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Journal of Cell Science, Vol 108, Issue 5 1831-1841, Copyright © 1995 by Company of Biologists
JOURNAL ARTICLES |
S Chevalier, JP Tassan, R Cox, M Philippe and C Ford
Departement de Biologie et Genetique du Developpement, CNRS, URA 256, Universite de Rennes I, France.
Xenopus egg extracts induce S phase DNA replication in added sperm pronuclei in a highly regulated manner, similar to events in vivo. Removal of cyclin-dependant kinases (cdks) or cdk2 from these extracts using affinity matrices severely inhibits initiation of S phase. We have used p13suc1 beads to remove both cdk2 and cdc2 proteins from egg extracts and developed a method to replace either protein alone to assess their capacity to initiate DNA replication. Re-addition of either cdk2 or cdc2 proteins to depleted extracts, through translation of their respective mRNAs, restimulated replication, judged by both total synthesis and labelling index. An ATP-binding-site mutant cdk2 mRNA (cdk2.R33) failed to stimulate replication and inhibited S phase initiation in mock-depleted extracts. Both human and Xenopus cdc2 mRNAs rescued replication in this system. Human mutant mRNAs have been used to show that the stimulation induced requires cdc2 catalytic activity, though not its mitotically active form. Rescue of replication by p34cdc2 is also observed in extracts depleted of cdks with a cdk2 antibody, which still retain much of their endogenous cdc2 protein. We conclude that newly synthesised p34cdc2, but not the inherited 'old' form, can induce S phase and in this form may overlap in function with p33cdk2.
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C Roghi, R Giet, R Uzbekov, N Morin, I Chartrain, R Le Guellec, A Couturier, M Doree, M Philippe, and C Prigent The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly J. Cell Sci., January 3, 1998; 111(5): 557 - 572. [Abstract] [PDF]
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 A. Fotedar, D. Cannella, P. Fitzgerald, T. Rousselle, S. Gupta, M. Doree, and R. Fotedar Role for Cyclin A-dependent Kinase in DNA Replication in Human S Phase Cell Extracts J. Biol. Chem., December 6, 1996; 271(49): 31627 - 31637. [Abstract] [Full Text] [PDF]
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 J F Diffley Once and only once upon a time: specifying and regulating origins of DNA replication in eukaryotic cells. Genes & Dev., November 15, 1996; 10(22): 2819 - 2830. [PDF]
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S Chevalier, A Couturier, I Chartrain, R Le Guellec, C Beckhelling, K Le Guellec, M Philippe, and C. Ford Xenopus cyclin E, a nuclear phosphoprotein, accumulates when oocytes gain the ability to initiate DNA replication J. Cell Sci., January 6, 1996; 109(6): 1173 - 1184. [Abstract] [PDF]
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U. Strausfeld, M Howell, P Descombes, S Chevalier, R. Rempel, J Adamczewski, J. Maller, T Hunt, and J. Blow Both cyclin A and cyclin E have S-phase promoting (SPF) activity in Xenopus egg extracts J. Cell Sci., January 6, 1996; 109(6): 1555 - 1563. [Abstract] [PDF]
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H. Lindsay, M. Whitaker, and C. Ford Calcium requirements during mitotic cdc2 kinase activation and cyclin degradation in Xenopus egg extracts J. Cell Sci., January 11, 1995; 108(11): 3557 - 3568. [Abstract] [PDF]
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