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• W2159203886 abstract "Genetic instability investigations on three triplet repeat sequences (TRS) involved in human hereditary neurological diseases (CTG·CAG, CGG·CCG, and GAA·TTC) revealed a high frequency of small expansions or deletions in 3-base pair registers in Escherichia coli. The presence of G to A polymorphisms in the CTG·CAG sequences served as reporters for the size and location of these instabilities. For the other two repeat sequences, length determinations confirmed the conclusions found for CTG·CAG. These studies were conducted in strains deficient in methyl-directed mismatch repair or nucleotide excision repair in order to investigate the involvement of these postreplicative processes in the genetic instabilities of these TRS. The observation that small and large instabilities for (CTG·CAG)175 fall into distinct size classes (1–8 repeats and approximate multiples of 41 repeats, respectively) leads to the conclusion that more than one DNA instability process is involved. The slippage of the complementary strands of the TRS is probably responsible for the small deletions and expansions in methyl-directed mismatch repair-deficient and nucleotide excision repair-deficient cells. A model is proposed to explain the observed instabilities via strand misalignment, incision, or excision, followed by DNA synthesis and ligation. This slippage-repair mechanism may be responsible for the small expansions in type 1 hereditary neurological diseases involving polyglutamine expansions. Furthermore, these observations may relate to the high frequency of small deletionsversus a lower frequency of large instabilities observed in lymphoblastoid cells from myotonic dystrophy patients. Genetic instability investigations on three triplet repeat sequences (TRS) involved in human hereditary neurological diseases (CTG·CAG, CGG·CCG, and GAA·TTC) revealed a high frequency of small expansions or deletions in 3-base pair registers in Escherichia coli. The presence of G to A polymorphisms in the CTG·CAG sequences served as reporters for the size and location of these instabilities. For the other two repeat sequences, length determinations confirmed the conclusions found for CTG·CAG. These studies were conducted in strains deficient in methyl-directed mismatch repair or nucleotide excision repair in order to investigate the involvement of these postreplicative processes in the genetic instabilities of these TRS. The observation that small and large instabilities for (CTG·CAG)175 fall into distinct size classes (1–8 repeats and approximate multiples of 41 repeats, respectively) leads to the conclusion that more than one DNA instability process is involved. The slippage of the complementary strands of the TRS is probably responsible for the small deletions and expansions in methyl-directed mismatch repair-deficient and nucleotide excision repair-deficient cells. A model is proposed to explain the observed instabilities via strand misalignment, incision, or excision, followed by DNA synthesis and ligation. This slippage-repair mechanism may be responsible for the small expansions in type 1 hereditary neurological diseases involving polyglutamine expansions. Furthermore, these observations may relate to the high frequency of small deletionsversus a lower frequency of large instabilities observed in lymphoblastoid cells from myotonic dystrophy patients. Neurogenetic diseases including myotonic dystrophy, fragile X syndrome, Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia type 1, and Friedreich's ataxia result from expanded triplet repeat sequences (TRS) 1The abbreviations used are: TRS, triplet repeat sequence(s); MMR, methyl-directed mismatch repair; NER, nucleotide excision repair system; PAGE, polyacrylamide gel electrophoresis; SSED, small slipped register expansions and deletions. CTG·CAG, CGG·CCG, or GAA·TTC within their genes (1Paulson H.L. Fischbeck K.H. Annu. Rev. Neurosci. 1996; 19: 79-107Crossref PubMed Scopus (301) Google Scholar, 2Campuzano 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 (2322) Google Scholar). Also, instabilities (expansions and deletions) of TRS have been associated with limb developmental diseases, including human synpolydactyly, hypodactyly in mice, and hereditary nonpolyposis colon cancer (4Fishel R. Lescoe M.K. Rao M.R.S. Copeland N.G. Jenkins N.A. Garber J. Kane M. Kolodner R. Cell. 1993; 75: 1027-1038Abstract Full Text PDF PubMed Scopus (2608) Google Scholar, 5Mortlock D.P. Post L.C. Innis J.W. Nat. Genet. 1996; 13: 284-289Crossref PubMed Scopus (148) Google Scholar, 6Muragaki Y. Mundlos S. Upton J. Olsen B.R. Science. 1996; 272: 548-551Crossref PubMed Scopus (491) Google Scholar). The earlier age of onset and the increased severity of the neurological diseases through family pedigrees (clinically referred to as anticipation) are influenced by the lengths of the TRS. Long tracts of TRS are unstable and show repeat size polymorphisms in successive generations and in different tissues. In addition to observations in humans, TRS instabilities have been demonstrated inEscherichia coli (7Bacolla A. Gellibolian R. Shimizu M. Amirhaeri S. Kang S. Ohshima K. Larson J.E. Harvey S.C. Stollar B.D. Wells R.D. J. Biol. Chem. 1997; 272: 16783-16792Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 8Bowater R.P. Jaworski A. Larson J.E. Parniewski P. Wells R.D. Nucleic Acids Res. 1997; 25: 2861-2868Crossref PubMed Scopus (87) Google Scholar, 9Bowater R.P. Rosche W.A. Jaworski A. Sinden R.R. Wells R.D. J. Mol. Biol. 1996; 264: 82-96Crossref PubMed Scopus (64) Google Scholar, 10Jaworski A. Rosche W.A. Gellibolian R. Kang S. Shimizu M. Bowater R.P. Sinden R.R. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11019-11023Crossref PubMed Scopus (148) Google Scholar, 11Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar, 12Kang S. Ohshima K. Jaworski A. Wells R.D. J. Mol. Biol. 1996; 258: 543-547Crossref PubMed Scopus (55) Google Scholar, 13Rosche W.A. Jaworski A. Kang S.F. Kramer S. Larson J.E. Geidroc D.P. Wells R.D. Sinden R.R. J. Bacteriol. 1996; 178: 5042-5044Crossref PubMed Google Scholar, 14Shimizu M. Gellibolian R. Oostra B.A. Wells R.D. J. Mol. Biol. 1996; 258: 614-626Crossref PubMed Scopus (74) Google Scholar, 15Ohshima 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), yeast (16Freudenreich C.H. Stavenhagen J.B. Zakian V.A. Mol. Cell Biol. 1997; 17: 2090-2098Crossref PubMed Scopus (201) Google Scholar, 17Maurer D.J. O'Callaghan B.L. Livingston D.M. Mol. Cell. Biol. 1996; 16: 6617-6622Crossref PubMed Scopus (109) Google Scholar), transgenic mice (18Monckton D.G. Coolbaugh M.I. Ashizawa K.T. Sicilano M.J. Caskey C.T. Nat. Genet. 1997; 15: 193-196Crossref PubMed Scopus (116) Google Scholar), and cultured cells from patients (19Ashizawa T. Monckton D.G. Vaishnav S. Patel B.J. Voskova A. Caskey C.T. Genomics. 1996; 36: 47-53Crossref PubMed Scopus (36) Google Scholar, 20Steinbach P. Wohrle D. Glaser D. Vogel W. Wells R.D. Warren S.T. Genetic Instabilities and Hereditary Neurological Diseases. Academic Press, Inc., San Diego, CA1998: 509-528Google Scholar) and are influenced by factors including host strain genotypes (11Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar, 12Kang S. Ohshima K. Jaworski A. Wells R.D. J. Mol. Biol. 1996; 258: 543-547Crossref PubMed Scopus (55) Google Scholar), DNA replication (7Bacolla A. Gellibolian R. Shimizu M. Amirhaeri S. Kang S. Ohshima K. Larson J.E. Harvey S.C. Stollar B.D. Wells R.D. J. Biol. Chem. 1997; 272: 16783-16792Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 8Bowater R.P. Jaworski A. Larson J.E. Parniewski P. Wells R.D. Nucleic Acids Res. 1997; 25: 2861-2868Crossref PubMed Scopus (87) Google Scholar, 9Bowater R.P. Rosche W.A. Jaworski A. Sinden R.R. Wells R.D. J. Mol. Biol. 1996; 264: 82-96Crossref PubMed Scopus (64) Google Scholar, 11Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar, 12Kang S. Ohshima K. Jaworski A. Wells R.D. J. Mol. Biol. 1996; 258: 543-547Crossref PubMed Scopus (55) Google Scholar, 13Rosche W.A. Jaworski A. Kang S.F. Kramer S. Larson J.E. Geidroc D.P. Wells R.D. Sinden R.R. J. Bacteriol. 1996; 178: 5042-5044Crossref PubMed Google Scholar), methyl-directed mismatch repair (10Jaworski A. Rosche W.A. Gellibolian R. Kang S. Shimizu M. Bowater R.P. Sinden R.R. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11019-11023Crossref PubMed Scopus (148) Google Scholar), growth conditions (8Bowater R.P. Jaworski A. Larson J.E. Parniewski P. Wells R.D. Nucleic Acids Res. 1997; 25: 2861-2868Crossref PubMed Scopus (87) Google Scholar, 9Bowater R.P. Rosche W.A. Jaworski A. Sinden R.R. Wells R.D. J. Mol. Biol. 1996; 264: 82-96Crossref PubMed Scopus (64) Google Scholar), transcription (8Bowater R.P. Jaworski A. Larson J.E. Parniewski P. Wells R.D. Nucleic Acids Res. 1997; 25: 2861-2868Crossref PubMed Scopus (87) Google Scholar), and nucleotide excision repair. 2P. Parniewski, A. Jaworski, and R. D. Wells, manuscript in preparation. Simple repetitive sequences are known to cause misalignment-mediated DNA synthesis errors that give rise to instabilities (22Kunkel T.A. Biochemistry. 1990; 29: 8003-8011Crossref PubMed Scopus (309) Google Scholar, 23Lechner R.L. Engler M.J. Richardson C.C. J. Biol. Chem. 1983; 258: 11174-11184Abstract Full Text PDF PubMed Google Scholar, 24Ripley L.S. Annu. Rev. Genet. 1990; 24: 189-213Crossref PubMed Google Scholar, 25Tautz D. Schlötterer C. Curr. Opin. Genet. 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Nature. 1993; 365: 274-276Crossref PubMed Scopus (953) Google Scholar). Numerous prior investigations demonstrated the occurrence of DNA instabilities caused by inactivation of the methyl-directed mismatch repair (MMR) or nucleotide excision repair system (NER) system (29Bronner C.E. Baker C.M. Morrison P.T. Warren G. Smith L.G. Lescoe M.K. Kane M. Earabino C. Lipford J. Lindblom A. Tanergard P. Bollag R.J. Godwin A.R. Ward D.C. Nordenskjold M. Fishel R. Kolodner R. Liskay R.M. Nature. 1994; 369: 258-261Crossref Scopus (1935) Google Scholar, 30Papadopoulos N. Nicolaides N.C. Wei Y. Ruben S.M. Carter K.C. Rosen C.A. Haseltine W.A. Fleischmann R.D. Fraser C.M. Adams M.D. Venter J.C. Hamilton S.R. Petersen G.N. Watson P. Lynch H.T. Peltomaki P. Mecklin J. de la Chapelle A. Kinzler K.W. Vogelstein B. Science. 1994; 263: 1625-1629Crossref PubMed Scopus (1773) Google Scholar, 31Umar A. Boyer J.C. Kunkel T.A. Science. 1994; 266: 814-816Crossref PubMed Scopus (123) Google Scholar, 32Umar A. 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Dominska M. Greenwell P. Petes T.D. Mol. Cell. Biol. 1997; 17: 2851-2858Crossref PubMed Scopus (333) Google Scholar, 41Strauss B.S. Sagher D. Acharya S. Nucleic Acids Res. 1997; 25: 806-813Crossref PubMed Scopus (54) Google Scholar). Much less is known about the involvement of these repair systems in the instability of TRS (10Jaworski A. Rosche W.A. Gellibolian R. Kang S. Shimizu M. Bowater R.P. Sinden R.R. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11019-11023Crossref PubMed Scopus (148) Google Scholar, 42Kramer P.R. Pearson C.E. Sinden R.R. Hum. Genet. 1996; 98: 151-157Crossref PubMed Scopus (38) Google Scholar,43Schweitzer J.K. Livingston D.M. Hum. Mol. Genet. 1997; 6: 349-355Crossref PubMed Scopus (88) Google Scholar). The mechanism of TRS expansion that is responsible for the hereditary neurological diseases has not been fully elucidated. However, the mechanism responsible for large expansions (scores or hundreds of TRS), such as for CTG·CAG in myotonic dystrophy and CGG·CCG for the fragile X syndrome, is DNA replication errors caused by the slipped register of the DNA complementary strands along with transient formation of hairpin loops and DNA polymerase pausing with primer relocation and strand elongation (11Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar, 12Kang S. Ohshima K. Jaworski A. Wells R.D. J. Mol. Biol. 1996; 258: 543-547Crossref PubMed Scopus (55) Google Scholar, 16Freudenreich C.H. Stavenhagen J.B. Zakian V.A. Mol. Cell Biol. 1997; 17: 2090-2098Crossref PubMed Scopus (201) Google Scholar, 17Maurer D.J. O'Callaghan B.L. Livingston D.M. Mol. Cell. Biol. 1996; 16: 6617-6622Crossref PubMed Scopus (109) Google Scholar, 44Richards R.I. Sutherland G.R. Nat. Genet. 1994; 6: 114-116Crossref PubMed Scopus (290) Google Scholar, 45Samadashwily G.M. Gordana R. Mirkin S.M. Nat. Genet. 1997; 17: 298-304Crossref PubMed Scopus (288) Google Scholar, 46Wells R.D. J. Biol. Chem. 1996; 271: 2875-2878Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). Also, genetic recombination is a robust and efficient way to achieve large expansions of CTG·CAG. 3J. Jakupciak and R. D. Wells, submitted for publication. Small expansions are also extremely important for certain types of inherited neurological diseases that involve polyglutamine expansions that are encoded by CTG·CAG repeats. Normal gene products seem to tolerate a rather wide variation in the size of a polyglutamine tract (between 10–35 glutamines without any detectable adverse effects beyond a threshold of 35–40 glutamines) in five of the eight known diseases. However, when the tracts are beyond this threshold, pathological properties are observed. For all of the eight diseases known to share this mechanism, a strong inverse correlation exists between the length of the polyglutamine tract and the age of onset of clinical symptoms; for each added glutamine residue beyond the threshold, on average, a 1.5–2-year earlier onset age is found (48Trottier Y. Zeder-Lutz G. Mandel J.-L. Wells R.D. Warren S.T. Genetic Instabilities and Hereditary Neurological Diseases. Academic Press, Inc., San Diego, CA1998: 447-453Google Scholar). Furthermore, in lymphoblastoid cell lines from myotonic dystrophy patients, two types of mutations of the expanded CTG·CAG repeat alleles were detected: frequent mutations that showed small changes in the repeat size and relatively rare mutations with large changes in the CTG·CAG repeat. We believe that the large changes in repeat size observed in this human cell line are due to replication or recombination as elucidated in simpler cells (11Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar, 12Kang S. Ohshima K. Jaworski A. Wells R.D. J. Mol. Biol. 1996; 258: 543-547Crossref PubMed Scopus (55) Google Scholar, 16Freudenreich C.H. Stavenhagen J.B. Zakian V.A. Mol. Cell Biol. 1997; 17: 2090-2098Crossref PubMed Scopus (201) Google Scholar, 17Maurer D.J. O'Callaghan B.L. Livingston D.M. Mol. Cell. Biol. 1996; 16: 6617-6622Crossref PubMed Scopus (109) Google Scholar, 44Richards R.I. Sutherland G.R. Nat. Genet. 1994; 6: 114-116Crossref PubMed Scopus (290) Google Scholar, 46Wells R.D. J. Biol. Chem. 1996; 271: 2875-2878Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). However, the molecular mechanism responsible for the small instabilities has not been identified and may be rather different from that involved with the large instabilities. As part of our ongoing program to elucidate the molecular events involved in genetic instabilities as related to human neurological diseases, we have investigated small slipped register genetic instabilities. These small expansions and deletions found in E. coli were studied optimally in the absence of certain repair functions (methyl-directed mismatch repair or nucleotide excision repair), since these activities would be expected to recognize the looped structures formed in the slipped TRS conformation. All plasmids used in these experiments containing repeating (CTG·CAG)n inserts are shown in Fig. 1. The inserts may also be designated (TGC·GCA)n or (GCT·AGC)n. pRW3248 is a derivative of pUC19 NotI and was described previously (10Jaworski A. Rosche W.A. Gellibolian R. Kang S. Shimizu M. Bowater R.P. Sinden R.R. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11019-11023Crossref PubMed Scopus (148) Google Scholar). The plasmid contains (CTG)175 as the leading strand template for replication, termed orientation I. This sequence is not homogenous but contains two G to A interruptions at repeats 28 and 69. pRW3297, pRW3296, and pRW3294 are derivatives of pUC19 containing different lengths of (CTG·CAG) in orientation I and were constructed and characterized in this laboratory. 4K. Ohshima, unpublished results. pRW3297 contains (CTG·CAG)73 and G to A interruptions at repeats 28 and 59; pRW3296 contains (CTG·CAG)57 and G to A interruptions at repeats 28 and 43; pRW3294 contains (CTG·CAG)42 and a G to A interruption at repeat 28. pRW3024 is a pUC19NotI-based plasmid containing the (CGG·CCG)24insert cloned into the BamHI site, where the CGG is in the lagging strand template (14Shimizu M. Gellibolian R. Oostra B.A. Wells R.D. J. Mol. Biol. 1996; 258: 614-626Crossref PubMed Scopus (74) Google Scholar). pRW3808 is a derivative of pUC18NotI (49Herrero M. de Lorenzo V. Timmis K.N. J. Bacteriol. 1990; 172: 6568-6572Crossref PubMed Google Scholar) containing the (GAA·TTC)176 fragment inserted into the BamHI site of the vector (50Ohshima K. Montermini L. Wells R.D. Pandolfo M. J. Biol. Chem. 1998; 273: 14588-14595Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). The plasmid was obtained by the in vivo expansion technique (11Kang S. Jaworski A. Ohshima K. Wells R.D. Nat. Genet. 1995; 10: 213-218Crossref PubMed Scopus (317) Google Scholar), and the GAA strand is in the leading strand template (50Ohshima K. Montermini L. Wells R.D. Pandolfo M. J. Biol. Chem. 1998; 273: 14588-14595Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar). pRW3832 and pRW3821 are derivatives of pSPL3 vector (51Church D.M. Stotler C.J. Rutter J.L. Murrel J.R. Trofatter J.A. Buckler J.A. Nat. Genet. 1994; 6: 98-105Crossref PubMed Scopus (270) Google Scholar) and contain (GAA·TTC)15 and (GAA·TTC)59, respectively. These plasmids were constructed and characterized in this laboratory.4 The following E. coli MMR mutator phenotype strains were used. KA796 (ara, thi, Δpro-lac) is a parent (wild type) of the MMR-deficient strains; NR8039 is isogenic with KA796 but is also mutH101; NR8040 is isogenic with KA796 but is also mutL101; and NR8041 is isogenic with KA796 but is also mutS101. These strains were the kind gift of Dr. R. Schaaper (NIEHS, National Institutes of Health, Research Triangle Park, NC) and were used previously (10Jaworski A. Rosche W.A. Gellibolian R. Kang S. Shimizu M. Bowater R.P. Sinden R.R. Wells R.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11019-11023Crossref PubMed Scopus (148) Google Scholar). To study the influence of the NER on the frequency of mutations of (CTG·CAG)n sequences, the following E. colistrains were used. AB1157 (thr-1, ara-14, leuB6, Δ(gpt-proA)62, lacY1, tsx-33, qsr' − , glnV44(AS), galK2, λ, arc − hisG4(Oc), rfbD1, mgl-51, rpsL31(Sm R ), kdgK51, xylA5, mtl1, argE3(Oc), thi-1) is a parent (wild type) of the NER-deficient strains; E. coli AB1886 is isogenic with AB1157 exceptuvrA6; E. coli AB1885 is isogenic with AB1157 except uvrB5; E. coli AB2421 is isogenic with AB1157 except uvrA6uvrB45. Strains AB1157, AB1886, and AB1885 were obtained from the E. coli Genetic Stock Center. E. coli AB2421 was a gift of Dr. E. Tang (University of Texas System Cancer Center Science Park-Research Division, Smithville, TX). For the recloning of the plasmids containing expansion and deletion products, we used E. coli HB101 (Life Technologies, Inc.): mcrB, mrr, hsdS20, (rB− , mB− ), recA13, supE44, ara14, galK2, lacY1, proA2, rplS20 (SmR), xyl5, λ − , leuB6, mtl-1. The plasmids containing various lengths of repeating (CTG·CAG) sequences were transformed into the appropriate E. coli strain and grown for a number of generations, as described (9Bowater R.P. Rosche W.A. Jaworski A. Sinden R.R. Wells R.D. J. Mol. Biol. 1996; 264: 82-96Crossref PubMed Scopus (64) Google Scholar). Briefly, E. coli cells were transformed with plasmids, and an aliquot of this mixture was inoculated into 10 ml of LB containing ampicillin at 100 μg/ml. Incubations of the liquid cultures were continued overnight at 37 °C at a shaking rate of 250 rpm. The bacteria then were subcultured into fresh liquid media with a dilution factor of 107. The cells from each culture were harvested, and plasmids were isolated. Plasmids with deletions in the (CTG·CAG)175 insert were obtained as follows. TheE. coli parental MMR proficient strain and themutS, mutL, and mutH mutants harboring pRW3248 were grown for approximately 100 generations. Plasmids were isolated and cleaved with SacI and HindIII to release the triplet repeat-containing inserts, which were analyzed by PAGE at room temperature (data not shown). Bands corresponding to fragments with fewer than 175 triplet repeat units were purified from the gels and recloned into theSacI/HindIII-linearized pUC19 NotI. The ligated DNA was electroporated into E. coli HB101. ThisE. coli strain was chosen because it provided greater stability for the TRS-containing plasmids. Cells were then spread on ampicillin plates. In order to minimize further deletions in the inserts, single colonies were grown to early logarithmic phase (A600 ≤ 0.7) (9Bowater R.P. Rosche W.A. Jaworski A. Sinden R.R. Wells R.D. J. Mol. Biol. 1996; 264: 82-96Crossref PubMed Scopus (64) Google Scholar) in LB medium containing 100 μg/ml of ampicillin, and plasmids were isolated. These TRS instabilities were experienced in the primary MMR-proficient and -deficient strains rather than in the secondary cloning in E. coli HB101; only one of the 25 clones analyzed from the wild type cells (Tables Table I, Table II, Table III) showed any small instabilities. The number of triplet repeats and the positions of the interruptions in these clones were determined by DNA sequencing using the dideoxy chain termination method and by restriction analyses. The same technique was used to analyze the repeat composition of (CTG·CAG)73 from pRW3297. pRW3294 and pRW3296 were very stable and contained only nondeleted (CTG·CAG) inserts.Table IRepeat composition of deletion products of (CTG · CAG)175 in wild type and MMR-deficient strainsCloneNumber of repeats in the insertPosition of interruption(s)Number of pure repeats between interruptionsObserved changes in repeat compositionExpansionsDeletionsabcabcWild type 1∼15028, 6940∼25 210528, 694070 310128, 694074 4,5>7028<105 645130* 741134* 840135* 939136* 1035139* 11–1432143* 1527148* 1626149*mutL 1713628, 32, 723, 394142 1813328, 7748850 198928, 7546692 20812894 216528110 2251124* 2349126* 2443132*mutS 25>13628, 68391<39 268828, 7546693 2769271105 286928106 296728108 305028125 3149271125mutH 3214028, 32, 723, 394138 33, 34>13628, 6940<39 3513328, 6839141 36, 376828107 386528110 3965291111 406128114 416028115 425828117 4349126*Forty-three clones harboring deleted inserts were isolated from the MMR-proficient strain (wild-type), as well as the mutL,mutS, and mutH strains (see “Experimental Procedures”). The number of repeats found by DNA sequencing for each of the clones is tabulated in the second column. The quality of the sequencing data enabled the precise determination of the number of repeats except for the cases otherwise designated (clones 1, 4, 5, 25, 33, and 34). However, in all cases, the locations of the interruptions, presented in the third column, were unambiguous. The fourth column lists the number of CTG units between two or more interruptions. The size and location of the expansions and deletions are shown in the fifth column, where a represents the region from the 1st to the 28th triplet repeat, b from the 29th to 69th, and c from the 70th to 175th. Expansions and deletions within these deletion products occurred within the region specified (numbers under a, b, or c) as well as in region b plus c (numbers between letters b and c) and in a to c (numbers with asterisks). Open table in a new tab Table IIRepeat composition of clones of (CTG · CAG)73 in wild type and MMR-deficient strainsCloneStrainNumber of repeats in the insertPosition of interruptionsNumber of pure repeats between interruptionsObserved changes in repeat compositionExpansionsDeletionsabcabc44Wild type7328, 593045mutS7328, 593046mutL7328, 58291147mutL6627, 57291548mutL6419, 5030949mutL6328, 59301050mutL5612, 422916151mutL5510, 413018Eight clones harboring full-size or deleted inserts were isolated from the MMR-proficient strain (wild type), as well as the mutSand mutL mutants. The size and location of the deletions and the expansion are shown in the sixth column, where a represents the region from the 1st to the 28th triplet repeat, b from the 29th to 59th, and c from the 60th to 73rd. Deletions and the expansion occurred within the regions specified (numbers under a, b, or c). Open table in a new tab Table IIIRepeat composition of (CTG · CAG)175 clones in wild type and NER-deficient strainsCloneNumber of repeats in the insertPosition of interruption(s)Number of pure repeats between interruptionsObserved changes in repeat compositionExpansionsDeletionsabcabcWild type 52–55∼17528, 6940 5617228, 66373 5713428, 69, 11040, 404182 586228113 5925150*uvrA 60–65∼17528, 6940uvrB 66–69∼17528, 6940 7017427, 68401 7117326, 67402 7217026, 674023 73, 74∼12028, 694055 75842891uvrA uvrB 76–78∼17528, 6940 7917028, 69405 8067108* 8130145*Thirty clones harboring full-size and deleted inserts were isolated from the NER-proficient strain (wild type) as well as theuvrA, uvrB, and uvrA uvrB strains (see “Experimental Procedures”). The quality of the sequencing data enabled the precise determination of the number of repeats except for the cases designated otherwise (clones 52–55, 60–69, 73, 74, and 76–78). However, in all cases, the location of the G to A interruptions was unambiguous. The fourth column lists the number of CTG units between two or more interruptions. The size and location of the expansions and deletions is shown in the fifth column, where a represents the region from the 1st to the 28th triplet repeat, b from the 29th to 69th, and c from the 70th to 175th. An expansion and the deletions occurred within the specified regions (numbers under a, b, or c) as well as in the region b plus c (numbers between letters b and c) and a to c (numbers with asterisks). Open table in a new tab Forty-three clones harboring deleted inserts were isolated from the MMR-proficient strain (wild-type), as well as the mutL,mutS, and mutH strains (see “Experimental Procedures”). The number of repeats found by DNA sequencing for each of the clones is tabulated in the second column. The quality of the sequencing data enabled the precise determination of the number of repeats except for the cases otherwise designated (clones 1, 4, 5, 25, 33, and 34). However, in all cases, the locations of the interruptions, presented in the third column, were unambiguous. The fourth column lists the number of CTG units between two or more interruptions. The size and location of the expansions and deletions are shown in the fifth column, where a represents the region from the 1st to the 28th triplet repeat, b from the 29th to 69th, and c from the 70th to 175th. Expansions and deletions within these deletion products occurred within the regio" @default.
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• W2159203886 date "1998-07-01" @default.
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• W2159203886 title "Small Slipped Register Genetic Instabilities in Escherichia coli in Triplet Repeat Sequences Associated with Hereditary Neurological Diseases" @default.
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• W2159203886 doi "https://doi.org/10.1074/jbc.273.31.19532" @default.
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