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Volumn 283, Issue 5403, 1999, Pages 851-854

Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in xenopus egg extracts

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIN DEPENDENT KINASE; CYCLIN E;

EID: 0033525007     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5403.851     Document Type: Article
Times cited : (444)

References (38)
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    • note
    • Cycling Xenopus egg extracts were prepared as described (76) and used fresh. Demembranated sperm nuclei were prepared as described (79) and used at a final concentration of 400 heads/μl. Aphidicolin (Sigma) was added (final concentration of 10 μg/ml). Histone HI kinase assays on egg extracts were done as described (6, 16). Histone H1 phosphorylation was analyzed by a phosphoimager (Molecular Dynamics, Sunnydale, CA) as described (6). To visualize asters, we placed extract in an FC-47 chamber [G. Sluder et al., Meth. Cell Biol. 61, 439 (1998)] and viewed it with a modified Zeiss ACM microscope (Zeiss) with polarization optics and a charge-coupled device camera (Hammamatsu, East Bridgewater, NJ, or Dage-MTI, Michigan City, IL) at 20°C. Time-lapse video images were written directly to the hard drive of a PC, as described (6). Video sequence playback of aster doubling was done with Adobe Premiere Software (Mountain View, CA).
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    • note
    • Glutathione 5-transferase-tagged fusion proteins of Δ34Xic1 and C-Xic1 were prepared and purified as described (13, 24). Recombinant Xenopus Cdk2-E complex was expressed, purified, and tested for kinase activity in vitro as described (13, 24).
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    • note
    • Immunofluorescence staining of Xenopus embryos was done as described (8), with affinity-purified polyclonal antibody to cyclin E (9) and monoclonal antibodies to α tubulin or γ tubulin (Sigma). Confocal microscopy was performed on an MRC-600 system (Bio-Rad, Hercules, CA). The confocal images presented represent projections of Z-series scans.
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    • note
    • We thank F. J. Miller for help developing the observation chambers, C. Wilkerson and M. Cham for assistance in building the image acquisition computers, and R. Pope, T. Pederson, G. Witman, and B. Luna for comments on the manuscript Supported by the NIH (J.L.M. and G.S.), the Trustees of the Worcester Foundation (G.S.), and the Cabot Family Charitable Trust (G.S.). J.L.M. is an investigator of the Howard Hughes Medical Institute. E.H.H. is supported by an NIH postdoctoral training fellowship. The authors would like to dedicate this work to the memory of Dan Mazia.


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