Nuclear envelope cell-free systems

Burke, B., and Gerace, L. (1986). A cell free system to study reassembly of the nuclear envelope at the end of mitosis. Cell (Cambridge, Mass.) 44, 639-652. 

Baricheva, E. A., Berrios, M., Bogachev, S. S., Borisevich, 1. V., Lapik, E. R., Sharakhov, 1. V., Stuurman, N., and Fisher, P. A. (1996). DNA from Drosophila melanogasier B-helerochromatin binds specifically to nuclear lamins in vitro and the nuclear envelope in situ. Gene 171, 171-176. Berrios. M and Avilion, A. A. (1990). Nuclear formation in a Drosophila cell-free system. Exp. Cell Re.s. 191, 64-70. 

Dessev, G., Palazzo, R., Rebhun, L., and Goldman, R. (1989). Disassembly of the nuclear envelope of Spisula oocytes in a cell-free system. Dev. Biol. 131, 496-504. 

An advantage of cell-free systems is the potential to evaluate independently cytosolic and membrane vesicle (MV) contributions to nuclear development. Membrane-free cytosol is obtained after ultracentrifugation of crude lysates and MVs can be recovered from the pellets. Both cytosolic extracts and MVs can be stored frozen without detectable loss of envelope assembly activity. They can also be manipulated easily by chemical or enzymatic treatments. Such manipulations have enabled the identification of distinct steps of male pronuclear formation and of factors required for each of these steps, notably in Xenopus (Lohka and Masui, 1984 Wilson and Newport, 1988 Vigers and Lohka, 1 1 Boman et al., 1992) and the sea urchin (Cameron and Poccia, 1994 Collas and Poccia, 1995a,b Collas etal., 1995). Studies in the sea urchin and surf clam have indicated that decondensation of sperm chromatin in vitro meets several criteria established by microinjection of sperm nuclei into living eggs (Cothren and Poccia, 1993) and by electron microscopy observations of normal pronuclear formation in vivo (Longo and Anderson, 19( 1970). 

The procedures reported here have been used to investigate the development of male pronuclei in cell-free systems of sea urchins and, to a lesser extent, surf clams. Pronuclear formation in vitro is a slower process than that in vivo. This property has been used advantageously to examine the steps of pronuclear formation. Each step can be easily manipulated, but the methods described here may require some adjustments for other organisms. Sea urchin and surf clam male pronuclei formed in vitro are virtually complete, with decondensed chromatin, nuclear envelopes, pores, and lamina. 

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