Spindle length was measured in cells with unseparated chromosomes, where both spindle poles were in focus, using either IPLab or Metamorph software. followed by fivefold dilutions. Plates were incubated at 25C for 3C7 d. Assessment of growth on YES at 36C and YES containing 0, 10, or CCT241736 15 mg/ml TBZ at 25C. (D) RT-PCR of transcripts from random integrant (insertion sites, compared with a minigene control (are expressed relative to the wild type at 25C for each sequences) and the outer repeat regions form two distinct domains (Fig. 1 A). The outer repeats are assembled in chromatin, which resembles centromeric heterochromatin in metazoa. Genes placed within fission yeast centromeres are transcriptionally silenced (Allshire et al., 1994, 1995), and this is likely to CCT241736 reflect packaging into heterochromatin and the assembly of the kinetochore complex. Several mutants have been identified that alleviate transcriptional silencing and concomitantly affect centromere CCT241736 function (Allshire et al., 1995; Ekwall et al., 1995, 1997; Partridge et al., 2000). Most of these affect only the heterochromatic domain and include mutants in and silencing (Partridge et al., 2000; Jin et al., 2002). The existence of two classes of mutants is reflected in the regions of centromeric DNA with which the encoded proteins associate (Partridge et al., 2000). Swi6 and Chp1 are specifically associated with the region, whereas Mis6 and Mal2 associate with the central core region (Saitoh et al., 1997; Partridge et al., 2000; Jin et al., 2002). Ultrastructural studies also reveal two physically distinct domains (Kniola et al., 2001). Nucleosomes of the outer repeats, like other heterochromatin, are Rabbit Polyclonal to CAPN9 underacetylated on histone H3 and H4 CCT241736 tails (Ekwall et al., 1997) and methylated on lysine 9 of H3. Recent observations suggest a model in which Clr4, guided by RNAi activity, methylates histone H3, promoting Swi6 binding and the assembly of transcriptionally silent heterochromatin (Bannister et al., 2001; Nakayama et al., 2001; Partridge et al., 2002; Volpe et al., 2002, 2003), which is required for the recruitment of a high density of Rad21-cohesin to the centromere (Bernard et al., 2001; Nonaka et al., 2002). Central core chromatin is unusual; limited digestion with micrococcal nuclease (MNase) generates a smear rather than a nucleosomal ladder typical of most chromatin (Polizzi and Clarke, 1991; Takahashi et al., 1992). Mutants in central coreCassociated proteins disrupt this central coreCspecific chromatin structure (Saitoh et al., 1997; Goshima et al., 1999; Takahashi et al., 2000; Jin et al., 2002). The composition of central core chromatin is also distinct, as evidenced by the specific association of the H3 variant Cnp1 (fission yeast CENP-A; Takahashi et al., 2000). Mis6 is required for the incorporation of newly synthesized Cnp1CGFP at centromeres (Takahashi et al., 2000). It is likely that the kinetochore proper assembles at the central core, and the outer repeats provide an important but auxiliary role, possibly affecting kinetochore conformation in addition to centromeric cohesion. In support of such a model, the MT-associated protein Dis1 is associated with the central core region in a mitosis-specific manner (Nakaseko et al., 2001). However, another MAP, Alp14/Mtc1, associates with and in CCT241736 mitosis, but not with (Garcia et al., 2001; Nakaseko et al., 2001). Each fission yeast kinetochore, like those of many metazoa, contains multiple MT-binding sites (Ding et al., 1993). In contrast, budding yeast centromeres associate with one MT (Winey et al., 1995). An additional level of organization must operate at the more complex kinetochores so that all MT attachment sites are oriented in a concerted fashion toward the same pole. Metazoan kinetochores are not easily dissected by genetic screens, and much knowledge has stemmed from the use of autoimmune sera (Pluta et al., 1995). An alternative route to the identification of mammalian kinetochore proteins is by homology to proteins discovered in genetically tractable organisms (Wigge and Kilmartin, 2001; Nishihashi et al., 2002). Screens based on minichromosome stability performed in budding and fission yeasts have garnered mutants affecting cohesion, replication, as well as kinetochore function (for review see Pidoux and Allshire, 2000b). The phenomenon of silencing at fission yeast.