Structure of S. pombe centromeres. (A) Schematic diagram of the centromeres. Each of the three centromeres contains central core cnt and flanking imr and otr repeat regions. imr and otr have repeat sequences so that a probe designed for these regions will hybridize to DNA fragments from multiple loci. Cross-hybridization thus renders nucleosome mapping unfeasible for imr and otr. Similarly, cnt1 and cnt3 on chromosomes 1 and 3 share >70% identical sequences, and thus, only the partial non-overlapping portions can be tiled unambiguously. On the other hand, cnt2 contains unique sequences and can be tiled densely for nucleosome mapping. (B) Gel electrophoresis and Southern. Chromatin or naked genomic DNA was treated with MNase digestion (see Methods for the details of digestion conditions), and the resulting DNA fragments were run on gel. Southern hybridization was performed using a cnt DNA fragment (pKT110) or an otr DNA fragment (pYC148) (Takahashi et al. 1992). Bands containing mono-nucleosomes or DNA of similar size from naked DNA digestion (white boxes) were extracted and hybridized onto the microarray. The otr region shows ladders for both the wild type and the mis12 mutant. In cnt, the mis12 mutant still exhibits distinct ladders, while the wild type shows a smear with only faint mono- and di-nucleosome bands.
