FIG. 7.

Increased telomere length of the est2^LT alleles is dependent on TEL1 but independent of YKU70, RIF1, and RIF2. (A) Epistasis analysis of yku70Δ and est2^E76K. Gene replacement was used to generate strains with YKU70 (yku70::KAN) deleted and expressing either wild-type (WT) EST2 (YKF203; lane 5) or est2^E76K (YKF203-E76K; lanes 3 and 4) from the endogenous, chromosomal locus. After isolation of the appropriate yku70 disruption, the est2^E76K yku70::KAN strain was restreaked nine times to confirm that telomere length had stabilized. DNA isolated after restreak 1 (lane 3) and restreak 9 (lane 4) is shown. Telomere length was visualized by Southern blotting of PstI-digested genomic DNA with a telomeric probe. DNA isolated from isogenic YKU70 strains expressing wild-type EST2 (YKF201, lanes 1 and 6) or est2^E76K (YKF201-E76K, lane 2) was included for comparison. The positions of molecular weight markers are indicated (M). (B) Epistasis analysis of tel1Δ and est2^E76K. Gene replacement was used to generate strains with tel1 deleted (tel1::KAN) and expressing either wild-type EST2 (YKF202; lanes 9 and 17) or est2^E76K (YKF202-E76K; lanes 3 to 8 and lanes 11 to 16) from the endogenous, chromosomal locus. Because the telomere length phenotype of tel1Δ takes many generations to reach maximum expression, newly transformed strains were sequentially restreaked on plates. For the double mutant, two independent transformants were restreaked and DNA was prepared from restreaks 1, 3, 5, 7, 9, and 10 as indicated. Only restreak 10 of the tel1Δ strain is shown. Genomic DNA was digested with PstI, and telomere length was measured by Southern blotting. DNA isolated from isogenic TEL1 strains expressing wild-type EST2 (YKF201, lanes 1 and 18) or est2^E76K (YKF201-E76K, lanes 3 and 10) was included for comparison. The positions of molecular weight markers are indicated (M). (C) Epistasis analysis of rif1Δ and rif2Δ with est2^E76K. Gene replacement was used to generate strains with rif2 deleted (rif2::KAN) and expressing either wild-type EST2 (YKF205; lane 3) or est2^E76K (YKF205-E76K; lane 4) from the endogenous, chromosomal locus. Strains with rif1 deleted (rif1::KAN) and expressing wild-type EST2 (YKF204; lane 5) or est2^E76K (YKF204-E76K; lane 6) from the chromosomal locus were also created. Genomic DNA was digested with XhoI, and telomere length was measured by Southern blotting. DNA isolated from isogenic RIF1 and RIF2 strains expressing wild-type EST2 (YKF201, lane 1) or est2^E76K (YKF201-E76K, lane 2) was included for comparison. The positions of molecular weight markers are indicated (M).