Senescence and emergence of survivors in trt1- cells. (a) Growth curves. YES cultures (200 ml) were inoculated at 2.5 × 104 cells/ml with either trt1+ or trt1- cells. Cell density is shown for trt1+ cells (open circles) and trt1- cells (filled squares) at the end of each 24-h period, after which a new culture was inoculated at 2.5 × 104 cells/ml. When cells were counted on day 1, they had already undergone about 45 generations after germination. Note that when the culture density reached 3-5 × 106 cells/ml, a portion of the cells was harvested for microarray analysis and Southern hybridization. Cells appeared enlarged near day 8 and were morphologically normal by day 11. (b) Restriction-enzyme sites in the TAS of one chromosome arm cloned into the plasmid pNSU70 . Locations of the probes used for Southern hybridization are indicated by the bottom bars. These probes hybridize to multiple chromosome arms because the TASs are found on the four arms of chromosomes I and II and, depending upon the strain background, on one or both arms of chromosome III. (c) Telomere length in wild-type and trt1- strains from the growth curve. DNA (~15 μg) was digested with EcoRI, subjected to electrophoresis, transferred to a nylon membrane and probed with the 32P-labeled telomere fragment shown in (b) that was expected to report the state of the telomere end. As a loading control, a probe for the single-copy gene pol1+ was included. Signals arising from the telomeres are labeled. (d) As in (c), but DNA was digested with HindIII and the blot probed with TAS2 and a fragment of pol1+. The TAS2 probe was expected to hybridize to sequences at least 2 kb, and up to 6 kb, from the telomere end.