FIG. 2.
(A) Telomerase does not contribute significantly to the creation of G-rich overhangs in Yku− mutants. Strain SGY51 (yku70Δ tlc1Δ pKu70-30ts) and the same strain carrying in addition a plasmid-borne copy of TLC1(pAZ1) were grown at 23°C and then shifted for 8 h to 37°C. DNA was extracted, digested with XhoI, and analyzed by in-gel hybridization as described for Fig. 1B. Top, native gel; bottom, the same gel hybridized after denaturation. Lane M, end-labeled 1-kb ladder DNA serving as a size standard; lanes ss and ds, DNA with telomeric TG1-3 repeats in single-stranded and double-stranded forms, respectively. Note that in yku70-30ts cells telomere shortening occurs during the 30 generations of growth at 23°C that were necessary to produce the yku70-30ts tlc1Δ strain. (B) Telomere length analysis for pol1-17 yku70Δ double mutants. Strain SGY56 (MATa/α pol1-17/POL1 yku70Δ::URA3/YKU70) was sporulated and microdissected. Spores derived from a tetratype were grown, and total genomic DNA was extracted and digested with XhoI. The denatured gel from an in-gel analysis using the CA probe is shown. Lane M, end-labeled 1-kb ladder DNA serving as a size standard; lane 1, yku70Δ pol1-17 cells grown at 23°C; lane 2, the same strain as in lane 1 after a shift to 30°C for 16 h; lane 3, YKU70 POL1 wt cells grown at 23°C; lane 4, the same strain as in lane 3 after a shift to 30°C for 16 h; lane 5, yku70Δ POL1 cells grown at 23°C; lane 6, the same strain as in lane 5 after a shift to 30°C for 16 h; lane 7, YKU70 pol1-17 cells grown at 23°C; lane 8, the same strain as in lane 7 after a shift to 30°C for 16 h.