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Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

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Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins. / Gillespie, Peter J.; Gambus, Agnieszka; Blow, J. Julian.

In: Methods, Vol. 57, No. 2, 06.2012, p. 203-13.

Research output: Contribution to journalArticle

Harvard

Gillespie, PJ, Gambus, A & Blow, JJ 2012, 'Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins' Methods, vol 57, no. 2, pp. 203-13.

APA

Gillespie, P. J., Gambus, A., & Blow, J. J. (2012). Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins. Methods, 57(2), 203-13doi: 10.1016/j.ymeth.2012.03.029

Vancouver

Gillespie PJ, Gambus A, Blow JJ. Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins. Methods. 2012 Jun;57(2):203-13.

Author

Gillespie, Peter J.; Gambus, Agnieszka; Blow, J. Julian / Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins.

In: Methods, Vol. 57, No. 2, 06.2012, p. 203-13.

Research output: Contribution to journalArticle

Bibtex - Download

@article{846fc8bb6b1a41cd8dfe0e8fb63c3ce6,
title = "Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins",
author = "Gillespie, {Peter J.} and Agnieszka Gambus and Blow, {J. Julian}",
year = "2012",
volume = "57",
number = "2",
pages = "203--13",
journal = "Methods",
issn = "1046-2023",

}

RIS (suitable for import to EndNote) - Download

TY - JOUR

T1 - Preparation and use of Xenopus egg extracts to study DNA replication and chromatin associated proteins

A1 - Gillespie,Peter J.

A1 - Gambus,Agnieszka

A1 - Blow,J. Julian

AU - Gillespie,Peter J.

AU - Gambus,Agnieszka

AU - Blow,J. Julian

PY - 2012/6

Y1 - 2012/6

N2 - The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication.

AB - The use of cell-free extracts prepared from eggs of the South African clawed toad, Xenopus laevis, has led to many important discoveries in cell cycle research. These egg extracts recapitulate the key nuclear transitions of the eukaryotic cell cycle in vitro under apparently the same controls that exist in vivo. DNA added to the extract is first assembled into a nucleus and is then efficiently replicated. Progression of the extract into mitosis then allows the separation of paired sister chromatids. The Xenopus cell-free system is therefore uniquely suited to the study of the mechanisms, dynamics and integration of cell cycle regulated processes at a biochemical level. In this article we describe methods currently in use in our laboratory for the preparation of Xenopus egg extracts and demembranated sperm nuclei for the study of DNA replication in vitro. We also detail how DNA replication can be quantified in this system. In addition, we describe methods for isolating chromatin and chromatin-bound protein complexes from egg extracts. These recently developed and revised techniques provide a practical starting point for investigating the function of proteins involved in DNA replication.

KW - LICENSING SYSTEM

KW - Chromatin

KW - REQUIRES

KW - Cell-free system

KW - SPERM PRONUCLEI

KW - In vitro

KW - GEMININ

KW - Egg extract

KW - PREVENTING RE-REPLICATION

KW - CHROMOSOMAL DNA

KW - Xenopus

KW - CELL-CYCLE

KW - COMPONENT

KW - NUCLEI

KW - DNA replication

KW - INHIBITION

U2 - 10.1016/j.ymeth.2012.03.029

DO - 10.1016/j.ymeth.2012.03.029

M1 - Article

JO - Methods

JF - Methods

SN - 1046-2023

IS - 2

VL - 57

SP - 203

EP - 213

ER -

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