Xenopus egg extract

Blow, J. J., and Nurse, P. (1990). Acdc2-like protein is involved in the initiation of DNA replication in Xenopus egg extracts. Cell 62 855-862. 

Clarke, P. R., Hoffman, I., Draetta, G., and Karsenti, E. (1993). Dephosphorylation of cdc25-C by a type-2A protein phosphatase specific regulation during the cell cycle in Xenopus egg extracts. Mol. Biol. Cell 4 397—411. 

The relationship between MAP kinase activity and/or its direct substrates (e.g. p90rsk) and CSF inactivation is unclear. Abrieu and colleagues (1996) found that in cell-free Xenopus eggs extracts MAP kinase remains active when MPF is inactivated. Thus, despite the fact that MAP kinase remains continuously active and phosphorylated, the CSF activity seems to be inactivated. A similar pattern... 

Abrieu A, Lorca T, Labbe J-C, Morin N, Keyse S, Doree M 1996 MAP kinase does not inactivate, but rather prevents the cyclin degradation pathway from being turned on in Xenopus egg extract. J Cell Sci 109 239—246... 

Elledge SJ 1996 Cell cycle checkpoints preventing an identity crisis. Science 274 1664—1672 Evans EK, Kombluth S 1998 Regulation of apoptosis in Xenopus egg extracts. Adv Enzyme Regul 38 265-280... 

Luger K, Richmond TJ (1998) DNA binding within the nucleosome core. Curr Opin Struct Biol 8 33-40 MacCallum DE, Losada A, Kobayashi R, Hirano T (2002) ISWl remodeling complexes in Xenopus egg extracts identification as major chromosomal components that are regulated by INCENP-aurora B. Mol Biol Cell 13 25-39... 

Kumar A, Brown DT, Leno GH (2004) DNA intercalators differentially affect chromatin structure and DNA repUcation in Xenopus egg extract. Anticancer Drugs 15(6) 633—639 Kuo MT (1981) Preferential damage of active chromatin by bleomycin. Cancer Res 41(6) 2439—2443 Kuo MT, Sarny TS (1978) Effects of neocarzinostatin on mammalian nuclei release of nucleosomes. Biochim Biophys Acta 518(1) 186—190... 

MacCallum DE, Losada A, Kobayashi R, Hirano T (2002) ISWI remodeling complexes in Xenopus egg extracts identification as major chromosomal components that are regulated by INCENP-aurora B. 

Scrittori L, Hans F, Angelov D, Charra M, Prigent C, Dimitrov S (2001) pEg2 aurora-A kinase, histone H3 phosphorylation, and chromosome assembly in Xenopus egg extract. J Biol Chem 276(32) 30002-30010... 

Fig. 4. Schematic of the single-molecule chromatin assembly experiments, (a) The fluorescence videomicroscopy experiments of Ladoux et al. [64] (for details see text), (b) The chromatin assembly experiments of Bennink et al. [65] a single >.-DNA molecule suspended between two micron-sized beads is being assembled with the help of Xenopus egg extract containing core histones and assembly factors (for details see text), (c) Example assembly curve at 1 pN (redrawn from Ref. [65]).

Hinchcliffe EH, Li C, Thompson EA, Mailer JL, Sluder G (1999) Requirement of Cdk2-cyclin E activity for repeated centrosome reproduction in Xenopus egg extracts. Science 283 851-854... 

Keratin IFs exhibit different motile behaviors compared with type III and IV IFs, and continue to move in the absence of MT in cells treated with MT inhibitors, such as nocodazole. It has been proposed that actin may be involved in their transport (Yoon et al., 2001). Time lapse imaging of keratin-GFP has revealed that keratin IF formation begins in the cell cortex in a region enriched in actin and required for both intact MT and actin, and that actin governs the organization and movement of keratin in Xenopus egg extracts (Weber and Bement, 2002). Less is known about specific mediators of these interactions, although the fact that myosin Va associates with NFs raises the interesting possibility that other such interactions may exist for keratins. 

Shirasu-Hiza, M., Coughlin, P., and Mitchison, T. (2003). Identification of XMAP215 as a microtubule-destabilizing factor in Xenopus egg extract by biochemical purification. J. Cell Biol. 161, 349-358. 

T Ohba, M. Nakamura, H. Nishitani, and T. Nishimoto. Self-organizanon of microtubule asters induced by Xenopus egg extracts by GTP-bound Ran. Science, 284, 1356-1358, 1999. 

Ellis, D.J., Jenkins, H., Whitfield, W.G. and Hutchison, C.J. (1997) GST-lamin fusion proteins act as dominant negative mutants in Xenopus egg extract and reveal the function of the lamina in DNA replication. J. Cell Sci. 110, 2507-2518. 

Guo Z, Kumagai A, Wang SX, Dunphy WG. Requirement for Atr in phosphorylation of Chkl and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev. 2000 14 2745-2756. 

Tirnauer JS, Grego S, Salmon ED, Mitchison TJ. EBl-microtubule interactions in Xenopus egg extracts role of EBl in microtubule stabilization and mechanisms of targeting to microtubules. Mol. Biol. Cell 2002 13 3614-3626. 

D.D. Newmeyer, D.M. Farschon, and J.C. Reed, Cell-free apoptosis in Xenopus egg extracts inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochodria. Cell 79 (1994), 353-364. 

Discovery In the course of screening mitotic spindle assembly inhibitors in Xenopus egg extract from the combinatorial library of 2,6,9-trisubstituted purines... 

When the duplicated centrosomes have become aligned, formation of the spindle proceeds, driven by simultaneous events at centrosomes and chromosomes. As just discussed, the centrosome facilitates spindle formation by nucleating the assembly of the spindle microtubules. In addition, the (—) ends of microtubules are gathered and stabilized at the pole by dynein-dynactin working with the nuclear/mitotic apparatus protein. The role of dynein in spindle pole formation has been demonstrated by reconstitution studies in which bipolar spindles form in Xenopus egg extracts in the presence of centrosomes, microtubules, and sperm nuclei. The addition of antibodies against cytosolic dynein to this in vitro system releases and splays the spindle microtubules but leaves the cen-trosomal astral microtubules in position (Figure 20-35). 

Cyclin B Levels and Kinase Activity of Mitosis-Promoting Factor (MPF) Change Together in Cycling Xenopus Egg Extracts... 

Studies with this egg extract experimental system were aided by development of a new assay for MPF activity. Using MPF purified with the help of the oocyte injection assay (see Figure 21-6), researchers had found that MPF phosphory-lates histone HI. This HI kinase activity provided a much simpler and more easily quantitated assay for MPF activity than the oocyte injection assay. Armed with a convenient assay, researchers tracked the MPF activity and concentration of cyclin B In cycling Xenopus egg extracts. These studies showed that MPF activity rises and falls In synchrony with the concentration of cyclln B (Figure 21-9a). The early events of mitosis—chromosome condensation and nuclear envelope disassembly—occurred when MPF activity reached its highest levels in parallel with the rise In cyclln B concentration. Addition of cycloheximide, an Inhibitor of protein synthesis, prevented cyclin B synthesis and also prevented the rise in MPF activity, chromosome condensation, and nuclear envelope disassembly. 

A EXPERIMENTAL FIGURE 21-9 Cycling of MPF activity and mitotic events in Xenopus egg extracts depend on synthesis and degradation of cyclin B. In all cases, MPF activity and cyclin B concentration were determined at various times after addition of sperm nuclei to a Xenopus egg extract treated as Indicated In each panel. Microscopic observations determined the occurrence of early mitotic events (blue shading),... 

In these experiments, chromosome decondensation and nuclear envelope assembly (late mitotic events) coincided with decreases in the cyclin B level and MPF activity. To determine whether degradation of cyclin B is required for exit from mitosis, researchers added a mutant mRNA encoding a nondegradable cyclin B to a mixture of RNase-treated Xenopus egg extract and sperm nuclei. As shown in Figure 2 l-9d, MPF activity Increased in parallel with the level of the mutant cyclin B, triggering condensation of the sperm chromatin and nuclear envelope disassembly (early mitotic events). However, the mutant cyclin B produced in this reaction never was degraded. As a consequence, MPF activity remained elevated, and the late mitotic events of chromosome decondensation and nuclear envelope formation were both blocked. This experiment demonstrates that the fall in MPF activity and exit from mitosis depend on degradation of cyclin B. 

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