Embryo extract

Figure 3. The stability of the nucleosome is affected by the length and the superhelicity of DNA. (a-b) The chromatin fibers were reconstituted from the purified plasmids and the histone octamers by a salt-dialysis method and observed under AFM. The 3 kb (a) or 106 kb (e) supercoiled circular plasmid was used as a template, (c) Relationship between the plasmid length and the frequency of nucleosome formation in the reconstitution process. The nucleosome frequency is represented as the number of base pairs per nucleosome and plotted against the length of the template DNA in supercoiled (filled circle) and linear (open circle) forms, (d) AFM image of the chromatin fiber reconstituted on the topoisomerase 1-treated plasmid, (e) Chromatin fiber reconstituted with Drosophila embryo extract. The chromatin fiber was reconstituted from plasmid DNA of 10kband the embryo extract of Drosophila, and was observed by AFM...

Hershko, A., Ganoth, D., Pehrson, J., Palazzo, R.E., and Cohen, L.H. (1991). Methylated ubiquitin inhibits cyclin degradation in clam embryo extracts. J. Biol. Chem. 266, 16376-16379. 

Prepare 50 pL of translational reaction mixture by combining 10 pL of wheat embryo extract (thus 20%), 2 pL of 1 mg/mL creatine kinase, 10 pL of 5X WEB, and 28 pL of dissolved mRNA from 25 pL of transcription mixture. 

Key Words Cell-free protein synthesis wheat embryo extract 5 and 3 ORF design pEU construction bilayer reaction method robotic automation. 

Fig. 2. Removal of tritin from embryos. Extracts were prepared from unwashed or washed embryos (A) and the depurination assay was performed (B). Translation mixtures prepared with the extract from unwashed embryos were incubated for 0, 1, 2, 3, 4 h (lanes 1-5, respectively) mixtures with washed embryos were incubated for 0, 2, 4 h (lanes 10-12, respectively). Isolated RNA was treated with acid/aniline, and then separated on 4.5% polyacrylamide gels. Additionally, RNA was directly extracted from embryos with guanidine isothiocyanate-phenol and analyzed before (lane 7) and after (lane 8) treatment with acid/aniline. For the fragment marker, incubation was carried out in the presence of gypsophilin, a highly active ribosome-inactivating protein from Gypsophila elegance the arrow indicates the aniline-induced fragment.

There are a number of media available which are not based on a detailed investigation of growth requirements, but rather include crude mixtures of nutrients added to promote cell growth. These include lactalbumin hydrolysate (Appendix 1 Table 9) or yeast extract (Appendix 4) to provide an inexpensive source of amino acids or vitamins. Thus Melnick s monkey kidney media A and B (Melnick, 1955) contain lactalbumin hydrolysate and calf serum in Hanks and Earle s BSS, respectively. Chick embryo extract and tryptose phosphate broth (Appendix 1, Tables 11 and 12) are also used occasionally and their use is referred to where appropriate throughout the book. Mitsuhashi and Maramorosch mosquito cell medium contains lactalbumin hydrolysate, yeast extract and foetal calf serum in a specially developed saline (Mitsuhashi and Maramorosch, 1964 Singh, 1967). 

Myoblasts may differentiate and fuse without undergoing DNA synthesis (Nadel-Ginard, 1978), but fusion does not occur in calcium-free medium. Thus, if chick embryo myoblasts are set up in calcium-free (< 20/xM Ca2+) DMEM containing 5% heat inactivated FBS and 2% chick embryo extract (Appendix 1) the fusion which normally starts at around 24 h fails to occur. Addition of 1.4 mM CaCl 2 after 50 h will now produce synchronised fusion of cells (Wakelam and Pette, 1982). 

Figure 2 1H NMR data of stage 38 medaka embryo extracts, including (a) 1D spectrum, (b) the 1D skyline projection (p-JRES) of (c) a 2D JRES spectrum, and (d) the preprocessed p-JRES spectrum. Resonances due to citrate (Cit), taurine (Tau), and alanine (Ala) are indicated. Reprinted from M. R. Viant, Biochem. Biophys. Res. Commun. 2003, 310 (3), 943-948. Copyright (2003), with permission from Elsevier.

Table 15.2 Composition of media and buffers for E. coli and wheat embryo extract preparations.

Selected strains of E. coli can be grown under controlled conditions in a highly reproducible maimer. Wheat embryo extracts suffer from batch-to-batch variation due to differences in the conditions under which the wheat is grown, harvested, milled, and stored. This source of variation in the activity of wheat embryo extracts can be offset by the simplicity of the preparation protocol. Small amounts of extract can be quickly prepared from embryos obtained from different sources, and the activity of each assessed. Large batches of extract can then be prepared from the best embryo source and stored at -80 °C until required. As with E. coli extracL wheat embryo extracts are stable for more than a year if stored in hquid nitrogen, or at -80 °C. 

The conditions for performing a wheat embryo cell-free translation reaction from an mRNA template are listed in Table 15.4. Points discussed for E. coli optimization in Section 15.6.1 are relevant to expression in wheat embryo extracts. However, it is worth noting that the wheat embryo system is better suited to the translation of added mRNA template, whereas coupled transcription/translation is better in the E. coli system. The principal reason for this difference is that transcription with bacteriophage RNA polymerases requires a relatively high Mg concentration (ca. 16 mM) E. coli translation-only reac-... 

By the use of no-flow cytometry, Barald (1989) has isolated this subpopulation of cells from neural crest cultures and studied its behaviour under a variety of different culture conditions. The cells proliferate in the presence of 15% fetal bovine serum and high concentrations of chick embryo extract, but do not differentiate. However, in chick serum, elevated K+ or heart-, iris- or lung-conditioned medium, the cells stopped proliferating and all of the cells became neuron-like within 10 days (Barald, 1989). These cells also stained positively for choline acetyl transferase (ChAT). 

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. 

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. 

Permeable Xenopus sperm cells, referred to as nuclei, are most often used form vitro remodeling studies with homologous egg (Philpotte/a/., 1991 Philpott and Leno, 1992 Leno et al., 1996), heterologous egg (Ohsumi and Katagiri, 1991b), or heterologous embryo extracts (Berrios and Avilion, 1990 Kawasaki... 

Fresh or previously frozen Drosophila egg or embryo extract is supplemented with an energy-regenerating system (6 miW phosphocreatine, 150/tg/ml creatine kinase) and Xenopus sperm nuclei to a final concentration of 2 x 10 //tl extract. Samples are then incubated at 25"C (Kawasaki et ai, 1994) for various times. 

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. 

Nucleosome Assembly and Remodeling in Preblastoderm Embryo Extracts... 

Typically, 1.5 pg plasmid DNA is incubated with embryo extract (3 mg protein) containing 30 mM creatine phosphate, 3 mM MgCl2,3 mM ATP (pH 8.0) 0.1 pg/ ml creatine phosphokinase (type 1, Sigma), 1 mM DTT, and EX buffer to a total volume of 200 /u.1. The conductivity of the assembly reaction mix is equivalent to 65 mM KCl. The reaction is carried out for up to 6 hr at 26°C. Preblastoderm embryo extracts contain a maternal stockpile of core histones that are utilized in the assembly reaction. The linker histone, HI, on the other hand, is absent from the early embryo and, hence, from the reconstituted chromatin. However, exogenous HI can be incorporated into chromatin during the assembly reaction, where it increases the nucleosome repeat length from 180 to 197 bp DNA (Becker and Wu, 1992). 

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