INDICADORES Y MÉTRICAS

Subcloning efficiency com petent E. coli (DH5a) w ere purchased from GibcoBRL (#18265-017) and transform ation followed instructions

provided by the m anufacturer. Briefly, a 50 [xl aliquot of com petent ceUs

was thaw ed on ice, mixed thoroughly w ith 2 pi of the ligation reaction and

left to incubate on ice for 30 m inutes. Cells were heat-shocked for 30

seconds at 37° C and then placed on ice for two minutes. After the heat

shock cells were grow n in a shaking incubator set at 225 rpm in 0.8 m l of

SOC m edia (GibcoBRL #15544-042) for 1 hour at 37° C. As a general rule,

0.2 ml of the transform ation was plated onto LB-agar plates (LB m edium

plus 20 g/1 bacto agar) containing 100 p g /m l Carbenicillin (SIGMA #C-

3416) or 50 p g /m l of Kanamycin (SIGMA #K-0254). Plates were incubated

overnight at 37° C to allow grow th of the transform ed cells. For DNA

purification, colonies were picked and grow n overnight in 2 mis of LB plus

II.1.7- DNA P lasm id constructs 11.1.7.1- Beta-catenin constructs

GFP fusions were m ade using GFP derived from pEGFP vectors

(Clontech, Palo Alto, CA). To make GFP-p-catenin, an EcoRI fragm ent

encoding Xenopus p-catenin w ith a carboxyl-terminal m yc-epitope tag (pMT23-pmyc) (Kypta et al., 1996) was cloned into the EcoRI site of pEGFP-

C l. GFP-p-catenin-AR (which lacks the arm adillo repeats) and GFP p-catenin-

AC were m ade in the same way using pMT23-pARmyc and pMT23-pACmyc,

respectively. GFP-p-catenin-AN and GFP-p-catenin-ANAC w ere m ade by

cloning the XhoI-EcoRI fragm ents from pMT23-pmyc and pMT23-pACmyc,

respectively into pEGFP-Cl that was cut w ith XhoI-EcoRI. Both plasmids

encode GFP fused to residue 151 of p-catenin. To m ake GFP-p-catenin-AN2,

pMT23-p-43-165 was first m ade by PGR. This encodes the amino acid

sequence MEVMN followed by residues 43-165 of p-catenin. The XhoI-EcoRI

fragm ent from pMT23-pmyc (containing residues 151-781) was then cloned

into pMT23-p-43-165 cut w ith Xhol, to m ake pMT23-AN2-pmyc. The EcoRI

fragm ent from pMT23-AN2-pmyc was then inserted into pEGFP-Cl resulting

in a GFP fusion containing the sequence MEVMN followed by residues 43-781

of p-catenin and a myc-epitope tag. To make GFP-p-catenm-Aa, pMT23-p-120

was first m ade by PGR. This encodes residues 1-120 of p-catenin w ith a 3' Xhol site. The Xhol fragm ent containing these residues was cloned into

pMT23-pANmyc to make pMT23-pAamyc, which lacks residues 122-150 of

Pmyc. This was subcloned into pEGFP-Gl to make GFP-p-catenin-Aa. Beta-

catenin-GFP encodes residues 1-761 w ith a carboxyl-terrninal GFP tag. It was

m ade by first inserting a BgUI fragm ent containing residues 7-761 of p-catenin

into the BamHI site of pEGFP-Nl. The missing aniino-term inal sequences of

365 w ith the Hindm fragm ent from wild-type p-catenin encoding residues 1- 365. To m ake GFP-p-1-420 (GFP-p-1/7), an EcoRI-PvuII fragm ent from

pMT23-pmyc was inserted into pEGFP-Cl cut w ith EcoRI-Smal. To make p-

398-761-GFP (p-8/ 12-GFP), p-catenin-GFP w as cut w ith H in d in and re-ligated

to rem ove the sequence encoding residues 1-365. The fusion protein begins at

the next m ethionine, residue 398.

11.1.7.2- APC, Tcfs, A xin and Reporter plasm ids

Myc-epitope tagged APC cDNA (Vleminckx et al., 1997) was kindly

provided by Dr. Barry Gumbiner, Memorial Sloan-Kettering Cancer Center,

NY. HA-tagged Tcf-4 cDNAs, p45 Tcf-1, p33 Tcf-1, pTOPFlash and pFOPFlash

reporters (Korinek at al., 1997) were gifts from Dr. Marc van de W etering and

Hans Clevers, U trecht University, The Netherlands. An EcoRI/Sail treated

HA-Tcf-4 was cloned into pEGFP-Nl treated w ith the same restriction

enzymes to create a GFP-Tcf-4 fusion protein. The Cyclin D l reporter plasm id

include the pA3 luciferase plasm id attached to the full length cyclin D l

prom oter (1745-luc) and was a gift from Dr. Richard PesteU, Albert Einstein

College of Medicine, Yeshiva University, N ew York. Axin DNA was a gift

from Dr. Akira Kikuchi, Hiroshima University, Japan. Axin was excised w ith

Smal/EcoRV and sub-cloned into the pECFP-Cl Smal site to create a CFP-

axin fusion protein, w ith the GFP tagged to the amino term inus of axin.

11.1.7.3- Cadherin, a-catenin, Vinculin and ADF/cofilin constructs

Truncated myc-epitope tagged Xenopus N-cadherin cDNAs (Riehl et al. 1996) were provided by Dr. Christine Holt, University of California San

Diego, CA, and cloned into pSC5 (Stratagene) for expression in COS-7 ceUs.

Plakoglobin-CFP was m ade by inserting the EcoRI/Xbal fragm ent from

pCS2mt-dC-XPKC-CFP (Rubenstein et al., 1997) (a gift from Dr. Mike

Xenopus plakoglobin lacking the first 39 residues (similar to the deletion m ade in p-catenin to produce GFPAN2-p-catenin) w ith GFP at the carboxyl

terminus. H um an aN -catenin cDNA (Claverie et al., 1993) was provided by

Drs. Jean-Pierre HardeHn and Christine Petit, Institute Pasteur, Paris. The

coding sequence was rem oved using Xbal-Nsil and cloned into pMT23 cut

w ith Spel-Pstl. Avian aN -catenin fused to GFP (Sehgal et al., 1997) was

provided by Drs. Ravinder Sehgal and Louis Reichardt, (University of

California, San Francisco). Plasmids encoding GST-a-caterdn constructs (full-

length, GST-a N-228 and GST-a C-447; Rimm et al., 1995) and respective

purified fusion proteins were generously provided by Dr. Vania Braga

(Imperial CoUege, London), w ith perm ission from Dr. David Rimm (Yale

University).

To m ake GFP-a-catenin-NLS (GFP-a-NLS), a double-stranded

oligonucleotide (MWG Biotech, Germany) encoding the nuclear localisation

signal from SV40 large T antigen (Fisher et al., 1988) and appropriate

restriction sites was ligated into GFP-aN-catenin cut w ith AfUI and H indm .

This resulted in the insertion of the sequence AARDPKKKRKV after residue

902 of avian aN-catenin, followed by a stop codon. GFP-aA-catenin-NLS

(GFP-aA-NLS) was m ade by deleting internal sequences in GFP-a-catenin-

NLS using Xhol, resulting in a fusion protein containing residues 1-214 of aN -

catenin followed by the nuclear localisation signal.

GFP-vinculin consists of full-length chicken vinculin cDNA inserted

into pGZ21 vector that contains a CMV prom oter and enhanced GFP

protein. This construct was a gift from Dr. Benjamin Geiger (The

W eizm ann Institute of Science, Rehovot, Israel).

XAC(A3)-GFP consists of Xenopus ADF (actin depolym erising factor) in w hich serine residue 3 has been m utated into an alanine. This

m utation prevents phosphorylation and consequent inactivation of ADF

by LIM kinase (Abe et al., 1993 and 1996) and w as a gift of Dr. James

Bamburg (University of Colorado).

II.8- Cloning o f a-catenin NES into Rev(lA)-G FP

Oligonucleotides were synthesised that encoded the amino acid

sequences of the two putative NES present in avian aN-catenin. The

oligonucleotides were 5' phosphorylated and had a 5" BamHI site and a 3'

Age I site to facilitate cloning into the Rev(1.4)-GFP vector. The

oligonucleotides were annealed by heating for 30 seconds at 95°C and then

cooling at 37^C for 15 m inutes. Annealing was checked by agarose

electrophoresis. Purified vector and oligonucleotides were ligated as

described above and the ligation used to transform com petent E.coli ll.S.l-O ligonucleotide sequences

aN-catenin-NESl

Amino acid sequence(126-143) AARA LLSA VTRLLILADM (NES in bold)

Oligonucleotide sequence:

5'-GATCCAGCTGCTCGTGCCTTGCTTTCTGCTGTTACCCGCTTGCTCATCCTGGCTGATATGA-3'

3'-GTCGACGAGCACGGAACGAAAGACGACAATGGGCGAACGAGTAGGACCGACTATACTGCC-5'_________

aN-catenin-NES2

Amino acid sequence(142-159) DMADVMRLLSHLKIVEEA (NES in bold)

Oligonucleotide sequeno

5'-GATCCAGATATGGCGGACGTCATGAGGCrTCTTTCACATCTGAAAATTGTAGAGGAAGCA-3' 3'-GTCTATACCGCCTGCAGTACrCCGAAGAAAGTGTAGACTTTTAACATCTCCTTCGTGGCC-5'

These oligonucleotides were annealed and ligated to the Rev(1.4)-GFP

to create Rev-a-NESl and Rev-a-NES2, respectively. The insertion was

confirmed by sequencing using specificpEGFP primers.