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Analysis of the Cell Cycle Using Xenopus Egg Extracts

Sally Kornbluth1,  Jing Yang1,  Maureen Powers2

1Duke University Medical Center, Durham, North Carolina
2Emory University School of Medicine, Atlanta, Georgia


Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
Unit 11.11
DOI: 
10.1002/0471143030.cb1111s29
Online Posting Date: 
January, 2006
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Abstract

In this unit, Xenopus eggs are isolated from hormonally primed female frogs, and then the extract is treated with cyclohexamide so it remains in interphase of the cell cycle. In the presence of sperm chromatin and ATP, membrane vesicles in the extract fuse to assemble nuclei, making the extract suitable for studies of DNA replication and nuclear transport.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Preparation of the Cycling Extract
  • Basic Protocol 2: Preparation of CSF-Arrested Extracts
  • Basic Protocol 3: Preparing a Mitotic Extract
  • Basic Protocol 4: Driving Interphase Extracts into Mitosis
  • Alternate Protocol: Generating a Replication Checkpoint In Vitro
  • Support Protocol 1: Monitoring the Cell Cycle State of Extracts
  • Support Protocol 2: Assaying Histone H1 Kinase Activity
  • Support Protocol 3: Release of CSF-Arrested Extracts and Their Progression into Interphase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of the Cycling Extract

 Materials
  • 1× and 0.2× MMR (see recipe)
  • Unfertilized eggs laid in 1× MMR (see recipe)
  • Versilube F-50/silicone oil (see note in Basic Protocol 2)
  • XB buffer (see recipe)
  • CL protease inhibitor cocktail (see recipe)
  • 2% (w/v) cysteine (see recipe)
  • 5-ml polyallomer centrifuge tubes for Beckman SW 55 Ti rotor
  • Activation chamber (see Fig. 11.11.1)
  • 12 V (AC) power supply with a toggle switch to rapidly control the delivery of current
  • Wide-bore Pasteur pipet
  • 15-ml centrifuge tubes
  • Clinical centrifuge
  • Ice-water bath
  • Beckman floor model ultracentrifuge (with a SW 55 Ti rotor or equivalent)
  • 18-G needle attached to a 5-ml syringe
  • 1.5-ml microcentrifuge tubes and microcentrifuge at 4°C
  • Additional reagents and equipment for frog injection and egg collection (unit 11.10)

NOTE: Versilube F-50 silicon oil is the oil traditionally used in preparing cycling extracts. However, the company has recently replaced this with an M-20 oil, which purportedly has the same properties; this has not yet been tested extensively.

Basic Protocol 2: Preparation of CSF-Arrested Extracts

 Materials
  • Xenopus eggs in 1× MMR (see recipe)
  • 2% (w/v) cysteine (see recipe)
  • 0.2× MMR (see recipe)
  • XB buffer (see recipe)
  • Versilube F-50/silicon oil (Andpak-EMA)
  • 0.5 M EGTA stock solution, pH 8.0
  • 1 M MgCl2 stock solution
  • CL protease inhibitor cocktail (see recipe)
  • 2% (w/v) cytochalasin B (Calbiochem)
  • 5-ml polyallomer tubes for Beckman SW 55 Ti rotor (or equivalent)
  • Sawed-off and fire-polished Pasteur pipet
  • 15-ml centrifuge tube
  • Clinical centrifuge
  • Floor model high-speed centrifuge (e.g., Sorvall, Beckman)
  • Beckman SW 55 Ti swinging-bucket rotor, or equivalent
  • 5-ml syringe with an 18-G needle attached
  • Polycarbonate ultraclear microcentrifuge tubes (Beckman)
  • Microcentrifuge, 4°C

NOTE: Versilube F-50 silicon oil is the oil traditionally used in cycling extracts. However, the company has recently replaced this with an M-20 oil, which purportedly has the same properties; this has not yet been tested extensively.

Basic Protocol 3: Preparing a Mitotic Extract

 Materials
  • Xenopus eggs laid in 100 mM NaCl
  • 2% (w/v) cysteine (see recipe)
  • Mitotic buffer (see recipe)
  • AL protease inhibitor cocktail (see recipe)
  • 5 mg/ml cytochalasin B stock (see recipe)
  • Mitotic buffer (see recipe) supplemented with 250 mM sucrose (optional)
  • 15-ml polycarbonate Ultraclear centrifuge tubes
  • Clinical centrifuge
  • High-speed floor model centrifuge (e.g. Sorvall or Beckman)
  • Large-capacity swinging-bucket rotor (e.g. Sorvall HB-4 or HB-6)
  • 18-G needle attached to a 5-ml syringe

Basic Protocol 4: Driving Interphase Extracts into Mitosis

 Materials
  • Interphase extract (crude or fractionated) containing newly formed nuclei (see unit 11.10)
  • Recombinant cyclin protein (Desai et al., 1995)
  • Additional reagents and equipment for nuclear disassembly (unit 11.10)

Alternate Protocol: Generating a Replication Checkpoint In Vitro

 Materials
  • Sperm chromatin (unit 11.10)
  • 20× energy-regenerating mix (see recipe)
  • Cycling extract (see Basic Protocol 1)
  • 5 mg/ml aphidicolin stock solution (Sigma) in high-quality DMSO (e.g., Pierce) (see recipe)

Support Protocol 1: Monitoring the Cell Cycle State of Extracts

 Materials
  • Cycling extract
  • Sperm chromatin (unit 11.10)
  • 20× energy regenerating mix (see recipe)

Support Protocol 2: Assaying Histone H1 Kinase Activity

 Materials
  • Extract: cycling (see Basic Protocol 1) or interphase driven into mitosis (see Basic Protocol 4)
  • EB buffer (see recipe)
  • Liquid nitrogen
  • 10× kinase buffer (see recipe)
  • 1 mg/ml histone H1 (Roche Diagnostics)
  • 500 µM protein kinase inhibitor peptide (PKI; Sigma)
  • 0.2 M ATP
  • 5 mCi/ml [-32P]-ATP (3000 Ci/mmol)
  • 2× SDS sample loading buffer (appendix 2A)

Support Protocol 3: Release of CSF-Arrested Extracts and Their Progression into Interphase

 Materials
  • CSF-arrested extract (see Basic Protocol 2)
  • 20× energy-regenerating mix (see recipe)
  • Sperm chromatin (unit 11.10)
  • 1 M calcium chloride
  • Fixative for visualizing nuclear assembly (see recipe)
  • Cycloheximide (optional)
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 11.11.1
    The activation chamber. The chamber is 11 cm on a side and 5 cm high and made of Plexiglas. The bottom of the chamber and the underside of the lid are covered with stainless steel plate electrodes. There is a pad of 0.2% (w/v) agarose in 0.2× MMR buffer covering the bottom of the chamber. Eggs and 0.2× MMR buffer are poured onto the agarose pad at the base of the chamber. The electrodes are attached via leads to a 12-V AC power supply. It is critical that the buffer make contact with the upper plate electrode in order for current to pass through the chamber, thereby activating the eggs.

    View Image

Literature Cited

Literature Cited
    Dasso, M. and Newport, J.W. 1990. Completion of DNA replication is monitored by a feedback system that controls the initiation of mitosis in vitro: Studies in Xenopus. Cell 61:811-823.
    Desai, D., Wessling, H.C., Fisher, R.P., and Morgan, D.O. 1995. Effects of phosphorylation by CAK on cyclin binding by CDC2 and CDK2. Mol. Cell. Biol. 15:345-350.
    Glotzer, M., Murray, A., and Kirschner, M. 1991. Cyclin is degraded by the ubiquitin pathway. Nature 349: 132-138.
    Hirano, T. and Mitchison, T. 1994. A heterodimeric coiled-coil protein required for mitotic chromosome condensation in vitro. Cell 79:449-458.
    Minshull, J., Sun, H., Tonks, N.K., and Murray, A.W. 1994. A MAP kinase-dependent spindle assembly checkpoint in Xenopus egg extracts. Cell 79:475-486.
    Murray, A. and Kirschner, M. 1989. Cyclin synthesis drives the early embryonic cell cycle. Nature 339:275-280.
    Murray, A., Solomon, M.J., and Kirschner, M.W. 1989. The role of cyclin synthesis and degradation in the control of MPF activity. Nature 339: 280-286.
    Solomon, M., Glotzer, M., Lee, T., Philippe, M., and Kirschner, M. 1990. Cyclin activation of p34cdc2. Cell 63: 1013-1024.
     
 
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