The mak11-2 ts mutant shows alterations in 25S synthesis, cell cycle, and preribosome export. (A) Growth curves at 37°C for the mak11-2 mutant (open circles) compared with a wild-type strain (crosses). The number of cells was estimated using the absorbance of the culture at 600 nm. (B) The cell cycle distribution of yeast cells was estimated by FACS analysis. Wild-type and mak11-2 haploid cells in rich medium were shifted from 27°C to 37°C for 0, 1, 3, and 6 h; aliquots were fixed with 70% ethanol and stained with propidium iodide. The ratios of cells having 1n DNA content to those having 2n DNA content were estimated in triplicate experiments (wild-type strain, white bars; mak11-2 strain, gray bars); error bars are standard deviations of the measured ratios. (C) Total RNA was extracted from a wild-type strain or the mak11-2 strain at time zero and 4 and 12 h after shift to the nonpermissive temperature, separated on a 1% agarose gel (glyoxal denaturation), and stained with ethidium bromide. (D) GFP fluorescence of the mak11-2 ts or isogenic wild-type cells, each type expressing Rpl25-eGFP, was detected after a shift to 37°C for 8 h. DNA was stained with Hoechst 33342. Arrowheads indicate the relative positions of the DNA-stained regions of the nuclei. wt, wild type.
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