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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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Synthetic growth defect in the ndc80-1, ndc10-1 and nuf2-61, ndc10-1 double mutants when cells were spotted on plates with increasing dilutions at 30°C. This effect was not observed when the ndc10-2 allele was used or the double mutants were transformed with a plasmid containing NDC10.
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Figure 5: Synthetic growth defect in the ndc80-1, ndc10-1 and nuf2-61, ndc10-1 double mutants when cells were spotted on plates with increasing dilutions at 30°C. This effect was not observed when the ndc10-2 allele was used or the double mutants were transformed with a plasmid containing NDC10.

Mentions: We also looked for genetic interactions with genes encoding some of the centromere-associated components in S. cerevisiae. This is because of the defect in chromosome segregation and because a conditional synthetic lethal effect was detected between ndc80-1 and a deletion of CTF19, which encodes a centromere component (Hyland et al. 1999). Since this deletion of CTF19 shows synthetic lethality (Hyland et al. 1999) with mutants in genes encoding all four components of the centromere–binding factor 3 (CBF3) complex which directly binds CEN DNA (Lechner and Carbon 1991), we checked for similar synthetic lethal effects with the four ts mutants in the Ndc80p complex. We looked for interactions with ts mutants in three of the CBF3 components: ndc10-1 (Goh and Kilmartin 1993), ndc10-2 (Kopski and Huffaker 1997), ctf13-30 (Doheny et al. 1993), and cep3-1 (Strunnikov et al. 1995). No interactions were detected with ndc10-2 (Fig. 5), ctf13-30, or cep3-1 (data not shown). However, a synthetic growth defect was detected between ndc10-1 and ts mutants in all four components of the Ndc80p complex. This varied between the different ts mutants, being strongest with ndc80-1, intermediate with nuf2-61 (Fig. 5) and spc25-1, and weakest with spc24-1 (data not shown). These results suggest an association between the Ndc80p complex and the yeast centromere.


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)

Synthetic growth defect in the ndc80-1, ndc10-1 and nuf2-61, ndc10-1 double mutants when cells were spotted on plates with increasing dilutions at 30°C. This effect was not observed when the ndc10-2 allele was used or the double mutants were transformed with a plasmid containing NDC10.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199619&req=5

Figure 5: Synthetic growth defect in the ndc80-1, ndc10-1 and nuf2-61, ndc10-1 double mutants when cells were spotted on plates with increasing dilutions at 30°C. This effect was not observed when the ndc10-2 allele was used or the double mutants were transformed with a plasmid containing NDC10.
Mentions: We also looked for genetic interactions with genes encoding some of the centromere-associated components in S. cerevisiae. This is because of the defect in chromosome segregation and because a conditional synthetic lethal effect was detected between ndc80-1 and a deletion of CTF19, which encodes a centromere component (Hyland et al. 1999). Since this deletion of CTF19 shows synthetic lethality (Hyland et al. 1999) with mutants in genes encoding all four components of the centromere–binding factor 3 (CBF3) complex which directly binds CEN DNA (Lechner and Carbon 1991), we checked for similar synthetic lethal effects with the four ts mutants in the Ndc80p complex. We looked for interactions with ts mutants in three of the CBF3 components: ndc10-1 (Goh and Kilmartin 1993), ndc10-2 (Kopski and Huffaker 1997), ctf13-30 (Doheny et al. 1993), and cep3-1 (Strunnikov et al. 1995). No interactions were detected with ndc10-2 (Fig. 5), ctf13-30, or cep3-1 (data not shown). However, a synthetic growth defect was detected between ndc10-1 and ts mutants in all four components of the Ndc80p complex. This varied between the different ts mutants, being strongest with ndc80-1, intermediate with nuf2-61 (Fig. 5) and spc25-1, and weakest with spc24-1 (data not shown). These results suggest an association between the Ndc80p complex and the yeast centromere.

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.

Show MeSH
Related in: MedlinePlus