Article Title: The Length of the Shortest Telomere as the Major Determinant of the Onset of Replicative Senescence
Figure Lengend Snippet: Telomere length distribution by simulation and single-telomere Southern blot. (A) The protein-counting mechanism is based on the measurement of telomere length by Rap1/Rif1/Rif2 binding to telomeric repeats and an inhibition of telomerase elongation depending on the number/concentration of these complexes. (B) Trajectories of 10 independent telomeres over 100 divisions. Depending on the length, a telomere (blue trajectory) can shorten by length a over several consecutive divisions (solid arrowhead) and then be elongated by a random length b (shaded arrowhead). (C) Telomere length distribution at equilibrium. Equation 1 was iterated 500 times starting with 100,000 telomeres drawn from an uniform distribution between 200 and 400 bp, and the resulting telomere length distribution was plotted (bin size, 3 bp). (D) Representative single-telomere Southern blot. Six independent TLC1 wild-type spores derived from TLC1/tlc1 Δ heterozygous diploids were grown for 24 hr on YPD plates and then transferred to liquid culture for another 24 hr at exponential growth (for a total of ∼30 population doublings). The Southern blot was performed on Nde I- and Bst EII-digested genomic DNA using simultaneously oT355 (green) and oT360 (red) fluorescent probes, designed to detect I-L and VI-R telomeres, respectively. (E) Single-telomere Southern blot with oT355 and oT360 fluorescent probes performed on telomerase-positive (wild type, WT) or negative ( tlc1 Δ) cells. TLC1/tlc1 Δ diploid cells were sporulated, and the four spores from a tetrad were grown for 24 hr on a plate and then in liquid YPD medium for the total indicated population doublings (PD). (F) Comparison of experimental and simulated data of telomere length distribution. Plot of 117 experimental measurements of telomere length from single-telomere Southern blot as in D (black line, bin size, 20 bp) and 1000 simulated values L i ¯ ∈ [ | 1 : 1000 | ] (colored lines, bin size, 20 bp) for 20, 30, …, 70 divisions. The latter were obtained as follows: after drawing randomly 100 initial lengths L i 0 from the theoretical distribution in C, we applied the dynamics of Equation 1 to 1000 telomeres with initial length L i 0 to get, after 50 divisions, a mean length L i ¯ . This was done for each plot, corresponding to 20, 30,…, or 70 divisions (blue to orange lines).
Article Snippet: Nde I, Bst EII, and Bst NI enzymes were purchased from New England Biolabs.
Techniques: Southern Blot, Binding Assay, Inhibition, Concentration Assay, Derivative Assay