The rDNA tandem array is contracted in Δctk null mutant strains. (A–D) Chromosome XII analysis. FY1679-18B and W303-1B derived cells were grown in YPD. After cell digestion, agarose plugs were subjected to CHEF electrophoresis. (A) Gels were photographed after ethidium bromide straining (EtBr), and further analysed by Southern blotting with a 35S probe and autoradiographed (35S probe). Arrows point to chromosome XII. (B) rDNA was quantified using probes specific for the rDNA and for genes located on the extremities of chromosome XII (VPS13 and SIR3). Genomic DNA was analysed either by Southern blotting and signals were quantified with a PhosphorImager (light grey boxes), or by quantitative real-time PCR (dark grey boxes). Ratios of rDNA over non-rDNA values were reported. Units were set relative to the measurement for the FY1679 strain (left panel) or the wild-type isogenic strain (right panel), performed using Southern blotting. (C) Chromosomes from W303-1B (WT) and isogenic Δpdr13 cells were analysed by CHEF electrophoresis and ethidium bromide straining. The arrow points to chromosome XII species. (D) Five independent cultures of Δctk1 cells (FY1679-18B) were grown for 100 generations in YPD prior to chromosome analysis. Bracket points to chromosome XII. (E) Homologous recombination analysis. Wild-type and Δctk1 cells were spotted on YPD prior to replica-plating on SD-ARG. The number of papillae was scored and reported for each strain (ARG+).

The rDNA contraction is reversible and specific. FY1679-18B and W303-1B derived cells were grown in YPD. (A) Complementation analysis. Chromosomes from two independent Δctk1/CTK1 (FY1679-18B) and Δctk2/CTK2 (W303-1B) clones, respectively, were analysed by CHEF electrophoresis and ethidium bromide staining. Size markers are Hansenula wingei chromosomes (M), and their sizes are indicated in megabase pairs. (B) Telomeres analysis. After digestion, genomic DNA was analysed by Southern blotting with a probe specific for telomeres. Bands are visualized by autoradiography.

Analysis of Mcd1 association to rDNA by chromatin immunoprecipitation. After cross-linking, cell lysis and sonication, wild-type MCD1-HA (light grey boxes) and Δctk1 MCD1-HA (dark grey boxes) extracts were immunoprecipitated with anti-HA antibodies. Total and immunoprecipitated DNA were analysed by real-time PCR using four sets of primers: RFB, CAR, CEN3 and CUP1, respectively. Ratio of immunoprecipitated over total DNA was reported.

Fob1 is required for rDNA expansion, but not for contraction. Chromosomes from FY1679-18B (A and C) and W303-1B (B) derived cells grown in YPD were analysed by CHEF electrophoresis and ethidium bromide staining. (A and B) Contraction analysis. CTK1 was deleted in Δfob1 cells, and independent clones were analysed either after growth for 100 generations (A, a–d and B) or for 200 generations (A, a+100, b+100). (C) Expansion analysis. FOB1 was deleted in Δctk1 cells prior to re-introduction of the CTK1 gene born on a CEN, LEU2 plasmid. Five independent clones were analysed after growth for 100 generations. Size markers are Hansenula wingei chromosomes (M), and their sizes are indicated in megabase pairs.

ERC analysis. DNA was extracted from W303-1B (WT), Δctk1, Δfob1 and Δsgs1 cells, respectively, prior to electrophoresis on an agarose gel and Southern blot analysis with a 35S probe. The chromosomal band is indicated by an arrowhead, and ERC species by arrows.

Pol I transcription analysis. (A–C) Psoralen cross-linking experiments. After psoralen treatment, genomic DNA from wild type (WT), Δctk1, Δctk2 and Δfob1−Δctk1 extracts were analysed by Southern blotting with either an intergenic rDNA probe (A and C) or a 35S rDNA probe (B). (B) As a control, non-crosslinked wild-type DNA was loaded (WT-UN). Arrows indicate the 2.8 kb-EcoRI slow (s) and fast (f) migrating fragments, respectively. Quantification from two independent experiments was performed with a PhosphorImager. Ratios of slow to fast fragments are reported. (D) Primer extension analysis. RNA was extracted from wild type (WT), Δctk1 and Δfob1−Δctk1 cells. Primer extensions were performed with oligonucleotides specific for the 35S and the U6 RNA, respectively. Two independent experiments were analysed by electrophoresis and autoradiography. Signals were quantified with a PhosphorImager and reported as the ratio of 35S over U6 signals.

Analysis of Pol II silencing at rDNA (A) Sir2 expression analysis. After extraction from wild-type SIR2-HA (WT) and Δctk1 SIR2-HA (Δctk1) cells, proteins were analysed by western blotting with anti-HA (upper panel) and polyclonal anti-A43 (lower panel) antibodies, respectively. Chemoluminescence was quantified with a Fluorchem FC and reported as the ratio of Sir2-HA over A43 signal. (B) mURA3 silencing analysis. 5 μl of 10-fold serial dilutions of the indicated strain grown in YPD were spotted on SD+URA (−HIS) and SD-URA (+HIS), respectively.