Cytoplasmic extract materials

This procedure describes the preparation of nuclear and cytoplasmic extracts from cells grown in suspension (2-201) and is essentially as described by Dignam et al.1 and modified by Jamison and Garcia-Bianco.2 A procedure for small-scale preparation of extracts from HeLa cells grown as monolayer has been described by Lee and Green.3 The latter procedure is recommended when the cell material is scarce, if radioactively labelled extracts are made, if several extracts from parallel cultures, or if expensive growth conditions are necessary for cell propagation. 

Fig. 12. Informosomes from loach embryo extracts labeled for 3 hours with uridine. Dotted lines, radioactivity solid lines, optical density. A. Sucrose density gradient centrifugation of cytoplasmic extract (22,000 rpm for 18 hours). B. Recentrifugation of the material of 73 to 62S peak in CsCI density gradient. C. The same but of 45 to 36S zone. (From Ovchinnikov et al. 1960a. Molec. Biot. (U.S.S.RJ, 3 448 3.)...

The important bacterial storage material poly-hydroxybutyric acid is related metabolically and structurally to the lipids. This highly reduced polymer is made up of D-(3-hydroxybutyric acid units in ester linkage, about 1500 residues being present per chain. The structure is that of a compact right-handed coil with a twofold screw axis and a pitch of 0.60 nm.a Within bacteria it often occurs in thin lamellae 5.0 nm thick. Since a chain of 1500 residues stretches to 440 nm, there must be 88 folds in a single chain. Present in both cytoplasmic granules and in membranes,b polyhydroxybutyrate can account for as much as 50% of the total carbon of some bacterial In E. coli and many other bacteria polyhydroxybutyrate is present in a lower molecular mass form bound to calcium polyphosphates, proteins, or other macromolecules.d e It has also been extracted from bovine serum albumin and may be ubiquitous in both eukaryotes and prokaryotes.d/e The polymer may function in formation of Ca2+ channels in membranes.b/d... 

Following such treatment, the cultured cells in monolayer are washed with phosphate-buffered saline and extracted in 4 ml 0.5% Triton X-100 in saline/EDTA (100 mM NaCl, 10 mM EDTA, pH 8.0) for 2 min at room temperature. This releases most of the cytoplasmic material whilst the nuclei remain attached to the culture dish. 0.5% sodium dodecyl sulphate and 40 //g/ml pancreatic RNAse (preincubated at 80°C for 10 min, to inactivate DNAse) in saline/EDTA (above) is then added and the mixture incubated for 20 min at 37°C. One volume of chloroform/isoamyl alcohol (20 5, v/v) is then added and the phases mixed gently. The aqueous phase is separated by centrifugation and extracted again with chloroform/isoamyl alcohol. DNA is precipitated from the aqueous phase with 2 vol 95% ethanol and resuspended in 0.01 M Tris, pH 7.5. Alkaline sucrose density gradients (5-20%) are prepared in 0.1 M NaCl, 0.1 M NaOH with a final volume of 4.1 ml. Samples of DNA (max 3 fig) are layered on the top of these gradients and spun at 32 000 rpm at 20°C in a SW.50.1 Beckman rotor for 120 min. Fractions are collected and the [3H]DNA precipitated... 

The extraneous materials are a heterogeneous group of non-structural constituents most of which are organic compounds extractable in neutral solvents such as ether, acetone, ethyl alcohol, ethyl alcohol-benzene, and water. They include waxes, fats, essential oils, tannins, resin and fatty acids, terpenes, alkaloids, starch, soluble saccharides (gums), and various cytoplasmic constituents such as amino acids, proteins, and nucleic acids. An excellent review on the general subject of wood extractives is that of Hillis (26). The distribution of cytoplasmic constituents in wood has also been reviewed recently (11, 46). 

Gros As far as the percentage of transcripts that show alternative splicing goes, I had a couple of questions that relate to the primary material used for the analysis. For example, these alternatively spliced exons could also be viewed as incompletely spliced introns. The fact that on average you find them to be longer will go in that direction, at least in part. In terms of quality control for the RNA that is used as the initial material to create the cDNAs from which these sequence data are extracted, how much quahty control is there to avoid contamination by partially sphced RNA Are people looking at cytoplasmic RNA fractions versus total RNA fraction, for example Also, I assume that when the sequences are analysed. 

Two questions arise. First, is the informosome-forming protein (IFF) also found in cytoplasmic informosomes and nuclear D-RNPs Second, are cytoplasmic informosomes preexisting structures, or are they produced artificially by the interaction of IFF with mRNA during the preparation of the cellular extract The answer to the first question has not yet been obtained, since the isolation of pure IFF meets with a number of difficulties. The first results obtained by Baltimore and Huang (1970) indicate the possibility of separating active material into a number of fractions by chromatography on DEAE-cellulose. 

Cell-free system. The first experiment with cell-free systems containing labeled nuclei and nonlabeled cytoplasm (Schneider, 1959 Scholtissek and Potter, 1960) indicated the removal of newly formed RNA from the nuclei during the incubation. The newly synthesized RNA was found in the incubation medium in particles with different sedimentation coefficients. These results have been confirmed by others (Samarina and Zbarsky, 1964 Ishikawa et al., 1969 Lukanidin, 1969). Sometimes ATP and other donors of energy enhance the loss of RNA from nuclei (Ishikawa et al., 1969). However, this process seems not to differ from the simple extraction of D-RNP from nuclei and thus may not be a model of transport. Lukanidin (1969) analyzed the particles in a CsCl density gradient and found that almost all labeled material leaving the nucleus has a buoyant density of 1.40 g/cm and does not interact with the cytoplasmic ribosomes which band at p = 1.55 to 1.58 g/cm. Only a small amount of labeled material banded in the intermediate zone, but it had a base composition similar to rRNA and thus may represent the ribosomal RNA precursors. The addition to the system of a variety of factors necessary for protein synthesis did not influence the results. 

PHA are a family of linear polyesters of 3, 4, 5 and 6-hydroxyacids, synthesised by a wide variety of bacteria through the fermentation of sugars, lipids, alkanes, alkenes and alkanoic acids. In the presence of an abundant carbon source when other essential nutrients such as oxygen (Oi), phosphorous (P) or nitrogen (Ni) are limited, many microorganisms usually assimilate and store PHA for future consumption [1]. They are found as discrete cytoplasmic inclusions in bacterial cells. These polymeric materials are able to be stored at high concentrations within the cell since they do not substantially alter its osmotic state [2]. Once extracted from the cells, PHA exhibit... 

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