Use of Mass Spectrometry to Study the Centromere and Kinetochore
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A number of paths have led to the present list of centromere proteins, which is essentially complete for constitutive structural proteins, but still may be only partial if we consider the many other proteins that briefly visit the centromere and kinetochore to fine-tune the chromatin and adjust other functions. Elegant genetics led to the description of the budding yeast point centromere in 1980. In the same year was published the serendipitous discovery of antibodies that stained centromeres of human mitotic chromosomes in antisera from CREST patients. Painstaking biochemical analyses led to the identification of the human centromere antigens several years later, with the first yeast proteins being described 6 years after that. Since those early days, the discovery and cloning of centromere and kinetochore proteins has largely been driven by improvements in technology. These began with expression cloning methods, which allowed antibodies to lead to cDNA clones. Next, functional screens for kinetochore proteins were made possible by the isolation of yeast centromeric DNAs. Ultimately, the completion of genome sequences for humans and model organisms permitted the coupling of biochemical fractionation with protein identification by mass spectrometry. Subsequent improvements in mass spectrometry have led to the current state where virtually all structural components of the kinetochore are known and where a high-resolution map of the entire structure will likely emerge within the next several years.
KeywordsCentromere Protein Stable Isotope Labeling Of Amino Acids In Cell Culture (SILAC) Ndc80 Complex Mis12 Complex Outer Kinetochore
We thank Shinya Ohta and Juri Rappsilber for allowing us to reproduce their unpublished data. Work in the Earnshaw lab is funded by the Wellcome Trust, of which W.C.E. is a Principal Research Fellow (grant number 073915). The Wellcome Trust Centre for Cell Biology is supported by core grant numbers 077707 and 092076.
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