Chromatin embedding

Transcription is the first and often most critically regulated step in gene expression. In eukaryotes, multisubunit RNA polymerases transcribe genes, whose DNA is packaged together with histones and other nonhistone chromosomal proteins into chromatin, which is notoriously intractable and not easily transcribed by those polymerases in vitro. A central question driving research on eukaryotic transcription has therefore been, How is timely and efficient transcription of chromatin-embedded genes achieved in eukaryotic cells ... 

Like HATs, most functional HDACs are embedded in large multifunctional protein complexes, which also contain other chromatin modifying enzymes and coregulator proteins [1]. 

Fig. 1.—Nucleus (N) from D-Galactose-treated Orchid Seedling (Phalaenopsis cv. Doris Fi) Showing Dispersed Chromatin with Nuclear Envelope Envaginated (Arrows) into the Cytoplasm. [After treatment with D-galactose, seedlings were fixed in 2% glu-taraldehyde for 2 h followed by 2% OsO, for 12 h. Tissue was dehydrated in a graded concentration, embedded in Epon 812, sectioned with a diamond knife, and photographed with a Zeiss EM9A electron microscope x 13,340 (reproduced, by permission, from Ref. 533).]...

Thin-sectioning for EM observation is a standard technique that provides important information about meiotic cells, especially when three-dimensional (3D) reconstructions are performed (Wettstein and Sotelo, 1967 Solari, 1970 Holm and Rasmussen, 1977). The use of this technique is especially suitable for establishing topographical relationships among nuclear structures and for observation of variations in chromatin packing. A wide variety of fixatives, embedding, and staining procedures may be used for this purpose and are available in the literature (Hayat, 1989). Methods have been previously described (Solari, 1970). 

The fate of other nuclear components can concomitantly be analyzed using double-label immunofluorescence microscopy. Cells were fixed and air dried (see above), then incubated with antiguinea pig-specific secondary antibodies conjugated to FITC (10 min), and afterward washed in PBS (10 min). In a second step, the cover slips were incubated for 20 min with an antibody specific for a different nuclear protein (for example, a mouse monoclonal antibody against nucleolar protein fibrillarin). After washing in PBS (10 min), the cells were incubated with an appropriate secondary antibodies (in this case, antimouse-speciflc antibodies) conjugated to Texas Red (Dianova) for 10 min. For the visualization of chromatin, specimens were stained with the DNA-specific fluo-rochrome Hoechst 33258 (5 jug/ml in PBS) simultaneously with the secondary antibody. Finally, the cover slips were washed for 10 min in PBS, dehydrated in ethanol, and embedded in Mowiol (Hoechst, Frankfurt, Germany). 

Fig. 313. Renal proximal tubule cell (block BNh 3405) from a male Wistar rat (No. 2569) 90 min after a single intraperitoneal injection of 5 mg N-benzyl-N -p-hydroxybenzylpiper-azine HCl per kg body weight. Under pentobarbital anaesthesia (30 mg/kg), the animal was perfu from the abdominal aorta with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Postfixation with 1 % osmium tetroxide in sodium cacodylate buffer. Embedded in Epon 812 and sectioned at 50 nm. Lead citrate and uranyl acetate. Plate 2478. -Nucleus most of the chromatin is condensed into large, confluent heterochromatin masses forming a perforated shell immediately under the nuclear envelope. Several microbodies, some with a prominent nucleoid...

Two major techniques have been used to study the dimension of chromosome fibers by electron microscopy. The first is the surface spreading method, in which the cells are spread on a water-air interphase, and all the intracellular components are dispersed. But the chromatin and the spindle remain close together and can be picked up on a grid, which can then be prepared for electron microscopic examination. The second technique is the thin-sectioning method. After fixation the tissue is embedded in plastic and cut less than 1000 A thick. Refer to specialized texts for descriptions of these techniques [119]. The important finding is that with the first method, the dimension of the fibers diameter is estimated to be 200-300 A, whereas it is only 80-180 A with the second. 

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