0.35 ml/min. Fractions of 500 ml were collected. An amount of 250 ml of every second fraction were TCA precipitated and analysed by western blotting. Co-immunoprecipitations To characterize the Pol I–Rrn3p interaction in yeast strains pNOP1-RRN3-Prot.A-RPA43-HA3 and pNOP1- RRN3- N-Prot.A-RPA43-HA3, cells were crosslinked and lysed as described [(27) and below]. The HA3-tagged Pol I subunit A43 was immunoprecipitated from 250 ml of the resulting extracts with 1.5 mg of an anti-HA antibody (3F10, Roche) bound to 50 ml of Protein G sepharose (Amersham). For the following washing steps the chroma- tin IP washing buﬀers were used (see below). To elute the immunoprecipitated proteins and to reverse the crosslink, the beads were mixed with SDS sample buﬀer and incubated for 20 min at 95 C. Fifty percent of the eluates were analysed by western blotting. To determine the amounts of co-precipitated Rrn3p, both the Rrn3p and the A43 signals were detected with the LAS 3000 imaging system and quantiﬁed using the AIDA software (Raytest). Then, the Rrn3p levels were normalized to the A43 levels and the values of the growth arrested cells were related to the values obtained with extracts of growing cells which were put arbitrarily to 100%. Chromatin immunoprecipitations Chromatin immunoprecipitations were performed as described (27). An amount of 45 ml yeast cultures (OD600 = 1.0) were crosslinked for 15 or 30 min with 1% (v/v) formaldehyde at 30 C. The cells were harvested by centrifugation and suspended in the same volume of cold lysis buﬀer [50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) DOC, 1 mM PMSF, 2 mM benzamidine]. Glass beads (Ø 0.75– 1.0 mm, Roth) were added and cells were disrupted on a VXR basic Vibrax (IKA) for 45 min with 2000– 2200 r.p.m. at 4 C. After discarding the glass beads, the lysates were soniﬁed to obtain an average DNA fragment size of 500–1000 bp. Cell debris was removed from the lysates by centrifugation (20 min, 13 000 r.p.m., 4 C). The resulting chromatin extracts were incubated for 2 h at 4 C with 1.5 mg of a monoclonal a-HA antibody (3F10, Roche) bound to 50 ml of Protein G sepharose (Amersham) to enrich the HA3-tagged Pol I subunit A43. After immunoprecipitation, the beads were washed three times with lysis buﬀer (without protease inhibitors), twice with washing buﬀer I [50 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) DOC] and twice with washing buﬀer II [10 mM Tris–HCl (pH 8.0), 250 mM LiCl, 1 mM EDTA, 0.5% (v/v) Nonidet P40, 0,5% (w/v) DOC] followed by a ﬁnal washing step with TE buﬀer [10 mM Tris–HCl (pH 8.0), 1 mM EDTA]. Immunoprecipitated material was eluted from the beads by incubating the samples for 10 min at 65 C in the presence of 140 ml of buﬀer TE con- taining 1% (w/v) SDS. To reverse the crosslink, 120 ml of the eluates (IPs) as well as 40 ml of the chromatin extracts mixed with 80 ml of TE buﬀer containing 1% SDS (inputs) Nucleic Acids Research, 2010, Vol. 38, No. 16 5317 were heated over night at 65 C. Then, proteins were digested by mixing the samples with 130 ml of TE buﬀer containing 1.54 mg/ml Proteinase K (Sigma). After 2 h of incubation at 37 C, DNA was isolated by phenol–chloro- form extraction and ethanol-LiCl precipitation. Both, the input and the IP DNA were suspended in 50 ml of TE buﬀer. DNA dilutions of 1: 2000 (inputs) and 1: 400 (IPs) were analysed in triplicates by quantitative real-time PCR using a RotorGene RG-3000 system (Corbett Research) and the SYBR Green I dye (Roche) for detection. To characterize the rDNA occupancy of Pol I subunit A43, a rDNA promoter fragment, a part of the 18S and 25S rRNA coding region and a 5S rRNA gene fragment were ampliﬁed using the following oligonucleotides: #969 (50-TCA TGG AGT ACA AGT GTG AGG A-30) and #970 (50-TAA CGA ACG ACA AGC CTA CTC-30), #710 (50-TGG AGC AAA GAA ATC ACC GC-30) and #711 (50-CCG CTG GAT TAT GGC TGA AC-30), #712 (50-GAG TCC TTG TGG CTC TGG GC-30) and #713 (50-AAT ACT GAT GCC CCC GAC C-30), #920 (50-GCC ATA TCT ACC AGA AAG CAC C-30) and #921 (50-GAT TGC AGC ACC TGA GTT TCG-30). Data were evaluated using the comparative quantitation module of the RotorGene analysis software (version 6.1, Corbett Research) and the MS Oﬃce program Excel 2003. After normalizing the IP to the respective input values, relative occupancies were obtained by relating data to the promoter occupancy of the growing cells, which was put arbitrarily to 1.0. Fractionation Nuclear and cytoplasmic fractions were prepared as described (28) according to the method of (29) with the modiﬁcation that 12 ml of yeast cultures were grown to an OD of 0.8 before they were harvested. Psoralen crosslinking analyses Psoralen crosslinking was carried out as previously described (30). Southern blot analysis Blot analysis and quantitation have been performed as described elsewhere (30). Templates for probe prepar- ation, detecting the rDNA locus were an Eco RI fragment spanning nt 323–2277 of the 35S rDNA, and a 3.5 kb Nco I-fragment spanning nt 1045–4491 of the 35S rDNA. Immunolocalization Immunoﬂuorescence microscopy was performed accord- ing (28). Specimen preparation for electron microscopy Yeast chromatin spreading was performed as described in (31).