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

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 e...

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Bibliographic Details
Published in:Methods (San Diego, Calif.) Calif.), 2012-06, Vol.57 (2), p.203-213
Main Authors: Gillespie, Peter J., Gambus, Agnieszka, Blow, J. Julian
Format: Article
Language:English
Subjects:
Amphibia
Animals
Cell cycle
Cell Extracts - genetics
Cell Extracts - isolation & purification
Cell Nucleus - metabolism
Cell-free system
Chemical Precipitation
Chromatin
Chromatin - genetics
Chromatin - isolation & purification
Chromatin - metabolism
DNA - genetics
DNA - isolation & purification
DNA - metabolism
DNA biosynthesis
DNA Replication
DNA-Binding Proteins - isolation & purification
DNA-Binding Proteins - metabolism
Egg extract
Eggs
Female
In vitro
Integration
Male
Mitosis
Nuclear Transfer Techniques
Nuclei
Oocytes - chemistry
Oocytes - cytology
Replication
sister chromatids
Sperm
Spermatozoa - cytology
Testis - cytology
Trichloroacetic Acid - chemistry
Xenopus
Xenopus laevis
Xenopus Proteins - isolation & purification
Xenopus Proteins - metabolism
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Summary: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.
ISSN:1046-2023
1095-9130
DOI:10.1016/j.ymeth.2012.03.029