Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
This protocol describes a cell-free system for studying vertebrate centromere and kinetochore formation. We reconstitute tandem arrays of centromere protein A (CENP-A) nucleosomes as a substrate for centromere and kinetochore assembly. These chromatin substrates are immobilized on magnetic beads and then incubated in Xenopus egg extracts that provide a source for centromere and kinetochore proteins and that can be cycled between mitotic and interphase cell cycle states. This cell-free system lends itself to use in protein immunodepletion, complementation and drug inhibition as a tool to perturb centromere and kinetochore assembly, cytoskeletal dynamics, DNA modification and protein post-translational modification. This system provides a distinct advantage over cell-based investigations in which perturbing centromere and kinetochore function often results in lethality. After incubation in egg extract, reconstituted CENP-A chromatin specifically assembles centromere and kinetochore proteins, which locally stabilize microtubules and, on microtubule depolymerization with nocodazole, activate the mitotic checkpoint. A typical experiment takes 3 d.
Akiyoshi,
Tension directly stabilizes reconstituted kinetochore-microtubule attachments.
2010,
Pubmed
Akiyoshi,
Quantitative proteomic analysis of purified yeast kinetochores identifies a PP1 regulatory subunit.
2009,
Pubmed
Barnhart,
HJURP is a CENP-A chromatin assembly factor sufficient to form a functional de novo kinetochore.
2011,
Pubmed
Black,
Structural determinants for generating centromeric chromatin.
2004,
Pubmed
Blow,
Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs.
1986,
Pubmed
,
Xenbase
Carroll,
Centromere assembly requires the direct recognition of CENP-A nucleosomes by CENP-N.
2009,
Pubmed
Carroll,
Dual recognition of CENP-A nucleosomes is required for centromere assembly.
2010,
Pubmed
Cheeseman,
Molecular architecture of the kinetochore-microtubule interface.
2008,
Pubmed
Desai,
The use of Xenopus egg extracts to study mitotic spindle assembly and function in vitro.
1999,
Pubmed
,
Xenbase
Desai,
A method that allows the assembly of kinetochore components onto chromosomes condensed in clarified Xenopus egg extracts.
1997,
Pubmed
,
Xenbase
Gascoigne,
Induced ectopic kinetochore assembly bypasses the requirement for CENP-A nucleosomes.
2011,
Pubmed
Guse,
In vitro centromere and kinetochore assembly on defined chromatin templates.
2011,
Pubmed
,
Xenbase
Hannak,
Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts.
2006,
Pubmed
,
Xenbase
Harrington,
Formation of de novo centromeres and construction of first-generation human artificial microchromosomes.
1997,
Pubmed
Heald,
Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts.
1996,
Pubmed
,
Xenbase
Huynh,
A method for the in vitro reconstitution of a defined "30 nm" chromatin fibre containing stoichiometric amounts of the linker histone.
2005,
Pubmed
Hyman,
Microtubule-motor activity of a yeast centromere-binding protein complex.
1992,
Pubmed
Ikeno,
Construction of YAC-based mammalian artificial chromosomes.
1998,
Pubmed
Kim,
Multisite M-phase phosphorylation of Xenopus Wee1A.
2005,
Pubmed
,
Xenbase
Kingsbury,
Centromere-dependent binding of yeast minichromosomes to microtubules in vitro.
1991,
Pubmed
Lowary,
New DNA sequence rules for high affinity binding to histone octamer and sequence-directed nucleosome positioning.
1998,
Pubmed
Luger,
Characterization of nucleosome core particles containing histone proteins made in bacteria.
1997,
Pubmed
,
Xenbase
Luger,
Expression and purification of recombinant histones and nucleosome reconstitution.
1999,
Pubmed
Luger,
Preparation of nucleosome core particle from recombinant histones.
1999,
Pubmed
,
Xenbase
Maresca,
Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.
2006,
Pubmed
,
Xenbase
Mendiburo,
Drosophila CENH3 is sufficient for centromere formation.
2011,
Pubmed
Minshull,
A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts.
1994,
Pubmed
,
Xenbase
Murray,
Cyclin synthesis drives the early embryonic cell cycle.
1989,
Pubmed
,
Xenbase
Nakano,
Inactivation of a human kinetochore by specific targeting of chromatin modifiers.
2008,
Pubmed
Nakashima,
Assembly of additional heterochromatin distinct from centromere-kinetochore chromatin is required for de novo formation of human artificial chromosome.
2005,
Pubmed
Ohzeki,
Breaking the HAC Barrier: histone H3K9 acetyl/methyl balance regulates CENP-A assembly.
2012,
Pubmed
Okada,
CENP-B controls centromere formation depending on the chromatin context.
2007,
Pubmed
Sandall,
A Bir1-Sli15 complex connects centromeres to microtubules and is required to sense kinetochore tension.
2006,
Pubmed
Selleck,
Recombinant protein complex expression in E. coli.
2008,
Pubmed
Sorger,
Factors required for the binding of reassembled yeast kinetochores to microtubules in vitro.
1994,
Pubmed
Tan,
A modular polycistronic expression system for overexpressing protein complexes in Escherichia coli.
2001,
Pubmed
Thåström,
Sequence motifs and free energies of selected natural and non-natural nucleosome positioning DNA sequences.
1999,
Pubmed
Vary,
Assembly of yeast chromatin using ISWI complexes.
2004,
Pubmed
