FIG. 1.
(A) Identification of a TS allele of YKU70. The entire YKU70 gene was randomly mutagenized, and the mutant alleles were expressed in a yku70Δ strain carrying the ADE2 and URA3 genes as reporter genes for telomere position effect (strain UCC3505/yku70Δ). Cells containing an empty vector (yku70Δ) or the vector containing the wt YKU70 gene (pKu70; YKU70) were used as controls for silencing status and growth at high temperature. Patches of cells were grown at 23°C on SC-Trp and then rereplica plated on the same medium at 37°C (ADE2 assay, top), or on FOA-Trp at 23 and 37°C (URA3 assay, bottom) and incubated for 3 days. (B) yku70-30ts cells grown at 37°C exhibit telomeric single-stranded G-rich DNA. Strain BYyku70Δ transformed with the indicated plasmids was grown in liquid culture at 23°C and then shifted to 37°C for 8 h. Genomic DNA was extracted and digested with XhoI, and telomeres were analyzed for G tails by nondenaturing in-gel hybridization with a CA oligonucleotide probe (top). To control for DNA loading, the DNA was denatured in the gel and rehybridized to the same probe (bottom). 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, as described previously (21).