Drosophila cell-free extract

Stage 14 Drosophila egg chambers (hereafter referred to as oocytes) are arrested in first meiotic metaphase. A cell-free extract of these oocytes catalyzes apparent disassembly of purified Drosophila nuclei (purified from either embryos or tissue culture cells) as well as of nuclear lamin polymers formed in vitro from isolated interphase lamins (Maus et al., 1995). Biochemically, the oocyte extract catalyzes lamin solubilization and phosphorylation as well as characteristic changes in one- and two-dimensional gel mobility. Cell-free nuclear lamina disassembly is ATP dependent and addition of calcium to extracts blocks disassembly as judged both morphologically and biochemically. 

Crevel, G., and Cotterill, S. (1991). DNA replication in cell-free extracts from Drosophila melanogas-ter. EM BO J. 10, 4361-4369. 

Ulitzur. N., Harel, A., Feinstein, N., and Gruenbaum, Y. (1992). Lamin activity is essential for nuclear envelope assembly in a Drosophila embryo cell-free extract. J. Cell Biol. 119,17-25. 

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. 

Screens for small-molecule modulators of biological pathways typically utilize cultured cell lines, purified proteins, or, recently, model organisms (e.g., zebrafish. Drosophila C. elegans). Herein, we describe a method for using Xenopus laevis egg extract, a biologically active and highly tractable cell-free system that recapitulates a legion of complex chemical reactions foimd in intact cells. Specifically, we focus on the use of a luciferase-based fusion system to identify small-molecule modulators that affect protein turnover. 

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