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Preparation of human cDNAs encoding expanded polyglutamine repeats
Neuroscience Letters 275 (1999) 129±132 www.elsevier.com/locate/neulet
Preparation of human cDNAs encoding expanded polyglutamine repeats Matthew F. Peters a, Christopher A. Ross a, b, c,* a
Laboratory of Molecular Neurobiology, Division of Neurobiology, Department of Psychiatry, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205±2196, USA b Department of Neuroscience, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205±2196, USA c The Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205±2196, USA Received 27 July 1999; accepted 10 September 1999
Abstract At least eight neurodegenerative diseases result from expansions of polyglutamine tracts encoded by CAG trinucleotide repeats. Although polyglutamine diseases typically have onset after age 50 in humans, these diseases can be modeled in animals and in cell culture by using highly expanded repeats to accelerate the pathogenesis. Unfortunately, current methods for preparing recombinant constructs with large glutamine tracts either alter the coding region adjacent to the repeat or yield highly unstable pure CAG repeats. We have developed a technique for expanding repeats that results in a more stable mix of CAG and CAA glutamine codons. We expect this technique to allow rapid preparation of highly expand repeats suitable for stable animal and cell culture models for any of the polyglutamine repeat diseases. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Polyglutamine; Trinulceotide repeat; CAG repeat; Huntington's disease; Type IIS restriction enzyme
Polyglutamine repeat diseases are inherited neurodegenerative disorders resulting from expansion of CAG trinucleotide repeats (reviewed in Refs. [7,12]). For each of these diseases the normal gene contains approximately 5± 35 CAG triplets while disease alleles harbor repeats in excess of 35±40. Polyglutamine repeat diseases are typically adult onset conditions that progress inexorably over 10±20 years. Rare expansions longer than 70 repeats result in juvenile onset often with rapid neurodegeneration. The apparent accelerated pathogenesis resulting from long CAG repeats is useful for generating recombinant animal and cell culture models that develop features similar to these diseases. An obstacle in generating animal and cell culture models is the instability of long CAG repeats. Long repeats isolated from patients typically contain pure CAG tracts, which tend to contract. A patient-derived huntingtin with a (CAG)180 repeat was originally isolated in lambda phage but became unstable when transferred to plasmids [9]. Lines of transgenic mice expressing this construct contained repeats lengths ranging from 111 to 157 in the founders with addi* Corresponding author. Tel.: 11-410-614-0011; fax: 11-410614-0013. E-mail address: [email protected] (C.A. Ross)
tional variation in their offspring [4]. Recently prepared stable cell lines expressing huntingtin have encountered similar repeat length instability [3]. Long repeats encoded by a mix of CAG and CAA codons are more stable. Current techniques are not well suited for preparation of such long repeats in the context of disease genes. CAA-containing repeats can be prepared synthetically using polymerase chain reaction (PCR)-based techniques [6,11]. However, inserting these synthetic repeats requires creation of a blunt end restriction site that also changes the sequence ¯anking the repeat. Alternatively, an existing CAG repeats can be expanded by propagating constructs in bacterial strains prone to repeat expansion [5,8]. However, since the repeats in many disease genes contain CAAs located at the ends of the polyglutamine encoding tract, expansions in bacteria are more likely to occur in the pure CAG repeat and not duplicate the CAAs. Here, we develop a PCR technique for rapidly expanding CAG repeats while increasing the number of CAA interruptions. To approximately double the trinucleotide repeat length in a recombinant construct, we combine two PCR products each containing the same repeat length. One PCR reaction ampli®es the N-terminal coding region up to
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 75 8- 2
130
M.F. Peters, C.A. Ross / Neuroscience Letters 275 (1999) 129±132
Fig. 1. Schematic outlining a method for expanding trinucleotide repeats. (A) (I) Two PCR reactions are performed amplifying the repeat region plus either the 5 0 or 3 0 ¯anking region. (II) PCR products are digested with BsgI (New England BioLabs) and restriction enzymes cutting sites in the vector convenient for subcloning (not shown). (III) Products from reactions 1 and 2 are cleaved by BsgI in the terminal CAG to generate compatible cohesive ends. (IV) Products are joined with a vector in a single ligation reaction. Note that digested reaction products contain the CAAs (indicated by 1 and * for huntingtin and atrophin, respectively). Thus, the ®nal product contains double the number of CAAs. (B) Gene speci®c (GS) primers ¯anking the CAG repeats are comprised of a four base leader sequence, a BsgI recognition site, 13 or 14 bases ¯anking the CAG repeat, and several bases within the repeat. Primers V1 and V2 anneal in the vector pcDNA 3.1. EcoN57 sites can be substituted for BsgI. All PCR reactions were ampli®ed for thirty cycles (30 s at 628C, 90 s at 728C, 30 s at 948C) with expand high ®delity DNA polymerase mix (Boehringer Mannheim) supplemented with 5% DMSO. No mutations were detected in isolated clones.
and including the repeat while the other ampli®es the repeat plus the C-terminal coding region (Fig. 1A; I, reactions 1 and 2, respectively). The primers annealing near the repeat ends are comprised of unique sequence immediately ¯anking the repeats plus a recognition sequence for a restriction enzyme that has its cleavage site a precise distance outside its recognition site (type IIS enzymes, reviewed in Ref. [10]). This primer organization allows the enzyme recognition site to be located 5 0 of the repeat ¯anking sequence and still direct cleavage within the CAG repeat located at the 3 0 end of the primer. After ampli®cation and restriction digestion, each of the two PCR products contains the CAG repeat
but they have the ¯anking sequence removed from opposite sides. To facilitate subcloning, the positions of the type IIS restriction sites are coordinated so that the two cleaved products have compatible cohesive ends within their terminal repeats. Speci®cally, in the reaction to amplify the Nterminal coding region, a BsgI recognition site is positioned 14 bases downstream of the terminal repeat in order to cleave the last CAG leaving an AG overhang (Fig. 1A; II). In the C-terminal reaction, a BsgI site is separated from the repeats by 13 bases of gene-speci®c sequence thereby directing cleavage with compatible overhang. The
M.F. Peters, C.A. Ross / Neuroscience Letters 275 (1999) 129±132
PCR products are also cleaved with enzymes convenient for subcloning and joined with a suitable vector in a single ligation reaction (Fig. 1A; III). The completed construct has a repeat length equal to one less than the sum of repeats in the two templates and has no trace of the BsgI sites used for subcloning (Fig. 1A; IV). We expanded the polyglutamine/CAG repeat in human huntingtin and atrophin, the proteins mutated in Huntington's disease and Dentatorubral±Pallidoluysian Atrophy (DRPLA), respectively. Constructs encoding the N-terminal 63 amino acids of huntingtin with either 75 or 97 glutamines (HD-N63-75Q, -97Q) were used as PCR templates [1]. Amplifying N63±75Q in both PCR reactions yielded a construct with a 149 glutamine repeat (Fig. 2A). Combining PCR products containing 75 and 97 repeats produced an N63±171Q construct (Fig. 2A). Atrophin was expanded from full-length templates with either 22 or 65 glutamines (Atr-22Q, -65Q). Combining the N-terminal coding region ampli®ed from Atr-65Q with the C-terminal coding region ampli®ed from Atr-22Q or Atr-65Q yielded Atr-86Q and 129Q, respectively (Fig. 2B). All constructs were con®rmed by sequence. The PCR primers used are shown in Fig. 1B. The polyglutamine tracts in normal huntingtin and atrophin are encoded by CAG repeats with CAA interruptions at the extreme 3 0 and 5 0 , respectively (indicated by asterisk and
131
plus, respectively, in Fig. 1A). Repeats expanded by our technique contain centrally located CAA triplets. Data from bacteria indicate that a limited number of interruptions may by suf®cient to increase stability. Compared with an uninterrupted (CAG)130 repeat, a sequence identical in length that contained one TAG interruption at repeat 28 was ®ve times less likely to expand in bacteria [2]. Our method doubles the number of CAAs with each completed expansion. Thus, starting with a normal length repeat and completing several expansions will result in frequent CAA interruptions. Moreover, a wide range of stable repeat lengths can generated by additional rounds of expansion in which various combinations of the original and expanded repeats are joined. Using PCR may constrain the length expanded repeats that can be produced. Repeats .100 tend to amplify nonspeci®cally suggesting a limit of 200 triplets in the ®nal products. An advantage of using PCR is the ability to introduce CAAs even when expanding a pure CAG repeat. We have introduced additional CAAs into the cDNA encoding the amino terminus of huntingtin by including a CAG to CAA point substitution in one of the ¯anking primers (primer GS-2, data not shown). This resulted in two centrally located CAAs with each completed expansion. This method for expanding trinucleotide repeats has signi®cant advantages over previous approaches. Unlike insertion of long synthetic repeats, the sequence ¯anking the repeat is not altered. Instead, type IIS restriction enzymes are used for subcloning, thereby allowing this technique to be applied to any trinucleotide repeat-containing construct. In contrast to repeats expanded in bacteria, the resulting constructs have regular CAA interruptions. We expect repeats comprised of mixed CAG/CAA triplets to reduce the instability of highly expanded repeats and allow preparation of stable animal and cellular models for polyglutamine diseases [3,4]. We thank Holly Seamen for technical assistance and Jillian Cooper for preparing N63±97Q. This work was supported by NINDS grant NS16375 to and the HDSA Coalition for the Cure C.A.R., and M.F.P. has been supported by NIH training grant MH15330 and an HDSA fellowship award.
Fig. 2. Huntingtin and atrophin constructs with highly expanded CAG repeats. (A) Huntingtin constructs N63±75Q and N63±97Q were used as templates (lanes 1 and 2). Combining the 5 0 PCR product from N63±75Q with 3 0 product of N63±75Q or -97Q yielded N63±149Q and -171Q, respectively (lanes 3 and 4). (B) Products encoding the N-terminus of Atr-65Q were joined with products from the C-terminus of Atr-22 and -65 resulting in Atr90Q and -129Q (lanes 1±4, respectively). HD-N63s and atrophins digested with BamHI/XhoI and Nhe I/Age I, respectively, were resolved on a 1% agarose gel. Markers are in kb.
[1] Cooper, J.K., Schilling, G., Peters, M.F., Herring, W.J., Sharp, A.H., Kaminsky, Z., Masone, J., Khan, F.A., Delanoy, M., Borchelt, D.R., Dawson, V.L., Dawson, T.M. and Ross, C.A., Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture. Hum. Mol. Genet., 7 (1998) 783± 790. [2] Kang, S., Ohshima, K., Jaworski, A. and Wells, R.D., CTG triplet repeats from the myotonic dystrophy gene are expanded in Escherichia coli distal to the replication origin as a single large event. J. Mol. Biol., 258 (1996) 543±547. [3] Li, S.H., Cheng, A.L. and Li, X.J., Cellular defects and altered gene expression in PC12 cells stably expressing mutant huntingtin. J. Neurosci., 19 (1999) 5159±5172.
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M.F. Peters, C.A. Ross / Neuroscience Letters 275 (1999) 129±132
[4] Mangiarini, L., Sathasivam, K., Mahal, A., Mott, R., Seller, M. and Bates, G.P., Instability of highly expanded CAG repeats in mice transgenic for the Huntington's disease mutation. Nat. Genet., 15 (1997) 197±200. [5] Ohshima, K., Kang, S. and Wells, R.D., CTG triplet repeats from human hereditary diseases are dominant genetic expansion products in Escherichia coli. J. Biol. Chem., 271 (1996) 1853±1856. [6] Ordway, J.M. and Detloff, P.J., In vitro synthesis and cloning of long CAG repeats. Biotechniques, 21 (1996) 609± 612. [7] Ross, C.A., Intranuclear neuronal inclusions: a common pathogenic mechanism for glutamine-repeat neurodegenerative diseases? Neuron, 19 (1997) 1147±1150. [8] Sarkar, P.S., Chang, H.C., Boudi, F.B. and Reddy, S., CTG
[9]
[10] [11] [12]
repeats show bimodal ampli®cation in E. coli. Cell, 95 (1998) 531±540. Sathasivam, K., Amaechi, I., Mangiarini, L. and Bates, G., Identi®cation of an HD patient with a (CAG)180 repeat expansion and the propagation of highly expanded CAG repeats in lambda phage. Hum. Genet., 99 (1997) 692±695. Szybalski, W., Kim, S.C., Hasan, N. and Podhajska, A.J., Class-IIS restriction enzymes ± a review. Gene, 100 (1991) 13±26. Takahashi, N., Sasagawa, N., Suzuki, K. and Ishiura, S., Synthesis of long trinucleotide repeats in vitro. Neurosci. Lett., 262 (1999) 45±48. Wells, R.D. and Warren, S.T., Genetic Instabilities and Hereditary Neurological Diseases, Academic Press, San Diego, CA, 1998.
Preparation of human cDNAs encoding expanded polyglutamine repeats Matthew F. Peters a, Christopher A. Ross a, b, c,* a
Laboratory of Molecular Neurobiology, Division of Neurobiology, Department of Psychiatry, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205±2196, USA b Department of Neuroscience, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205±2196, USA c The Program in Cellular and Molecular Medicine, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205±2196, USA Received 27 July 1999; accepted 10 September 1999
Abstract At least eight neurodegenerative diseases result from expansions of polyglutamine tracts encoded by CAG trinucleotide repeats. Although polyglutamine diseases typically have onset after age 50 in humans, these diseases can be modeled in animals and in cell culture by using highly expanded repeats to accelerate the pathogenesis. Unfortunately, current methods for preparing recombinant constructs with large glutamine tracts either alter the coding region adjacent to the repeat or yield highly unstable pure CAG repeats. We have developed a technique for expanding repeats that results in a more stable mix of CAG and CAA glutamine codons. We expect this technique to allow rapid preparation of highly expand repeats suitable for stable animal and cell culture models for any of the polyglutamine repeat diseases. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Polyglutamine; Trinulceotide repeat; CAG repeat; Huntington's disease; Type IIS restriction enzyme
Polyglutamine repeat diseases are inherited neurodegenerative disorders resulting from expansion of CAG trinucleotide repeats (reviewed in Refs. [7,12]). For each of these diseases the normal gene contains approximately 5± 35 CAG triplets while disease alleles harbor repeats in excess of 35±40. Polyglutamine repeat diseases are typically adult onset conditions that progress inexorably over 10±20 years. Rare expansions longer than 70 repeats result in juvenile onset often with rapid neurodegeneration. The apparent accelerated pathogenesis resulting from long CAG repeats is useful for generating recombinant animal and cell culture models that develop features similar to these diseases. An obstacle in generating animal and cell culture models is the instability of long CAG repeats. Long repeats isolated from patients typically contain pure CAG tracts, which tend to contract. A patient-derived huntingtin with a (CAG)180 repeat was originally isolated in lambda phage but became unstable when transferred to plasmids [9]. Lines of transgenic mice expressing this construct contained repeats lengths ranging from 111 to 157 in the founders with addi* Corresponding author. Tel.: 11-410-614-0011; fax: 11-410614-0013. E-mail address: [email protected] (C.A. Ross)
tional variation in their offspring [4]. Recently prepared stable cell lines expressing huntingtin have encountered similar repeat length instability [3]. Long repeats encoded by a mix of CAG and CAA codons are more stable. Current techniques are not well suited for preparation of such long repeats in the context of disease genes. CAA-containing repeats can be prepared synthetically using polymerase chain reaction (PCR)-based techniques [6,11]. However, inserting these synthetic repeats requires creation of a blunt end restriction site that also changes the sequence ¯anking the repeat. Alternatively, an existing CAG repeats can be expanded by propagating constructs in bacterial strains prone to repeat expansion [5,8]. However, since the repeats in many disease genes contain CAAs located at the ends of the polyglutamine encoding tract, expansions in bacteria are more likely to occur in the pure CAG repeat and not duplicate the CAAs. Here, we develop a PCR technique for rapidly expanding CAG repeats while increasing the number of CAA interruptions. To approximately double the trinucleotide repeat length in a recombinant construct, we combine two PCR products each containing the same repeat length. One PCR reaction ampli®es the N-terminal coding region up to
0304-3940/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 9 9) 00 75 8- 2
130
M.F. Peters, C.A. Ross / Neuroscience Letters 275 (1999) 129±132
Fig. 1. Schematic outlining a method for expanding trinucleotide repeats. (A) (I) Two PCR reactions are performed amplifying the repeat region plus either the 5 0 or 3 0 ¯anking region. (II) PCR products are digested with BsgI (New England BioLabs) and restriction enzymes cutting sites in the vector convenient for subcloning (not shown). (III) Products from reactions 1 and 2 are cleaved by BsgI in the terminal CAG to generate compatible cohesive ends. (IV) Products are joined with a vector in a single ligation reaction. Note that digested reaction products contain the CAAs (indicated by 1 and * for huntingtin and atrophin, respectively). Thus, the ®nal product contains double the number of CAAs. (B) Gene speci®c (GS) primers ¯anking the CAG repeats are comprised of a four base leader sequence, a BsgI recognition site, 13 or 14 bases ¯anking the CAG repeat, and several bases within the repeat. Primers V1 and V2 anneal in the vector pcDNA 3.1. EcoN57 sites can be substituted for BsgI. All PCR reactions were ampli®ed for thirty cycles (30 s at 628C, 90 s at 728C, 30 s at 948C) with expand high ®delity DNA polymerase mix (Boehringer Mannheim) supplemented with 5% DMSO. No mutations were detected in isolated clones.
and including the repeat while the other ampli®es the repeat plus the C-terminal coding region (Fig. 1A; I, reactions 1 and 2, respectively). The primers annealing near the repeat ends are comprised of unique sequence immediately ¯anking the repeats plus a recognition sequence for a restriction enzyme that has its cleavage site a precise distance outside its recognition site (type IIS enzymes, reviewed in Ref. [10]). This primer organization allows the enzyme recognition site to be located 5 0 of the repeat ¯anking sequence and still direct cleavage within the CAG repeat located at the 3 0 end of the primer. After ampli®cation and restriction digestion, each of the two PCR products contains the CAG repeat
but they have the ¯anking sequence removed from opposite sides. To facilitate subcloning, the positions of the type IIS restriction sites are coordinated so that the two cleaved products have compatible cohesive ends within their terminal repeats. Speci®cally, in the reaction to amplify the Nterminal coding region, a BsgI recognition site is positioned 14 bases downstream of the terminal repeat in order to cleave the last CAG leaving an AG overhang (Fig. 1A; II). In the C-terminal reaction, a BsgI site is separated from the repeats by 13 bases of gene-speci®c sequence thereby directing cleavage with compatible overhang. The
M.F. Peters, C.A. Ross / Neuroscience Letters 275 (1999) 129±132
PCR products are also cleaved with enzymes convenient for subcloning and joined with a suitable vector in a single ligation reaction (Fig. 1A; III). The completed construct has a repeat length equal to one less than the sum of repeats in the two templates and has no trace of the BsgI sites used for subcloning (Fig. 1A; IV). We expanded the polyglutamine/CAG repeat in human huntingtin and atrophin, the proteins mutated in Huntington's disease and Dentatorubral±Pallidoluysian Atrophy (DRPLA), respectively. Constructs encoding the N-terminal 63 amino acids of huntingtin with either 75 or 97 glutamines (HD-N63-75Q, -97Q) were used as PCR templates [1]. Amplifying N63±75Q in both PCR reactions yielded a construct with a 149 glutamine repeat (Fig. 2A). Combining PCR products containing 75 and 97 repeats produced an N63±171Q construct (Fig. 2A). Atrophin was expanded from full-length templates with either 22 or 65 glutamines (Atr-22Q, -65Q). Combining the N-terminal coding region ampli®ed from Atr-65Q with the C-terminal coding region ampli®ed from Atr-22Q or Atr-65Q yielded Atr-86Q and 129Q, respectively (Fig. 2B). All constructs were con®rmed by sequence. The PCR primers used are shown in Fig. 1B. The polyglutamine tracts in normal huntingtin and atrophin are encoded by CAG repeats with CAA interruptions at the extreme 3 0 and 5 0 , respectively (indicated by asterisk and
131
plus, respectively, in Fig. 1A). Repeats expanded by our technique contain centrally located CAA triplets. Data from bacteria indicate that a limited number of interruptions may by suf®cient to increase stability. Compared with an uninterrupted (CAG)130 repeat, a sequence identical in length that contained one TAG interruption at repeat 28 was ®ve times less likely to expand in bacteria [2]. Our method doubles the number of CAAs with each completed expansion. Thus, starting with a normal length repeat and completing several expansions will result in frequent CAA interruptions. Moreover, a wide range of stable repeat lengths can generated by additional rounds of expansion in which various combinations of the original and expanded repeats are joined. Using PCR may constrain the length expanded repeats that can be produced. Repeats .100 tend to amplify nonspeci®cally suggesting a limit of 200 triplets in the ®nal products. An advantage of using PCR is the ability to introduce CAAs even when expanding a pure CAG repeat. We have introduced additional CAAs into the cDNA encoding the amino terminus of huntingtin by including a CAG to CAA point substitution in one of the ¯anking primers (primer GS-2, data not shown). This resulted in two centrally located CAAs with each completed expansion. This method for expanding trinucleotide repeats has signi®cant advantages over previous approaches. Unlike insertion of long synthetic repeats, the sequence ¯anking the repeat is not altered. Instead, type IIS restriction enzymes are used for subcloning, thereby allowing this technique to be applied to any trinucleotide repeat-containing construct. In contrast to repeats expanded in bacteria, the resulting constructs have regular CAA interruptions. We expect repeats comprised of mixed CAG/CAA triplets to reduce the instability of highly expanded repeats and allow preparation of stable animal and cellular models for polyglutamine diseases [3,4]. We thank Holly Seamen for technical assistance and Jillian Cooper for preparing N63±97Q. This work was supported by NINDS grant NS16375 to and the HDSA Coalition for the Cure C.A.R., and M.F.P. has been supported by NIH training grant MH15330 and an HDSA fellowship award.
Fig. 2. Huntingtin and atrophin constructs with highly expanded CAG repeats. (A) Huntingtin constructs N63±75Q and N63±97Q were used as templates (lanes 1 and 2). Combining the 5 0 PCR product from N63±75Q with 3 0 product of N63±75Q or -97Q yielded N63±149Q and -171Q, respectively (lanes 3 and 4). (B) Products encoding the N-terminus of Atr-65Q were joined with products from the C-terminus of Atr-22 and -65 resulting in Atr90Q and -129Q (lanes 1±4, respectively). HD-N63s and atrophins digested with BamHI/XhoI and Nhe I/Age I, respectively, were resolved on a 1% agarose gel. Markers are in kb.
[1] Cooper, J.K., Schilling, G., Peters, M.F., Herring, W.J., Sharp, A.H., Kaminsky, Z., Masone, J., Khan, F.A., Delanoy, M., Borchelt, D.R., Dawson, V.L., Dawson, T.M. and Ross, C.A., Truncated N-terminal fragments of huntingtin with expanded glutamine repeats form nuclear and cytoplasmic aggregates in cell culture. Hum. Mol. Genet., 7 (1998) 783± 790. [2] Kang, S., Ohshima, K., Jaworski, A. and Wells, R.D., CTG triplet repeats from the myotonic dystrophy gene are expanded in Escherichia coli distal to the replication origin as a single large event. J. Mol. Biol., 258 (1996) 543±547. [3] Li, S.H., Cheng, A.L. and Li, X.J., Cellular defects and altered gene expression in PC12 cells stably expressing mutant huntingtin. J. Neurosci., 19 (1999) 5159±5172.
132
M.F. Peters, C.A. Ross / Neuroscience Letters 275 (1999) 129±132
[4] Mangiarini, L., Sathasivam, K., Mahal, A., Mott, R., Seller, M. and Bates, G.P., Instability of highly expanded CAG repeats in mice transgenic for the Huntington's disease mutation. Nat. Genet., 15 (1997) 197±200. [5] Ohshima, K., Kang, S. and Wells, R.D., CTG triplet repeats from human hereditary diseases are dominant genetic expansion products in Escherichia coli. J. Biol. Chem., 271 (1996) 1853±1856. [6] Ordway, J.M. and Detloff, P.J., In vitro synthesis and cloning of long CAG repeats. Biotechniques, 21 (1996) 609± 612. [7] Ross, C.A., Intranuclear neuronal inclusions: a common pathogenic mechanism for glutamine-repeat neurodegenerative diseases? Neuron, 19 (1997) 1147±1150. [8] Sarkar, P.S., Chang, H.C., Boudi, F.B. and Reddy, S., CTG
[9]
[10] [11] [12]
repeats show bimodal ampli®cation in E. coli. Cell, 95 (1998) 531±540. Sathasivam, K., Amaechi, I., Mangiarini, L. and Bates, G., Identi®cation of an HD patient with a (CAG)180 repeat expansion and the propagation of highly expanded CAG repeats in lambda phage. Hum. Genet., 99 (1997) 692±695. Szybalski, W., Kim, S.C., Hasan, N. and Podhajska, A.J., Class-IIS restriction enzymes ± a review. Gene, 100 (1991) 13±26. Takahashi, N., Sasagawa, N., Suzuki, K. and Ishiura, S., Synthesis of long trinucleotide repeats in vitro. Neurosci. Lett., 262 (1999) 45±48. Wells, R.D. and Warren, S.T., Genetic Instabilities and Hereditary Neurological Diseases, Academic Press, San Diego, CA, 1998.