SUPPLEMENTAL MATERIAL Materials and Methods Primers for ChIP/qPCR (Fig. 3): CG7939 (RpL32) Rp49+10F Rp49+110R Rp49+241F Rp49+341R Rp49+549F Rp49+613R TCTGGTTTCCGGCAAGGTATGT GCAGTTCAACTCGAAACCGCCAAA ATACTGCCCAAGAAGCTAGCCCAA GCACTGACCCACTGGAAATATCAC CCCAAGGGTATCGACAACAGA CGATGTTGGGCATCAGATACTG CG2922 (elf-5c) exba+20f exba+99r exba+374f exba+527r exba+1841f exba+1991r exba+5104f exba+5190r GACACCTTAGTGAGCTCTGTACCA TTGGGCTGCTTCTTCTGGATGACT TCGGCCTTTGTTCGACTGTACCTT ATACACTGACATGGCGAAAGCGGA TGCTATCGGGTCAACGCATCAAGA TGTTGCCCGCGCTGTCTAGATATT ATGAGGATGAACACAAACGGAGCC TATAGTTCGACTGGCTGTTGCAGGTG CG9277 (betatub56d, Beta-1-tubulin) βtub56d+5f GCTCTCCAAAGCGAATGCACTA βtub56d+82r CGCTTATAGCAGTCGAACACAACA βtub56d+261f ACTTCGCTGTCCATGTGGCAAA βtub56d+357r CACACACACGCACACAACTTGGTA βtub56d+714f TGCCCAGATGGTGAAGGGTACTTT βtub56d+793r ATGAGCAGCAAGCTCTTATGTGCG βtub56d+1899f AACAACCGGTGATGTAAGCCGCAT βtub56d+2009r TGTCTATATGTCTACGTGCTGCCC βtub56d+2648f TGTATGCAGCAGATGGTCTAGGCT βtub56d+2723r GACTTGGCCAATGAGTCATCACAG CG31366 (Hsp70Aa), CG18743 (Hsp70Ab), CG31449 (Hsp70Ba), CG31359 (Hsp70Bb), CG5834 (Hsp70Bbb), CG6489 (Hsp70Bc) Hsp70Ab-1159F GCAGGAAGGGATATTGAACTACAT specific to the Ab copy
Hsp70Ab-1036R ATCTGTAAAGCTCCCAGATTATACA specific to the Ab copy Hsp70Ab-836F CTTGCCTCATTGACTGGAGCTAT specific to the Ab copy Hsp70Ab-719R AGGATACAATTTAAGGATCCGGAAA specific to the Ab copy Hsp70 200F TGGCAGAAAGAAAACTCGAGAAA Hsp70 108R GACAGAGTGAGAGAGCAATAGTACAGAGA Hsp70+56F ACAAGCGCAGCTGAACAAGCTA Hsp70+137R ACTTGGTTGTTGGTTACTTT Hsp70+334F CACCACGCCGTCCTACGT Hsp70+423R GGTTCATGGCCACCTGGTT Hsp70+645F ATATCTGGGCGAGAGCATCACA Hsp70+718R GTAGCCTGGCGCTGGGAGTC Hsp70+872F CATCGACGAGGGATCTCTGTTC Hsp70+1019R GGCGCGAGGGTTGGA Hsp70+1363F CTGTGCAGGCCGCTATCC Hsp70+1490R GCGCTCGATCAGCTTGGT Hsp70+1649F GGGTGTGCCCCAGATAGAAG Hsp70+1754R TGTCGTTCTTGATCGTGATGTTC Hsp70+1925F TGGACGAGGCTGACAAGAACT Hsp70+1978R ACCGGATAGTGTCGTTGCACTT Hsp70Ab+2155F GGTCGACTAAGGCCAAAGAGTCTA specific to the Ab copy Hsp70Ab+2266R TCGATCGAAACATTCTTATCAGTCTCA specific to the Ab copy Hsp70Ab+2631F TCGCAGACACCGCATTTGT specific to the Ab copy Hsp70Ab+2706R ACCAATTGCAACAGAGACTGGAA specific to the Ab copy Hsp70Ab+4035F TGGAAACTGCCTCCAACAACTG specific to the Ab copy Hsp70Ab+4124R AGACGCACGAGACCAATCTGTA specific to the Ab copy Background-F TTGCACTCACCGTGATTGGAATG 32kb from the 87C locus Background-R GTCACAATGCTAACATCTCCTTAT 32kb from the 87C locus CG3845 NAT1+134F NAT1+259R NAT1+1616F NAT1+1720R NAT1+3998F NAT1+4127R NAT1+6980F NAT1+7084R GGAAAGTACACTTCTTTGCAGGTGG AAACGAGGACGTTCACACAAGCAC CACCAACACTTGCACACATGCGAT AACCTCGGATGAGCACCTTCAAGT TCCAGGAACTGAGCGATGAACTGT ACAGCTGCGCATACATAGACGAGT GGCAAAGGATCCGCTTTGTTCCAA ATTATGCGGGACTGCGTTGCTTTC Yeast strains:
Strain Relevant characteristics Reference FY23 MATa ura3-52 trp1δ63 leu2δ1 Fred Winston Z26 MAT α his3 Δ 200 leu2-3,112 ura3- Richard Young 52 rpb1 Δ 187::HIS3/pRP112 (LEU2 CEN4 RPBI-XTPH) GY2002 bur1-8 MATa his4-912d lys2-128d Greg Prelich suc2duas(-1900/-390) trp1d63 bur1-8::kan BY4742 MATα his3a1 leu2a0 lys2a0 ura3a0. Plasmids: Plasmid Size (bp) Relevant characteristics pgex-5x-1 4900 E. coli N-terminal GST tag plasmid pgex-ctk1n 5446 N-terminal part of CTK1 cloned between EcoNI and BamHI sites in pgex-5x-1. pgex-ctk1nc 5308 Both N- and C-terminal parts of CTK1 cloned between EcoNI and XhoI sites in pgex-5x-1. pgex-ctk1/13 6520 CTK1/13 chimera in pgex-5x-1 plasmid. pgex-ctk1/12 6520 CTK1/12 chimera in pgex-5x-1 plasmid. pgex-ctk1/9 6523 CTK1/9 chimera in pgex-5x-1 plasmid. pgex-ctk1/0 5854 N- and C-terminal of CTK1 but not the kinase domain cloned in pgex-5x-1 plasmid. pvd45 6543 puc19 based plasmid containing the 5, 3 flanking region and the whole CTK1 sequence with 3HA tags at its C-terminus and a TRP1 yeast selectable marker. pvd45-ctk1/13 6861 pvd45 plasmid with replacement of CTK1 kinase domain with aligned hcdk13 kinase domain. pvd45-ctk1/12 6861 pvd45 plasmid with replacement of CTK1 kinase domain with aligned hcdk12 kinase domain. pvd45-ctk1/9 6864 pvd45 plasmid with replacement of CTK1 kinase domain with aligned hcdk9 kinase domain. pvd45-ctk1/0 6199 pvd45 plasmid without CTK1 kinase domain pvd62 7158 puc19 based plasmid containing the 5, 3 flanking region and the whole CTK1 sequence with 3HA tags at its C-terminus and a URA3 yeast selectable marker. pvd62-ctk1/13 5472 pvd62 plasmid with replacement of CTK1 kinase domain with aligned hcdk13 kinase domain. pvd62-ctk1/12 5472 pvd62 plasmid with replacement of CTK1 kinase domain with aligned hcdk12 kinase domain. pvd62-ctk1/1 5475 pvd62 plasmid with replacement of CTK1 kinase domain with aligned hcdk9 kinase domain. pvd62-ctk1/0 4806 pvd62 plasmid without CTK1 kinase domain. pag415gal-ccdb-tap 9304 Gateway destination vector with LEU2 marker. pag415gal-ctk1/13-11421 After LR rxn with TOPO entry vector, ccdb cassette
pag415gal-ctk1/12- pag415gal-ctk1/9- pag415gal-ctk1/0- was replaced by CTK1 promoter, CTK1/13 sequence 11421 After LR rxn with TOPO entry vector, ccdb cassette was replaced by CTK1 promoter, CTK1/12 sequence 11424 After LR rxn with TOPO entry vector, ccdb cassette was replaced by CTK1 promoter, CTK1/9 sequence and TRP1 marker. 10755 After LR rxn with TOPO entry vector, ccdb cassette was replaced by CTK1 promoter, CTK1NC sequence Primers: Primer Sequence Use CTK1 NF GCCTATACTAGGCATGCTTGTGGATACTGG FP: amplification of CTK1 N-terminal sequence CTK1 NR CATTAGGATCCAACTGACGTGCTCCTTTG RP: amplification of CTK1 N-terminal sequence CTK1 CF CATATGGATCC TTCAAGATTATGGGAACTCC FP: amplification of CTK1 C-terminal sequence CTK1 CR CATTA CTCGAG CGTCGTAAGGGTAAGCGT RP: amplification of CTK1 C-terminal sequence CDK13 F CTGCGTGGATCCAAATTTGATATCATCGGA FP: amplification of hcdk13 kinase domain CDK13 R CAATA GGATCC TAT TAA TTC TAG TTG TGC RP: amplification of hcdk13 kinase domain CDK9 F CTGCGTGGATCCTACGAGAAGCTCGCCAAG FP: amplification of hcdk9 kinase domain CDK9 R CAATA GGATCC GATGAGGGCGAGTTGGTG RP: amplification of hcdk9 kinase domain CDK12 F CTGCGTGGATCC TTTGACATTATTGGGATTATTGGAG FP: amplification of hcdk12 kinase domain CDk12 R CAATA GGATCC GAT CAG TTC TAG CTG AGC RP: amplification of hcdk12 kinase domain PVD45 SEQF ACCAGGATGATACAGAAGA FP: sequencing of CTK1/X chimera construction PVD45 SEQ R CGATGTAAGTGGAGGAGT RP: sequencing of CTK1/X chimera construction YTPVD45 F GCGGATGAGGTCCACAAT FP: amplification of CTK1/X and 5 and 3 flanking sequence YTPVD45 R CGTTATCCCCTGATTCTG RP: amplification of CTK1/X and 5 and 3 flanking sequence His6 MluI F C GCG TAC CAT CAC CAT CAC CAT CAC TA FP: coding for His tag with MluI
His6 MluI R CGCGTA GTG ATG GTG ATG GTG ATG GTA RP: coding for His tag with MluI Strep BstPI F GT TAC CAG TGG AGC CAC CCG CAG TTC GAA AAG G FP: coding for Strep tag with BstPI Strep BstPI R GTA ACC CTT TTC GAA CTG CGG GTG GCT CCA CTG RP: coding for Strep tag with BstPI SUPPLEMENTAL FIGURE LEGENDS Figure S1. Evolutionary relationships among CDKs. (A) Depiction of the Ctk1 group (newer nomenclature indicated in blue) and the Bur1 group (orange) of CDKs as determined by Liu and Kipreos (Liu and Kipreos 2000). (B) Depiction of the Ctk1 group (newer nomenclature indicated in blue) and the Bur1 group (orange) of CDKs as determined by Guo and Stiller (Guo and Stiller 2004). Figure S2. Western blots using previously undescribed antibodies. Whole cell (W) or nuclear (N) extracts were subjected to SDS PAGE (along with pre-stained marker proteins, m) followed by immunoblotting using affinity-purified antibody against the indicated protein (typical final IgG concentration about 0.2 µg/ml). (A) Rabbit anti-dcdk12 IgG; detection with alkaline phosphatase color reagent substrates (Bio-Rad, 170-6432). (B) Sheep anti-dcyclin T; detection by ECL. (C) Rabbit anti-hcdk13; detection by Odyssey infrared scanner (LiCor). (D) Rabbit anti-hcdk12; detection by Odyssey scanner. Figure S3. Primary structure diagrams of CDKs and chimeric kinases. (A) Overview of relevant CDKs. Green rectangles indicate approximate positions of peptides used for generating and affinity-purifying antibodies. (B) Diagrammatic overview of scheme used to construct kinase chimeras. Figure S4. Chimeric kinase purification and kinase specificity assays. (A) Western blot of (His- and HA-tagged) chimera 1/13 fractions 1 5 eluted from Ni ++ affinity resin by imidazole. Blot was reacted with mouse anti-ha tag mab (green) and affinity-purified rabbit anti-yctk3 antibody (Sterner et al. 1995) (red). (B) Kinase assay of fractions in (A) using GST-yCTD as substrate. Phosphorimage of SDS gel shown; molecular weight markers indicated at right. (C) Kinase specificity assays. The kinases were assayed under identical conditions using GST- (variant)ctd fusion protein substrates as indicated (the 1/12 kinase was purified from a 1/12 strain as for the 1/13 kinase). SDS gels of terminated reactions were stained with Coomassie blue (blue panels), showing that very similar amounts of each CTD substrate were used, and analyzed by phosphorimager (grayscale panels). The ratio of phosphorimage signal to Coomassie staining intensity was determined, and within each set of assays the value of this ratio for the WT CTD substrate was set to 1 (bottom panels).