Biomedical subjects
Publications and source records attributed to P A Wigge.
The complete genome sequence of the flowering plant Arabidopsis thaliana has been determined. New insights have come from comparisons between this sequence and genome sequences of other species, including those of cyanobacteria, yeast, worms and flies.
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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A highly enriched spindle pole preparation was prepared from budding yeast and fractionated by SDS gel electrophoresis. Forty-five of the gel bands that appeared enriched in this fraction were analyzed by high-mass accuracy matrix-assisted laser desorption/ ionization (MALDI) peptide mass mapping combined with sequence database searching. This identified twelve of the known spindle pole components and an additional eleven gene products that had not previously been localized to the spindle pole. Immunoelectron microscopy localized eight of these components to different parts of the spindle. One of the gene products, Ndc80p, shows homology to human HEC protein (Chen, Y., D.J. Riley, P-L. Chen, and W-H. Lee. 1997. Mol. Cell Biol. 17:6049-6056) and temperature-sensitive mutants show defects in chromosome segregation. This is the first report of the identification of the components of a large cellular organelle by MALDI peptide mapping alone.