Embryo Nuclear extract

Additional biochemical characterization of in vitro disassembly products can be achieved through two-dimensional gel analysis. After either silver staining (Maus et ai, 1995) or immunoblot analysis (Smith et al., 1987 Havel et at., 1992), interphase lamins Dmi and Dm2 can be detected as multiple spots, indicating multiple posttranslationally modified forms. Meiotic lamin Dmmit in the stage 14 oocyte extract exhibits an entirely different pattern (Maus et ai, 1995). When embryo nuclei, for example, are added to an oocyte disassembly extract and incubated for 90 min, a pattern distinct from the interphase lamins and indistinguishable from that of the extract alone should be seen, indicating that embryo nuclear lamin was converted to forms highly similar to, if not identical to, meiotic lamin Dm ,ii. 

Fig. 17. Properties of RNP containing RNA synthesized in isolated cell nuclei of rat liver. A. Sucrose gradient sedimentation of nuclear extract obtained after in-vitro Incubation of isolated nuclei in the presence of four nucleoside-triphosphates. (SW-39 rotor, 4 hours, 15 to 30 percent sucrose gradient). —, radioactivity — —, OD26O. B. CsCI density gradient ultracentrifugation of 30S peak obtained in experiment A. —, OD260 — —1 radioactivity (cpm). (From Georgievand Samarina. 1969. An/7. Embryo . Morph., (Suppl. 1) 81-87.)...

Preparation of Nuclear Extracts from Drosophila Embryos and In Vitro Transcription Analysis ... 

Pool the nuclear extract, and quick-freeze in 300- j,l aliquots in liquid nitrogen. Store at -80°C. A typical yield from 200 g of embryos is approximately 30 ml of SNF. 

Chick embryo erythroblasts at 14 days actively synthesize globin, whereas cultured undifferentiated MSB cells do not. (a) Nuclei from each type of cell were isolated and exposed to increasing concentrations of DNase I. The nuclear DNA was then extracted and treated with the restriction enzyme SamHI, which cleaves the DNA around the globin sequence and normally releases a... 

Nuclear assembly is initiated in vitro by the addition of demembranated sperm (sperm chromatin) to a supplemented embryo extract. The extract should be thawed immediately before nuclear assembly and supplemented with an ATP-regenerating system and protease inhibitors such that it contains, finally, 2 mM Mg -ATP, 20 mM phosphocreatine, 50 /xg/ml creatine kinase, and 2 figl ml each of antipain, aprotinin, chymostatin, leupeptin, and pepstatin A. Typically, supplementation is accomplished by addition of one volume of freshly prepared lOx ATP-regenerating system plus protease inhibitors to nine volumes newly thawed extract. One volume newly thawed lOx sperm is then added to nine volumes supplemented extract such that, finally, about 2.4 x 10 demembranated sperm are incubated in 50 p at 24°C. For microscopic analysis, incubation should be stopped by addition of an equal volumes of 8% (w/v) paraformaldehyde in 154 mM PIPES-NaOH, pH 7.5 (Berrios and Avilion, 1990 Berrios and Colflesh,... 

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. 

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. 

Some degree of controversy exists as to whether nuclear envelope formation precedes, parallels, or follows the assembly of a nuclear lamina (see Georgatos et ai, 1994 Lourim and Krohne, 1994). Formation of the nuclear envelope in Drosophila embryo extracts is lamin dependent (Ulitzur et ai, 1992). In contrast, lamin-independent nuclear envelope assembly in vitro has been reported in Xenopus (Newport et ai, 1990 Meier et ai, 1991) and sea urchin (Collas et ai, 1995). The latter studies corroborate immunofluorescence observations of nuclear reconstitution after mitosis in somatic mammalian cells in vivo (Chaudhary and Courvalin, 1993). Assembly of a nuclear lamina in vitro can be monitored by immunofluorescence and immunoblotting using anti-lamin antibodies. 

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. 

In addition, we present methods for the preparation of HeLa cell extracts that can support import in vitro. In general, we find for both proteins and snRNPs that the Xenopus egg extract is the most efficient in terms of the speed of nuclear import and the final level of intranuclear accumulation. The Xenopus oocyte extract, HeLa cell extract, and reticulocyte lysates are generally less efficient in our hands. We have not been able to reconstitute nuclear import using extracts from Drosophila early embryos (C. Dingwall, unpublished). 

The amount of jSRNA began to increase in the early blastula stage which correlated with the accumulation of the cytoplasmic mRNA which contained poly (A). The maximum amount of aRNA (25% of the total heterogenous nuclear RNA) was obtained in the middle blastula stage (Dubroff and Nemer, 1975/76). The ratio of mRNA s with and without poly (A) sequences correlated with the increasing of size of free polysomes in the sea urchin embryo (Nemer and Surrey, 1975/76 Nemer et al., 1975). It is possible to translate different mRNA fractions extracted from sea urchin and amphibian embryos in the cell-free system (Ruder-man and Pardu, 1977). 

From 20 ml heparinized venous blood lymphocytes were isolated and one part of these was suspended in TC 199 medium, supplemented with calf serum, chicken embryo extract, antibiotics, and cultured for 72 hours in the presence of phytohaemagglutinin (PHA). The other part was used for experiments immediately. Before incubation, the cells were washed twice with isotonic Na,K-phosphate buffer pH 7.4 and resuspended in incubation medium, containing hypoxanthine-8- H (10 uCi/ml). After incubation for three hours at 37 , cells were spun down and washed three times. After hypotonic treatment and fixation, the cells were brought on slides, spread by flame drying, and stained with orceine. The slides were coated with Kodak Nuclear Track emulsion and developed after exposurefor 7 days. Details of the procedure are reported elsewhere (5) ... 

Kamakaka R.T. and Kadonaga J.T. 1994. The soluble nuclear fraction, a highly efficient transcription extract from Drosophila embryos. Methods Cell Biol. 44 225-235. 

The first method takes advantage of the cortical location of mitoses 11-13. Edgar et al. (1994) first identified live embryos that were undergoing mitosis by the disappearance of the nuclear envelope under DIG (differential interference contrast) optics. The embryos were then aged for appropriate time intervals to reach desired points in the following division cycle, prior to homogenization. Half of the extract from one embryo was used for HI kinase assays and the other half was immunoblotted to correlate kinase activity with phosphorylation on Cdkl and with cyclin levels. 

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