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. 2011 Jan 10;31(6):1263–1274. doi: 10.1128/MCB.00831-10

FIG. 3.

FIG. 3.

Targeting Est1 to telomeres suppresses synthetic lethality of YKU80 and EST2 double deletion. (A) Growth analyses of the yku70Δ or yku80Δ mutants. The pRS415-CDC13-EST2 or pRS415 plasmid was transformed into the yku70Δ or yku80Δ cells. Five-fold serial dilutions were spotted on Leu− medium to select for the presence of plasmid at 30°C and 37°C, respectively. (B) Growth analyses of the yku70Δ mutant. The plasmid pRS415-CDC13-EST1 (CDC13-EST1) was transformed into the yku70Δ cells. Five-fold dilutions were spotted on the medium (Leu−) at 30 and 37°C, respectively. The parallel transformation and growth analyses were done with pRS315-YKU70 and pRS315 vector alone for controls. (C) Western blot analyses of Cdc13-Est1 and Cdc13-Est2 expression. pRS415 vector, pRS415-Myc-CDC13-EST1, or pRS415-Myc-CDC13-EST2 plasmid was transformed into YPH499 cells, and the total cell lysate was subjected to Western blot analyses with anti-Myc antibody (α-Myc) (upper panel). A nonspecific band shown in the same film was set as loading control (LC) (lower panel). (D) Growth analyses of the est2Δ yku80Δ cells in the presence of CDC13-EST1 fusion expression. The est2Δ/pRS316-EST2/pRS415, yku80Δ est2Δ/pRS316-EST2/pRS415, yku80Δ est2Δ/pRS316-EST2/pDBL-CDC13 (63), est2Δ cdc13Δ/pRS316-EST2/pRS415-CDC13-EST1, and yku80Δ est2Δ cdc13Δ/pRS316-EST2/pRS415-CDC13-EST1 haploid cells were generated from tetrad dissection. Five-fold serial dilutions were spotted at 30°C on Leu− Ura− medium to select for the presence of both pRS415-CDC13-EST1 (or pRS415) and pRS316-EST2 plasmids (left panel) and on Leu− 5′-FOA+ medium (right panel) that selected for eviction of pRS316-EST2 plasmid. (E) QAOS analysis of the est2Δ yku80Δ mutant. Strains generated from the 5′-FOA plate described in panel D were cultured at 30 or 37°C as described. Genomic DNA was extracted by the Zymolyase digestion method and monitored by QAOS analysis. The single-stranded DNA of the TG strand at the genomic locus of 600 bp from the right telomere in chromosome V was measured (3), and the error bars represent the standard deviation (SD) from three independent experiments. (F) Liquid cell viability assay for the est2Δ yku80Δ mutant. est2Δ and est2Δ yku80Δ/pRS415-CDC13-EST1 cells were grown in selective medium to saturation, and each culture was diluted to an OD600 of 0.05 every 24 h. The total cell number was monitored. The error bars represent the results from two independent clones. (G) Analysis of telomere length of est2Δ yku80/pRS415-CDC13-EST1 cells. est2Δ and cdc13Δ est2Δ yku80Δ/pRS415-CDC13-EST1 haploid cells were passaged in liquid medium for the indicated time in days and collected for telomere Southern blot analysis using a TG1-3 telomeric probe.