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Cold Spring Harb Symp Quant Biol. 2010;75:433-8. doi: 10.1101/sqb.2010.75.022. Epub 2011 Feb 2.

Proteomics of isolated mitotic chromosomes identifies the kinetochore protein Ska3/Rama1.

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Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, United Kingdom.


Despite many decades of study, mitotic chromosomes remain poorly characterized with respect to their structure and composition. Here, we have purified mitotic chromosomes from nocodazole-treated chicken DT40 cells. These chromosomes have a 0.7:1:1 ratio of nonhistone proteins to histones to DNA. They also contain a significant content of RNAs that have yet to be characterized. Overall, the isolated chromosomes contained >4000 polypeptides, >500 of which are either novel or uncharacterized. Elsewhere, we have developed an approach for comparing the results of multiple proteomics experiments. As a validation of this approach, one of 13 novel centromere proteins identified was found to occur in a complex with the previously described proteins Ska1 and Ska2. This novel protein, now known as Ska3/Rama1, occupies a unique domain in the outer kinetochore and was revealed by RNA interference (RNAi) experiments to be essential for cell cycle progression in human cells. The approach presented here offers a powerful way to define the functional proteome of complex organelles and structures whose composition is not simple or fixed.

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