The centromeric binding of kinetochore components is partially impaired in mutants

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Actin-related protein Arp4 functions in kinetochore assembly"</p><p></p><p>Nucleic Acids Research 2007;35(9):3109-3117.</p><p>Published online 22 Apr 2007</p><p>PMCID:PMC1888834.</p><p>© 2007 The Author(s)</p> 3HA-tagged or untagged wild-type and cells were grown in YPAD at 23°C for 3 h with 15 μg/ml nocodazole to ensure that both populations had an equivalent cell cycle distribution since a higher proportion of cells are in G2/M phase. The culture was shifted to 37°C and incubated in the presence of nocodazole for 1 h. Cells were fixed with 1% formaldehyde for 15 min and subjected to ChIP. Input DNA and DNA coimmunoprecipitated with the anti-HA antibody (IP) were amplified with primer sets corresponding to sequences around centromeres (). Quantitative data were obtained by real-time PCR. To ensure the linearity of the PCR signal, appropriate dilutions of IP samples were used in PCR amplifications. In each case, ChIP enrichment is expressed relative to that for a subtelomeric region of chromosome V (9716–9823). Results are expressed as the mean and SD of two independent ChIP experiments. Dashed lines indicate the background level of ChIP signal intensity in an untagged strain. () Schematic of kinetochore components. () The centromere-specific histone H3 variant Cse4p (wild-type cells: YHO805; cells: YHO825), a representative protein of the inner kinetochore Mif2p (wild-type cells: YHO806; cells: YHO826) and Ndc10p (wild-type cells: YHO807; cells: YHO827) were analyzed by ChIP at . () A representative protein of the outer kinetochore Mtw1p (wild-type cells: YHO808; cells: YHO828), Nuf2p (wild-type cells: YHO809; cells: YHO829) and Ctf3p (wild-type cells: YHO810; cells: YHO830) were analysed by ChIP at . () The cohesin component Scc1p (wild-type cells: YHO811; cells: YHO831) was analysed by ChIP at