Supplementary Figure 4: The behavior of spontaneous revertants of Δcen3-NCs.

(a) A color image of a typical colony showing the emergence of spontaneous revertants from the Δcen3-NC strain. Δcen3-NC cells (cd3-386) were grown continuously for 30 generations and were allowed to form colonies on YES plates containing phloxine B (PhB). (b) The proportions of revertants that emerged from the indicated Δcen3-NC strains during continuous cell culture. (c) PFGE separation of the chromosomes in the type-F revertants showing the reduction in chromosome number. m, molecular size marker. (d) SfiI-digested chromosomes of the type-F revertants separated by PFGE and stained with ethidium bromide (EtBr) or subjected to Southern blot (SB) analysis with the chr3L, chr3R, tel1L, and tel2R probes to observe telomere-to-telomere fusion. (e) PFGE separation of the chromosomes in the type-R revertants showing the three-chromosome configurations. m, molecular size marker. (f) The percentages of normal (gray) and defective (magenta) chromosome segregation patterns in anaphase cells. The percentages were determined as described in Figure 3e (n > 100). Error bars represents s.e.m. of triplicate cultures. Note that segregation failure occurs less frequently in the type-R revertants than in the heterochromatin-deficient Δswi6 cells and Δclr4 cells (see Fig. 3e). Furthermore, the Δswi6 mutation in the type-R revertant conferred a low level of lagging chromosomes (cd3-389-r1 Δswi6). This level was similar to that observed in the Δswi6 mutant, but different from that observed in the original Δcen3-NC^rDNA cells (cd3-389). (g) SfiI-digested chromosomes of the type-R revertants separated by PFGE and stained with EtBr or subjected to Southern blot analysis with the indicated probes to show the reduction in the number of rDNA repeats in the left arm. (h) PstI-digested chromosomes of the type-R revertants separated by PFGE and subjected to Southern blot analysis with the rDNA probe. Given that there is no PstI restriction site in the 10.8-kb rDNA repeat units and the non-rDNA repeat region of the left-most PstI fragment in chromosome III encompasses approximately 3.4 kb (chrIII, 24571–28056), the rDNA 15-kb PstI fragment in cd3-386-r1 and cd3-390-r1 that hybridized to the probe most likely harbors only one copy of the rDNA repeat, and the 25-kb PstI fragment in cd3-389-r1 most likely harbors two copies.