FIGURE 1.

Loss of telomerase activity causes immediate death of yeast cells with terminal vertebrate telomeric repeats. A, strains in which either the WT or the tlc1h allele of TLC1 are controlled by the tetO promoter (respectively tetO₂-TLC1 or tetO₂-tlc1h) and that also contained a centromeric plasmid, as indicated, were plated on selective media on which the tetO-controlled gene was expressed (left panels) or repressed (right panels). Note that the alleles on the plasmid are not controlled by tetO. B, cells with deletions of both EST2 and TLC1 (est2::HIS3 tlc1::KanMX4) contained either a WT TLC1 (left panels) or the allele templating vertebrate repeats (tlc1h) on a plasmid (right panels). For viability, all strains initially also contained a WT EST2 gene on a pRS316(URA3) plasmid as positive controls (bottom panels). Finally, strains contained pRS415(LEU2) plasmids harboring the EST2 alleles as indicated. Exponentially growing cultures were diluted by steps of 10-fold, and an equal number of cells were spotted onto plates. Cells on the top plates only harbor the EST2 alleles on the pRS415 plasmid as indicated on top (rightmost panel; lanes 5 and 10, negative control, empty pRS415; lanes 4 and 9, positive control with pRS415-EST2wt). Pictures were taken after incubating the plates for 3 days at 30 °C. Note that for the plates showing cells after pRS316-EST2 loss, only the row with the highest cell number is shown (top panels). C, genomic DNA was extracted from the above strains cultivated in synthetic complete drop-out media, digested with XhoI, and analyzed by Southern blotting using a telomere Y'-specific probe. All strains do harbor the WT allele of EST2 (as indicated with the pRS316-EST2 plasmid) and also express a second allele on the pRS315 plasmid as indicated on top of the lanes. Lanes 1–5, DNA derived from strains that express the WT allele of TLC1. Note the telomere elongation with the Cdc13-EST2 fusion protein (compare lanes 1 and 4). Lanes 6–10, DNA derived from strains that express the tlc1h allele. Note a slight telomere elongation with the Cdc13-EST2 fusion protein (compare lanes 6 and 9).