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Cold Spring Harb Protoc. 2018 Feb 23. doi: 10.1101/pdb.prot097121. [Epub ahead of print]

Chromosome Cohesion and Condensation in Xenopus Egg Extracts.

Author information

1
Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104.
2
Program in Cell Cycle and Cancer Biology, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104 susannah-rankin@omrf.org.
3
Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104.

Abstract

Chromosome structure in both interphase and M-phase cells is strongly influenced by the action of the cohesin and condensin protein complexes. The cohesin complex tethers the identical copies of each chromosome, called sister chromatids, together following DNA replication and promotes normal interphase chromosome structure and gene expression. In contrast, condensin is active largely in M phase and promotes the compaction of individual chromosomes. The Xenopus egg extract system is uniquely suited to analyze the functions of both complexes. Egg extracts, in which the cell cycle state can be manipulated, contain stockpiles of nuclear proteins (including condensin and cohesin) sufficient for the assembly of thousands of nuclei per microliter. Extract prepared from unfertilized eggs is arrested by the presence of cytostatic factor (CSF) in a state with high levels of M-phase kinase activity, but can be stimulated to enter interphase, in which DNA replication occurs spontaneously. For cohesion assays, demembranated sperm nuclei are incubated in interphase extract, where they undergo rapid and synchronous DNA replication and cohesion establishment through the recruitment of proteins and other factors (e.g., nucleotides) from the extract. Sister chromatid cohesion is assessed by then driving the extract into M phase by the addition of fresh CSF-arrested extract. In contrast, because chromosome condensation occurs spontaneously in M-phase extracts, sperm nuclei are added directly to CSF extracts to assay condensation.

PMID:
29475994
DOI:
10.1101/pdb.prot097121

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