Patent Application: US-34509203-A

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:
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 - 1573 ) is a human embryonic kidney fibroblast cell line . 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 . 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 . 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 ). 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 . 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 co ( 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 . 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 ). 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 fig4 and 5 ). 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 )). 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 : clone 164n16 was found to comprise these first three exons , as confirmed by southern blot analysis . 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 . these primers correspond to intronic sequences and are flanking an exon , as deduced from the sequence of a genomic clone ( genbank no aq163827 ) ( table 1 ; fig5 ). 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 . 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 . 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 . 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 ). 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 . 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 ). 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 ). 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 . 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 . 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 . 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 . 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 . 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 . 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 ). 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 ). 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 ). 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 . 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 . 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 ( 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 . 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 )). 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 )). 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 ). 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 .). 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 . 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 . 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 . 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 . 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 . 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 ). 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 . 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 .). 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 so 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 .). 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 ). 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 . 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 ( fig1 a ), 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 % ( fig1 b ). 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 ( fig2 ). 5 ′ race and rt - pcr experiments provided us with a full - length mouse αt - catenin cdna sequence of 2979 bp ( fig1 ; 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 ( fig1 a ). the overall identity of the mouse αt - catenin to the human αt - catenin protein ( fig1 b 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 . 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 ( fig3 a 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 ( fig3 c ). 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 fig4 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 ( fig4 and 5 ). 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 ( fig6 a 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 ( fig6 b ) and indeed demonstrated for the mef2c binding site ( fig2 ). 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 fig4 ) 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 . 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 ). 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 ( fig7 a ). 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 ( fig7 a ), suggesting that α - catenins bind to β - catenin with the following decreasing strength : αt - catenin & gt ; αn - catenin & gt ; αe - catenin . 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 ( fig7 b ). 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 ( fig7 c ), and also by coimmunoprecipitation from lysates of mouse heart and testis tissues ( fig7 d ). thus , the interaction between αt - catenin and β - catenin occurs also in vivo . the αt - catenin protein is preferentially expressed in heart and testis tissues 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 ) ( fig8 a ). in comparison to the ubiquitously expressed αe - catenin ( fig8 a ), 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 ( fig8 a ). these findings were confirmed by rt - pcr analysis ( fig8 b ) of several mouse tissues . the brain - specific expression of mouse αn - catenin mrna is in line with the literature ( hirano et al ., 1992 ). 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 ( fig8 c ). this confirms the observed tissue - specificity of the αt - catenin mrna ( fig8 b ) 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 ( fig1 b ). 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 ( fig2 ). also in western blot analysis , some bands with smaller apparent molecular weights ( of about 43 kda , 66 kda and 86 kda ) were detected in heart and testis lysates ( fig2 a ). immunodetection of all these bands is competed out by addition of the αt - catenin - specific immunogenic peptide ( fig2 b ). 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 . 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 ( fig9 a 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 ( fig1 a and 11b ). these results suggest that αt - catenin protein is expressed in specific contractile cells of heart and testis tissues . in double labeling experiments , it co - localizes with αe - catenin ( fig1 a ) as well as n - cadherin ( fig1 b ). the muscle marker desmin can be detected at both intercalated discs and sarcomeric z - lines , whereas αt - catenin expression is confined to intercalated discs ( fig1 c ). in human testis , αt - catenin protein was detected mainly in spindle - shaped cells surrounding testicular tubuli ( fig1 ). interestingly , αt - catenin did not co - localize here with αe - catenin , as the latter showed an abundant intratubular expression pattern ( fig1 a ). the αt - catenin expressing cells in testis correspond to desmin - positive cells , and therefore could be identified as peritubular myoid cells ( fig1 b ). these stainings strongly suggest that αt - catenin expression is confined to specific muscle cell types 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 ( fig1 a ). 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 ( fig1 b ). however , when αt - catenin was overexpressed for longer time periods , it tended to form cytoplasmic rod - like aggregates . 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 - 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