Figure 1. Mismatch repair increases the occurrence of large deletions from (CTG·CAG) repeats in E. coli. Plasmids containing (CTG·CAG) tracts were transformed into E. coli strains KA796 (MMR⁺) and NR8041 (mutS⁻) and grown overnight on LB-agar plus ampicillin (100 μg/ml) at 37 °C. After growth of liquid cultures as specified, DNA was purified, and the length of the triplet-repeat was determined by electrophoresis on 1.25 % agarose in TAE. Gels were photographed after staining with ethidium bromide. 1 kbp refers to a 1 kbp DNA ladder. (a) Single colonies of bacteria transformed with pRW3248 (containing (CTG·CAG)₁₇₅{28, 69}) were inoculated into 5 ml of LB plus ampicillin (100 μg/ml) and gro?wn overnight at 37 °C and a shaking rate of 200 rpm. Supercoiled DNA was electrophoresed on the agarose gel. DNA fragments migrating at different mobilities have deletions within the TRS. (b) Plasmids pRW4026, pRW4027, pRW3246, pRW3230, pRW3218 and pRW3248 were transformed and approximately 1000–10,000 transformants were obtained after overnight growth on each plate. Colonies were washed from the agar plate and an aliquot was grown overnight in 5 ml of LB plus ampicillin (100 μg/ml) at 37 °C and a shaking rate of 200 rpm. DNA was digested with BglI before electrophoresis. After digestion, the (CTG·CAG) tract is contained within the larger fragments (approximately 1800–2100 bp), and deletions from the repeat migrate faster than the main band.

Table 1. Plasmids used in this study

The top strand of each duplex sequence is shown. Sequences are shown for the equivalent top strand of pUC18NotI, with the 5′-end of the insert closest to bp numbered 1 [Bowater et al 1996]. The 5′-ends are on the left.

Note that pRW3247 and pRW3248 contain exactly the same repeat tract in opposite orientations with respect to the direction of replication. Plasmids with the CTG strand on the leading strand template (pRW3248) are defined as orientation I, and those with the CAG strand as the leading strand template (pRW3247) are defined as orientation II [Kang et al 1995 and Bowater et al 1996]. For the nomenclature of the TRS (third column), the numbers in the brackets designate the location(s) of G to A interruptions; if no bracketed numbers are shown, the repeat tract does not contain interruptions.