancer is provided. The method comprises the step of: testing a human tissue to determine if the tissue expresses less EBl gene product than a normal human tissue or no EBl gene product. In another embodiment of the invention a method is provided to assess treatment options for a cancer. The method comprises the step of: contacting a lysate of cancer cells with EBl protein and detecting the formation of protein complexes comprising said EBl protein, a lysate which fails to form complexes indicating cancer cells which are good candidates for treatment with cyclooxygenase inhibitors. In yet another embodiment of the invention, a method is provided to assess the status of APC alleles in a cell. The method comprises the step of contacting a lysate of cells with EBl protein, a lysate which fails to form complexes indicating cancer cells which may lack wild-type APC. These and other embodiments of the invention provide the art with the identity of a gene and a protein which are involved in the suppression of neoplasia. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the nucleotide and predicted amino acid sequences of EBl. The arrowheads above the sequences indicate the 5' termini of different EBl cDNA clones isolated by yeast two hybrid screening. The predicted amino acid sequence begins at nucleotide 65 and ends at the nucleotide 868. The nucleotide .sequence 1ms been deposited with Genbank (# U24166). Figure 2 shows in vitro Binding of EBl to APC. Figure 2A shows binding of cellular APC to GST-EB1 (glutathione S-transferase = GST) fusion protein. SW480 and HCTl 16 are human colorectal cancer cell lines that express truncated and full length APC, respectively (19). Protein from total cell lysates (-) or protein bound by GST-EB1 fusion protein (GST-EB1) were analyzed by Western blot analysis with APC-specific monoclonal antibody FE9 (19). Figure 2B shows the binding of EBl to GST-APC fusion protein. GST-CTN has been described (19) and was used as a negative control. SW480 and HCTl 16 cells were metabolically labelled with 35 S-Met and incubated with the GST fusion proteins as indicated. In vitro transcribed and translated EBl (in vitro) was run on gel directly (-) or following binding to GST-APC(X) fusion protein as indicated. Proteins were detected by fluorography. Figure 2C shows one dimensional peptide mapping. Cellular (SW480, HCTl 16) and in vitro translate (in vitro) EBl proteins were isolated by binding to GST-APC(X) and subjected to one dimensional peptide mapping as described (19). Figure 3 shows in vivo association of APC and EBl. SW480 cells were transiently transfected with expression vectors for EBl or APC as indicated. The parental expression vector pCMV-NEO-BAM (pCMV) was used to equalize the total amount of DNA transfected. Lysates prepared from these transfected cells were used directly (total), or after immunoprecipitation with a monoclonal antibody against hemagglutinin (HA) as negative control or an EBl -specific monoclonal antibody (EBl). Detection of APC was carried out by immunoblotting using APC specific monoclonal antibody FE9. MT and FL indicate truncated and full length APC, respectively. Figure 4 shows the localization of EBl to chromosome 20qll.2 by fluorescence in situ hybridization (FISH). The left panel shows an ideogram of a G-banded human chromosome 20 with the band ql 1.2 bracketed. The top right panel shows the fluorescent signals localizing EBl to chromosome 20. The bottom right panel shows a G-banded human chromosome 20 localizing EBl to 20qll.2. Figure 5 shows human and yeast EBl homologues. Figure 5A shows an amino acid sequence comparison among human EBl homologues. EB2 represents the amino acid sequence predicted from the nucleotide sequence of a contig of 3 different EST's (Z46175, T17004 and Z42534.) The Z19434 and M85402 lines show the predicted amino acid sequences of these two EST's, respectively. Because of the lack of overlap between Z 19434 and M85402, we could not determine whether they represented one or two genes. "-" indicates that no sequence information was available at that position. Figure 5B shows an amino acid sequence comparison between human EBl and a potential yeast EBl homolog. The sequence of Yer016p is predicted from an open reading frame (ORF) from yeast chromosome V as described in the text. "-" indicates gap introduced to allow the best alignment between the two sequences. In both Figures 5A & 5B, blocks of homology are capitalized and shaded according to their mean scores. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS We have identified a cDNA that codes for a protein that interacts with the carboxyl terminus of APC. This interaction was clearly demonstrated by binding of cellular APC to recombinant EBl and by binding of cellular EBl to recombinant APC. The association between EBl and APC in mammalian cells was also demonstrated in cells cotransfected with vectors expressing these two proteins. Because almost all previously identified APC mutations result in the truncation of the APC protein, these mutant APC proteins cannot associate with EBl . This observation strongly suggests that the interaction between APC and EBl is important for the normal function of APC and that loss of this association is essential for the development of colorectal cancer. Mutation of EBl is one way that a cell can lose this association. EBl nucleic acid molecules according to the present invention include both ribonucleic acids and deoxyribonucleic acids. They may be incorporated as a part of a vector, such as a virus, phage, plasmid, minichromosome, etc. A vector typically contains an origin of replication which allows for independent replication of the nucleic acids of the vector and any insert it may be carrying. Suitable vectors may be chosen for a particular purpose, as is well within the skill of the art. Isolation and purification of nucleic acid molecules from other nucleic acid molecules and from other cellular components can be accomplished as is well known in the art. Nucleic acid molecules comprising at least about 12, 18, or 20 nucleotides of EBl coding sequence can be used inter αliα as probes and primers. Probes are typically labelled with a detectable label such as a radionuclide, an enzyme, or ligand. Primers may have restriction enzyme sites or promoters appended, as may be desirable for cloning or in vitro protein synthesis. Nucleic acid molecules encoding at least about 6 or 20 contiguous amino acids of EBl can be used for expressing fragments of EBl, for example for use in fusion proteins or as antigens or immunogens. The nucleotide sequence of wild-type EBl is provided in SEQ ID NO: l. The amino acid sequence of EBl protein is provided in SEQ ID NO:2. EBl protein may be isolated and purified from human cells, from transformed mammalian, other eukaryotic, or prokaryotic cells. Purification may be accomplished employing antibodies which are specific for EBl, such as AE9, EA3, and GD10, as provided herein. Other antibodies can be used which are made using all or a portion of EBl as an immunogen. Affinity methods may also be used which take advantage of the binding of EBl to APC. EBl may also be synthesized chemically or in an in vitro system, as described in more detail below. Portions of EBl which contain at least 6 or 20 contiguous amino acids according to SEQ ID NO:2 can be used in assays and as immunogens. These can be synthesized and isolated according to established techniques with the benefit of the sequence information provided herein. Predisposition to colorectal and other neoplasms can be determined by examination of a sample for a mutation in an EBl gene. Such other cancers include, but are not limited to desmoid tumors, osteomas, glioblastomas, medulloblastomas and other tumors of the central nervous system. Examination can be done by comparison with the wild-type sequence provided in SEQ ID NO: 1 or to the EBl found in human tissues which are normal. It can also be done by determining diminished expression of EBl protein or message, or failure of EBl to form complexes with APC. Methods for determining mutations include PCR, sequencing, restriction mapping, SI nuclease mapping, and hybridization with allele-specific probes. Any method known in the art can be used. Methods for determining diminished EBl expression or failure to form complexes with APC can be determined using techniques such as immunoprecipitation, immunoblotting, immunohistochemistry, etc. Antibodies which are particularly useful for such purposes are monoclonal antibodies AE9, EA3 and GD10, whose isolation and production are discussed in more detail below. Polyclonal antibodies can also be used, especially if purified to render a preparation monospecific. Samples which may be tested for assessing susceptibility to colorectal cancer include blood, chorionic villi, fetal trophoblasts, amniotic fluid, and blastomeres of pre- implantation embryos. Solid tissues can also be tested to determine predisposition and/or diagnosis. Assays using EBl can be used to assess the status of APC alleles, since according to the present invention EBl and APC interact. Thus, for example, a lysate of cells can be contacted with EBl protein and the formation of protein complexes comprising EBl protein can be detected. If the lysate fails to form complexes with EBl the cells are likely cancer cells which lack wild-type APC. Other means for measuring the interaction of EBl with APC can be used to provide such information. The drug sulindac has been shown to inhibit the growth of benign colon tumors in patients with familial adenomatous polyposis (FAP), presumably by virtue of its activity as a cyclooxygenase inhibitor (Waddell et al., J. Surg. Oncology 24(1), 83 (1983); Wadell, et al., Am. J. Surg., 157(1), 175 (1989); Charneau et al., Gastroenterologie Clinique at Biologique 14(2), 153 (1990)). Cyclooxygenase is required to convert arachidonic acid to prostaglandins and other biologically active molecules. Since FAP is attributed to mutations in APC, treatment options for a cancer may be assessed using EBl. EBl can be used as described above to assess the status of APC alleles. Cells which fail to form protein complexes with EBl are likely cancer cells which are good candidates for treatment with cyclooxygenase inhibitors, such as sulindac. EXAMPLES Example 1 This example describes the isolation of a gene which encodes a protein which interacts with the carboxy terminus of APC. We used a modified yeast two hybrid system (22,23) to screen a HeLa cDNA library for proteins interacting with the carboxyl terminus (codons 2167 to 2843) of APC. A total of 90 positive clones with the appropriate phenotype were identified after screening one million transformants. The cDNAs isolated from 67 out of these 90 clones were able to confer the correct phenotype when retransformed into the test strain of yeast. The nucleotide sequences of both ends of each cDNA were determined and were coπψared to each other. Forty-eight of these cDNAs were found to be derived from a same gene and could be separated into 11 groups according to their length (Figure 1). We chose to characterize this cDNA in detail and named it EBl (for EcoRI fragment binding protein I). The fusion proteins encoded by two independent cDNA clones did not interact with amino proximal residues 6 to 1013 when tested in the two hybrid assay. Northern blot analysis with probes to EBl identified a single 2.4 kb transcript. Because the largest EBl cDNA isolated by interaction trap method was 1.4 kb, we screened a human fetal brain cDNA library to isolate the full length cDNA. None of the newly isolated cDNA clones had additional 5' nucleotide sequence but many of them had additional 3' nucleotide sequence extending the length of the cloned message to 2.4 kb. Furthermore, no additional 5' sequence was obtained after screening three 5'-RACE cDNA libraries. Together, these results suggest that the full-length message for EBl had been isolated. Nucleotide sequence analysis of the overlapping cDNA clones revealed an ORF extending from nucleotide 1 to 868 (Figure 1). If translation initiated at the first methionine, EBl would be predicted to encode a 268 amino acid protein with a predicted molecular weight of 30 kD. Methods: Two hybrid screening. The modified yeast two hybrid system, the cDNA library and screening the cDNA library using this system have been described (22, 23). The bait was made by inserting a 2.5 Kb EcoRI fragment of APC containing nucleotides nucleotide 6498 to 8950 into the Smal site of LexA(l- 202)+ PL (24) .after making the EcoRI fragment blunt-ended using the Klenow fragment of DNA polymerase I. Example 2 This example demonstrates the in vitro and in vivo binding of APC to EBl. To confirm and extend the two hybrid results, we tested the direct interaction between EBl and APC using an in vitro binding assay. The carboxyl terminjil 163 residues of EBl were expressed as a glutathione-S-transferase fusion protein in E. coli. This fragment was expected to bind APC because it included more of ΕB1 than several of the EBl cDNA clones originally isolated by the yeast interaction trap method. As expected, this fusion protein was able to associate with the full-length APC from cell lysates, but was unable to bind to mutant APC that lacked the putative ΕB1 binding region (Figure 2A). This result clearly showed that ΕB1 interacts with endogenous APC and that this interaction requires the carboxyl terminus of APC. To test whether APC could bind endogenous ΕB1 , we expressed amino acid codons 2167 to 2843 of APC as a GST fusion protein (GST-APCΕ) and incubated the purified fusion protein bound on the glutathione agarose with lysates prepared from metabolically labeled colon cancer cell lines. The APC fusion protein bound a 30 kD cellular protein bound which had identical mobility to the ΕB1 expressed in vitro (Figure 2B). To confirm that this 30 kD protein was indeed ΕB1, we compared the one-dimensional peptide map of this 30 kD protein with that of ΕB1 expressed in vitro. The peptide maps of these proteins were identical (Figure 2C). This result also provided additional evidence that the first codon for methionine in the ΕB1 cDNA is the translational initiation codon. Methods: GST fusion proteins. The pGSTagΕBIA expression vector was constructed using an ΕcoRI fragment (nucleotides 317 to 899 of ΕB1) of an ΕB1 cDNA clone isolated by interaction trap screening. After subcloning into the ΕcoRI site of pBluescript SK II, the ΕcoRI fragment was excised as a BamHI-Sall fragment and inserted into the BamHI and Xhol sites of pGSTag (25). The pGSTagEBlB expression vector constructed by inserting a 1.8 Kb Sall-Hindlll fragment (nucleotides 40 to 2091) of an ΕB1 cDNA clone isolated from human fetal brain cDNA library into the Sail and Hindlll sites of pGSTag. The pGSTa APCΕ expression vector was constructed by inserting the 2.5 Kb ΕcoRI fragment of APC cDNA, identical to that used for making the bait for two hybrid screening, into the ΕcoRI site of pGSTag. The expression and purification of fusion proteins were carried out as described (19). Methods: PCR and in vitro expression of EBl. The EBl coding region w a s a m p l i f i e d b y u s i n g t h e u p s t r e a m p r i m e r 5 ' - GGATCCTAATACGACTCACTATAGGGAGACCACCATGGCAGTGAACG TATAC-rC ' and the downstream primer 5'-ATTTCTCCACTGAGGTCGC-3\ The upstream primer contains the sequence of the promoter for the T7 DNA polymerase and the first 20 nucleotides of the EBl coding sequence. The downstream primer locates at the 3' untranslated region of EBl. The PCR reaction was carried out using an isolated cDNA clone as the template with 35 cycles of 30 sec at 95°C, 1 min at 50°C, and 1 min at 70°C. The PCR product was using directly in a coupled in vitro transcription and translation reaction as described (26). Methods: in vitro binding assay. Metabolically labelled protein extracts from the human colorectal cancer cell lines SW480 and HCTl 16 were used for the in vitro binding assay. Metabolic labeling, preparation of cell lysates, in vitro binding, .and peptide mapping were carried out as described (19). Example 3 This example demonstrates the in vivo association of EBl and APC by co- immunoprecipitation. In order to further characterize the association APC and EBl, three monoclonal antibodies (AE9, EA3 and GD10) against EBl were generated. Western blot analysis with all three of these antibodies detected a 30 kD protein in total cell lysates which associated with GST-APCE, but not with a control protein GST-CTN. EBl protein was detected in several human colon cancer cell lines including a human kidney fibroblast cell line 293, the canine kidney epithelial cell line MDCK, and the mouse fibroblast cell line NIH3T3. To demonstrate an in vivo association between EBl and APC in mammalian cells, SW480 cells were transiently transfected with vectors expressing APC or EBl. The association between these two proteins was examined by immunoprecipitation using the EBl - specific antibody EA3 followed by immunoblotting with the APC-specific antibody FE9. The co-immunoprecipitation of APC and EBl was clearly demonstrated when cells were transfected with both expression vectors but not when either one was omitted. (Figure 3.) We have not been able to detect the association between endogenous full- length APC and EBl by co-immunoprecipitation experiments. The reason for this may be purely technical. This is consistent with our inability to co- immunoprecipitate APC and EBl from cell lysates prepared from yeast clones with clear functional evidence of an association between these two proteins as reflected by the two-hybrid assay. Similar reasons have also been suggested for the failure to demonstrate an association between pRB and RBP2 by co-immunoprecipitation (26, 27). Methods: Monoclonal antibodies. The three EBl monoclonal antibodies, AE9, EA3, and GD10, were derived from mice immunized with GST-EB1 fusion protein. Immunization of mice, cell fusion, and the preparation of monoclonal antibodies were carried out as described (27). The EA3 monoclonal was found to specifically recognize EBl by both Western blot and immunoprecipitation. Methods: in vivo Binding Assay. SW480 cell lines were transiently transfected with pCMV-APC or pCMV-EBl. The pCMV-APC was as described (20) and the pCMV-EBl vector was derived by cloning a PCR product containing EBl nucleotides 62 to 871 into the BamHl site of pCMV-NEO-BAM. PCR was performed with following primers which were engineered to include the underlined Bglll sites: 5'-CGAGAT£TAAGATGGCAGTGAACGTATAC-3' and 5'- GCA^ATCTTTAATACTCTTCTTGATCCTCC-3'). To eliminate the possibility of PCR errors, the sequence of the EBl fragment cloned into PCMV-EBl was verified by nucleotide sequencing. Transient transfections, preparation of cell lysates, immunoprecipitation and western blot analysis were performed as described (16, 19, 20). Example 4 This example demonstrates the chromosomal mapping of EBl. The chromosomal localization of EBl was determined by fluorescence in situ hybridization (FISH). Three PI clones for EBl were isolated from a PI library by PCR. One of these PI clones was used as the probe in the FISH analysis as previously described (24). Sixteen out of a total of 17 metaphase cell examined displayed double fluorescent signals (i.e. one on each chromatin) on the proximal short arm of chromosome 20. The same cells hybridized for FISH had been previously G-banded and photographed to allow direct comparisons of the results. The result demonstrated that the sequences hybridizing to EBl can be localized to 20qll.2 (Figure 4). Methods: Chromosomal localization. Three EBl genomic clones (EB- 922, EB1-923, EB1-924) were obtained by PCR screening of A PI library (Genome Systems, Inc.) using primers (5'-AAAACAGAGAGGCTGACCG-3 and S'-ATTTCTCCACTGAGGTCGC-S') designed to amplify EBl nucleotides 1102 to 1205. Total EB1-923 DNA was labeled with Biotin-16-dUTP by nick translation and used for FISH. For FISH, about 100 ng of probe was used in 10 μl hybridization mixture (55% formamide, 2X SSC, and 1 μg human Cot 1 DNA) which was denatured at 75°C for 5 minutes. Hybridization was carried out using a modified procedure of Pinkel et al. (28) as previously described (29). Example 5 This example analyzes the nucleotide and amino acid sequences of EBl. Searches of the National Center for Biotechnology Information (NCBI) non- redundant nucleotide and EST (expressed sequence tag) databases indicated that EBl had not been previously characterized although there were several ESTs that were almost identical to parts of the 3' untranslated region. Interestingly, there were also iϊve ESTs which were similar but not identical to the coding region of EBl . These ESTs likely represented novel EBl-related genes rather than sequencing mistakes as there were numerous nucleotide substitutions that preserved the encoded amino acids of EBl in these ESTs. These five ESTs could be divided into three contigs which represented at least two different EBl related proteins (Figure 5A). Searches of NCBI's non-redundant protein database with EBl identified three proteins with statistically significant (P < 0.05) multiple regions of homology. These were a calcium channel protein from caφ (PIR# A37860, P = .0075), a bacterial RNA polymerase sigma chain homolog (PIR # JN0445, P = .0028) and Yer016ρ (P = 2.4 x 10 "53 ). YerOlόp is a putative gene identified in a 66,030 bp Saccharomyces cerevisiae chromosome V cosmid contig (Genbank #U18778). The predicted Yer016p protein shared five blocks of similarity with EBl and could represent a ye^t homolog of EBl (Figure 5B). Together, these data suggest that EBl is a member of a highly conserved multi-gene family. Methods: Database searches and alignments. The NCBI's non- redundant nucleotide, non-redundant protein and DBEST databases (1/19/95 releases) were searched using the BLASTN, BLASTP and TBLASTN basic local alignment search software, respectively (30). Multiple alignments were performed using the MACAW multiple alignment construction and analysis software version 2.03 (31). References 1. 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Generating yeast transcriptional activators containing no yeast protein sequences. Nature, 350: 250-252, 1991. 25. Ron, D., and Dressier, H. pGSTag~A versatile bacterial expression plasmid for enzymatic labeling of recombinant proteins. BioTechniques, 13: 866-869, 1992. 26. Powell, S. M., Petersen, G. M., Krush, A. J., Booker, S., Jen, J., Giardiello, F. M., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. Molecular diagnosis of familial adenomatous polyposis. New Engl. J. Med. , 329: 1982-1987, 1993. 27. Smith, K. J., Johnson, K. A., Bryan, T. M., Hill, D. E., Markowitz, S., Wilson, J. K. V., Paraskeva, C, Petersen, G. M., Hamilton, S. R., Vogelstein, B., and Kinzler, K. W. The APC gene product in normal and tumor cells. Proc. Natl. Acad. Sci. USA, 90: 2846-2850, 1993. 28. Pinkel, D. , Landegent, J., Collins, C, Fuscoe, J., Segraves, R., Lucas, J., and Gray, J. Fluorecence in situ hybridization with human chromosome- specific libraries: Detection of trisomy 21 and translocation of chromosome 4. Proc. Natl. Acad. Sci. USA, 85: 9138-9142, 1988. 29. Meltzer, P. S. , Guan, X.-Y., Burgess, A., and Trent, J. M. Micro-FISH: a novel stategy to identify cryptic chromosomal rearrangements. Nature Genet. , 1: 24-28, 1992. 30. Altschul, S. F., Gish, W., Miller, W., Myers, E.W., and Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215: 403-410, 1990. 31. Schuler, G. D., Altschul, S. F., and Lipman D. J. A workbench for multiple alignment construction and analysis. Proteins Struct. Funct. Genet. 9: 180-190, 1991. 32. Fattaey, A. R., Helin, K., Dembski, M. S., Dyson, N., Harlow, E., Vuosolo, G. A., Hanobik, M. G., Haskell, K. M., Oliff, A., Defeo-Jones, D., and Jones, R. E. Characterization of the retinoblastoma binding proteins RBP1 and RBP2. Oncogens, 8: 3149-3156, 1993. 33. Kim, Y. W., Otterson, G. A., Kratzke, R. A., Coxon, A. B., and Kaye, F. J. Differential specificity for binding of retinoblastoma binding protein 2 to RB, pl07, and TATA-binding protein. Mol. Cell. Biol. , 14: 7256- 7264, 1994. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: The Johns Hopkins University (ii) TITLE OF INVENTION: EBl Gene Product Binds to APC (iii) NUMBER OF SEQUENCES: 12 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Banner & Allegretti, Ltd. (B) STREET: 1001 G Street, N.W. (C) CITY: Washington (D) STATE: D.C (E) COUNTRY: U.S. (F) ZIP: 20001-4597 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: 22-MAY-1996 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Kagan, Sarah A. (B) REGISTRATION NUMBER: 32,141 (C) REFERENCE/DOCKET NUMBER: 01107.49255 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 202.508.9100 (B) TELEFAX: 202.508.9299 (2) INFORMATION FOR SEQ ID Nθ:l: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2540 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: EBl (viii) POSITION IN GENOME: (A) CHROMOSOME/SEGMENT: 20gll.2 ( ix ) FEATURE : (A) NAME/KEY: CDS (B) LOCATION: 65..868 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ACGAGACGAA GACGGAACCG GAGCCGGTTG CGGGCAGTGG ACGCGGTTCT GCCGAGAGCC 60 GAAG ATG GCA GTG AAC GTA TAC TCA ACG TCA GTG ACC AGT GAT AAC CTA 109 Met Ala Val Asn Val Tyr Ser Thr Ser Val Thr Ser Asp Asn Leu 1 5 10 15 AGT CGA CAT GAC ATG CTG GCC TGG ATC AAT GAG TCT CTG CAG TTG AAT 157 Ser Arg His Asp Met Leu Ala Trp lie Asn Glu Ser Leu Gin Leu Asn 20 25 30 CTG ACA AAG ATC GAA CAG TTG TGC TCA GGG GCT GCG TAT TGT CAG TTT 205 Leu Thr Lys lie Glu Gin Leu Cys Ser Gly Ala Ala Tyr Cys Gin Phe 35 40 45 ATG GAC ATG CTG TTC CCT GGC TCC ATT GCC TTG AAG AAA GTG AAA TTC 253 Met Asp Met Leu Phe Pro Gly Ser lie Ala Leu Lys Lys Val Lys Phe 50 55 60 CAA GCT AAG CTA GAA CAC GAG TAC ATC CAG AAC TTC AAA ATA CTA CAA 301 Gin Ala Lys Leu Glu His Glu Tyr lie Gin Asn Phe Lys lie Leu Gin 65 70 75 GCA GGT TTT AAG AGA ATG GGT GTT GAC AAA ATA ATT CCT GTG GAC AAA 349 Ala Gly Phe Lys Arg Met Gly Val Asp Lys lie lie Pro Val Asp Lys 80 85 90 95 TTA GTA AAA GGA AAG TTT CAG GAC AAT TTT GAA TTC GTT CAG TGG TTC 397 Leu Val Lys Gly Lys Phe Gin Asp A3n Phe Glu Phe Val Gin Trp Phe 100 105 110 AAG AAG TTT TTC GAT GCA AAC TAT GAT GGA AAA GAC TAT GAC CCT GTG 445 Lys Lys Phe Phe Asp Ala Asn Tyr Asp Gly Lys Asp Tyr Asp Pro Val 115 120 125 GCT GCC AGA CAA GGT CAA GAA ACT GCA GTG GCT CCT TCC CTT GTT GCT 493 Ala Ala Arg Gin Gly Gin Glu Thr Ala Val Ala Pro Ser Leu Val Ala .130 135 140 CCA GCT CTG AAT AAA CCG AAG AAA CCT CTC ACT TCT AGC AGT GCA GCT 541 Pro Ala Leu Asn Lys Pro Lys Lys Pro Leu Thr Ser Ser Ser Ala Ala 145 150 155 CCC CAG AGG CCC ATC TCA ACA CAG AGA ACC GCT GCG GCT CCT AAG GCT 589 Pro Gin Arg Pro lie Ser Thr Gin Arg Thr Ala Ala Ala Pro Lys Ala 160 165 170 175 GGC CCT GGT GTG GTG CGA AAG AAC CCT GGT GTG GGC AAC GGA GAC GAC 637 Gly Pro Gly Val Val Arg Lys Asn Pro Gly Val Gly Asn Gly Asp Asp 180 185 190 GAG GCA GCT GAG TTG ATG CAG CAG GTC AAC GTA TTG AAA CTT ACT GTT 685 Glu Ala Ala Glu Leu Met Gin Gin Val Asn Val Leu Lys Leu Thr Val 195 200 205 GAA GAC TTG GAG AAA GAG AGG GAT TTC TAC TTC GGA AAG CTA CGG AAC 733 Glu Asp Leu Glu Lys Glu Arg Asp Phe Tyr Phe Gly Lys Leu Arg Asn 210 215 220 ATT GAA TTG ATT TGC CAG GAG AAC GAG GGG GAA AAC GAC CCT GTA TTG 781 lie Glu Leu lie Cys Gin Glu Asn Glu Gly Glu Asn Asp Pro Val Leu 225 230 235 CAG AGG ATT GTA GAC ATT CTG TAT GCC ACA GAT GAA GGC TTT GTG ATA 829 Gin Arg lie Val Asp lie Leu Tyr Ala Thr Asp Glu Gly Phe Val He 240 245 250 255 CCT GAT GAA GGG GGC CCA CAG GAG GAG CAA GAA GAG TAT TAACAGCCTG 878 Pro Asp Glu Gly Gly Pro Gin Glu Glu Gin Glu Glu Tyr 260 265 GACCAGCAGA GCAACATCGG AATTCTTCAC TCCAAATCAT GTGCTTAACT GTAAAATACT 938 CCCTTTTGTT ATCCTTAGAG GACTCACTGG TTTCTTTTCA TAAGCAAAAA GTACCTCTTC 998 TTAAAGTGCA CTTTGCAGAC GTTTCACTCC TTTTCCAATA AGTTTGAGTT AGGAGCTTTT 1058 ACCTTGTAGC AGAGCAGTAT TAACATCTAG TTGGTTCACC TGGAAAACAG AGAGGCTGAC 1118 CGTGGGGCTC ACCATGCGGA TGCGGGTCAC ACTGAATGCT GGAGAGATGT ATGTAATATG 1178 CTGAGGTGGC GACCTCAGTG GAGAAATGTA AAGACTGAAT TGAATTTTAA GCTAATGTGA 1238 AATCAGAGAA TGTTGTAATA AGTAAATGCC TTAAGAGTAT TTAAAATATG CTTCCACATT 1298 TCAAAATATA AAATGTAACA TGACAAGAGA TTTTGCGTTT GACATTGTGT CTGGGAAGGA 1358 AGGGCCAGAC CTTGGAACCT TTGGAACCTG CTGTCAACAG GTCTTACAGG GCTGCTTGAA 1418 CCCTCATAGG CCTAGGCTTT GGTCTAAAAG GAACATTTAA AAAGTTGCCC TGTAAAGTTA 1478 TTTGGTGTCA TTGACCAATT GCATCCCAGC TAAAAAGCAA GAGGCATCGT TGCCTGGATA 1538 ATAGAGGATG TGTTTCAGCC CTGAGATGTT ACAGTTGAAG AGCTTGGTTT CATTGAGCAT 1598 TTCTCTATTT TTCCAGTTAT CCCGAAATTT CTATGTATTA TTTTTTGGGG AAGTGAGGTG 1658 TGCCCAGTTT TTTAATCTAA CAACTACTTT TGGGGACTTG CCCACATCTC TGGGATTTGA 1718 ATGGGGATTG TATCCCATTT TACTGTCTTT TAGGTTTACA TTTACCACGT TTCTCTTCTC 1778 TGCTCCCCTT GCCCACTGGG ACTCCTCTTT GGCTCCTTGA AGTTTGCTGC TTAGAGTTGG 1838 AAGTGCAGCA GGCAGGTGAT CATGCTGCAA GTTCTTTCTG GACCTCTGGC AAAGGGAGTG 1898 GTCAGTGAAG GCCATCGTTA CCTTGGGATC TGCCAGGCTG GGGTGTTTTC GGTATCTGCT 1958 GTTCACAGCT CTCCACTGTA ATCCGAATAC TTTGCCAGTG CACTAATCTC TTTGGAGATA 2018 AAATTCATTA GTGTGTTACT AAATGTTAAT TTTCTTTTGC GGAAAATACA GTACCGTGTC 2078 TGAATTAATT ATTAATATTT AAAATACTTC ATTCCTTAAC TCTCCCTCAT TTGCTTTGCC 2138 CACAGCCTAT TCAGTTCCTT TGTTTGGCAG GATTCTGCAA AATGTGTCTC ACCCACTACT 2198 GAGATTGTTC AGCCCCTGAT GTATTTGTAT TGATTTGTTT CTGGTGGTAG CTTGTCCTGA 2258 AATGTGTGTA GAAAGCAAGT ATTTTATGAT AAAAATGTTG TGTAGTGCAT GCTCTGTGTG 2318 GAATTCAGAG GAAAACCCAG ATTCAGTGAT TAACAATGCC AAAAAATGCA AGTAACTAGC 2378 CATTGTTCAA ATGACAGTGG TGCTATTTCT CTTTTGTGGC CTTTTAGACT TTTGTTGCCC 2438 TAAAATTCCA TTTTATTGGG AACCCATTTT CCACCTGGTC TTTCTTGACA GGGTTTTTTT 2498 CTACTTTAAA CAGTTTCTAA ATAAAATTCT GTATTTCAAA AA 2540 (2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 268 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ( i) SEQUENCE DESCRIPTION: SEQ ID Nθ:2: Met Ala Val Asn Val Tyr Ser Thr Ser Val Thr Ser Asp Asn Leu Ser 1 5 10 15 Arg His Asp Met Leu Ala Trp He Asn Glu Ser Leu Gin Leu Asn Leu 20 25 30 Thr Lys He Glu Gin Leu Cys Ser Gly Ala Ala Tyr Cys Gin Phe Met 35 40 45 Asp Met Leu Phe Pro Gly Ser He Ala Leu Lys Lys Val Lys Phe Gin 50 55 60 Ala Lys Leu Glu His Glu Tyr He Gin Asn Phe Lys He Leu Gin Ala 65 70 75 80 Gly Phe Lys Arg Met Gly Val Asp Lys He He Pro Val Asp Lys Leu 85 90 95 Val Lys Gly Lys Phe Gin Asp Asn Phe Glu Phe Val Gin Trp Phe Lys 100 105 110 Lys Phe Phe Asp Ala Asn Tyr Asp Gly Lys Asp Tyr Asp Pro Val Ala 115 120 125 Ala Arg Gin Gly Gin Glu Thr Ala Val Ala Pro Ser Leu Val Ala Pro 130 135 140 Ala Leu Asn Lys Pro Lys Lys Pro Leu Thr Ser Ser Ser Ala Ala Pro 145 150 155 160 Gin Arg Pro He Ser Thr Gin Arg Thr Ala Ala Ala Pro Lys Ala Gly 165 170 175 Pro Gly Val Val Arg Lys Asn Pro Gly Val Gly Asn Gly Asp Asp Glu 180 185 190 Ala Ala Glu Leu Met Gin Gin Val Asn Val Leu Lys Leu Thr Val Glu 195 200 205 Asp Leu Glu Lys Glu Arg Asp Phe Tyr Phe Gly Lys Leu Arg Asn He 210 215 220 Glu Leu He Cys Gin Glu Asn Glu Gly Glu Asn Asp Pro Val Leu Gin 225 230 235 240 Arg He Val Asp He Leu Tyr Ala Thr Asp Glu Gly Phe Val He Pro 245 250 255 Asp Glu Gly Gly Pro Gin Glu Glu Gin Glu Glu Tyr 260 265 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 149 amino acids (B) TYPE: .amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: EB2 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: He Ala Trp Val Asn Asp He Val Ser Leu Asn Tyr Thr Lys Val Glu 1 5 10 15 Gin Leu Cys Ser Gly Ala Ala Tyr Cys Gin Phe Met Asp Met Leu Phe 20 25 30 Pro Gly Cys He Ser Leu Lys Lys Val Lys Phe Gin Ala Lys Leu Glu 35 40 45 His Glu Tyr He His Asn Phe Lys Leu Leu Gin Ala Ser Phe Lys Arg 50 55 60 Met Asn Val Asp Lys Val He Pro Val Glu Lys Leu Val Lys Gly Arg 65 70 75 80 Phe Gin Asp Asn Leu Asp Phe He Gin Trp Phe Lys Lys Phe Tyr Asp 85 90 95 Ala Asn Tyr Asp Gly Lys Glu Tyr Asp Pro Val Glu Ala Arg Gin Gly 100 105 110 Gin Asp Ala He Pro Pro Pro Asp Pro Gly Glu Gin He Phe Asn Leu 115 120 125 Pro Lys Lys Ser His His Ala Asn Ser Pro Thr Ala Gly Ala Ala Lys 130 135 140 Phe Lys Phe Gin Xaa 145 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 344 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Saccharomyces cerevisiae (vii) IMMEDIATE SOURCE: (B) CLONE: Yer016p (xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:4: Met Ser Ala Gly He Gly Glu Ser Arg Thr Glu Leu Leu Thr Trp Leu 1 5 10 15 Asn Gly Leu Leu Asn Leu Asn Tyr Lys Lys He Glu Glu Cys Gly Thr 20 25 30 Gly Ala Ala Tyr Cys Gin He Met Asp Ser He Tyr Gly Asp Leu Pro 35 40 45 Met Asn Arg Val Lys Phe Asn Ala Thr Ala Glu Tyr Glu Phe Gin Thr 50 55 60 Asn Tyr Lys He Leu Gin Ser Cys Phe Ser Arg His Gly He Glu Lys 65 70 75 80 Thr Val Tyr Val Asp Lys Leu He Arg Cys Lys Phe Gin Asp Asn Leu 85 90 95 Glu Phe Leu Gin Trp Leu Lys Lys His Trp He Arg His Lys Asp Glu 100 105 110 Ser Val Tyr Asp Pro Asp Ala Arg Arg Lys Tyr Arg Pro He He Thr 115 120 125 Asn Asn Ser Ala Thr Lys Pro Arg Thr Val Ser Asn Pro Thr Thr Ala 130 135 140 Lys Arg Ser Ser Ser Thr Gly Thr Gly Ser Ala Met Ser Gly Gly Leu 145 150 155 160 Ala Thr Arg His Ser Ser Leu Gly He Asn Gly Ser Arg Lys Thr Ser 165 170 175 Val Thr Gin Gly Gin Leu Val Ala He Gin Ala Glu Leu Thr Lys Ser 180 185 190 Gln Glu Thr He Gly Ser Leu Asn Glu Glu He Glu Gin Tyr Lys Gly 195 200 205 Thr Val Ser Thr Leu Glu He Glu Arg Glu Phe Tyr Phe Asn Lys Leu 210 215 220 Arg Asp He Glu He Leu Val His Thr Thr Gin Asp Leu He Asn Glu 225 230 235 240 Gly Val Tyr Ly Phe Asn Asp Glu Thr He Thr Gly His Gly Asn Gly 245 250 255 Asn Gly Gly Ala Leu Leu Arg Phe Val Lys Lys Val Glu Ser He Leu 260 265 270 Tyr Ala Thr Ala Glu Gly Phe Glu Met Asn Asp Gly Glu Asp Glu Leu 275 280 285 Asn Asp Lys Asn Leu Gly Glu His Gly Thr Val Pro Asn Gin Gly Gly 290 295 300 Tyr Ala Asn Ser Asn Gly Glu Val Asn Gly Asn Glu Gly Ser Asn His 305 310 315 320 Asp Val He Met Gin Asn Asp Glu Gly Glu Val Gly Val Ser Asn Asn 325 330 335 Leu He He Asp Glu Glu Thr Phe 340 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 112 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: zl9434 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: Asp Glu Asp Pro Pro Pro Arg Ser Arg Arg Pro Glu Pro Gin Pro Leu . 1 5 10 15 Pro Gin Arg Pro Arg His Leu Ser Pro Pro Pro Pro Pro Pro Pro Glu 20 25 30 Pro Pro Arg Ala Leu Trp Gly Met Ala Val Asn Val Tyr Ser Thr Ser 35 40 45 Val Thr Ser Glu Asn Leu Ser Arg His Asp Met Leu Ala Trp Val Asn 50 55 60 Asp Ser Leu His Leu Asn Tyr Thr Lys He Glu Gin Leu Cys Ser Gly 65 70 75 80 Ala Ala Tyr Cys Gin Phe Met Asp Met Leu Phe Pro Gly Cys Val His 85 90 95 Leu Arg Lys Val Lys Phe Gin Gly Lys Leu Gly His Xaa Tyr He His 100 105 110 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 120 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: YES (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Homo sapiens (vii) IMMEDIATE SOURCE: (B) CLONE: M85402 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Asn Phe Lys Val Leu Gin Xaa Ala Phe Lys Lys Met Gly Val Asp Lys 1 5 10 15 He He Pro Val Glu Lyε Leu Val Lys Gly Lys Phe Gin Asp Asn Phe 20 25 30 Xaa Phe He Gin Trp Phe Lyε Lys Xaa Phe Asp Ala Asn Tyr Asp Gly 35 40 45 Lys Asp Tyr Asn Pro Leu Leu Ala Arg Gin Gly Gin Asp Val Ala Pro 50 55 60 Pro Pro Asn Pro Val Pro Gin Arg Thr Ser Pro Thr Gly Pro Lys Asn 65 70 75 80 Met Gin Thr Ser Gly Arg Leu Ser Asn Val Ala Pro Pro Cys He Leu 85 90 95 Arg Lys Xaa Pro Pro Ser Ala Arg Asn Gly Gly His Glu Thr Cys Pro 100 105 110 Asn Ser Leu Asn Ser Asn Gin Gin 115 120 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 54 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGATCCTAAT ACGACTCACT ATAGGGAGAC CACCATGGCA GTGAACGTAT ACTC 54 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: ATTTCTCCAC TGAGGTCGC 19 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGAGATCTAA GATGGCAGTG AACGTATA 28 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: GCAGATCTTT AATACTCTTC TTGATCCTCC 30 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: AAAACAGAGA GGCTGACCG 19 (2) INFORMATION FOR SEQ ID NO:12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOG : 1inear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: ATTTCTCCAC TGAGGTCGC 1

Claims: CLAIMS 1. A nucleic acid molecule comprising an EBl DNA according to SEQ ID NO: l. 2. The nucleic acid molecule of claim 1 further comprising a vector containing an origin of replication. 3. An isolated and purified nucleic acid molecule comprising at least 18 contiguous nucleotides of EBl coding sequence according to SEQ ID NO: l. 4. An isolated and purified nucleic acid molecule which comprises a coding sequence which encodes at least 20 contiguous amino acids of EBl according to SEQ ID NO:2. 5. An isolated and purified EBl protein according to SEQ ID NO:2. 6. An isolated and purified EBl polypeptide comprising at least 20 contiguous amino acids according to SEQ ID NO: 2. 7. An isolated and purified nucleic acid molecule consisting at least 12 contiguous nucleotides of EBl coding sequence according to SEQ ID NO:l. 8. An isolated and purified nucleic acid molecule consisting of a coding sequence for at least 6 contiguous amino acids of EBl according to SEQ ID NO:2. 9. An isolated and purified EBl polypeptide consisting of at least 6 contiguous amino acids according to SEQ ID NO:2. 10. A method for determining a predisposition to neoplasms, comprising the step of: determining a mutation in an EBl allele of a human tissue, wherein wild-type EBl is as shown in SEQ ID NO: l. 11. The method of claim 10 wherein said human tissue is selected from the group consisting of blood, chorionic villi, fetal trophoblasts, amniotic fluid, and a blastomere of a pre-implantation embryo. 12. A method for determining a predisposition to neoplasms, comprising the step of: assaying protein complexes in a cell, wherein said protein complexes comprise APC and EBl , wherein reduction of said complexes in the cell relative to a cell which contains wild-type APC and wild-type EBl alleles indicates a predisposition to neoplasms. 13. The method of claim 12 wherein said step of assaying is performed by immunoprecipitation followed by immunoblotting. 14. The method of claim 13 wherein immunoprecipitation is performed with an antibody specifically immunoreactive with APC. 15. The method of claim 13 wherein immunoprecipitation is performed with an antibody specifically immunoreactive with EBl. 16. The method of claim 14 wherein immunoblotting is performed with an antibody specifically immunoreactive with EBl . 17. The method of claim 15 wherein immunoblotting is performed with an antibody specifically immunoreactive with APC. 18. An antibody preparation which is specifically immunoreactive with EBl. 19. The preparation of claim 17 wherein said antibody is monoclonal. 20. The preparation of claim 17 wherein said antibody is polyclonal. 21. A method for determining a predisposition to cancer, comprising the steps of: testing a human tissue to determine if the tissue expresses less EBl gene products than a normal human tissue. 22. The method of claim 21 wherein the step of testing utilizes an antibody which is specifically immunoreactive with EBl protein. 23. The method of claim 21 wherein the step of testing utilizes a nucleic acid probe which specifically hybridizes to an EBl mRNA, said probe having a sequence of at least 12 contiguous nucleotides selected from SEQ ID NO:l. 24. A method for diagnosing a neoplasm, comprising the step of: determining mutations in EBl alleles of a human tissue, wherein wild-type EBl is as shown in SEQ ID NO: 1. 25. A method for diagnosing a neoplasm, comprising the step of: assaying protein complexes in a cell, wherein said protein complexes comprise APC and EBl, wherein absence of said complexes indicates a neoplasm. 26. The method of claim 25 wherein said step of assaying is performed by immunoprecipitation followed by immunoblotting. 27. The method of claim 26 wherein immunoprecipitation is performed with an antibody specifically immunoreactive with APC. 28. The method of claim 26 wherein immunoprecipitation is performed with an antibody specifically immunoreactive with EBl. 29. The method of claim 27 wherein immunoblotting is performed with an antibody specifically immunoreactive with EBl . 30. The method of claim 28 wherein immunoblotting is performed with an antibody specifically immunoreactive with APC. 31. A method for diagnosing a neoplasm, comprising the step of: testing a human tissue to determine if the tissue expresses EBl gene products, wherein a tissue which expresses no EBl gene products is neoplastic. 32. The method of claim 31 wherein the step of testing utilizes an antibody which is specifically immunoreactive with EBl protein. 33. The method of claim 31 wherein the step of testing utilizes a nucleic acid probe which specifically hybridizes to an EBl mRNA, said probe having a sequence of at least 12 contiguous nucleotides selected from SEQ ID NO: l. 34. A method to assess treatment options for a cancer, comprising the step of: contacting a lysate of cancer cells with EBl protein and detecting the formation of protein complexes comprising said EBl protein, a lysate which fails to form complexes indicating cancer cells which are good candidates for treatment with cyclooxygenase inhibitors. 35. A method to assess the status of APC alleles in a cell, comprising the step of: contacting a lysate of cells with EBl protein and detecting the formation of protein complexes comprising said EBl protein, a lysate which fails to form complexes indicating cancer cells which may lack wild-type APC.

Inventors: KINZLER KENNETH, VOGELSTEIN BERT, KINZLER, KENNETH, VOGELSTEIN, BERT
Applicants: UNIV JOHNS HOPKINS, THE JOHNS HOPKINS UNIVERSITY
Application year: 1996
Main classification: C12N15
IPC codes: A61K38, C07K14, C07K16, C12N1, C12N15, C12P21, C12Q1, G01N33