Quantitative Fitness Analysis Shows That NMD Proteins and Many Other Protein Complexes Suppress or Enhance Distinct Telomere Cap Defects
A) Transcript levels of four telomere-binding factors measured in upf2Δ and ebs1Δ mutants. Four strains of each genotype were grown exponentially in liquid culture at 23°C. RNA was isolated and transcript levels were determined by SYBR Green RT-PCR. Each measurement was performed in triplicate and error bars indicate standard deviation from four independent measurements. RNA concentrations of the samples were normalized to the loading control BUD6. A single wild type sample was given the value of 1 and all other values were corrected relative to this. Strains measured are 640, 2824, 3001, 4763, 4764, 4765, 4766, 4780, 4781, 4782, 4783 and 4784; B) Western blot analysis of Stn1 protein levels using antibodies against Stn1-13Myc tagged strains. Strains shown are 5757, 5758, 5759, 5760, 5761, 5763, 5764, 5765 and 5766; C) Growth analysis of yku70Δ or cdc13-1 mutants over-expressing STN1 using the centromeric plasmid pVl1045. The empty vector Ycplac111 was used as a control. Strains 5046, 5047, 5051 and 5052 were spot tested on –LEU medium; D) upf2Δ and ebs1Δ mutants were combined with yku70Δ or cdc13-1 mutations in the W303 genetic background and assessed for growth by spot test. Strains shown are 640 and 3001 (wild type), 2764 and 2824 (ebs1Δ), 2787 and 4309 (yku70Δ), 2889 and 2890 (ebs1Δ yku70Δ), 5007 and 5008 (nmd2Δ yku70Δ), 5251 and 5242 (ebs1Δ nmd2Δ yku70Δ), 1195 and 4557 (cdc13-1), 4576 and 4577 (ebs1Δ cdc13-1), 4624 and 4625 (nmd2Δ cdc13-1), 5238 and 5239 (ebs1Δ nmd2Δ cdc13-1); E) ChIP analysis of Est2-13Myc association to the VI-R telomere and the internal locus PDI1 on Chromosome III using primers previously described . Duplicate cultures were grown and harvested in exponential phase. Individual ChIP samples were measured in triplicate and group means are shown with 95% confidence bars derived from a two-way ANOVA. Strains shown are 6977 (Est2-13Myc), 6978 (nmd2Δ Est2-13Myc), 6979 (nmd2Δ yku70Δ Est2-13Myc) and 6980 (yku70Δ Est2-13Myc).