Patent Abstract:
the apc tumor suppressor protein binds to β - catenin , a protein recently shown to interact with tcf / lef transcription factors . here , the gene encoding a tcf family member that is expressed in colonic epithelium was cloned and characterized . htcf - 4 transactivates transcription only when associated with β - catenin . nuclei of apc -/- colon carcinoma cells were found to contain a stable β - catenin - htcf - 4 complex that was constitutively active , as measured by transcription of a tcf reporter gene . reintroduction of apc removed β - catenin from htcf4 and abrogated the transcriptional transactivation . constitutive transcription of tcf target genes , caused by loss of apc function , may be a crucial event in the early transformation of colonic epithelium . it is also shown here that the products of mutant apc genes found in colorectal tumors are defective in regulating β - catenin / tcf - 4 transcrpitional activation . furthermore , colorectal tumors with intact apc genes were shown to contain subtle activating mutations of β - catenin that altered functionally significant phosphorylation sites . these results indicate that regulation of β - catenin is critical to apc &# 39 ; s tumor suppressive effect and that this regulation can be circumvented by mutations in either apc or β - catenin .

Detailed Description:
it is a discovery of the present invention that htcf - 4 binds to β - catenin and activates transcription in colorectal epithelial cells . moreover , it has now been found that apc regulates this transcriptional activation , at least in part by binding to β - catenin . in colorectal cancer cells this regulation is frequently abrogated , either by mutation of apc or by mutation of β - catenin . two alternative splice forms of human tcf - 4 have been found . one form ( htcf - 4e ) is homologous to htcf - 1e and the other ( htcf - 4b ) is homologous to htcf - 1b . the sequence of the nucleotide and amino acid sequences are shown in seq id nos : 1 , 2 , 5 , and 6 . the coding sequences and proteins can be used in assays as described below . intron - free dna molecules are provided which are originally made by reverse transcription of a mrna molecule . they can be propagated in cells or amplified as is desired . isolated tcf - 4 proteins can be provided substantially free of other human proteins if , for example , the nucleotide sequences are expressed in non - human cells . methods and vectors for achieving such expression are well known in the art . choice of such expression means is made by the skilled artisan according to the desired usage and convenience . cells can be tested to determine if they have a wild - type apc or a wild - type downstream protein in the apc transcription regulatory pathway , called herein the crt pathway ( β - catenin / tcf - regulated transcription ). one protein within the crt pathway which has been identified as a target of mutations in human cancers is β - catenin ( encoded by the ctnnb1 gene ). other parts of the pathway are also likely to be targets . although the target genes of the crt pathway have not been identified , they can be readily identified using the system disclosed here . genes which are differentially transcribed in the presence of wild - type and mutant ctnnb1 , for example , can be identified . tcf - responsive reporter genes are those constructs which comprise a readily detectable or assayable gene ( such as luciferase , β - galactosidase , chloramphenicol acetyltransferase ) linked in cis to a tcf - responsive element . such responsive elements are known in the art ( 7 ) and any such elements can be used . an optimal tcf motif contains the sequence cctttgatc . from one to twenty copies , and preferably from three to six copies , of the motif may be used . mutation of the sequence to cctttggcc abrogates responsiveness . another necessary part of such constructs is a minimal promoter , such as the c - fos or the herpes virus thymidine kinase promoter . transcription of the reporter gene may be performed by any means known in the art , usually by assaying for the activity of the encoded gene , although immunological detection methods can also be used . in addition , transcription can be monitored by measuring the transcribed mrna directly , typically using oligonucleotide probes . as shown below , a cell which has a wild - type apc protein will inhibit crt . however , most mutations in apc render apc unable to inhibit crt . similarly , certain mutations in ctnnb1 render β - catenin super - active and / or refractory to the inhibition by apc . thus measuring tcf - responsive reporter gene transcription is an indication of the status of apc and ctnnb1 . mutations in both of these genes are associated with cancers and therefore provides diagnostic and prognostic information . assays for crt can be accomplished in vitro or in cells . if the assay is to be accomplished in cells , then a tcf - responsive reporter gene must be introduced into the cell . any means for introducing genetic material into cells can be used , including but not limited to infection , transfection , electroporation . if the assay is to be performed in vitro then the components for transcription must be present . these include suitable buffers , rna polymerase , as well as ribonucleotides . if the protein product is to be assayed , then the components for translation must also be present , such as ribosomes , and amino acids . these assays can also be used to screen compounds for potential as anti - cancer therapeutic agents . using either the in vitro or cell form of the assay , test compounds can be introduced to determine whether they are able to mimic the effect of wild - type apc or to convert a mutant apc into a form which is able to inhibit crt or a mutant β - catenin into a form which is regulatable by apc . in addition , compounds can be tested for the ability to inhibit the binding of β - catenin and tcf - 4 , thus mimicking the action of apc . such a test can be conducted in vitro or in vivo , for example using a two hybrid assay . a means for diagnosis of cancers is the result of the observation that ctnnb1 mutations are found in tumor cells , especially those which have wild - type apc . such mutations can be found , inter alia , by sequencing either the gene or the protein found in a sample . functional assays can also be used , such as whether β - catenin binds to apc or tcf - 4 , or whether it is capable of mediating crt . sequences can be compared to those found in a normal tissue of a human , especially the same human who provided the sample being tested . suitable tumors for testing include , but are not limited to those which are associated with fap . suitable tumors include colorectal cancer , thyroid cancer , brain cancer , medulloblastoma , desmoid tumor , osteoma , breast cancer , and head and neck cancer . because apc mutations are so frequent , and because it appears that apc mutations do not occur in the same tumors as ctnnb1 mutations , one can prescreen samples for apc mutations before performing a ctnnb1 determination . the portion of the apc gene which encodes the β - catenin binding site can be used in a gene therapy format . suitable techniques are known in the art for administering genes to tumors , and any such technique can be used . suitable expression vectors are also known in the art and it is within the skill of the artisan to select an appropriate one . upon expression in a tumor cell of the β - catenin binding portion of apc , β - catenin will be bound and titrated away from binding to tcf - 4 , thus preventing unregulated expression of the crt target genes . similarly , a polypeptide portion of apc containing the β - catenin binding site can be administered to cells to perform a titration of β - catenin . techniques for such administration to cells is well known in the art . cells which are treated with either the polynucleotide or the polypeptide can be used to study the interaction between apc and β - catenin , and for developing drugs which interfere with such binding . the above disclosure generally describes the present invention . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . this example identifies tcf - 4 as the expressed family member in colorectal epithelial cells and provides the complete sequence of the cloned cdna . there are four known members of the tcf / lef family in mammals : the lymphoid - specific factors tcf - i and lef - 1 ( 7 , 8 ), and the less well characterized tcf - 3 and 4 ( 9 ). we performed a qualitative reverse transcriptase - polymerase chain reaction assay for expression of the four tcf / lef genes on 43 colon tumor cell lines . while most colon cell lines expressed more than one of the genes , only htcf - 4 mrna was expressed in essentially all lines . we then screened a human fetal cdna library and retrieved clones encoding full - length htcf - 4 ( fig1 ). a genomic fragment encoding , the hmg box region of htcf - 4 ( 7 ) was used to probe a human 12 week - fetal cdna library in lambda gt - 11 . positive clones were subcloned into pbluescriptsk and sequenced . see seq id nos : 1 and 2 . the predicted sequence of htcf - 4 was most similar to that of htcf - 1 . alternative splicing yielded two cooh - termini that were conserved between htcf - i and htcf - 4 . the nh 2 - terminus , which in htcf - 1 , mlef - 1 and xenopus tcf - 3 mediates binding to β - catenin ( 6 ), was also conserved in htcf - 4 . northern blot analysis of selected colon carcinoma cell lines revealed high - level expression of htcf - 4 ( fig2 a ). northern blot hybridizations ( 7 ) were performed with full - length htcf - 1 , hlef - i and htcf - 4 cdna . colon epithelial cells were freshly prepared from a mucosal preparation dissected from a healthy surgical colon sample . the sample was minced , and incubated with 1 mm dithiothreitol ( dtt ) in hanks &# 39 ; medium to remove mucus . single - cell suspensions were prepared by incubation at rt in 0 . 75 mm edta in hanks &# 39 ; medium . epithelial cells were separated from lymphocytes by percoll gradient centrifugation . as evidenced by in situ hybridization ( fig2 b and c ) and northern blotting ( fig2 a ), htcf - 4 mrna was readily detectable in normal colonic epithelium , whereas htcf - i and hlef - i were not detectable . in situ hybridization of 6μ frozen sections of healthy colon biopsy samples were performed as described ( 10 ). htcf - 4 cdna encoding amino acids 200 to 310 was amplified and labeled with dig - 11 - dutp ( boehringer mannheim , germany ) by pcr . after hybridization and washing , the sections were sequentially incubated with mouse anti - dig antibody ( boehringer ) and a horseradish peroxidase conjugated rabbit antibody to mouse immunoglobulin ( dako , glostrup , denmark ). the signal was visualized with diaminobenzidine , which produces a reddish - brown precipitate . blue counterstining was performed with haematoxyline . this example demonstrates the interaction of tcf - 4 and β - catenin and their function as a transcriptional activating factor . to investigate whether htcf - 4 functionally interacts with β - catenin , we used two sets of reporter constructs in a β - catenin - tcf reporter gene assay ( 7 ). one contained three copies of the optimal tcf motif cctttgatc , or three copies of the mutant motif cctttggcc , upstream of a minimal c - fos promoter driven - luciferase expression ( ptopflash and pfopflash ). the second set contained three copies of the optimal motif , or three copies of the mutant motif , upstream of a minimal herpes virus thymidine kinase promoter driven - chloramphenicol acetyl transferase ( cat ) expression ( ptopcat and pfopcat , respectively ). reporter gene assays were performed as in ( 7 ). in brief , 2 × 10 6 cells were transfected with plasmids by electroporation . after 24 hours , cells were harvested and lysed in 1 mm dtt , 1 % triton x - 100 , 15 % glycerol , 25 mm tris ph 7 . 8 and 8 mm mgcl 2 . cdnas encoding myc - tagged versions of β - catenin and htcf - 4 were inserted into the mammalian expression vector pcdna ( invitrogen ). pcatcontrol , encoding the cat enzyme under the control of the sv40 promoter , was purchased from promega . epitope - tagged htcf - 4 and a deletion mutant lacking , the nh 2 - terminal 30 amino acids ( δnhtcf - 4 ) were cloned into the expression vector pcdna . transient transfections were performed in a murine b cell line ( iia1 . 6 ), that does not express any of the tcf genes ( 6 ). the topflash reporter was strongly transcribed upon cotransfection with the combination of β - catenin and htcf - 4 plasmids , but not with the individual plasmids or with the combination of β - catenin and δnhtcf - 4 plasmids . no enhanced transcription was detected in cells transfected with the negative control pfopflash ( fig3 a ). these results show that interaction of the nh 2 - terminus of htcf - 4 with β - catenin results in transcriptional activation . this example demonstrates the functional regulation of crt transcriptional activation by wild - type apc . in three apc -/- carcinoma cell lines , sw480 , sw620 and dld - 1 ( fig3 b ), the ptopflash reporter was 5 - 20 fold more actively transcribed than pfopflash . importantly , transfection of sw480 cells with the reporter gene and an apc - expression vector abrogated the transcriptional activity in a dose - dependent manner ( fig3 b ). in contrast apc had no effect on a cotransfected internal control ( pcatcontrol ), or on the basal transcription of pfopflash ( fig3 b ). the use of ptopcat and pfopcat instead of ptopflash and pfopflash led to comparable observations . the constitutive transcriptional activity of tcf reporter genes in apc -/- colon carcinoma cells was in stark contrast to the inactivity of these genes in non - colonic cell lines , including iia1 . 6 b cells ( fig3 a ), the c57mg breast carcinoma cell line ; the jurkat and bw5147 t cell lines ; the daudi and ns1 b cell lines ; the k562 erythromyeloid cell line ; the hela cervical carcinoma line ; the hepg2 hepatoma cell line ; 3t3 , 3t6 , and rat - i fibroblasts ; and the kidney derived sv40 - transformed cos cell line ( 7 , 16 ). this example demonstrates that a functional β - catenin - htcf - 4 complex exists constitutively in apc -/- cells . we used ht29 - apc -/- colon carcinoma cells ( 12 ), in which apc is controlled by a metallothionein promoter . induction by zn ++ restores wild - type levels of apc , and leads to apoptosis ( 12 ). ht29 - gal cells which carry a zn ++ - inducible lacz gene were used as a control . the only tcf family member expressed in ht29 is htcf - 4 ( fig2 c ). in nuclear extracts from uninduced ht29 derived transfectants , we readily detected htcf - 4 by gel retardation ( fig4 ). an additional band of slightly slower mobility was also observed . the addition of a β - catenin antibody resulted in the specific retardation of the latter band , indicating that it represented a β - catenin - htcf - 4 complex ( fig4 ) ( 12 ). after zn ++ induction for 20 hours , the β - catenin - htcf - 4 complex was diminished sixfold relative to uncomplexed htcf - 4 in ht29 - apc1 , while no significant change was observed in ht29 - gal cells ( fig4 ). importantly , the overall levels of cellular β - catenin do not change during the induction period in ht29 - apc1 cells ( 12 ). gel retardation assays were performed as described elsewhere ( 7 ). extracts were prepared from intact nuclei that were washed four times to avoid contamination with cytoplasmic β - catenin . as the optimal tcf / lef probe , we used a double - stranded 15 - mer ccctttgatcttacc ( seq id no : 3 ); the control probe was ccctttggccttacc ( seq id no : 4 ). ( all oligonucleotides were from isogene , holland ). the β - catenin antibody was purchased from transduction laboratories lexington , ky .). a typical binding reaction contained 3 μg nuclear protein , 0 . 1 ng radiolabeled probe , 100 ng of didc , in 25 μl of binding buffer ( 60 mm kcl , 1 mm edta , 1 mm dtt , 10 % glycerol ). samples were incubated for 20 min at room temperature , antibody was added , and the samples incubated 20 min further . on the basis of these data , we propose the following model . in normal colonic epithelium htcf - 4 is the only expressed member of the tcf family . the interaction of β - catenin with htcf - 4 is regulated by apc . when appropriate extracellular signals are delivered to an epithelial cell , β - catenin accumulates in a form that is not complexed with gsk3β - apc , and that enables its nuclear transport and association with htcf - 4 . the hmg domain of htcf - 4 binds in a sequence - specific fashion to the regulatory sequences of specific target genes ; β - catenin supplies a transactivation domain . thus , transcriptional activation of target genes occurs only when htcf - 4 is associated with β - catenin . the htcf - 4 target genes remain to be identified . however , the link with apc and catenin suggests that these genes may participate in the generation and turnover of epithelial cells . upon loss of wild - type apc , monomeric β - catenin accumulates in the absence of extracellular stimuli , leading to uncontrolled transcription of the htcf - 4 target genes . the apparent de novo expression of other members of the tcf family in some colon carcinoma cell lines might lead to a further deregulation of tcf target gene expression by the same mechanism . the control of β - catenin - tcf signaling is likely to be an important part of the gatekeeper function of apc ( 19 ), and its disruption an early step in malignant transformation . this example demonstrates that mutant apc protein does not regulate crt and that a complete set of 20 - aa repeats in apc is required to mediate inhibition of crt . we tested four apc mutants ( fig5 a ) for their ability to inhibit β - catenin / tcf - regulated transcription ( crt ) in transfection assays . the first mutant , apc331δ represents a type of mutation found in the germline of familial adenomatous polyposis ( fap ) patients as well as in sporadic tumors ( 15 ). the apc331δ protein is truncated at codon 331 , amino - terminal to the three 15 - amino - acid ( aa ) β - catenin - binding repeats between codons 1020 and 1169 . the second mutant , apc1309δ , is the most common germline apc mutation ( 15 ), a 5 - bp deletion that produces a frameshift at codon 1309 and truncation of the protein . the apc1309δ protein retains the 15 - aa β - catenin repeats but lacks the seven 20 - aa repeats between codons 1323 and 2075 that have been implicated in binding and phosphorylation of β - catenin ( 18 ). the third mutant , apc1941δ , represents one of the most distal somatic mutations observed in colorectal tumors ( 25 ). the apc1941δ protein is truncated at codon 1941 and therefore contains the 15 - aa repeats and all but the last two 20 - aa repeats . finally , apc2644δ represents a germline mutation resulting from a 4 - bp deletion in codon 2644 . patients with this type of unusual carboxyl - terminal mutation develop few polyps ( attenuated polyposis ) but have pronounced extracolonic disease , particularly desmoid tumors ( 26 ). each of the apc mutants was cotransfected with a crt reporter into the sw480 colorectal cancer cell line . sw480 cells have truncated apc and constitutively active crt which can be suppressed by exogenous wt apc . although all four mutants produced comparable levels of apc protein after transfection , they varied in their crt inhibitory activity . the three mutants found in patients with typical polyposis or cancer were markedly deficient in inhibition of crt ( fig5 b ). the reduced activity of apc1309δ and apc1941δ suggests that β - catenin binding is not sufficient for apc - mediated inhibition of crt and that the complete set of 20 - aa repeats is required . interestingly , the inhibitory activity of the apc2644δ mutant associated with attenuated polyposis was comparable to that of wt apc ( fig5 b ), suggesting that the dlg - binding domain at the carboxyl - terminus of apc is not required for down - regulation of crt . wt and mutant apc constructs ( 2 μg ) were transfected into 293 , sw480 , and hct116 cells using lipofectamine ( gibco / brl , gaithersburg ). protein was harvested 24 hours later and subjected to immunoblot analysis with apc monoclonal antibody fe9 ( 23 ). in hct116 and 293 cells , exogenous wt apc comigrated with the endogenous apc . in sw480 cells , apc1309δ comigrated with the endogenous mutant apc . in all other cases , the nonfunctional apc constructs ( apc331δ , apc 1309δ , and apc1941δ ) produced as much or more protein than the crt - functional forms of apc ( apc wt and apc 2644δ ). this example demonstrates that other components of the apc - regulatory pathway are affected in some cancer cells . we evaluated crt in two colorectal tumor cell lines ( hct116 and sw48 ) that express full - length apc ( fig6 a ). both hct116 and sw48 displayed constitutively active crt and , in contrast to cell lines with truncated apc ( dld1 and sw480 ), this activity was not inhibited by exogenous wt apc ( fig5 b , 6b ). other ( noncolorectal cancer ) cell lines expressing wt apc do not display constitutive crt activity . these transfection results suggested that the constitutive crt in hct116 and sw48 might be due to an altered downstream component of the apc tumor suppressor pathway . this example demonstrates a defect in the gene encoding β - catenin in some cancer cells , which affects crt . we evaluated the status of a likely candidate for a downstream component of the apc tumor suppressor pathway , β - catenin , in the same four lines . all four lines expressed similar amounts of apparently intact β - catenin , as assessed by immunoblots ( fig7 a ). however , sequence analysis revealed that both hct116 and sw48 harbored mutations in the β - catenin gene ( ctnnb1 ) ( fig7 b ). hct116 had a 3 - bp deletion that removed one aa ( ser - 45 ), and sw48 had a c to a missense mutation that changed ser - 33 to tyr . analysis of paraffin - embedded archival tissue from the hct116 patient confirmed the somatic nature of this mutation and its presence in the primary tumor prior to culture . interestingly , both mutations affected serines that have been implicated in the downregulation of β - catenin through phosphorylation by the zw3 / gsk3β kinase in xenopus embryos ( fig7 c ) ( 27 , 28 ). genomic dna was isolated from paraffin - embedded normal and tumor tissue from the patient from whom the hct116 cell line was derived . a 95 bp pcr product encompassing the mutation was then amplified by pcr and directly sequenced using thermosequenase ( amersham ). the 3 bp deletion was observed in tumor but not in normal tissue . to test the generality of this mutational mechanism , we evaluated five primary colorectal cancers in which sequencing of the entire coding region of apc revealed no mutations ( 25 ). three of these five tumors were found to contain ctnnb1 mutations ( s45f , s45f , and t44a ) that altered potential zw3 / gsk3β phosphorylation sites ( fig7 c ). each mutation appeared to affect only one of the two ctnnb1 alleles and to be somatic . genomic dna was isolated from frozen - sectioned colorectal cancers and a 1001 bp pcr product containing exon 3 of ctnnb1 was then amplified by pcr and directly sequenced using thermosequenase ( amersham ). an acc to gcc change at codon 41 ( t41a ) and a tct to ttt at codon 45 ( s45f ) was observed in one and two tumors , respectively . this example demonstrates dominant mutations of ctnnb1 that render crt insensitive to the effects of wt apc . because the β - catenin mutations were heterozygous , we hypothesized that the mutations might exert a dominant effect , rendering a fraction of cellular β - catenin insensitive to apc - mediated down regulation . to test this notion , we performed gel shift analyses with nuclear extracts from untransfected hct116 cells . in contrast to noncolorectal cancer cell lines with intact apc , hct116 cells contained a β - catenin / tcf complex that gel - shifted an optimized tcf - binding oligonucleotide , and this complex supershifted with anti - p - catenin ( fig8 a ). we also constructed β - catenin expression vectors and compared the biologic activity of the mutant β - catenin from hct116 ( β - cat δ45 ) and sw48 ( β - cat s33y ) with that of their wt counterpart . for these experiments , we used the 293 kidney epithelial cell line as it is highly transfectable , exhibits low endogenous crt , and contains a high level of endogenous apc ( fig6 a ). in the presence of endogenous apc , both mutant β - catenins were at least 6 - fold more active than the wt protein and this activity was inhibited by dominant - negative htcf - 4 ( fig8 b ). together , these results indicate that disruption of apc - mediated regulation of crt is critical for colorectal tumorigenesis . this is most commonly achieved by recessive inactivating mutations of both apc alleles but , as shown here , can also be achieved by dominant mutations of ctnnb1 that render crt insensitive to the effects of wt apc . our results suggest that apc inhibition of crt requires phosphorylation of β - catenin at multiple sites . these potential phosphorylation sites are consistent with the known specificity of zw3 / β sk3p ( 29 ) a serine kinase that negatively regulates β - catenin in xenopus and drosophila cells ( 27 ) and that interacts with apc and β - catenin in mammalian cells ( 23 ). these results also suggest a functional basis for the occasional ctnnb1 mutations observed in other tumor types ( 30 ) and illustrate how a critical pathway in human disease can be illuminated by the discovery of mutations in different components of the pathway . the next step in understanding apc function will be the identification of the genes that are activated by htcf - 4 / β - catenin complexes and inhibited by wt apc . these genes are likely to be related to apc &# 39 ; s ability to induce apoptosis in colorectal cancer cells ( 31 ). 1 . b . rubinfeld et al science , 262 , 1731 ( 1993 ); l . k . su , b . vogelstein , k . w . kinzler , ibid 262 , 1734 ( 1993 ). 4 . j . papkoff , b . rubinfeld , b . schryver , p . polakis , mol . cell . biol 16 , 2128 ( 1996 ). 5 . s . munemitsa , b . souza , i . albert , b . rubinfeld , p . polakis , proc . natl . acad sci . u . s . a . 92 , 3046 ( 1995 ); b . rubinfeld , b . souza , i . albert , s . muneinitsa , p . polakis , j biol chem . 270 , 5549 ( 1995 ). 6 . m . molenaar et al , cell 86 , 396 ( 1996 ); j . behrens et al , nature 382 , 638 ( 1996 ); o . huber et al ., mech . dev . 59 , 3 ( 1996 ). 7 . m 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contain apc mutations ; y . miyoshi et al ., hum mol genet 1 , 229 - 33 ( 1992 ); j . jen et al ., cancer res . 54 , 5523 ( 1994 ). 15 . h . nagase and y . nakamura , hum . mutation 2 , 425 ( 1993 ). 16 . k . w . kinzler and b . vogelstein , cell 87 , 159 ( 1996 ); s . m . prescott and r . l . white , ibid ., p . 783 . 17 . g . joslyn , d . s . richardson , r . white , t . alber , proc . natl . acad . sci . u . s . a . 90 , 11109 ( 1993 ); l . k . su et al ., cancer res . 53 , 2728 ( 1993 ). 18 . b . rubinfeld et al ., science 262 , 1731 ( 1993 ); l . k . su , b . vogelstein , k . w . kinzler , ibid ., p . 1734 . 19 . j . hulsken , j . behrens , w . birchmeier , curr . opin . cell . biol . 6 , 711 ( 1994 ); b . rubinfeld , b . souza , i . albert , s . munemitsu , p . polakis , j . biol . chem . 270 , 5549 ( 1995 ). 20 . s . munemitsu et al ., cancer res . 54 , 3676 ( 1994 ); k . j . smith et al ., ibid . p . 3672 . 21 . l . k . su et al ., cancer res . 55 , 2972 ( 1995 ). 24 . m . molenaar et al ., cell 86 , 391 ( 1996 ); j . behrens et al ., nature 382 , 638 ( 1996 ). 26 . d . m . eccles et al ., am . j . of hum . genet . 59 , 1193 ( 1996 ); w . friedl et al ., hum genet 97 , 579 ( 1996 ); r . j . scott et al ., human molecular genetics 5 , 1921 ( 1996 ). 28 . s . munemitsu , i . albert , b . rubinfeld , p . polakis , mol cell biol 16 , 4088 ( 1996 ). 29 . m . peifer , l . m . pai , m . casey , dev . biol . 166 , 543 ( 1994 ). 30 . d . j . kawanishi , et al ., mol . cell biol . 15 , 1175 ( 1995 ); p . f . robbins , et al ., j . exp . med . 183 , 1185 ( 1996 ). 31 . p . j . morin , b . vogelstein , k . w . kinzler , proc . natl . acad . sci . u . s . a . 93 , 7950 ( 1996 ). 32 . j . groden et al ., cell 66 , 589 ( 1991 ); g . joslyn et al ., ibid ., p . 601 ; k . w . kinzler et al , science 253 , 661 ( 1991 ); i . nishisho et al ., ibid ., p . 665 . 33 . m . peifer , s . berg , a . b . reynolds , cell 76 , 789 ( 1994 ). 34 . k . j . smith et al , proc . natl . acad . sci . u . s . a . 90 , 2846 ( 1993 ). 35 . s . e . goelz , s . r . hamilton , b . vogelstein , biochem . biophys . res . commun . 130 , 118 ( 1985 ) __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 11 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 2040 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 1 : 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( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 2444 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 2 : ggttttttttttttaccccccttttttatttattatttttttgcacattgagcggatcct60tgggaacgagagaaaaaagaaacccaaactcacgcgtgcagaagatctccccccccttcc120cctcccctcctccctcttttcccctccccaggagaaaaagacccccaagcagaaaaaagt180tcaccttggactcgtctttttcttgcaatattttttgggggggcaaaactttgagggggt240gattttttttggcttttcttcctccttcatttttcttccaaaattgctgctggtgggtga300aaaaaaaatgccgcagctgaacggcggtggaggggatgacctaggcgccaacgacgaact360gatttccttcaaagacgagggcgaacaggaggagaagagctccgaaaactcctcggcaga420gagggatttagctgatgtcaaatcgtctctagtcaatgaatcagaaacgaatcaaaacag480ctcctccgattccgaggcggaaagacggcctccgcctcgctccgaaagtttccgagacaa540atcccgggaaagtttggaagaagcggccaagaggcaagatggagggctctttaaggggcc600accgtatcccggctaccccttcatcatgatccccgacctgacgagcccctacctccccaa660gcgatccgtctcgcccaccgcccgaacctatctccagatgaaatggccactgcttgatgt720ccaggcagggagcctccagagtagacaagccctcaaggatgcccggtccccatcaccggc780acacattgtctctaacaaagtgccagtggtgcagcaccctcaccatgtccaccccctcac840gcctcttatcacgtacagcaatgaacacttcacgccgggaaacccacctccacacttacc900agccgacgtagaccccaaaacaggaatcccacggcctccgcaccctccagatatatcccc960gtattacccactatcgcctggcaccgtaggacaaatcccccatccgctaggatggttagt1020accacagcaaggtcaaccagtgtacccaatcacgacaggaggattcagacacccctaccc1080cacagctctgaccgtcaatgcttccgtgtccaggttccctccccatatggtcccaccaca1140tcatacgctacacacgacgggcattccgcatccggccatagtcacaccaacagtcaaaca1200ggaatcgtcccagagtgatgtcggctcactccatagttcaaagcatcaggactccaaaaa1260ggaagaagaaaagaagaagccccacataaagaaacctcttaatgcattcatgttgtatat1320gaaggaaatgagagcaaaggtcgtagctgagtgcacgttgaaagaaagcgcggccatcaa1380ccagatccttgggcggaggtggcatgcactgtccagagaagagcaagcgaaatactacga1440gctggcccggaaggagcgacagcttcatatgcaactgtaccccggctggtccgcgcggga1500taactatggaaagaagaagaagaggaaaagggacaagcagccgggagagaccaatgaaca1560cagcgaatgtttcctaaatccttgcctttcacttcctccgattacagacctcagcgctcc1620taagaaatgccgagcgcgctttggccttgatcaacagaataactggtgcggcccttgcag1680gagaaaaaaaaagtgcgttcgctacatacaaggtgaaggcagctgcctcagcccaccctc1740ttcagatggaagcttactagattcgcctcccccctccccgaacctgctaggctcccctcc1800ccgagacgccaagtcacagactgagcagacccagcctctgtcgctgtccctgaagcccga1860ccccctggcccacctgtccatgatgcctccgccacccgccctcctgctcgctgaggccac1920ccacaaggcctccgccctctgtcccaacggggccctggacctgcccccagccgctttgca1980gcctgccgccccctcctcatcaattgcacagccgtcgacttcttggttacattcccacag2040ctccctggccgggacccagccccagccgctgtcgctcgtcaccaagtctttagaatagct2100ttagcgtcgtgaaccccgctgctttgtttatggttttgtttcacttttcttaatttgccc2160cccacccccaccttgaaaggttttgttttgtactctcttaattttgtgccatgtggctac2220attagttgatgtttatcgagttcattggtcaatatttgacccattcttatttcaatttct2280ccttttaaatatgtagatgagagaagaacctcatgattggtaccaaaatttttatcaaca2340gctgtttaaagtctttgtagcgtttaaaaaatatatatatatacataactgttatgtagt2400tcggatagcttagttttaaaagactgattaaaaaacaaaaaaaa2444 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 15 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( xi ) sequence description : seq id no : 3 : ccctttgatcttacc15 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 15 base pairs ( b ) type : nucleic acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : cdna ( xi ) sequence description : seq id no : 4 : ccctttggccttacc15 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 442 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : none ( xi ) sequence description : seq id no : 5 : 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( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 596 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : none ( xi ) sequence description : seq id no : 6 : 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( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 2973 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : none ( xi ) sequence description : seq id no : 7 : 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( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 486 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : none ( xi ) sequence description : seq id no : 8 : 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( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 511 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : none ( xi ) sequence description : seq id no : 9 : 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( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 20 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : none ( xi ) sequence description : seq id no : 10 : sertyrleuaspserglyilehisserglyalathrthrthralapro151015serleusergly20 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 21 amino acids ( b ) type : amino acid ( c ) strandedness : single ( d ) topology : linear ( ii ) molecule type : none ( xi ) sequence description : seq id no : 11 : sertyrleuglyaspserglyilehisserglyalavalthrglnval151015proserleusergly20__________________________________________________________________________