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• W2137770909 abstract "Friedreich ataxia (FRDA) is associated with the expansion of a GAA·TTC triplet repeat in the first intron of the frataxin gene, resulting in reduced levels of frataxin mRNA and protein. To investigate the mechanisms by which the intronic expansion produces its effect, GAA·TTC repeats of various lengths (9 to 270 triplets) were cloned in both orientations in the intron of a reporter gene. Plasmids containing these repeats were transiently transfected into COS-7 cells. A length- and orientation-dependent inhibition of reporter gene expression was observed. RNase protection and Northern blot analyses showed very low levels of mature mRNA when longer GAA repeats were transcribed, with no accumulation of primary transcript. Replication of plasmids carrying long GAA·TTC tracts (∼250 triplets) was greatly inhibited in COS-7 cells compared with plasmids carrying (GAA·TTC)9 and (GAA·TTC)90. Replication inhibition was five times greater for the plasmid whose transcript contains (GAA)230than for the plasmid whose transcript contains (UUC)270. Our in vivo investigation revealed that expanded GAA·TTC repeats from intron I of the FRDA gene inhibit transcription rather than post-transcriptional RNA processing and also interfere with replication. The molecular basis for these effects may be the formation of non-B DNA structures. Friedreich ataxia (FRDA) is associated with the expansion of a GAA·TTC triplet repeat in the first intron of the frataxin gene, resulting in reduced levels of frataxin mRNA and protein. To investigate the mechanisms by which the intronic expansion produces its effect, GAA·TTC repeats of various lengths (9 to 270 triplets) were cloned in both orientations in the intron of a reporter gene. Plasmids containing these repeats were transiently transfected into COS-7 cells. A length- and orientation-dependent inhibition of reporter gene expression was observed. RNase protection and Northern blot analyses showed very low levels of mature mRNA when longer GAA repeats were transcribed, with no accumulation of primary transcript. Replication of plasmids carrying long GAA·TTC tracts (∼250 triplets) was greatly inhibited in COS-7 cells compared with plasmids carrying (GAA·TTC)9 and (GAA·TTC)90. Replication inhibition was five times greater for the plasmid whose transcript contains (GAA)230than for the plasmid whose transcript contains (UUC)270. Our in vivo investigation revealed that expanded GAA·TTC repeats from intron I of the FRDA gene inhibit transcription rather than post-transcriptional RNA processing and also interfere with replication. The molecular basis for these effects may be the formation of non-B DNA structures. Friedreich ataxia (FRDA) 1The abbreviations used are: FRDA, Friedreich ataxia; TRS, triplet repeat sequences; Pur·Pyr, polypurine·polypyrimidine; bp, base pair(s). is the first autosomal recessive neurodegenerative disease found to be caused by the hyperexpansion of a triplet repeat sequence (TRS) (1Campuzano V. Montermini L. Moltó M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.-L. Cocozza S. Koenig M. Pandolfo M. Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2323) Google Scholar), a GAA·TTC repeat in the first intron of the frataxin gene. The GAA·TTC expansion accounts for about 98% of all FRDA chromosomes, with the remaining ones carrying frataxin point mutations. The recessive inheritance, nature, and intronic localization of the expanded sequence make FRDA an unique case in TRS-related diseases (2Mandel J.-L. Nature. 1997; 386: 767-769Crossref PubMed Scopus (46) Google Scholar, 3Paulson H.L. Fischbeck K.H. Annu. Rev. Neurosci. 1996; 19: 79-107Crossref PubMed Scopus (301) Google Scholar, 4Wells R.D. Warren S.T. Genetic Instabilities and Hereditary Neurological Diseases. Academic Press, San Diego1998Google Scholar). However, the FRDA expanded GAA·TTC repeats show meiotic and mitotic instability as for other disease-associated TRS. In FRDA chromosomes, GAA·TTC repeat units vary from about 100 to more than 1,000 whereas less than 37 repeat units are found in normal chromosomes (1Campuzano V. Montermini L. Moltó M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.-L. Cocozza S. Koenig M. Pandolfo M. Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2323) Google Scholar, 5Dürr A. Cossée M. Agid Y. Campuzano V. Mignard C. Penet C. Mandel J.-L. Brice A. Koenig M. N. Engl. J. Med. 1996; 335: 1169-1175Crossref PubMed Scopus (894) Google Scholar, 6Montermini L. Andermann E. Labuda M. Richter A. Pandolfo M. Cavalcanti F. Pianese L. Iodice L. Farina G. Monticelli A. Turano M. Filla A. De Michele G. Cocozza S. Hum. Mol. Genet. 1997; 6: 1261-1266Crossref PubMed Scopus (187) Google Scholar). FRDA patients carrying two expanded GAA·TTC repeats show very low levels of mature frataxin transcript (1Campuzano V. Montermini L. Moltó M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.-L. Cocozza S. Koenig M. Pandolfo M. Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2323) Google Scholar, 7Cossée M. Campuzano V. Koutnikova H. Fischbeck K. Mandel J.-L. Koenig M. Bidichandani S.I. Patel P.I. Molt M.D. Cañizares J. De Frutos R. Pianese L. Cavalcanti F. Monticelli A. Cocozza S. Montermini L. Pandolfo M. Nat. Genet. 1997; 15: 337-338Crossref PubMed Scopus (80) Google Scholar, 8Bidichandani S.I. Ashizawa T. Patel P.I. Am. J. Hum. Genet. 1997; 60: 1251-1256PubMed Google Scholar) and of frataxin (9Campuzano V. Montermini L. Lutz Y. Cova L. Hindelang C. Jiralerspong S. Trottier Y. Kish S.J. Faucheux B. Trouillas P. Authier F.J. Dürr A. Mandel J.-L. Vescovi A. Pandolfo M. Koenig M. Hum. Mol. Genet. 1997; 6: 1771-1780Crossref PubMed Scopus (621) Google Scholar), indicating suppressed gene expression. Such a defect may be caused either by reduced transcription or by abnormal post-transcriptional processing (1Campuzano V. Montermini L. Moltó M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.-L. Cocozza S. Koenig M. Pandolfo M. Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2323) Google Scholar, 9Campuzano V. Montermini L. Lutz Y. Cova L. Hindelang C. Jiralerspong S. Trottier Y. Kish S.J. Faucheux B. Trouillas P. Authier F.J. Dürr A. Mandel J.-L. Vescovi A. Pandolfo M. Koenig M. Hum. Mol. Genet. 1997; 6: 1771-1780Crossref PubMed Scopus (621) Google Scholar, 10Rosenberg R.N. N. Engl. J. Med. 1996; 335: 1222-1224Crossref PubMed Scopus (37) Google Scholar). Together with the identification of frataxin point mutations resulting in a defective or truncated protein, this finding defines FRDA as a frataxin deficiency disease, in accordance with its recessive inheritance. The sizes of the GAA·TTC repeats carried by each patient correlate with the age of onset and the severity of the disease, particularly for the smaller one (5Dürr A. Cossée M. Agid Y. Campuzano V. Mignard C. Penet C. Mandel J.-L. Brice A. Koenig M. N. Engl. J. Med. 1996; 335: 1169-1175Crossref PubMed Scopus (894) Google Scholar). In addition, an inverse correlation between the length of the smaller GAA·TTC repeat and the residual amount of frataxin was observed in cultured cells from FRDA patients (9Campuzano V. Montermini L. Lutz Y. Cova L. Hindelang C. Jiralerspong S. Trottier Y. Kish S.J. Faucheux B. Trouillas P. Authier F.J. Dürr A. Mandel J.-L. Vescovi A. Pandolfo M. Koenig M. Hum. Mol. Genet. 1997; 6: 1771-1780Crossref PubMed Scopus (621) Google Scholar). The GAA·TTC tract is a polypurine·polypyrimidine (Pur·Pyr) sequence, which may form an intramolecular triple helix in vitro under appropriate conditions of pH, metal ions concentrations, and supercoiling (4Wells R.D. Warren S.T. Genetic Instabilities and Hereditary Neurological Diseases. Academic Press, San Diego1998Google Scholar, 11Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (227) Google Scholar). Increasing the length of the Pur·Pyr tract also promotes triplex formation (12Collier D.A. Wells R.D. J. Biol. Chem. 1990; 265: 10652-10658Abstract Full Text PDF PubMed Google Scholar). Ohshima et al. (13Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) demonstrated that plasmids containing 38, 58, and 103 GAA·TTC triplets, but not 16 triplets, showed supercoil-induced relaxations when examined by two-dimensional-agarose gel electrophoresis, even at pH 8.3, suggesting that they may adopt a triple helical structure in vivo. Such structures inhibit gene expression by blocking the progression of RNA polymerase, as shown to occur for Pur·Pyr tracts both in vitro (14Reaban M.E. Griffin J.A. Nature. 1990; 348: 342-344Crossref PubMed Scopus (195) Google Scholar, 15Reaban M.E. Griffin J.A. Nature. 1991; 351: 447-448PubMed Google Scholar, 16Reaban M.E. Lebowitz J. Griffin J.A. J. Biol. Chem. 1994; 269: 21850-21857Abstract Full Text PDF PubMed Google Scholar, 17Grabczyk E. Fishman M.C. J. Biol. Chem. 1995; 270: 1791-1797Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) andin vivo (18Sarkar P.S. Brahmachari S.K. Nucleic Acids Res. 1992; 20: 5713-5718Crossref PubMed Scopus (42) Google Scholar, 19Amirhaeri S. Wohlrab F. Wells R.D. J. Biol. Chem. 1995; 270: 3313-3319Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 20Kohwi Y. Kohwi-Shigematsu T. Genes Dev. 1991; 5: 2547-2554Crossref PubMed Scopus (84) Google Scholar). However, GAA-containing RNA may also adopt a secondary structure interfering with post-transcriptional processing (21Aoki T. Koch K.S. Leffert H.L. J. Mol. Biol. 1997; 267: 229-236Crossref PubMed Scopus (27) Google Scholar). Pur·Pyr sequences, including GAA·TTC tracts, can also interfere with DNA replication, since they have been shown to stall DNA polymerase in vitro, probably again as a consequence of intramolecular triplex formation (11Mirkin S.M. Frank-Kamenetskii M.D. Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 541-576Crossref PubMed Scopus (227) Google Scholar, 13Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). We used cloned GAA·TTC repeats to investigate the possible effect of this intronic sequence on gene expression in vivo. The cloned GAA·TTC tracts previously used by Ohshima et al.(13Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) contain interruptions and no FRDA-derived flanking sequence. Considering the effects of interruptions and of flanking sequences on the biological properties of TRS (3Paulson H.L. Fischbeck K.H. Annu. Rev. Neurosci. 1996; 19: 79-107Crossref PubMed Scopus (301) Google Scholar, 4Wells R.D. Warren S.T. Genetic Instabilities and Hereditary Neurological Diseases. Academic Press, San Diego1998Google Scholar, 6Montermini L. Andermann E. Labuda M. Richter A. Pandolfo M. Cavalcanti F. Pianese L. Iodice L. Farina G. Monticelli A. Turano M. Filla A. De Michele G. Cocozza S. Hum. Mol. Genet. 1997; 6: 1261-1266Crossref PubMed Scopus (187) Google Scholar, 22Mangiarini L. Sathasivam K. Mahal A. Mott R. Seller M. Bates G.P. Nat. Genet. 1997; 15: 197-200Crossref PubMed Scopus (262) Google Scholar, 23Wells R.D. J. Biol. Chem. 1996; 271: 2875-2878Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar, 24Bingham P.M. Scott M.O. Wang S. McPhaul M.J. Wilson E.M. Garbern J.Y. Merry D.E. Fischbeck K.H. Nat. Genet. 1995; 9: 191-196Crossref PubMed Scopus (126) Google Scholar, 25Monckton D.G. Neumann R. Guram T. Fretwell N. Tamaki K. MacLeod A. Jeffreys A.J. Nat. Genet. 1994; 8: 162-170Crossref PubMed Scopus (112) Google Scholar, 26Burright E.N. Clark H.B. Servadio A. Matilla T. Feddersen R.M. Yunis W.S. Duvick L.A. Zoghbi H.Y. Orr H.T. Cell. 1995; 82: 937-948Abstract Full Text PDF PubMed Scopus (528) Google Scholar, 27Lavedan C.N. Garrett L. Nussbaum R.L. Hum. Genet. 1997; 100: 407-414Crossref PubMed Scopus (31) Google Scholar, 28Andreassen R. Egeland T. Olaisen B. Am. J. Hum. Genet. 1996; 59: 360-367PubMed Google Scholar), we constructed new recombinant plasmids containing from 9 to 500 GAA·TTC triplets along with some frataxin gene-derived flanking sequence. These data evidence that such TRS inhibit transcription and possibly DNA replicationin vivo. Genomic DNA from a patient carrying ∼700 GAA·TTC repeats was amplified by polymerase chain reaction (1Campuzano V. Montermini L. Moltó M.D. Pianese L. Cossée M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticelli A. Zara F. Cañizares J. Koutnikova H. Bidichandani S.I. Gellera C. Brice A. Trouillas P. De Michele G. Filla A. De Frutos R. Palau F. Patel P.I. Di Donato S. Mandel J.-L. Cocozza S. Koenig M. Pandolfo M. Science. 1996; 271: 1423-1427Crossref PubMed Scopus (2323) Google Scholar) using primers Not-Bam (5′-GGAGGGAACCGTCTGGGCAAAGG-3′) and 2500-F (5′-CAATCCAGGACAGTCAGGGCTT-3′) and then digested withBamHI and BglII. The fragment containing (GAA·TTC)∼700 along with flanking sequences (352 bp 5′ and 250 bp 3′ to the TRS) was purified by a 1.2% agarose gel (13Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) and ligated into the BamHI site of pUC19. The ligation mixture was transformed into Escherichia coli SURE (Stratagene) by electroporation. The resultant recombinant plasmid contained 110 GAA·TTC triplets in orientation II (Fig.1 A). The SacI-HindIII digest was recloned into the SacI-HindIII site of pUC18NotI and pUC19NotI (29Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar) to give plasmids containing (GAA·TTC)70 and (GAA·TTC)65 in two different orientations, designating pRW3804 (orientation I) and pRW3803 (orientation II), respectively. To generate longer repeats, thein vivo expansion method was performed as described previously (13Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 29Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar). Briefly, after pRW3804 was grown in E. coli DH10B (Life Technologies) or SURE, theSacI-HindIII digest was loaded on an agarose gel and regions above 70 repeats were eluted and ligated into pUC18NotI. The procedure was repeated several times, obtaining cloned GAA·TTC repeats ranging from 70 to 360 triplets. To clone GAA·TTC repeats into pSPL3 (Life Technologies), inserts were excised from pUC18NotI-based plasmids by EcoRI and PstI digestion and cloned into the corresponding pSPL3 sites. The resulting constructs had the TRS in orientation I. To obtain pSPL3 constructs with (GAA·TTC)n in orientation II,Ecl136II-EcoRV fragments containing the TRS and flanking sequences were excised from the orientation I pSPL3-based plasmids and recloned in the opposite orientation into theEcoRV site of pSPL3. Using the in vivoexpansion-deletion method (13Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 29Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar), from 9 to 500 GAA·TTC repeats were eventually cloned in pSPL3. For the construction of pMP106, a cDNA fragment was amplified by reverse transcriptase-polymerase chain reaction from total RNA isolated from pSPL3-transfected COS-7 cells, using primers SD6 and SA2 (Life Technologies). The resulting 263-bp fragment containing parts of the two pSPL3 exons was cloned into an EcoRV-digested, T-tailed, pMOS vector (Amersham). To obtain pMP107 and pMP108, pMP106 was digested with either XbaI or AvaI and subsequently self-ligated. pMP107 and pMP108 contain a 73- and a 159-bp fragment of the first and second pSPL3 exon, respectively. As a positive hybridization control for RNA analysis, theEcoRI-KpnI digest of pTRI-β-Actin-125-Human (Ambion), which contains a 127-bp cDNA fragment of the human β-actin gene (820–946), was cloned into theEcoRI-KpnI site of pGEM-3Zf(−) (Promega) to produce pMP125. For the construction of pMP129, the HindIII-EcoRI fragment of pIND/lacZ (Invitrogen) containing the β-galactosidase gene was filled in the overhangs with the Klenow fragment of E. coli DNA polymerase I (New England Biolabs) and dNTPs, and cloned into the MspI site of pSPL3. pMP129-based plasmids containing (GAA·TTC)n were constructed by digesting the pSPL3-based plasmids with eitherApaI-PstI or HindIII and cloning the released inserts into the ApaI-PstI orHindIII sites of pMP129, respectively. Luciferase gene fragments (661 and 1340 bp), obtained by digesting pGL3-Control (Promega) with EcoNI or HindIII andHincII followed by filling in the overhangs, were cloned into the EcoRV site of pSPL3 in the antisense orientation to give pMP175 and pMP177, respectively. pMP183 was constructed by inserting the HindIII digest of pMP177 into theHindIII site of pMP129. For plasmid preparations, the recombinant plasmid DNA was transformed into E. coli SURE by electroporation and the transformant was grown in 1 liter or 100 ml of LB with 75 μg/ml ampicillin at 37 °C until the end of logarithmic phase (OD600 = ∼0.9). Plasmids were isolated by the alkali lysis method (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) and purified by CsCl density gradient centrifugation or the QIAprep Plasmid Kits (QIAGEN). Isolated plasmids were digested with appropriate restriction enzymes. The digest was analyzed on an agarose gel, stained with ethidium bromide, and photographed. The inserts in all of the recombinant plasmids were characterized by DNA sequencing on both strands to determine the repeat units. For plasmids containing more than 59 GAA·TTC repeats, the triplet repeat units were estimated from agarose gels and DNA sequencing to ±5 triplets. COS-7 cells were grown in Dulbecco's modified Eagle's medium (ICN Pharmaceuticals) containing 50 units/ml penicillin-streptomycin and supplemented with 2 mmglutamine and 10% fetal bovine serum (ICN Pharmaceuticals). Cultures were maintained at 37 °C in a 5% CO2 atmosphere. COS-7 cells were plated on 60-mm diameter plastic dishes at a density of 4 × 105 cells/dish. After 24 h, the cells achieved 40–70% confluence and were transfected with 1 μg of pSPL3-based plasmid using cationic liposomes (Lipofectace, Life Technologies) (7 μg), according to the manufacturer's protocol. 48 h after transfection, total RNA was isolated by the TRIzol method (Life Technologies): 800 μl of TRIzol and 200 μl of chloroform were added to the cells and the nucleic acids were subsequently recovered by precipitation with 2/3 volume of isopropyl alcohol. pMP106 and pMP125 were used as a template to make antisense RNA probes, R-MP106 and R-MP125, by in vitro transcription from the T7 and SP6 promoters, respectively, incorporating [α-32P]UTP (800 Ci/mmol, Amersham) with the MAXIscript kit (Ambion). The reaction mixtures were loaded on a 5% polyacrylamide electrophoresis gel containing 8 m urea, and the radiolabeled RNA products were excised and eluted according to the manufacturer's protocol. RNase protection assays were performed using the RPAII kit (Ambion): 1 μg of total RNA was hybridized to 1.2 × 105 cpm and 2.2 × 104 cpm RNA probes synthesized from pMP106 and pMP125, respectively, at 45 °C for 16 h in 20 μl of the hybridization buffer containing 80% deionized formamide, 100 mm sodium citrate (pH 6.4), 300 mm sodium acetate (pH 6.4), and 1 mm EDTA. The samples were digested with a mixture of RNase A (0.5 units) and RNase T1 (20 units) in 200 μl of digestion buffer for 30 min at 37 °C. RNA was precipitated with ethanol and dissolved in 8 μl of gel loading buffer. Samples were fractionated on a 8% denaturing polyacrylamide gel containing 8 m urea. After exposure of the dried gel to x-ray film at −80 °C for 48 h, the amounts of protected products was estimated using the AlphaImager version 3.0 (Alpha Innotech). Antisense probes R-MP107 and R-MP125 were synthesized by in vitro transcription using T7 and SP6 RNA polymerases from pMP107 and pMP125, respectively, as described above. 1 μg of total RNA was size-fractionated on a 1.0% agarose-formaldehyde gel (Ambion) and transferred to Hybond-N+ nylon membrane (Amersham) using the NorthernMax kit (Ambion). After UV cross-linking, blots were hybridized to either probe R-MP107 or R-MP125 at 65 °C for 16 h. The membrane was exposed to x-ray film at −80 °C for 24 h and 7 days for probes R-MP125 and R-MP107, respectively. COS-7 cells were plated on 100-mm diameter plastic dishes at a density of 8 × 105 cells/dish. After 24 h, pMP129-based plasmid (3 μg) and a luciferase reporter plasmid pGL3-Control (Promega) (1 μg) were introduced into COS-7 cells by cationic liposomes (LipofectAMINE, Life Technologies) (30 μg), according to the manufacturer's protocol. Transfected cells were harvested 48 h post-transfection and lysed by 900 μl of reporter lysis buffer (Promega). The β-galactosidase and luciferase assays were performed using the respective kits (Promega). Luciferase activity was measured using the AutoLumat LB953 (EG&G Berthold). Plasmid DNA was recovered from transfected COS-7 cells as follows: 3 μg of pMP141, pMP180, pRW3823, pMP145, pMP165, pRW3827, pMP175, and pMP177 were separately introduced, along with 1 μg of pGL3-Control, into COS-7 cells in 100-mm diameter plastic dishes using LipofectAMINE (30 μg) as described above. Transfected cells were washed twice with ice-cold phosphate-buffered saline 48 h after transfection and incubated with 900 μl of lysis solution containing 0.6% SDS and 10 mm EDTA for 20 min at room temperature. The lysate was transferred into two 1.5-ml microcentrifuge tubes, mixed with 450 μl of 2.5 m NaCl, and incubated at 4 °C for 16 h. After centrifugation at 4 °C for 4 min at 14,000 ×g, the supernatant was extracted with phenol twice and chloroform once and precipitated with ethanol. The pellet was resuspended in 250 μl of TE buffer (10 mm Tris·Cl, 1 mm EDTA, pH 8.0) and reprecipitated with 750 μl of ethanol in the presence of 25 μl of 3 m sodium acetate (pH 5.2). After centrifugation, plasmid DNA was isolated and resuspended in H2O. 10 μg of plasmid DNA were digested with the appropriate restriction enzymes in the presence of RNase A and the digests were separated on agarose gels in 1 × TBE buffer (90 mm Tris borate, 2 mm EDTA, pH 8.3). DNA was transferred onto nylon membranes (Hybond-N+, Amersham) (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). After UV cross-linking, blots were hybridized to32P-labeled probes indicated in the figures at 65 °C in 15 ml of hybridization solution (5 × SSC, 5 × Denhardt solution, 1% SDS) with 100 μg/ml herring sperm DNA. Membranes were washed with 2 × SSC, 0.1% SDS twice at 25 °C for 5 min and 0.2 × SSC, 0.1% SDS twice at 25 °C for 5 min, and then exposed to x-ray film at −80 °C for 5–10 days. Band intensities on autoradiograms were measured using the AlphaImager version 3.0 (Alpha Innotech). Attempts were made to clone a polymerase chain reaction product from a FRDA patient containing (GAA·TTC)700 in pUC19. E. coli SURE transformants harbored plasmids with a family of repeat lengths containing 110 GAA·TTC triplets at most. TTC triplets were in the leading strand template of all these recombinant plasmids (Fig. 1 A, orientation II). This first result indicated that GAA·TTC repeats cloned into pUC19 are unstable in E. coli, and the instability may be related to the direction of replication, as previously observed for CTG·CAG (29Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar) and CCG·CGG (31Shimizu M. Gellibolian R. Oostra B.A. Wells R.D. J. Mol. Biol. 1996; 258: 614-626Crossref PubMed Scopus (74) Google Scholar). The insert containing (GAA·TTC)110 was then subcloned in both orientations into pUC18NotI (Fig. 1 A, orientation I) and pUC19NotI (Fig. 1 A, orientation II). The resulting recombinant plasmids, pRW3804 and pRW3803, contained 70 and 65 GAA·TTC triplets, respectively (Fig. 1 A). These shorter GAA·TTC repeats were quite stable in both plasmids when grown inE. coli SURE, but pRW3804 was more stable than pRW3803 when grown in E. coli DH10B (data not shown). Hence, the stability of GAA·TTC repeats in E. coli is influenced by the direction of replication (see below, Fig. 1 C). To clone longer GAA·TTC repeats, we used the in vivoexpansion method (13Ohshima K. Kang S. Larson J.E. Wells R.D. J. Biol. Chem. 1996; 271: 16773-16783Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 29Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar, 32Ohshima K. Kang S. Wells R.D. J. Biol. Chem. 1996; 271: 1853-1856Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). pRW3804, in which (GAA·TTC)nis more stable, was chosen as the starting material. This repetitive procedure successfully generated repeats containing between 70 and 360 GAA·TTC triplets (Fig. 1 A). The instability of the longest repeats was evident during their propagation (Fig. 1 B). While shorter repeats as (GAA·TTC)70 (lane 1) and (GAA·TTC)150 (lane 3) were quite stable, (GAA·TTC)270 (lane 5) and (GAA·TTC)360 (lane 7) generated multiple deletion products, visible as distinct bands on a smeary background, as previously seen with CTG·CAG (29Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar). To investigate the effects of GAA·TTC triplet repeats on transcription and splicing (see below), we subcloned several such repeats from pUC vectors into the multicloning site of pSPL3 in both orientations (Fig. 2 A). The subsequent in vivo expansion-deletion procedure generated GAA·TTC repeats ranging from 9 to 500 triplets (Fig. 1 A). Surprisingly, GAA·TTC repeats were much more stable in pSPL3 than in pUC vectors (Fig. 1 B). pSPL3-based plasmids containing up to 270 repeats were completely stable (Fig. 1 B, lanes 2, 4, and 6), and pRW3824 containing 360 repeats showed only slight instability (lane 8). As expected, constructs containing 470 and 500 repeats showed an increasing frequency of deletions (data not shown). When comparing the stability of (GAA·TTC)360 in different orientations, the insert in orientation II (Fig. 1 C, lane 9) was less stable than the one in orientation I (lane 8), as observed for pUC-based plasmids. To avoid contamination by deleted products, we only used pSPL3-based plasmids in which the cloned GAA·TTC repeat was completely stable for further experiments. These contained up to 230 triplets in orientation II, and up to 270 triplets in orientation I (Fig. 1 C). pSPL3 (Fig.2 A) harbors a reporter gene, derived from the HIVgp120 gene, composed by two exons (exons 1 and 2) separated by an intron derived from the HIV tat gene (33Buckler A.L. Chang D.D. Graw S.L. Brook J.D. Haber D.A. Sharp P.A. Housman D.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4005-4009Crossref PubMed Scopus (443) Google Scholar). This intron contains a multiple cloning site where (GAA·TTC)n repeats were inserted. Transcription is controlled by the SV40 early promoter. (GAA·TTC)n repeat-containing pSPL3-based plasmids were transfected into COS-7 cells and the reporter gene transcripts were detected by RNase protection analysis (Fig. 2 B). We observed a reduction in the amount of the mature transcript containing both exons 1 and 2 (indicated as I in Fig. 2 B) as the length of GAA·TTC repeats increased. Such reduction was much greater when (TTC)n was in the template strand, i.e. when GAA-containing RNA was synthesized (Fig. 2 A, lower inset), than when (GAA)n was in the template strand, i.e.when UUC-containing RNA was formed (Fig. 2 A, upper inset). Specifically, the mature transcript derived from (GAA)230-containing RNA was 6 times less abundant than the one derived from the (UUC)270-containing RNA (3% (Fig.2 B, lane 7) versus 17% (lane 4) of pSPL3 (lane 8)). The protected fragment indicated as II in Fig. 2 B, whose size corresponds to exon 2 only, was also reduced as the repeat length increased, suggesting that accumulation of unspliced RNA was not occurring. The protected fragment indicated as III in Fig. 2 B was also reduced in amount as the repeat length increased and is of uncertain nature, possibly resulting from an alternate splicing of exon 1 with part of exon 2. In the same figure, IV indicates a protected fragment corresponding to exon 1 only. Interestingly, its abundance remained stable in all samples, suggesting that the initiation of transcription was not affected by the length of the TRS. Northern blot analysis of total RNA from transfected cells (Fig.2 C) confirmed that GAA·TTC triplet repeats caused" @default.
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• W2137770909 title "Inhibitory Effects of Expanded GAA·TTC Triplet Repeats from Intron I of the Friedreich Ataxia Gene on Transcription and Replicationin Vivo" @default.
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