Nuclear assembly

Montross L, Watkins S, Moreland RB, Mamon H, Caspar DL, Garcea RL (1991), Nuclear assembly of polyomavirus capsids in insect cells expressing the major capsid protein VP1,J. Virol. 65 4991-4998. 

The authors propose that this is the mechanism by which the S0 level is slowly oxidized to Si in the dark this may be necessary to oxidize a Mn2+ ion in the S0 level to Mn3+ to prevent dissociation of a tetra-nuclear assembly and would, therefore, be similar to the photoactivation by which Mn2+ is incorporated into the enzyme (279, 283). 

Although the presence of mixed bridging ligands may favour the formation of high nuclearity assemblies, it remains difficult to pinpoint the factors that give rise to these unusual cyclic products. In accordance with the previous discussion, it seems likely that these multi-component species may exist in solution in equilibrium with related oligomeric species their isolation will thus be influenced by both the position of the equilibrium as well as by the relative solubilities of the respective species in the chosen reaction solvent. 

Interestingly, the apoptotic activity of the nuclear assembly extracts had an absolute requirement for a subcellular fraction highly enriched in mitochondria. The system was used to test the effect of various treatments on apoptosis and it was found that the observed nuclear changes were inhibited by the addition of, amongst other things, inhibitors of calpain (a cysteine protease see Section 7). 

Table 2. Nuclear Assemblies for the Double- and Zero-Quantum Coherences Measured in...

The plant design would be based on production of many himdreds of essentially identical modules. Ideally, three or four major assemblies would be fabricated, quality inspected, and tested prior to shipment. With exception of the nuclear assembly and its auxiliary systems and components, these types of major production assemblies have been demonstrated in the large diesel and combined cycle plants operating under much the same technological complexity as the proposed nuclear plant. 

The first megaton-yield explosives (hydrogen bombs) were based on the application of x-rays produced by a primary nuclear device to compress and ignite a physically distinct secondary nuclear assembly. The process by which the time-varying radiation source is coupled to the secondary is referred to as radiation transport. 

Newmeyer, D. D., and Wilson, K. L. (1991). Egg extracts for nuclear import and nuclear assembly reactions. Methods Cell Biol. 36, 607-634. 

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. 

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

Sperm chromatin is the substrate for nuclear assembly and can be readily prepared after sperm collection from the testes of well-fed freshly killed adult male Xenopus. Two animals contain enough sperm for more than 50 experiments. Sperm should be demembranated with lysolecithin exactly as described by Lohka and Masui (1984). After demembranation, sperm heads (chromatin) should be aliquoted at about 2.4 x IdV/itl in a buffer containing 30% (v/v) glycerol, 200 mM sucrose, 2.4 mM MgCl , and 20 mMNa-maleate, pH 6.8. Once aliquoted into small polypropylene tubes, tubes should be immersed in liquid nitrogen and stored at -70°C. Xenopus sperm chromatin prepared in this way can be stored for at least 5 years without apparent deterioration. (For additional details, see the chapter by Lohka, this volume.)... 

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,... 

This chapter describes and discusses methods used in our laboratory to investigate each step of male pronuclear formation in a sea urchin egg cytoplasmic extract. When available, procedures previously reported for surf clam male pro-nuclear assembly in vitro are also indicated. These protocols may be applicable to other marine invertebrates as well. 

Nuclear-associated vesicles (either chromatin-bound or fused into an envelope) can be labeled. Alternatively, vesicles can be prelabeled prior to nuclear assembly reactions. To label chromatin-associated vesicles, a 10-/il extract containing nuclei is mixed with 10 ju.1 of DiOC6 or DilCis, each at 20 /xg/ml in egg lysis buffer (stock B), and incubated at room temperature for 20 min. Samples are visualized under the fluorescence microscope using the appropriate filter sets. Nuclear DNA can be simultaneously labeled at a final concentration of 0.1 /u.g/ml Hoechst 33342. If desired, samples can be fixed and stained simultaneously. A IO-/1I sample is mixed with 10 /x.1 of 7% paraformaldehyde containing 20 ju.g/ml of either of the lipophilic dyes. 

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