Analysis of the Cell Cycle Using Xenopus Egg Extracts

Sally Kornbluth1, Jing Yang1, Maureen Powers2

1 Duke University Medical Center, Durham, North Carolina, 2 Emory 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

  • 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 1: 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 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. )
  • 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 MgCl 2 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., )
  • Additional reagents and equipment for nuclear disassembly (unit 11.10)

Alternate Protocol 1: Generating a Replication Checkpoint In Vitro

  Materials
  • Sperm chromatin (unit 11.10)
  • 20× energy‐regenerating mix (see recipe)
  • Cycling extract (see 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 protocol 1) or interphase driven into mitosis (see 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 [γ‐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 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)
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Figures

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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