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The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation.

Wigge PA, Kilmartin JV - J. Cell Biol. (2001)

Bottom Line: Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere.Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells.Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.

ABSTRACT
We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

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ChIP assay of strains containing prA-tagged Ndc80p complex components together with controls, prA-tagged Spc110p and Spc34p, and wt cells (K699). Regions of DNA amplified were across CEN3, across 1 kb to either side of CEN3, and across an AT-rich region (see Materials and Methods).
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Figure 6: ChIP assay of strains containing prA-tagged Ndc80p complex components together with controls, prA-tagged Spc110p and Spc34p, and wt cells (K699). Regions of DNA amplified were across CEN3, across 1 kb to either side of CEN3, and across an AT-rich region (see Materials and Methods).

Mentions: We tested for the presence of the Ndc80p complex at the yeast centromere using the ChIP assay (Meluh and Koshland 1997). Here the cells are cross-linked with formaldehyde then sonicated to shear DNA. The tagged protein is precipitated and any associated centromere DNA detected by PCR. We used the prA-tagged components of the Ndc80p complex because the protein precipitation is particularly clean (Fig. 1). Centromere DNA coprecipitated with all four components of the Ndc80p complex (Fig. 6), while sequences 1 kb to either side or an AT-rich region were not detected. Centromere DNA was not detected with two control proteins (Fig. 6), prA-tagged Spc110p, a component of the SPB (Kilmartin et al. 1993), and prA-tagged Spc34p, a spindle component associated with all of the spindle microtubules (Wigge et al. 1998).


The Ndc80p complex from Saccharomyces cerevisiae contains conserved centromere components and has a function in chromosome segregation.

Wigge PA, Kilmartin JV - J. Cell Biol. (2001)

ChIP assay of strains containing prA-tagged Ndc80p complex components together with controls, prA-tagged Spc110p and Spc34p, and wt cells (K699). Regions of DNA amplified were across CEN3, across 1 kb to either side of CEN3, and across an AT-rich region (see Materials and Methods).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199619&req=5

Figure 6: ChIP assay of strains containing prA-tagged Ndc80p complex components together with controls, prA-tagged Spc110p and Spc34p, and wt cells (K699). Regions of DNA amplified were across CEN3, across 1 kb to either side of CEN3, and across an AT-rich region (see Materials and Methods).
Mentions: We tested for the presence of the Ndc80p complex at the yeast centromere using the ChIP assay (Meluh and Koshland 1997). Here the cells are cross-linked with formaldehyde then sonicated to shear DNA. The tagged protein is precipitated and any associated centromere DNA detected by PCR. We used the prA-tagged components of the Ndc80p complex because the protein precipitation is particularly clean (Fig. 1). Centromere DNA coprecipitated with all four components of the Ndc80p complex (Fig. 6), while sequences 1 kb to either side or an AT-rich region were not detected. Centromere DNA was not detected with two control proteins (Fig. 6), prA-tagged Spc110p, a component of the SPB (Kilmartin et al. 1993), and prA-tagged Spc34p, a spindle component associated with all of the spindle microtubules (Wigge et al. 1998).

Bottom Line: Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere.Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells.Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council, Laboratory of Molecular Biology, Cambridge CB2 2QH, United Kingdom.

ABSTRACT
We have purified a complex from Saccharomyces cerevisiae containing the spindle components Ndc80p, Nuf2p, Spc25p, and Spc24p. Temperature-sensitive mutants in NDC80, SPC25, and SPC24 show defects in chromosome segregation. In spc24-1 cells, green fluorescence protein (GFP)-labeled centromeres fail to split during spindle elongation, and in addition some centromeres may detach from the spindle. Chromatin immunoprecipitation assays show an association of all four components of the complex with the yeast centromere. Homologues of Ndc80p, Nuf2p, and Spc24p were found in Schizosaccharomyces pombe and GFP tagging showed they were located at the centromere. A human homologue of Nuf2p was identified in the expressed sequence tag database. Immunofluorescent staining with anti-human Nuf2p and with anti-HEC, the human homologue of Ndc80p, showed that both proteins are at the centromeres of mitotic HeLa cells. Thus the Ndc80p complex contains centromere-associated components conserved between yeasts and vertebrates.

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