mRNA analysis confirms suppression of transcriptional deregulation in not1-2 by the deletion of SPT3. (A) The results of the microarray experiments for the four indicated transcripts are given. (B) Cells were grown, as in Figure 1, to an OD₆₀₀ of 0.8, and 50 μg of total cellular RNA was analyzed by S1 digestion for the levels of the indicated transcripts. DED1 was always measured in the same reaction as the other transcripts as an internal control, since the microarray experiments indicated that it was not affected by not1-2. The RNA was sampled and analyzed the same day for SNZ1 and HSP12, and the results for DED1 were the same and are shown only once. The levels of ADK1 and GUA1 relative to DED1 were quantified using phosphoimager analysis and are indicated below the radiographs.

The presence of Taf3p at HSP12 and SNZ1 promoters increases in not1-2 and this is suppressed by the deletion of SPT3. The indicated strains were grown as in Figure 3 to an OD₆₀₀ of 0.8 at 37.5°, and the cells were crosslinked prior to total extract preparation and immunoprecpitation with the indicated antibodies. The amount of indicated promoter DNAs in the immunoprecipitates was evaluated by real-time PCR, expressed relative to input DNA, and then normalized to the amount of an intergenic unexpressed DNA. (A) The antibodies were against H3-K4-Me₃ and the results presented are the average of two experiments performed with independent cultures. (B) Antibodies were against Taf3p and the experiment was repeated three times with independent cultures. (C) Antibodies against Taf9p and Taf11p were used and the relative amount of DNA immunoprecipitated with Taf9p to that immunoprecipiated with Taf11p was calculated (Taf9p/Taf11p). The experiment was repeated two times with independent cultures. (D) The indicated strains expressed MYC-tagged Spt7p and antibodies against MYC were used. The experiment was repeated three times with independent cultures.

spt3Δ and spt7Δ display a synthetic phenotype. The indicated strains were grown exponentially for 24 hr and then (A) serially diluted and plated on glucose-rich plates that were placed at 30° or 37.5° as indicated for 3 days. (B) Strains shifted to high temperature, as in Figure 1, to an OD₆₀₀ of 0.8. Total extracts were made by post-alkaline lysis and analyzed by Western blot for the levels of histone H3. The Ponceau staining is shown for loading control. Lane 1, 14 μl of post-alkaline lysis corresponding to 0.7 OD₆₀₀; lane 2, 7 μl of post-alkaline lysis corresponding to 0.35 OD₆₀₀. (C) Strains shifted to high temperature, as in Figure 1, to an OD₆₀₀ of 0.8. Total cellular RNA was extracted and 50 μg were analyzed by S1 digestion for the levels of SNZ1 mRNAs and DED1 as an internal control. Results were quantified by phosphoimager analysis.

not1-2 temperature sensitivity is suppressed specifically by the deletion of SPT3. The indicated strains were grown exponentially in glucose-rich medium for 24 hr and then (A) serially diluted and plated on glucose-rich plates that were placed at 30° or 37.5° for 3 days or (B) were shifted to 37.5° after reaching an OD₆₀₀ of 0.2. Growth of the cells was measured over time.

The deletion of SPT3 specifically reduces increased HSP12 and SNZ1 mRNA in not1-2. (A) The indicated strains were grown exponentially for 24 hr and then shifted at an OD₆₀₀ of 0.2 to 37.5°. Total cellular RNA was prepared when cells reached an OD₆₀₀ of 0.8 as in Figure 2. The levels of HSP12 and SNZ1 mRNAs were measured simultaneously to that of DED1 in 50 μg of total cellular RNA by S1 analysis. Because of the unequal loading of some lanes, the results were quantified by phosphoimager analysis. (B) The levels of RPS9A in 50 μg of total RNA were measured for the indicated strains, and the levels of rRNA in 5 μg of total cellular RNA are indicated as a control.

The level of total histone H3 and H4 protein but not mRNA is decreased in not1-2 and this is suppressed by deletion of SPT3. The indicated strains were grown as in Figure 3 to an OD₆₀₀ of 0.8 at 37.5°. (A) Total protein extracts were prepared by post-alkaline lysis. Proteins were separated on 15% SDS–PAGE and analyzed by Western blot for the levels of histone H3. Lane 1, 14 μl of post-alkaline lysis corresponding to 0.7 OD₆₀₀; lane 2, 7 μl of post-alkaline lysis corresponding to 0.35 OD₆₀₀. (B) Total cellular RNA was extracted and 50 μg were analyzed by S1 digestion for the levels of HHT1/2 mRNAs. The probe cannot distinguish between the two different mRNAs that encode histone H3. The amount of rRNA is shown as the loading control. (C) Total protein extracts were prepared by post-alkaline lysis, as indicated in A, but also from wild-type or not1-2 cells carrying either an empty vector or a multicopy plasmid expressing histone H3 as indicated. (D) Total protein extracts were prepared by post-alkaline lysis. Proteins were separated on 15% SDS–PAGE and analyzed by Western blot for the levels of histone H4, using 28, 14 or 7 μl of post-alkaline lysis for each strain (left), total cellular RNA was extracted, and 50 μg was analyzed by S1 digestion for the levels of HHF1/2 mRNAs (right). (E) The indicated strains overexpressing or not overexpressing histone H3 and/or H4 as indicated were streaked on glucose-rich plates and placed at 30° or 37.5° as indicated.

Spt3p-dependent activation in not1-2 occurs at permissive temperature. The indicated strains were grown in glucose-rich medium to exponential phase and (A and B) total cellular RNA was extracted for analysis by S1 digestion of the indicated mRNAs. DED1 mRNA and rRNAs are shown as the loading control. (C) The cells were crosslinked prior to total extract preparation and immunoprecpitation with Taf9p polyclonal antibodies. The amount of indicated promoter DNAs in the immunoprecipitates was evaluated by real-time PCR, expressed relative to input DNA, and then normalized to the amount of an intergenic unexpressed DNA.