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. 2016 Apr 29;12(4):e1006021. doi: 10.1371/journal.pgen.1006021

Fig 5. Cohesin concentration in the pericentric region in GAL-CEN3, mcm21Δ and mcm21Δ, sir2Δ mutants.

Fig 5

A. Cells containing GAL-CEN3 as the only centromere in chromosome 3 were transferred from lactose to galactose and grown on galactose for over 6 hours to inactivate the centromere. Chromatin immunoprecipitation was performed as described in Snider et al., (2014) [27], using a ChIP grade antibody against GFP to immunoprecipitate the only copy of Smc3 fused to GFP at the C-terminus of Smc3 in the genome. Oligonucleotide primers against CEN3 (114,800), 8 kb (Stp22, position 105696..106853), 10 kb (Ilv6, position 104619..105548) and 87 kb (kar4 27929..28936) were utilized. Primers were designed to amplify a 600 bp fragment for each of the 4 reactions. Titrations of template were performed to ensure the analysis was in the linear range of amplification. For GAL-CEN3 glucose, the integrated intensity ranged from 5.5 x 10^6 to 6.1 x 10^6 (indicated by glucose text inset in graph)(n = 4). The average was 5.78 x 10^6 ± 2.5 x 10^5 (STD). For GAL-CEN3 galactose, integrated intensity ranged from 1.2 x 10^6 to 3.3 x 10^6 (indicated by galactose text inset in graph)(n = 4). The average was 2.33 x 10^6 ± 8.3 x 10^5 (STD). For STP22 (8 kb), ILV6 (10 kb) and KAR4 (87 kb) the galactose and glucose products ranged from 2.4 x 10^5 to 5.6 x 10^5 (n = 3). There was no significant difference between glucose and galactose grown samples for the 8, 10 or 87 kb products. B. The concentration of Smc3-GFP was determined in GAL-CEN3 WT, mcm21∆ and mcm21∆ sir2∆ mutants. The concentration of pericentric cohesin is reduced in mcm21∆ cells (from 30,540 to 18,509 arbitrary fluorescence units). In the double mutant, mcm21∆ sir2∆, the concentration of cohesin in the pericentromere is increased to almost wild-type levels (29,848 vs 30,540). C. Representative images of Smc3-GFP in GAL-CEN3 WT (left), mcm21∆ (middle) and mcm21∆ sir2∆ (right). Spindle poles are visualized using Spc29-RFP, cohesin with Smc3-GFP. The rightmost image in each strain is an overlay of the spindle poles with Smc3-GFP. White arrows indicate the cohesin barrel concentrated in the pericentric chromatin between the spindle poles (red). Note the absence of the cohesin barrel in mcm21Δ.