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GGA·TCC-interrupted Triplets in Long GAA·TTC Repeats Inhibit the Formation of Triplex and Sticky DNA Structures, Alleviate Transcription Inhibition, and Reduce Genetic Instabilities

2001; Elsevier BV; Volume: 276; Issue: 29 Linguagem: Inglês

10.1074/jbc.m101852200

1083-351X

Naoaki Sakamoto, Jacquelynn E. Larson, Ravi R. Iyer, Laura Montermini, Massimo Pandolfo, Robert D. Wells,

DNA Repair Mechanisms

Large expansions of GAA·TTC repeats in the first intron of the frataxin (X25) gene are the principal mutation responsible for Friedreich's ataxia (FRDA). Sticky DNA, based on R·R·Y triplexes, was found at the expanded GAA·TTC repeats from FRDA patients. The (GAAGGA·TCCTTC)65 repeat occurs in the same frataxin locus but is nonpathogenic and does not form sticky DNA. To elucidate the behavior of sticky DNA, we introduced various extents of GGA·TCC interruptions into the long GAA·TTC repeat. More than 20% of GGA·TCC interruptions abolished the formation of sticky DNA. However, the GAA·TTC repeats with less than 11% of GGA·TCC interruptions formed triplexes and/or sticky DNA similar to the uninterrupted repeat sequence. These triplexes showed different P1 nuclease sensitivities, and the GGA·TCC interruptions were slightly more sensitive than the surrounding GAA·TTC repeats. Furthermore, genetic instability investigations in Escherichia coli revealed that a small number (4%) of interruptions substantially stabilized the long GAA·TTC tracts. Furthermore, the greater the extent of interruptions of the GAA·TTC repeats, the less inhibition of in vitrotranscription was observed, as expected, based on the capacity of interruptions to inhibit the formation of sticky DNA. We propose that the interruptions introduce base mismatches into the R·R·Y triplex, which explains the observed chemical and biological properties. Large expansions of GAA·TTC repeats in the first intron of the frataxin (X25) gene are the principal mutation responsible for Friedreich's ataxia (FRDA). Sticky DNA, based on R·R·Y triplexes, was found at the expanded GAA·TTC repeats from FRDA patients. The (GAAGGA·TCCTTC)65 repeat occurs in the same frataxin locus but is nonpathogenic and does not form sticky DNA. To elucidate the behavior of sticky DNA, we introduced various extents of GGA·TCC interruptions into the long GAA·TTC repeat. More than 20% of GGA·TCC interruptions abolished the formation of sticky DNA. However, the GAA·TTC repeats with less than 11% of GGA·TCC interruptions formed triplexes and/or sticky DNA similar to the uninterrupted repeat sequence. These triplexes showed different P1 nuclease sensitivities, and the GGA·TCC interruptions were slightly more sensitive than the surrounding GAA·TTC repeats. Furthermore, genetic instability investigations in Escherichia coli revealed that a small number (4%) of interruptions substantially stabilized the long GAA·TTC tracts. Furthermore, the greater the extent of interruptions of the GAA·TTC repeats, the less inhibition of in vitrotranscription was observed, as expected, based on the capacity of interruptions to inhibit the formation of sticky DNA. We propose that the interruptions introduce base mismatches into the R·R·Y triplex, which explains the observed chemical and biological properties. Friedreich's ataxia dithiothreitol 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol base pairs kilobase pair(s) The clinical features as well as the molecular pathology of Friedreich's ataxia (FRDA)1are summarized in the accompanying paper (1Sakamoto N. Ohshima K. Montermini L. Pandolfo M. Wells R.D. J. Biol. Chem. 2001; 276: 27171-27177Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). The molecular mechanism of the biological effects of the long GAA·TTC repeat was proposed to be the formation of an unusual DNA structure (2Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 3Bidichandani S.I. Ashizawa T. Patel P.I. Am. J. Hum. Genet. 1998; 62: 111-121Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 4Ohshima K. Montermini L. Wells R.D. Pandolfo M. J. Biol. Chem. 1998; 273: 14588-14595Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). The GAA·TTC repeats have been known to form a triplex structure (5Wells R.D. Collier D.A. Hanvey J.C. Shimizu M. Wohlrab F. FASEB J. 1988; 2: 2939-2949Crossref PubMed Scopus (491) Google Scholar, 6Frank-Kamenetskii M.D. Mirkin S.M. Annu. Rev. Biochem. 1995; 64: 65-95Crossref PubMed Scopus (634) Google Scholar, 7Sinden R.R. DNA Structure and Function. Academic Press, Inc., San Diego1994Google Scholar, 8Soyfer V.N. Potaman V.N. Triple Helical Nucleic Acids. Springer-Verlag, New York1996Crossref Google Scholar, 9Hanvey J.C. Shimizu M. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 6292-6296Crossref PubMed Scopus (156) Google Scholar, 10Shimizu M. Hanvey J.C. Wells R.D. J. Biol. Chem. 1989; 264: 5944-5949Abstract Full Text PDF PubMed Google Scholar, 11Shimizu M. Hanvey J.C. Wells R.D. Biochemistry. 1990; 29: 4704-4713Crossref PubMed Scopus (33) Google Scholar). Recently, the sticky DNA structure was found specifically in the long GAA·TTC repeats from FRDA patients (12Sakamoto N. Chastain P.D. Parniewski P. Ohshima K. Pandolfo M. Griffith J.D. Wells R.D. Mol. Cell. 1999; 3: 465-475Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). This structure was based on the R·R·Y type of triplex and was hypothesized to be formed by exchanging the pyrimidine strands between two R·R·Y triplexes (12Sakamoto N. Chastain P.D. Parniewski P. Ohshima K. Pandolfo M. Griffith J.D. Wells R.D. Mol. Cell. 1999; 3: 465-475Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). The GAA·TTC repeat lengths required for the formation of sticky DNA correlated well with that required for the disease phenotype (12Sakamoto N. Chastain P.D. Parniewski P. Ohshima K. Pandolfo M. Griffith J.D. Wells R.D. Mol. Cell. 1999; 3: 465-475Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Furthermore, we found that sticky DNA inhibited T7 and SP6 RNA polymerase transcription effectively by sequestering RNA polymerases (1Sakamoto N. Ohshima K. Montermini L. Pandolfo M. Wells R.D. J. Biol. Chem. 2001; 276: 27171-27177Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar). Therefore, sticky DNA may be involved in the pathology of FRDA. On the other hand, the GAAGGA·TCCTTC repeat (65 units in length) was also found in the first intron of the frataxin gene but was demonstrated to be nonpathogenic (13Ohshima K. Sakamoto N. Labuda M. Poirier J. Moseley M.L. Montermini L. Ranum L.P.W. Wells R.D. Pandolfo M. Neurology. 1999; 53: 1854-1857Crossref PubMed Google Scholar). Unlike the GAA·TTC repeat, this hexamer repeat does not form a triplex and/or sticky DNA, does not inhibit transcription, and does not associate with the FRDA disease state (12Sakamoto N. Chastain P.D. Parniewski P. Ohshima K. Pandolfo M. Griffith J.D. Wells R.D. Mol. Cell. 1999; 3: 465-475Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 13Ohshima K. Sakamoto N. Labuda M. Poirier J. Moseley M.L. Montermini L. Ranum L.P.W. Wells R.D. Pandolfo M. Neurology. 1999; 53: 1854-1857Crossref PubMed Google Scholar). This strongly suggests that triplexes and/or sticky DNA may be involved in the pathology of FRDA. However, the reason why the hexamer repeat did not form a triplex or sticky DNA was unclear. In normal individuals with moderate lengths of GAA·TTC repeats, short GAGGAA·TTCCTC repeat interruptions were found (14Epplen C. Epplen J.T. Frank G. Miterski B. Santos E.J. Schols L. Hum. Genet. 1997; 99: 834-836Crossref PubMed Scopus (113) Google Scholar, 15Cossee M. Schmitt M. Campuzano V. Reutenauer L. Moutou C. Mandel J.L. Koenig M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7452-7457Crossref PubMed Scopus (269) Google Scholar). This repeat appeared to also be nonpathogenic, and thus it became evident that the length of the uninterrupted GAA·TTC repeat was important for the pathology of FRDA. Thus, interruptions in the long GAA·TTC repeats may influence the ability of the repeats to form sticky DNA and to inhibit transcription. These kinds of interruptions were shown to affect the genetic instabilities of other triplet repeat sequences (16Eichler E.E. Holden J.J. Popovich B.W. Reiss A.L. Snow K. Thibodeau S.N. Richards C.S. Ward P.A. Nelson D.L. Nat. Genet. 1994; 8: 88-94Crossref PubMed Scopus (412) Google Scholar, 17Nancarrow J.K. Holman K. Mangelsdorf M. Hori T. Denton M. Sutherland G.R. Richards R.I. Hum. Mol. Genet. 1995; 4: 367-372Crossref PubMed Scopus (34) Google Scholar, 18Parniewski P. Jaworski A. Wells R.D. Bowater R.P. J. Mol. Biol. 2000; 299: 865-874Crossref PubMed Scopus (66) Google Scholar). Furthermore, the GAAGGA·TCCTTC repeat was more stable genetically than the uninterrupted GAA·TTC repeat (13Ohshima K. Sakamoto N. Labuda M. Poirier J. Moseley M.L. Montermini L. Ranum L.P.W. Wells R.D. Pandolfo M. Neurology. 1999; 53: 1854-1857Crossref PubMed Google Scholar). Herein, we describe the preparation and characterization of a family of direct repeat sequences of composition intermediate between uninterrupted GAA·TTC and GAAGGA·TCCTTC repeats. Frequent GGA·TCC interruptions (more than 20%) in the long GAA·TTC repeat interfere with the formation of sticky DNA and triplexes, with the inhibitory effect on transcription of long GAA·TTC repeats, and with genetic instabilities. Mapping of P1 nuclease-cleaved sites revealed that the GAA·TTC repeats with less than 20% of GGA·TCC interruptions adopt R·R·Y triplexes similar to the pure GAA·TTC repeats but that the GGA·TCC interruptions may cause base mismatches in the triplexes. Hence, a high percentage of GGA·TCC interruptions in the GAA·TTC repeats inhibits the formation of sticky DNA by introducing base-mismatches into the triplexes, which makes them unstable. The GGA·TCC interruptions were introduced into the (GAA·TTC)150 repeat by site-directed mutagenesis (19Deng W.P. Nickoloff J.A. Anal. Biochem. 1992; 200: 81-88Crossref PubMed Scopus (1078) Google Scholar). pRW3546, which contains the (GAA·TTC)150 repeat in the pGEM-3Zf(−) vector (Promega), was constructed as described by Iyer and Wells (20Iyer R. Wells R.D. J. Biol. Chem. 1999; 274: 3865-3877Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The single-stranded circular pRW3546 containing the (TTC)150sequence was prepared using the pRW3546-transformed NM522Escherichia coli strain and M13K07 helper phage (21Sambrook J. Fritsch E.F. Maniatis Y. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To introduce GGA·TCC interruptions into the (GAA)150repeats, the following mutagenesis primers were synthesized by Genosys: GGA1/24, 5′-(GAA)12GGA(GAA)11-3′; GGA1/15, 5′-(GAA)7GGA(GAA)7-3′; GGA1/12, 5′-(GAA)6GGA(GAA)5-3′; GGA1/9, 5′-(GAA)4GGA(GAA)4-3′; GGA2/9, 5′-(GAA)2GGA(GAA)3GGA(GAA)2-3′. To eliminate the nonmutated original plasmid, elimination primer, 5′-GTGCCACCTGTCGACTAAGAAACC-3′, in which the unique AatII recognition site is mutated, was prepared. All of these primers were phosphorylated in 70 mm Tris-HCl (pH 7.6), 10 mm MgCl2, 5 mm DTT, 0.1 mm ATP, and 10 units of T4 polynucleotide kinase (New England Biolabs) at 37 °C. 25 pmol of one of the phosphorylated mutagenesis primers and 25 pmol of the phosphorylated elimination primer were annealed to the single-stranded pRW3546 in 20 mm Tris-HCl (pH 7.5), 10 mm MgCl2, and 50 mm NaCl in a 20-µl volume at 25 °C for 10 min. Then primer-directed DNA synthesis was performed by adding 3 µl of a solution containing 100 mm Tris-HCl (pH 7.5), 5 mm each of dNTPs, 10 mm ATP, 20 mmDTT, 5 µl of H2O, 1 µl (3 units) of T4 DNA polymerase (New England Biolabs), and 1 µl (400 units) of T4 DNA ligase (New England Biolabs) at 37 °C for 90 min. DNA was purified by phenol/chloroform extraction, precipitated by ethanol, and used for the transformation of the mismatch repair-defective (mutS)E. coli strain, XLmutS Kanr strain, Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 gyrA96 relA1 lac mutS::Tn10(Tetr) [F′ proAB lacI qZΔM15 Tn5(Kanr)] (Stratagene) by the CaCl2 method (21Sambrook J. Fritsch E.F. Maniatis Y. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The entire transformation mixture was grown in 10 ml of LB medium containing 20 µg/ml ampicillin, and the plasmid DNA was isolated by the orthodox alkaline-SDS method (21Sambrook J. Fritsch E.F. Maniatis Y. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The plasmids were digested byAatII to eliminate plasmids derived from the original pRW3546 single-stranded template, and then E. coli SURE strain was transformed by the AatII-digested plasmids. For the analyses of sticky DNA formation, theEcoRI–PstI fragments of the mutated plasmids were subcloned into the same sites in the pSPL3 vector (Life Technologies, Inc.) and transformed into E. coli SURE strain by the CaCl2 method (21Sambrook J. Fritsch E.F. Maniatis Y. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The transformants were cultured in 1 liter of LB medium at 37 °C to anA 600 of ∼0.5 (during logarithmic phase). Plasmids were isolated by the alkaline lysis method and purified by CsCl density gradient centrifugation (21Sambrook J. Fritsch E.F. Maniatis Y. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Three phenol/chloroform extractions were performed, followed by precipitation with ethanol and resuspension in 10 mm Tris-HCl (pH 8.0), 1 mm EDTA. For transcription studies, the EcoRI–XbaI fragments of the pSPL3-based plasmids were inserted into theEcoRI–NheI site of the pCR3.1 vector (Invitrogen) for the formation of the purine strand (interrupted or uninterrupted rGAA repeat) or into theEcoRI–XbaI site for the formation of the pyrimidine strand (interrupted or uninterrupted rUUC repeat). After the transformation of the E. coli SURE strain, the transformants were cultured in 1 liter of LB media. Plasmids were isolated by the alkaline lysis method and purified by the CsCl density gradient centrifugation method (21Sambrook J. Fritsch E.F. Maniatis Y. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar), followed by phenol/chloroform extraction, precipitation with ethanol, and resuspension in 10 mmTris-HCl (pH 8.0), 1 mm EDTA. All plasmids were characterized by restriction mapping and DNA sequencing on each complementary strand using the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing Kit (U.S. Biochemical Corp.). The pSPL3-based plasmids (1.5 µg) were linearized by XmnI (New England Biolabs) at 37 °C in NEBuffer 1 (10 mmBisTris propane-HCl, 10 mm MgCl2, 1 mm DTT (pH 7.0 at 25 °C)) supplemented with 100 µg/ml bovine serum albumin. The linearized plasmids were purified by phenol/chloroform extraction and ethanol precipitation and analyzed in a 0.7% agarose gel in 0.5× TBE buffer. The “weak” P1 nuclease digestion and analysis of P1 nuclease sensitivity on each DNA strand was performed as described (12Sakamoto N. Chastain P.D. Parniewski P. Ohshima K. Pandolfo M. Griffith J.D. Wells R.D. Mol. Cell. 1999; 3: 465-475Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar), except that P1 nuclease was purchased fromAmersham Pharmacia Biotech. The “strong” P1 nuclease digestion was performed as described by Hanvey et al. (22Hanvey J.C. Klysik J. Wells R.D. J. Biol. Chem. 1988; 263: 7386-7396Abstract Full Text PDF PubMed Google Scholar) with some modifications. Briefly, 1.5 µg of the supercoiled plasmids were treated with 10 units of P1 nuclease (Amersham Pharmacia Biotech) at 37 °C for 10 min in 10 mm Tris-HCl (pH 7.6), 10 mm MgCl2, and 50 mm NaCl. The reaction was terminated by phenol/chloroform extraction, and DNA was recovered by ethanol precipitation and analyzed by a 1.5% agarose gel electrophoresis in 0.5× TBE buffer. For high resolution mapping of P1 nuclease cleaved sites, 500 ng of P1 nuclease-cleaved plasmids were denatured in 200 mm NaOH, 0.2 mm EDTA in a volume of 40 µl at 65 °C for 10 min in the presence of one of the following primers: KO751 primer, 5′-ACCTGGCCAACATGGTGA-3′; KO753 primer, 5′-GTAGCTGGGATTACAGGC-3′. After neutralization by the addition of 13 µl of 3 m sodium acetate (pH 5.2), DNA was precipitated by ethanol. The DNA was dissolved in 10 µl of Sequenase buffer (40 mm Tris-HCl (pH 7.5), 20 mmMgCl2, 50 mm NaCl) and incubated at 42 °C for 10 min to anneal the primer to the denatured template. The primer extension reaction was started by the addition of 1 µl of 0.1m DTT, 2 µl of labeling mix (1.5 µm each of dGTP, dCTP, and dTTP), 1 µl of [α-32P]dATP (3000 Ci/mmol, 10 mCi/ml) (Amersham Pharmacia Biotech) and 2 µl of Sequenase version 2.0 (1.625 units/µl) (Amersham Pharmacia Biotech) and incubated at room temperature for 5 min. Then 14 µl of 80 µm dNTP mix were added and incubated at 37 °C for 5 min to complete the reaction. After purification of DNA, the extended products were analyzed in a 4% denaturing polyacrylamide gel in TBE buffer. As the marker, the sequencing reaction was performed according to the Sequenase version 2.0 (Amersham Pharmacia Biotech) sequencing protocol in parallel. The pCR3.1-based plasmids were linearized by XbaI to enable the run-off of transcription. The pCR3.1 vector was also linearized by XbaI as the internal control. 100 ng of the XbaI-linearized pCR3.1-based plasmid and 100 ng of the XbaI-linearized pCR3.1 vector were mixed and subjected to in vitro transcription using T7 RNA polymerase. Bacterial in vitro transcription forming the purine strand (uninterrupted or interrupted rGAA repeat) was performed in a 20-µl volume at 37 °C for 30 min in RNA polymerase buffer (40 mm Tris-HCl (pH 7.9), 6 mm MgCl2, 2 mm spermidine, 10 mm DTT) supplemented with 500 µm each of rATP, rGTP, and rUTP, 25 µmrCTP, 0.17 µm [α-32P]rCTP (3000 Ci/mmol, 10 mCi/ml) (Amersham Pharmacia Biotech), 40 units of RNasin (Promega), and 10 units of T7 RNA polymerase (New England Biolabs). For the formation of the pyrimidine strand (uninterrupted or interrupted rUUC repeat), 500 µm each of rATP, rUTP, and rCTP, 25 µm rGTP, and 0.17 µm[α-32P]rGTP (3000 Ci/mmol, 10 mCi/ml) (Amersham Pharmacia Biotech) were used. The RNA synthesized was purified by phenol/chloroform extraction, precipitated by ethanol, dissolved in formamide loading buffer (90% formamide, 20 mm EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol), and analyzed in a 4% polyacrylamide gel containing 7 m urea. The gels were exposed to x-ray film at room temperature after drying. Plasmids containing homogenous lengths of undeleted/unexpanded triplet repeat tracts were prepared as described previously (1Sakamoto N. Ohshima K. Montermini L. Pandolfo M. Wells R.D. J. Biol. Chem. 2001; 276: 27171-27177Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar) and transformed into E. coli AB1157 (thr-1, araC14, leuB6, Δ(gpt-proA)62, lacY1, tsx-33, qsr′-0, glnV44(AS), galK2(Oc), λ-, Rac-0, hisG4(Oc), rfbD1, mgl-51, rpoS396(Am), rpsL31(strR), kdgK51, xylA5, mtl-1, argE3(Oc), thi-1) by electroporation. The transformation mixture was plated on LB plates containing 100 µg/ml of kanamycin and incubated for 16 h at 37 °C. Liquid cultures were initiated from the colonies on the plates as follows. Washings from ∼100 single colonies were inoculated into 10 ml of L broth containing 10 µg/ml kanamycin. The culture was allowed to grow at 37 °C with shaking at 250 rpm. The growth of the culture was monitored periodically by measuring the absorbance at 600 nm. When the absorbance reached 1.0, an aliquot of the culture was inoculated into a fresh 10-ml L broth (with kanamycin as before) tube at a dilution of 10−6, resulting in a final cell concentration of 103 cells/ml. The fresh culture was incubated at 37 °C with shaking at 250 rpm until the absorbance was 1.0, a duration of ∼24 h. The original culture was centrifuged, and the plasmid DNA was isolated as per standard alkaline lysis procedures (2Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). The triplet repeat-containing fragment was excised by digestion withStuI and XmaI, labeled with [α-32P]dCTP, and analyzed by 6–8% native polyacrylamide gel electrophoresis. The dried gels were exposed to a PhosphorImager screen and scanned using a Molecular Dynamics PhosphorImager. The extents of the genetic instabilities of the repeat tracts were determined by measuring the signal intensity of the band corresponding to the full-length repeat tract as a percentage of the total signal in the lane from all triplet repeat-containing bands. For the analysis of the formation of sticky DNA, the amount of the retarded band (12Sakamoto N. Chastain P.D. Parniewski P. Ohshima K. Pandolfo M. Griffith J.D. Wells R.D. Mol. Cell. 1999; 3: 465-475Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar) was measured by a densitometric analysis of the negatives of the gel picture using the ImageQuant™ (Molecular Dynamics) on a Molecular Dynamics densitometer. The determination of the amount of the RNA synthesized in the transcription analysis was performed on the autoradiographs by ImageQuant™ (Molecular Dynamics) on a Molecular Dynamics PhosphorImager. Expanded GAA·TTC repeats from 22 Friedreich ataxia patients, including eight Acadians, were amplified from genomic DNA as described (13Ohshima K. Sakamoto N. Labuda M. Poirier J. Moseley M.L. Montermini L. Ranum L.P.W. Wells R.D. Pandolfo M. Neurology. 1999; 53: 1854-1857Crossref PubMed Google Scholar). The corresponding DNA fragments were separated on 1% agarose gels and purified on QIAGEN columns. To detect interruptions in GAA·TTC, the purified fragments were digested with the restriction enzymes EarI and MnlI, whose recognition sequences are GAAGAG and GAGG, respectively. In addition, the pyrimidine strand of 11 repeats was directly sequenced on a Licor automatic sequencer using a fluorescently labeled 2500R primer (28Campuzano V. Montermini L. Molto M.D. Pianese L. Cossee M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Canizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. Michele G.D. Filla A. Frutos R.D. Palau F. Patel P.I. Donato S.D. Mandel J.-L. Cocozza S. Koenig M. Pandolfo M. Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2250) Google Scholar) and the SequiTherm EXCEL™II sequencing kit (Epicentre Technologies). Readable sequence was obtained for up to 160 triplets.