Drosophila disassembly

Maus, N., Stuurman, N., and Fisher, P. A. (1995). Disassembly of the Drosophila nuclear lamina in a homologous cell-free system. J. Cell ScL 108,2027-2035. 

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

V. Cell-Free Disassembly of Drosophila Lamin Aggregates (Polymers)... 

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. 

Biochemical analyses can be performed in parallel with or instead of morphological studies. At various time points during the course of in vitro disassembly, aliquots of the reaction mixture should be separated into supernatant and pellet fractions by sedimentation at 12,000 g and proteins subjected to SDS-PAGE and immunoblot analysis. An extract of stage 14 Drosophila oocytes contains only lamin Dm i, (Smith and Fisher, 1989 see also Lin and Fisher, 1990 Mans et at., 1995). Purified Drosophila nuclei contain lamins Dmi and Dm2 (Smith et al, 1987 see also Lin and Fisher, 1990 Maus et al, 1995). Lamin Dmmit migrates with a gel mobility intermediate to lamins Dm, and Dm2 (Fig. 4). 

In general, the remodeling of sperm chromatin that accompanies stage I decondensation involves the replacement of sperm-specific basic proteins with histones from the egg and results in the formation of nucleosomes. It now seems clear, at least in amphibian egg and Drosophila embryo extracts, that this remodeling is mediated by specific factors that participate in both assembly and disassembly processes. These changes in chromatin composition and structure can be analyzed by one- or two-dimensional polyacrylamide gel electrophoresis and micrococcal nuclease digestion. 

Kadonaga J.T. 1990. Assembly and disassembly of the Drosophila RNA polymerase II complex during transcription. /. Biol. Chem. 265 2624—2631. 

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