Preparation and Use of Interphase Xenopus Egg Extracts

Maureen Powers1, Erica K. Evans2, Jing Yang2, Sally Kornbluth2

1 Emory University School of Medicine, Atlanta, Georgia, 2 Duke University Medical Center, Durham, North Carolina
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 11.10
DOI:  10.1002/0471143030.cb1110s09
Online Posting Date:  May, 2001
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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 Interphase Egg Extracts
  • Alternate Protocol 1: Preparing Fractionated Interphase Extracts
  • Support Protocol 1: Injection of Frogs to Obtain Eggs
  • Basic Protocol 2: Nuclear Assembly in the Interphase Egg Extract
  • Support Protocol 2: Preparation of Demembranated Sperm Chromatin to Use as Templates for Nuclear Assembly
  • Basic Protocol 3: Nuclear Protein Import In Vitro
  • Basic Protocol 4: DNA Replication with Continuous Labeling
  • Alternate Protocol 2: Pulse‐Labeling DNA to Assess Replication
  • Basic Protocol 5: Preparation of Oocyte Extract
  • Support Protocol 3: Immunodepletion of Extracts
  • Support Protocol 4: Addition of Protein to Extracts
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of Interphase Egg Extracts

  Materials
  • Eggs obtained from female frogs, in 100 mM NaCl (see protocol 3)
  • 2% (w/v) L‐cysteine free base (Sigma) in H 2O
  • 0.25× modified Ringer's solution (MMR), pH 7.7 (see recipe for 10×)
  • Egg lysis buffer, pH 7.7 (see recipe)
  • 5 mg/ml cytochalasin B (Calbiochem) in DMSO
  • 5 mg/ml aprotinin (Roche Diagnostics) in H 2O
  • 5 mg/ml leupeptin (Roche Diagnostics) in H 2O
  • 10 mg/ml cycloheximide in H 2O
  • 200‐ml beaker
  • 100‐mm glass petri dish
  • Pasteur pipets
  • Dissecting microscope
  • 15‐ml conical polypropylene centrifuge tubes
  • IEC Clinical centrifuge
  • Sorvall HB‐4 swinging bucket rotor
  • 18‐G needle attached to 5‐ml syringe

Alternate Protocol 1: Preparing Fractionated Interphase Extracts

  • Crude interphase extract (see protocol 1)
  • 2.5‐ml Ultraclear centrifuge tubes
  • TLS centrifuge and TLS‐55 rotor (Beckman) or equivalent

Support Protocol 1: Injection of Frogs to Obtain Eggs

  Materials
  • Female frogs, mature (e.g., Nasco, Xenopus I, or Xenopus Express)
  • 100 mM NaCl (25 g NaCl/5 liters H 2O)
  • 200 U/ml pregnant mare serum gonadotropin (PMSG; Calbiochem): reconstitute powder in sterile water to allow final injection volume of 0.5 ml (i.e., 200 U/ml final concentration); store in aliquots indefinitely at −20°C
  • 1000 U/ml human chorionic gonadotropin (HCG; Sigma or Amersham/USB): reconstitute powder in sterile water to allow final injection volume of 0.5 ml (i.e., 1000 U/ml final concentration); store up to 1 month at 4°C
  • 1× modified Ringer's solution (MMR; optional; see recipe)
  • Water tanks
  • 1‐ml syringes
  • 25‐G needles

Basic Protocol 2: Nuclear Assembly in the Interphase Egg Extract

  Materials
  • Interphase extract: crude (see protocol 1) or fractionated (see protocol 2)
  • 0.2 M phosphocreatine (store in small aliquots at −20°C)
  • 5 mg/ml creatine kinase (store in small aliquots at −20°C)
  • 0.2 M ATP (store in small aliquots at −20°C)
  • Sperm chromatin (see protocol 5)
  • Fixative for visualizing nuclear assembly (see recipe)
  • Microscope slides
  • 18‐mm2 glass coverslips
  • Fluorescence microscope

Support Protocol 2: Preparation of Demembranated Sperm Chromatin to Use as Templates for Nuclear Assembly

  Materials
  • 4 to 5 male Xenopus (e.g., Nasco, Xenopus I, or Xenopus Express)
  • 0.1% (w/v) Tricaine (see recipe)
  • Extraction buffer (see recipe), 4°C
  • Extraction buffer (see recipe) containing 200 mM, 2 M, 2.3 M, and 2.5 M sucrose
  • 5 mg/ml aprotinin (Roche Diagnostics) in H 2O
  • 5 mg/ml leupeptin (Roche Diagnostics) in H 2O
  • Dithiothreitol (DTT)
  • Triton X‐100
  • Sucrose, ultrapure
  • Bovine serum albumin (BSA; fraction V)
  • Dissecting tools including scissors and 2 pairs of sharp forceps
  • 60‐mm glass petri dish
  • 15‐ml conical polypropylene tubes
  • Tabletop centrifuge
  • Refrigerated centrifuge with Sorvall HB‐4 swinging‐bucket rotor or equivalent
  • 2.5 ml Beckman Ultraclear centrifuge tubes
  • TL‐100 tabletop centrifuge and TLS‐55 rotor (Beckman) or equivalent
  • Additional reagents and equipment for counting with a hemacytometer (unit 1.1)

Basic Protocol 3: Nuclear Protein Import In Vitro

  Materials
  • Interphase egg extract: crude (see protocol 1) or fractionated (see protocol 2)
  • ATP‐regenerating system (see protocol 4)
  • Xenopus sperm chromatin (see protocol 5)
  • Nuclear import substrate (unit 11.7)
  • Fix 1 (see recipe for fixatives for visualizing nuclear protein import)
  • 16% (w/v) paraformaldehyde
  • Fix 2 (see recipe for fixatives for visualizing nuclear protein import)
  • Microscope slides
  • Coverslips
  • Fluorescence microscope equipped with:
  •  64× objective (e.g., Zeiss 64× planapochromat, large aperture)
  •  CCD camera
  •  Frame grabber
  •  Computer running image analysis software (e.g., NIH Image)
  • Additional reagents and equipment for assembling nuclei (see protocol 4)

Basic Protocol 4: DNA Replication with Continuous Labeling

  Materials
  • Interphase extract (see protocol 1)
  • 0.2 M phosphocreatine (store in small aliquots at −20°C)
  • 5 mg/ml creatine kinase (store in small aliquots at −20°C)
  • 0.2 M ATP (store in small aliquots at −20°C)
  • Demembranated sperm chromatin (see protocol 5)
  • 10 µCi/µl [α–32P]dCTP (3000 Ci/mmol)
  • Replication stop buffer (see recipe)
  • 10 mg/ml proteinase K (Worthington)
  • Additional reagents and equipment for agarose gel electrophoresis ( appendix 3A) and autoradiography or phosphorimaging (unit 6.3)

Alternate Protocol 2: Pulse‐Labeling DNA to Assess Replication

  • XB buffer (see recipe)

Basic Protocol 5: Preparation of Oocyte Extract

  Materials
  • Female frogs, mature (e.g., Nasco, Xenopus I, or Xenopus Express)
  • 0.1% (w/v) Tricaine (see recipe)
  • Modified Barth's (MB) with and without calcium (see recipe)
  • 1 mg/ml collagenase A (Roche Diagnostics) in MB (see recipe) without calcium
  • EB buffer (see recipe)
  • 1000× CLAP protease inhibitor cocktail (see recipe)
  • 1000× (5 mg/ml) cytochalasin B in DMSO
  • Dissecting microscope
  • Dissecting scalpel
  • Sharp forceps
  • 50‐ml plastic tubes
  • Tube rotator
  • Clinical centrifuge
  • Tabletop ultracentrifuge and swinging‐bucket rotor
  • 1‐ml syringe
  • 18‐G needle

Support Protocol 3: Immunodepletion of Extracts

  Materials
  • Antibody/antiserum
  • Preimmune serum or control IgG
  • Protein A–Sepharose
  • Phosphate‐buffered saline(PBS; see recipe)
  • Bovine serum albumin (BSA), fraction V
  • XB buffer (see recipe)

Support Protocol 4: Addition of Protein to Extracts

  Materials
  • Recombinant protein of interest
  • XB buffer (see recipe)
  • Additional reagents and equipment for dialysis and microconcentration ( appendix 3C)
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Figures

Videos

Literature Cited

Literature Cited
   Blow, J.J. and Laskey, R.A. 1986. Initiation of DNA replication in nuclei and purified DNA by a cell‐free extract of Xenopus eggs. Cell 47:577‐587.
   Blow, J.J. and Laskey, R.A. 1988. A role for the nuclear envelope in controlling DNA replication within the cell cycle. Nature 332:546‐548.
   Carpenter, P.B., Mueller, P.R., and Dunphy, W.G. 1996. Role for a Xenopus Orc2–related protein in controlling DNA replication. Nature 379:357‐360.
   Finlay, D.R. and Forbes, D.J. 1990. Reconstitution of biochemically altered nuclear pores: Transport can be eliminated and restored. Cell 60:17‐29.
   Finlay, D.R., Newmeyer, D.D., Hartl, P.M., Horecka, J., and Forbes, D.J. 1989. Nuclear transport in vitro. J. Cell Sci. 11:225‐242.
   Forbes, D.J., Kirschner, M.W., and Newport, J.W. 1983. Spontaneous formation of nucleus‐like structures around bacteriophage DNA microinjected into Xenopus eggs. Cell 34:13‐23.
   Gorlich, D., Prehn, S., Laskey, R.A., and Hartmann, E. 1994. Isolation of a protein that is essential for the first step of nuclear protein import. Cell 79:767‐778.
   Hartl, P., Olson, E., Dang, T., and Forbes, D.J. 1994. Nuclear assembly with lambda DNA in fractionated Xenopus egg extracts: An unexpected role for glycogen in formation of a higher order chromatin intermediate. J. Cell Biol. 124:235‐248.
   Lew, D.J. and Kornbluth, S. 1996. Regulatory roles of cyclin‐dependent kinase phosphorylation in cell cycle control. Curr. Opin. Cell Biol. 8:795‐804.
   Macaulay, C. and Forbes, D.J. 1996. Assembly of the nuclear pore: Biochemically distinct steps revealed with NEM, GTP gamma S, and BAPTA. J. Cell Biol. 132:5‐20.
   Mechali, M. and Harland, R.M. 1982. DNA synthesis in a cell‐free system from Xenopus eggs: Priming and elongation on single‐stranded DNA in vitro. Cell 30:93‐101.
   Moore, M.S. and Blobel, G. 1993. The GTP‐binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365:661‐663.
   Newmeyer, D.D., Finlay, D.R., and Forbes, D.J. 1986. In vitro transport of a fluorescent nuclear protein and exclusion of nonnuclear proteins. J. Cell Biol. 103:2091‐2102.
   Nurse, P. 1990. Universal control mechanism regulating onset of M‐phase. Nature 344:503‐508.
   Philpott, A., Leno, G.H., and Laskey, R.A. 1991. Sperm decondensation in Xenopus egg cytoplasm is mediated by nucleoplasmin. Cell 65:569‐578.
   Powers, M.A., Forbes, D.J., Dahlberg, J.E., and Lund, E. 1997. The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways. J. Cell Biol. 136:241‐250.
   Sheehan, M.A., Mills, A.D., Sleeman, A.M., Laskey, R.A., and Blow, J.J. 1988. Steps in the assembly of replication‐competent nuclei in a cell‐free system from Xenopus eggs. J. Cell Biol. 106:1‐12.
   Wilson, K.L. and Newport, J. 1988. A trypsin‐sensitive receptor on membrane vesicles is required for nuclear envelope formation in vitro. J. Cell Biol. 107:57‐68.
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