Effects of H3 depletion on cell growth and rRNA synthesis. (A) NOY2022 and NOY2025 were grown in SG complete medium to a cell density of A₆₀₀ 0.06-0.08, glucose was then added to a final concentration of 2%, and incubation was continued for 4 h. Cell density increase was followed (Left). At indicated times, aliquots were taken, cells were pulse-labeled with [³H]uridine (5 μCi/ml; 39 Ci/mmol; 1 Ci = 37 GBq) for 5 min, and incorporation into a total trichloroacetic acid-insoluble RNA fraction was determined (Right). (B) In another experiment similar to above, cells were pulse-labeled with [methyl-³H]methionine (33 μCi/ml; 83 mCi/μmol) for 4 min at 4 h after the addition of glucose. Nonradioactive methionine (500 μg/ml) was then added (time 0) to chase [methyl-³H]methionine. At indicated times, samples were taken for measurements of incorporation of ³H-label into total trichloroacetic acid-insoluble fraction (mostly protein together with small amounts of RNA; data not shown, but mentioned in Results). ³H-labeled RNA was also isolated, and samples (derived from equal volumes of the original cultures) were subjected to a polyacrylamide/agarose composite gel electrophoresis followed by autoradiography (lanes 1-8). Lanes 9-12 are the same as lanes 5-8 but with a longer exposure time. The amount of ³H in radioactive rRNA bands (precursor 35S, 27S, 20S, and mature 25S and 18S) were quantified, and the sums of these values were used to calculate rRNA synthesized per unit amount of cell mass (A₆₀₀ unit). The values obtained for samples before and after chase were compared between the two strains and indicated below the 18S bands.

Representative electron micrographs of 35S rRNA genes from H3-depleted cells. (A) Control NOY2025 cells. A gene with ≈51 polymerases (close to the average polymerase density for this control strain) is shown. (B) H3-depleted NOY2022 cells. A gene with ≈34 polymerases (close to the average polymerase density for H3-depleted cells) is shown. (C) H3-depleted NOY2022 cells. A gene with 26 polymerases is shown. Schematic representations of the ends of the genes in A, B, and C are in A′, B′, and C′, respectively. Arrows indicate the longer uncleaved rRNA transcripts. (D) Decrease in polymerase density per gene in H3-depleted NOY2022 relative to control NOY2025 cells (at 4 h after the shift to glucose). The number of polymerases per gene was counted for 261 genes from H3-depleted cells and for 170 genes from control cells. Distribution of polymerases among transcribed genes is plotted by grouping with an increment of 5.

Analysis of UAF in H3-depleted cells. (A) Purification steps used to make UAF fractions. (B) H3 depletion strain NOY2020 and control strain NOY848 were grown in YEP-galactose. Cells at a midlog (A₆₀₀ ≈ 0.2) were harvested and resuspended in YEP-glucose and incubated for 4 h. Cells were then collected, and extracts were prepared. After total protein concentrations of the two extracts were adjusted to be equal, portions were used to determine the amount of myc-tagged H3 by Western blotting with anti-myc Ab (Upper). Remaining portions were used to obtain “crude UAF” fractions by Ni-column followed by elution with imidazole. The amounts of Rrn5p were then determined by Western blotting with anti-HA Ab (Lower). (C) Extracts were prepared from H3-depleted cells (NOY2020) and control cells (NOY848) as described in B. During the 4-h depletion of H3 in YEP-glucose, NOY2020 cell mass increased 2.0-fold and NOY848 cell mass increased 2.5-fold. After total protein concentrations of the two extracts were adjusted to the same values, equal volumes of the two extracts were used to purify UAF as described in A. Series of 2-fold diluted samples as indicated were subjected to gel electrophoresis. The gel was cut into two parts: Upper was used to determine the amount of HA-tagged Rrn5p by using anti-HA Ab, and Lower was used to determine myc-tagged H3 by using anti-myc Ab. Note that each of the control samples shown was 3-fold more diluted than each of the samples from H3-depleted cells. If the relative synthesis rate of UAF (per cell density) in glucose is similar to that in galactose, the theoretical maximum decrease in UAF concentration when H3-synthesis is halted upon transfer to glucose would be 2-fold. The observed 3-fold decrease strongly suggests an instability of UAF in the H3-depleted cells. (D) The purified UAF preparations used in the experiment shown in C were examined for their ability to stimulate Pol I transcription in vitro. Series of 2-fold diluted samples were analyzed. An autoradiogram of transcription products is shown. As in C, 3-fold differences in dilution factors were used to make comparison of H3-depleted samples and control samples easier. The UAF purified from NOY2020 and NOY848 activated transcription 2.7-fold and 2.8-fold compared with control without UAF, respectively, at the highest concentration.