ASSEMBLY OF CORRECT KINETOCHORE ARCHITECTURE IN XENOPUS EGG EXTRACT REQUIRES TRANSITION OF SPERM DNA
THROUGH INTERPHASE
E. Yu. Boyarchuk,1, 2 N. N. Nikolsky,1 M. Dasso,2
A. M. Arnaoutov 2, *
1 Institute of Cytology RAS, St. Petersburg, Russia, and
2 Laboratory of Gene Regulation and Development, NICHD, NIH, Bethesda, USA;
* e-mail: arnaouta@mail.nih.gov
Xenopus egg extracts provide a powerful tool for studying formation and function of chromosomes. Two alternative
protocols are generally used to obtain mitotic chromosomes. The first one employs direct assembly of
chromatin from sperm nuclei in CSF-arrested meiotic extracts, while the second is based on transition of sperm
DNA through a replication step, followed by re-establishing of CSF arrest. In this study we show that general kinetochore
structure is disrupted in chromosomes assembled directly in CSF egg extracts: the amounts of outer
kinetochore proteins such as Bub1, BubR1 and Dynactin subunit p150glued are reduced and the components of
the inner centromeric region (Aurora B kinase and Survivin) show compromised recruitment to centromeres. In
contrast, kinetochores on chromosomes assembled according to the second protocol closely resemble those in
somatic cells. Our results argue that transition of sperm nuclei through interphase is an essential step for proper
kinetochore assembly.
Key words: kinetochore, Xenopus laevis, Xenopus egg extract, chromosome
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