Gillespie PJ et al. (2012), Preparation and use of Xenopus egg extracts to ...

XB-ART-45188

Methods 2012 Jun 01;572:203-13. doi: 10.1016/j.ymeth.2012.03.029.

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

Gillespie PJ, Gambus A, Blow JJ.

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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.

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Species referenced: Xenopus laevis
Genes referenced: cdkn1b

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	Fig. 1. Xenopus eggs. (A and B) Unactivated, meiosis II metaphase arrested eggs, (C) activated egg and (D) apoptotic egg. A, C and D show top views; B shows side view.
	Fig. 2. Preparation of Xenopus egg extracts. (A) Egg extract, post-crushing spin. (i) lipid layer; (ii) crude cytoplasm; (iii) yolk platelets. Recovery of the crude cytoplasm by side puncture is shown. (B) Egg extract, post-clarifying spin. (i) lipid layer; (ii) cytoplasm; (iii) membranous layer, contains mitochondria; (iv) residual yolk platelets and insoluble material. (C and D) Drop-freezing the final extract in 20 μl aliquots in liquid nitrogen using a cut pipette tip and a plastic Petri dish.
	Fig. 3. Preparation of demembranated Xenopus sperm nuclei. (A) Schematic representation of the body portion of a frog; dashed box indicates the expected location of the testes internally. (B) Testes, in situ, post-incision. ∗testes; ∗∗digestive system. (C and D) Excised testis, pre- (C) and post- (D) removal of extraneous tissue and blood vessels. (E) Chopping the testes in a plastic Petri dish using a razor blade. (F) Filtered sperm solution.
	Fig. 4. Nuclear assembly and DNA replication in Xenopus egg extracts. (A) Timecourse of nuclear formation in Xenopus egg extracts. Sperm nuclei were incubated in metaphase arrested Xenopus egg extract released into interphase by addition of 0.3 mM CaCl2. Nuclear formation was followed over the course of 60 min by phase contrast (upper panels) and UV (lower panels) microscopy (i–vii). Sperm nuclei incubated in extract in the absence of CaCl2 were visualised after 60 min (viii). Bar = 10 μm. (B) The replication kinetics of sperm nuclei added to interphase egg extract as determined by the ‘TCA replication assay’.
	Fig. 5. Timecourse of replication factors association with chromatin. The replication reaction was assembled plus or minus 100 nM CDK inhibitor p27KIP1. (A) DNA synthesis was assayed by α32P-dATP incorporation. (B) Chromatin was isolated at the indicated times, separated by SDS–PAGE and immunoblotted with antibodies against the indicated replication proteins. The lower portion of the protein gel was stained with Coomassie for histones.
	Fig. 6. Release of native proteins from chromatin. Chromatin was isolated from egg extract in the middle of S-phase (when replisome proteins peak on chromatin) and proteins were optionally released from chromatin with benzonase. (Ex), 0.5 μl egg extract; (Ch), isolated chromatin, from 5 μl extract, after first centrifugation; other lanes correspond to insoluble pellet (P) and soluble supernatant (S) from 5 μl extract after a second centrifugation. Samples were separated by SDS–PAGE and immunoblotted with antibodies against indicated proteins.
	Fig. 7. Isolated intact nuclei. Nuclei were isolated from egg extract 40 min after replication reaction assembly. (A) Nucleus before isolation. (B) Isolated nucleus. (i) Phase contrast light microscopy; (ii) DNA was stained with Hoechst 33258 and visualised by UV fluorescence microscopy.

References [+] :

Blow, Preventing re-replication of DNA in a single cell cycle: evidence for a replication licensing factor. 1993, Pubmed, Xenbase