FIG. 4.

Effects of alanine substitutions in the Spt5 CTD. (A) S. pombe strains with the indicated chromosomal spt5-(1-800)-CTD alleles—in which 7 or 8 wild-type or mutated CTD nonamer repeats were fused to Spt5-(1-800)—were grown in liquid medium until the A₆₀₀ reached 0.3 to 0.5. The cultures were adjusted to equalize the A₆₀₀, and aliquots of serial 5-fold dilutions were spotted on YES agar medium. The plates were photographed after incubation for 8 days at 18°C, 5 days at 20°C, 3 days at 25 and 37°C, or 2 days at 30 and 32°C. WT, wild type. (B) Western blot analysis of Spt5. Whole-cell extracts of the indicated spt5-CTD strains were resolved by SDS-PAGE. The polypeptides were transferred to a membrane and probed by serial Western blotting with affinity-purified polyclonal anti-Spt5 antibody (top panel) and then with anti-Cdc2-p34 (PSTAIRE) antibody as a loading control (bottom panel). (C) Morphological phenotypes of spt5-CTD mutants. Cultures of the indicated spt5-CTD mutant strains were grown to mid-logarithmic phase at 30°C, and 300 to 500 individual cells were measured and sorted into the length bins specified. The percentage of cells in each bin is represented in the bar graph. (D) Effects of a phosphomimetic T1E change. Aliquots (3 μl) of serial 5-fold dilutions from exponentially growing cultures of the indicated strains were spotted onto YES agar medium. The plates were photographed after incubation for 8 days at 18°C, 6 days at 20°C, or 2.5 days at 30, 34, and 37°C.