Other known CTD modifications include phosphorylation at tyrosine, glycosylation, and perhaps isomerization of prolines (Dahmus, 1994; Morris et al., 1999; Wu et al., 2000). It will be very interesting to determine when the other CTD modifications occur, which enzymes are responsible for adding and removing modifications, and whether they act to regulate transcription initiation, elongation, and/or RNA processing.‘Materials and Methods’.Yeast strains used in this study:MATa; ur;YSB675 (MATa; ura3-52; leu2; trp1; his3∆200; ade2; taf40::LEU2; spt15::3)SPT15::URA3; [pRS313-TAF40]YSB756; rU2; ade2-1; ade3-22; can1-100; [pRS314-HA-kin28-T17D]MATa; ura3-52; his3∆200; rpb3-(HA)3::LEU2YMK13-11,15; ade2-1; can1-100; fcp1:LEU2; pMK86=
Komarnitsky: Page 12Chromatin immunoprecipitations were performed essentially as described in (Kuras and Struhl, 1999). Briefly, all yeast strains were grown to OD600~ 0.6 in synthetic complete medium supplemented with 2% glucose. Formaldehyde was added to a final concentration of 1% for 20 minutes. Crosslinking was quenched by addition of glycine to 240 mM. Cells were collected by centrifugation, washed in 1xTBS, and lysed with glass beads in FA lysis buffer (50 mM Hepes-KOH, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na Deoxycholate, 0.1% SDS, 1 mM PMSF). Chromatin was sheared by sonication so that the average fragment size was between 200 to 700 base pairs (as determined by agarose gel electrophoresis). Sheared chromatin was stored in aliquots at -70oC. Rabbit polyclonal antiserum recognizing Ceg1, Cet1, Abd1, Tfa2 (the small subunit of yeast TFIIE), and TBP were generated using standard methods by S.B., L. Fresco, T. Takagi, and N. Kuldell (Harvard Medical School). Anti-Hrp1 serum was the gift of C. Moore (Tufts). Anti-Kin28 polyclonal serum and monoclonal antibodies H14 and H5 were purchased from Covance. For immunoprecipitations all antibodies except H14 and H5 werepreincubated with Protein A-Sepharose CL-4B beads (Amersham/Pharmacia) and washed once with FA lysis buffer. Chromatin solution was then added and reactions incubated for 90minutes at room temperature. For the H5 immunoprecipitation, Anti-mouse IgM antibodies coupled to agarose beads (Sigma) were bound to the H5 antibody and used as described above. For H14 immunoprecipitations, Protein A-Sepharose CL-4B beads were pre-coated with anti-IgM/IgG (Sigma), and together with H14 antibody were added directly to the chromatin solution prior to the 90 minute incubation step. Immunoprecipitated chromatin was subsequently washed under stringent conditions, and subjected to protease treatment and reversal of crosslinks as described (Kuras and Struhl, 1999). A single preparation of immunoprecipitated DNA was used as template for all the PCR reactions within a given panel.
Komarnitsky: Page 13Conditions for PCR reactions were as follows: 0.25 M each primer, 0.1 mM each dNTP, 1x Gibco BRL PCR Buffer (no Mg2+), 1.5 mM MgCl2, 0.5 units Gibco BRL Platinum Taq polymerase, 0.06 mCi/ml -32P-dATP in 10 microliter reaction volume. PCR cycle was: once 90 s at 94oC, followed by 25 cycles of 30 s at 94oC, 30 s at 55oC, and 1 min at 72oC, and then once 10 min at 72oC. PCR products were resolved in 8% polyacrylamide-1xTBE gels. For the Input controls, 0.005% of the amount of chromatin
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