Cell-free nuclear assembly

Cell-Free Nuclear Assembly and Disassembly in Drosophila... 

Lohka. M. J., and Masui, Y. (1984). Roles of cytosol and cytoplasmic particles in nuclear envelope assembly and sperm pronuclear formation in cell free preparations from amphibian eggs. J. Cell Biol. 9si 1222-1230. 

Meier, J., Campbell, K. H. S., Ford, C. C., Stick, R., and Hutchison, C. (1991). The role of lamin LIII in nuclear assembly and DNA replication, in cell-free extracts of Xenopus eggs. J. Cell Set. 98,271-279. 

Ulitzur, N.. and Gruenbaum, Y. (1989). Nuclear envelope assembly around sperm chromatin in cell-free preparations from Drosophila embryos. FEBS Lett. 259,113-116. 

Fig.l Cell-free assembly of nuclei from demebranated Xenopus sperm in a Drosophila embryo extract. Time course through sperm decondensation and nuclear formation monitored by phase-contrast microscopy. Incubation was at 24°C for the times indicated (a) 0 min incubation (b and c) 15 min (d and e) 20 min (f and g) 30 min (h-k) 45-50 min (l-p) 60-70 min. The bar in panel p designates 25 (im and applies to all panels. 

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). 

Newport, J. (1987). Nuclear reconstitution in vitro Stages of assembly around protein-free DNA. Cell (Cambridge, Mass.) 48, 205-217. 

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