ISOLATION AND FRACTIONATION OF RAT BRAIN NUCLEI

A method for isolating pure and unaltered nuclei from rat brain by means of differential centrifugation is described. The isolated nuclei are further separated into discrete fractions of neuronal, astrocytic, and glial nuclei, with a yield amounting to 20 to 25% of the DNA of the original homogenate. Both the morphology and size of the nuclei remained unchanged. Problems concerning the composition of the isolation media, the use of detergents, as well as those raised by density gradient centrifugation in sucrose, Ficoll, and Dextran are discussed. Some values for the density of each type of brain nuclei are suggested.     

Purification of nuclei from a flagellate protozoan, Trypanosoma brucei

A simple and rapid technique is described for the isolation of nuclei from the flagellate protozoan Trypanosoma brucei. Cells were disrupted by nitrogen cavitation in the presence of hexylene glycol which enhances nuclear stability. Isolated nuclei were separated from nuclei trapped with cytoskeletal fragments and flagellae by Percoll density gradient centrifugation. Centrifugation in a medium with increased sucrose concentration greatly improved the separation of free nuclei from those trapped in cell debris. The isolation procedure resulted in the recovery of 34% of the nuclei present in the whole cell suspension. Electron microscopic and chemical analysis indicated that the recovered nuclei were in good condition and were highly purified.     

A rapid, simple method for nuclei isolation from plant protoplasts

A rapid, simple method for nuclei isolation and purification from soybean (Glycine max L. Merr.) protoplasts is described. The isolated nuclei exhibited active amino acid incorporation and RNA synthesis, but DNA synthesis was not detectable. Analysis by CsCl density gradient centrifugation showed that DNA isolated from nuclei had a single band, while DNA isolated from protoplasts consisted of three bands comprised of nuclear DNA, mitochondrial DNA, and chloroplast DNA.     

A simple procedure for the isolation of pure nuclei from carrot embryos in synchronized cultures

A simple method is presented for the isolation of nuclei from somatic embryos of carrot (Daucus carota L.), which is applicable to small amounts of material in synchronized culture. The method employs buffers containing a high concentration of glycerol to stabilize the structure of the nuclei. Purification was carried out by centrifugation using preformed Percoll gradients. Treatment with cell wall-degrading enzymes prior to homogenization improved the efficiency of isolation and permitted a reproducible yield of nuclei. The pure preparations were obtained with an efficiency of approximately 60%. The isolated nuclei retained their morphological characteristics as demonstrated by phase — contrast and electron microscopy. Nuclear proteins displayed the expected species of histones by two-dimensional gel electrophoresis. The isolated nuclei showed high RNA polymerase activity.     

A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM)

This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.     

A simple, efficient method for the separate isolation of RNA and DNA from the same cells.

A method for the isolation of total cytoplasmic RNA and high molecular weight DNA from the same cells is described. Cells are gently lysed with NP40 in the presence of vanadyl ribonucleoside complex and the nuclei pelleted by centrifugation. RNA is purified by phenol/CHCl3 extraction of the lysate supernatant followed by ethanol precipitation. Protein is removed from high molecular weight DNA by salt-precipitation after nuclei are digested with proteinase K in the presence of sodium dodecyl sulfate. High yields of clean, intact RNA and DNA are obtained. A major advantage of the method is that it can be scaled down to quantitatively extract RNA and DNA from as little as 1000 cells.     

The purification of cell nuclei from calf uterus by zonal centrifugation in reorienting density gradients

A method is described for isolation of substantial amounts of pure and enzymatically active nuclei from whole calf uterus. The technique involves a multistep sequential homogenization of the tissue and a zonal centrifugation of the crude nuclear preparation in a reorienting density gradient rotor. Electron and phase contrast microscopic observations show that the nuclei are intact and practically free from cytoplasmic contamination. Based on DNA recovery, the purified fraction contains 9% of the nuclei of the total tissue and more than 19% of the filtered homogenate. The pure nuclear fraction consists of 29% DNA, 7% RNA, and 64% protein, which parallels the composition of purified nuclei from other mammalian tissues.     

CHEMICALLY INDUCED ECDYSIS AND ISOLATION OF NUCLEI FROM THE DINOFLAGELLATES GONYAULAX POLYEDRA AND GONYAULAX TAMERENSIS

Armored dinoflagellates such as Gonyaulax sp. are not good candidates for isolation of intact clean nuclei, due to difficulty in breaking the cells. Previously, isolation of nuclei from Gonyaulax was achieved using vigorous cell disruption by ultrasonication (Rizzo & Hastings, unpublished observations). A detergent method for production of spheroplasts from Gonyaulax was published some time ago (Adamich & Sweeney, 1976), but was not reproducible (Rizzo & Hastings, Morris & Rizzo, unpublished observations). We describe here a modification of the method described by Adamich & Sweeney that is highly reproducible. Ecdysis and production of intact dinoflagellate spheroplasts with a yield of virtually 100% within 5 minutes, has been achieved through the use of anionic and nonionic detergents in hypotonic buffers. This method of ecdysis followed by a gentle spheroplast breakage in a hypotonic buffer allowed the release of nuclei for later biochemical analysis with minimal damage or breakage. The nuclei are subsequently purified by centrifugation in a Ficoll discontinuous gradient.     

Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

Simple procedure for rapid isolation of functionally intact mitochondria from human and rat skeletal muscle

A simple procedure for the rapid isolation of functionally intact skeletal muscle mitochondria is described. The method involves homogenization of muscle in a medium comprising sucrose (0.25 M) containing 50,000 units of heparin/liter, followed by differential centrifugation. Mitochondria so isolated are functionally and morphologically intact.     

Preparative scale isolation of basal-lateral plasma membranes from rat intestinal epithelial cells.

A simple, efficient procedure is described for the preparative scale isolation of basal-lateral membranes from the rat intestinal epithelium. The intestinal mucosa was mildly homogenized and soluble protein and RNA were separated from the homogenate by differential centrifugation. The basal-lateral membranes were then separated from nuclei, mitochondria, and brush border membranes by differential centrifugation in a medium close to the equilibrium density of the basal-lateral membranes. Final purification of the basal-lateral membranes was achieved on a linear density gradient in a high-capacity zonal rotor. The final product (usually at least 40 mg protein) represented a 34% yield of basal-lateral membranes purified 18-fold with respect to protein, 26-fold with respect to brush border membranes, and 53-fold with respect to mitochondria.     

Preparative Scale Isolation of Basal-Lateral Plasma Membranes from Rat Intestinal Epithelial Cells

A simple, efficient procedure is described for the preparative scale isolation of basal-lateral membranes from the rat intestinal epithelium. The intestinal mucosa was mildly homogenized and soluble protein and RNA were separated from the homogenate by differential centrifugation. The basal-lateral membranes were then separated from nuclei, mitochondria, and brush border membranes by differential centrifugation in a medium close to the equilibrium density of the basal-lateral membranes. Final purification of the basal-lateral membranes was achieved on a linear density gradient in a high-capacity zonal rotor. The final product (usually at least 40 mg protein) represented a 34% yield of basal-lateral membranes purified 18-fold with respect to protein, 26-fold with respect to brush border membranes, and 53-fold with respect to mitochondria.     

Digestion by micrococcal nuclease of mouse submandibular-salivary-gland chromatin.

Nucleic prepared from mouse submandibular salivary gland show marked fragility during isolation. Hwever, intact nuclei relatively free from cytoplasmic contamination were obtained by homogenization in buffers containing 0.88 M-sucrose, Ca2+, spermine, spermidine and the proteinase inhibitor aprotinin, followed by centrifugation through 2.2 M-sucrose. The kinetics of digestion by the micrococcal nuclease of chromatin in these nuclei are similar to those of chromatin from mouse liver nuclei. Base-pair size analysis of the solubilized DNA from both organs shows a stable high-molecular weight species of chromatin, which is further digested to mononucleosome and subnucleosome species. With extensive digestion the chromatin becomes insoluble. The mononucleosomes produced from salivary-gland chromatin after the inhibition of endogenous proteinase activity exhibit an s20,w value of 11S and contain histones H1, H2A, H2B, H3 and H4.     

THE ISOLATION OF CELL NUCLEI FROM RAT BRAIN

A method for preparing highly pure cell nuclei from adult rat brain, using both differential and isopycnic centrifugation in sucrose media, is described. The morphology of these preparations was examined by both phase contrast and electron microscopy. The isolated nuclei retained many aspects of their in situ morphology; in particular, the nuclear envelope was double layered and interrupted by pore-like discontinuities, and the nucleoli consisted of irregular masses of densely packed granules. Analyses of these nuclear preparations for cytochrome oxidase and cholinesterase activity, as well as RNA/DNA ratio, indicated minimal contamination with mitochondria and microsomes. Problems involving the homogenization technique, choice of ionic conditions in the homogenization medium, and choice of optimal density of the sucrose solution used for the final purification of nuclei are discussed. Results of application of the technique to isolation of adult rat liver nuclei are also reported.     

Fractionation of rat ventricular nuclei.

Myocardial cells were isolated after treatment with collagenase (0.05%) and hyaluronidase (0.1%) by discontinuous-gradient centrifugation on 3% Ficoll. Nuclei derived from these myocardial cells were then fractionated on a discontinuous sucrose density gradient with the following steps: (I) 2.0M/2.3M, (II) 2.3M/2.4M, (III) 2.4M/2.5M, (IV) 2.5M/2.6M, and (V) 2.6M/2.85M. The myocardial nuclei were sedimented in the interfaces of gradient fractions (II) and (III). Nuclei from whole ventricles that had been treated with the enzymes before isolation sedimented into five major subsets of nuclei. These findings suggest that nuclei sedimented in the isopycnic gradient at fractions (II) and (III) are most probably derived from myocardial cells. However, this procedure is laborious and lengthy, and the recovery of myocardial-cell nuclei is low. An alternative method was developed to isolate an enriched fraction of myocardial-cell nuclei from whole ventricular tissue without exposing the tissues to enzyme digestion. These ventricular nuclei could be fractionated into five nuclear subsets by using the same discontinuous sucrose density gradient as that described above. The content of DNA, RNA and protein per nucleus for each band was determined. Although the DNA content per nucleus was constant (10pg), that of RNA varied from 1.5 to 4.5pg and that of protein from 16 to 24pg. Nuclei from each band were examined by light-microscopy: large nuclei occurred in the ligher regions whereas smaller nuclei were found in the denser regions of the gradient. From the size distribution pattern of myocardial-cell nuclei compared with that of total ventricular nuclei, it was found that nuclear subsets (II), (III), and (IV) were similar to myocardial nuclei. Electrophoretic analyses of the proteins solubilized in sodium dodecyl sulphate/phenol or Tris/EDTA/2-mercaptoethanol/phenol obtained from each nuclear subset indicate that these fractions are similar, with limited qualitative differences. These findings indicate that isolation of an enriched fraction of myocardial-cell nuclei could be achieved by discontinuous-sucrose-density-gradient centrifugation.     

LARGE-SCALE ISOLATION AND FRACTIONATION OF ORGANS OF DROSOPHILA MELANOGASTER LARVAE

Methods for the mass isolation of diverse organs from small animals are described. They involve novel devices: a mechanical dissecting system, a centrifugal agitator for the separation of fibrillar from globular particles, and a settling chamber for the fractionation at unit gravity of particles with sedimentation velocities above the useful range for centrifugation. The application of these methods to the isolation of polytene and nonpolytene nuclei from Drosophila melanogaster larvae is described.     

ISOLATION AND PARTIAL CHARACTERIZATION OF MACRO- AND MICRONUCLEI FROM PARAMECIUM AURELIA

A method was developed for the isolation of macro- and micronuclei from Paramecium aurelia. This method utilized ionic and nonionic detergents to rupture the intact cells, calcium ions and spermidine were employed to protect the nuclei, and the nuclei were purified by centrifugation. Macronuclei consisted of 22% DNA, 10% RNA, and 68% protein. Micronuclei were composed of 9% DNA, 11% RNA, and 80% protein. DNA from both macro- and micronuclei had a density of 1.687 g/cc in CsCl and 1.417 g/cc in Cs₂SO₄. These values corresponded to G + C content of about 23%. The RNA of macronuclei was examined by gel electrophoresis, and two high molecular weight species were identified having molecular Weights of 1.3 x 10⁶ and 2.8 x 10⁶ daltons. Three syngens were studied, and in each case the conditions for isolation of the nuclei were the same and no differences were observed in the properties of the nuclei.     

Purification and characterization of chick intestine brush border membrane. Effects of 1alpha(OH) vitamin D3 treatment

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1alpha-hydroxyvitamin D3 treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.