Myotonic dystrophy type 1 (DM1) is caused by the expansion of a (CTG).(CAG) repeat in the DMPK gene on chromosome 19q13.3. At least 17 neurological diseases have similar genetic mutations, the expansion of DNA repeats. In most of these disorders, the disease severity is related to the length of the repeat expansion, and in DM1 the expanded repeat undergoes further elongation in somatic and germline tissues. At present, in this class of diseases, no therapeutic approach exists to prevent or slow the repeat expansion and thereby reduce disease severity or delay disease onset. We present initial results testing the hypothesis that repeat deletion may be mediated by various chemotherapeutic agents. Three lymphoblast cell lines derived from two DM1 patients treated with either ethylmethanesulfonate (EMS), mitomycin C, mitoxantrone or doxorubicin, at therapeutic concentrations, accumulated deletions following treatment. Treatment with EMS frequently prevented the repeat expansion observed during growth in culture. A significant reduction of CTG repeat length by 100-350 (CTG).(CAG) repeats often occurred in the cell population following treatment with these drugs. Potential mechanisms of drug-induced deletion are presented.

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Antigen receptor gene rearrangements are initiated by the RAG1/2 protein complex, which recognizes specific DNA sequences termed RSS (recombination signal sequences). The RAG recombinase can also catalyze transposition: integration of a DNA segment bounded by RSS into an unrelated DNA target. For reasons that remain poorly understood, such events occur readily in vitro, but are rarely detected in vivo. Previous work showed that non-B DNA structures, particularly hairpins, stimulate transposition. Here we show that the sequence of the four nucleotides at a hairpin tip modulates transposition efficiency over a surprisingly wide (>100-fold) range. Some hairpin targets stimulate extraordinarily efficient transposition (up to 15%); one serves as a potent and specific transposition inhibitor, blocking capture of targets and destabilizing preformed target capture complexes. These findings suggest novel regulatory possibilities and may provide insight into the activities of other transposases.

The generation of long uninterrupted DNA repeats is important for the study of repeat instability associated with several human genetic diseases, including myotonic dystrophy type 1. However, obtaining defined lengths of long repeats in vitro has been problematic. Strand slippage and/or DNA secondary structure formation may prevent efficient ligation. For example, a purified (CTG)140.(CAG)140 repeat fragment containing 4-bp AGCA/TGCT overhanging ends ligated poorly using T4 or Escherichia coli DNA ligase, although limited repeat ligation occurred using thermostable DNA ligase. Here we describe a general procedure for ligating multimers of DNA repeats. Multimers are efficiently ligated when slippage is prevented or when DNA repeats contain a single G/C overhang. A cloning vector is designed from which pure repeat fragments containing a G/C overhang can be generated for further ligation. (CAG)n.(CTG)n DNA molecules longer than 800 bp were generated using this approach. This approach also worked for (GAA)n.(TTC)n, (CCTG)n-(CAGG)n, and (ATTCT)n.(AGAAT)n tracts associated with Friedreich ataxia, DM2, and spinocerebellar ataxia type 10, respectively.

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(CAG) n ⅐(CTG) n expansion is associated with many neurodegenerative diseases. Repeat instability has been extensively studied in bacterial plasmids, where repeats undergo deletion at high rates. We report an assay for (CAG) n ⅐(CTG) n deletion from the chloramphenicol acetyltransferase gene integrated into the Escherichia coli chromosome. In strain AB1157, deletion rates for 25-60 (CAG)⅐(CTG) repeats integrated in the chromosome ranged from 6.88 ؋ 10 ؊9 to 1.33 ؋ 10 ؊10 , or ϳ6,300 to 660,000-fold lower than in plasmid pBR325. In contrast to the situation in plasmids, deletions occur at a higher rate when (CTG) 43 , rather than (CAG) 43 , comprised the leading template strand, and complete rather than partial deletions were the predominant mutation observed. Repeats were also stable on long term growth following multiple passages through exponential and stationary phase. Mutations in priA and recG increased or decreased deletion rates, but repeats were still greatly stabilized in the chromosome. The remarkable stability of (CAG) n ⅐(CTG) n repeats in the E. coli chromosome may result from the differences in the mechanisms for replication or the probability for recombination afforded by a high plasmid copy number. The integration of (CAG) n ⅐(CTG) n repeats into the chromosome provides a model system in which the inherent stability of these repeats reflects that in the human genome more closely.

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The plasmid profiles of 65 strains of Proteus penneri were analyzed to determine whether resistance was determined chromosomally or by plasmids. Only seven strains harboured one to three plasmids, although these strains exhibited resistance to a wide range of antibiotics. Markers for ampicillin and tetracycline resistance could be transferred to Escherichia coli by transformation. Plasmids carried resistance to chloramphenicol in two strains and resistance to sulfonamides in one strain. The result showed that resistance is determined chromosomally rather than by plasmids, however the possibility that these bacteria may acquire resistance plasmids which change their antibiotic susceptibility pattern cannot be excluded.