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+ transformants and analyzed by Southern blots for correct replacement and CTG tract length. Expansions must have occurred during or soon after transformation, because the vast majority of cells in the transformation colony had the expansion (Fig. 1B). Although we were unable to identify any single parameter of the transformation protocol that enhanced expansion, expansions were obtained in three of five independent transformations.
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20
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note
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R colony formation were determined by the method of the median (24). Values from starting colonies that had shortened tracts, as determined by Southern hybridization, were dropped.
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2 when grown at 37°C (26, 27). Unless otherwise indicated, rad27Δ strains were constructed and maintained at 25°C to minimize the rad27Δ phenotype.
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25
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6844264905
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note
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For pulsed-field gel analysis, isogenic strains lacking a CTG tract or with the CTG-250 tract were grown to early log phase in YC-Ura media to maintain the tract, then switched to complete media containing 0.05 M HU and grown for about two more doublings (∼18 hours). Cells were harvested, and chromosomes were prepared as in (28) and separated in a Bio-Rad apparatus. The 355-and 470-kb breakage products were observed in three of three experiments.
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For colony PCR of CTG tracts, a small number of cells was taken from individual colonies, resuspended in the PCR mix (Advantage-GC genomic PCR kit, Clontech, Palo Alto, CA), and heated at 94°C for 5 min. Primers (1 pmol/μl) were homologous to sequence flanking the CTG tract: T7 sequence (20-oligomer) and CTG-rev primer (GTG-GAGGATGGAACACGG). Cycle conditions were 94°C for 1 min, 54°C for 1 min, and 68°C for 3 min, for 35 cycles. To confirm the accuracy of the PCR data, we isolated DNA from a subset of colonies used for PCR and analyzed the tract length by Southern hybridization (33 were analyzed for CTG-40, 43 for CTG-70).
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note
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We thank R. Kolodner for his suggestion that we analyze CTG tracts in a rad27Δ strain; R. Wellinger and J. Stavenhagen for sharing unpublished data; and J. Haber, T. Vogt, L. Sandell, V. Schulz, and members of the Zakian lab for helpful comments. Supported by NIH grants GM26938 and GM43265. C.H.F. was supported by NIH postdoctoral fellowship AG05740-02 and S.M.K. by a grant from the New Jersey State Cancer Commission.
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