Chromatin fragments

Death by apoptosis is much less spectacular and is usually quicker than necrosis. Cells shrink, possibly to a quarter of their previous size, and vesicles appear in the cytosol and in the nucleus. As the cytosol contracts, the chromatin fragments into a number of distinct particles and the endoplasmic reticulum fuses with the plasma membrane. The dying cell breaks up into small vesicles (apop-totic bodies) which are removed by phagocytosis (Figure 20.34). Since little if any of the cytosolic contents escapes, there is little or no inflammation. That is, death by apotosis rather than reduces the risk of local or general inflammation. In fact, death by apoptosis is so inconspicuous that it was not discovered for many years. 

Less is known about the interaction of the nucleosomes between themselves or with free DNA. The nucleosome-nucleosome interaction has recently been parameterized by using the surface charge density of the known crystal structure [39] in a point-charge model [51]. While in that work only electrostatic interactions were considered and the quantitative influence of the histone tails on the interaction potential still remains obscure, simulations based on this potential allowed to predict an ionic-strength dependent structural transition of a 50-nucleosome chromatin fragment that occurred at a salt concentration compatible with known experimental data (Ref. [65], see below). 

For re-ChIP (or double ChIP), two sequential rounds of immunoprecipitations are performed using two different antibodies. Re-ChIP allows one to determine whether two antigens occur together on the same chromatin fragment or on different fragments and can be used to assemble proteins in complexes [23]. 

McConkey DJ, Hartzell P, Jondal M, Orrenius S (1989) Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that simulate protein kinase C.) Biol Chem 264 13399-13402 McConkey DJ, Nicotera P, Orrenius S (1994) Signalling and chromatin fragmentation in thymocyte apoptosis. Immunol Rev 142 343-363 McCoy C, Smith DE, Cornwell MM (1995) 12-0-tetradecanoylphorbol-13-acetate activation of the MDRl promoter is mediated by EGRl. Mol Cell Biol 15 6100-6108... 

Endonucleases. Endonucleases provide for DNA cleavage into small ( 200 base pairs) fragments, which is an essential step in apoptotic cascade (Wyllie 1980 Wyllie 1998). Endonucleases are stimulated by Ca2+ and their activation was detected in several cell types undergoing apoptosis (McConkey et al., 1988 Aw et al., 1990). The intimate mechanisms of endonucleases action remain not fully described at least in part they can be explained through the involvement of caspase-3 activated endonuclease (or caspase-activated DNAase - (Enari et al., 1998)). Nonetheless, the chromatin fragmentation was also observed in cell (and caspase)-free system, when isolated nuclei were treated with Ca2+ and ATP, suggesting the existence of caspase-independent DNA cleavage mechanism (Jones et al., 1989). 

This approach, also known as ChIP-on-chip, has been the most used technique to establish histone-maps. Briefly, as we have been detailing, the chromatin fragments are incubated in the presence of an antibody that recognizes a specific histone modification (e.g., methylation on histone 3 or acetylation on histone 4, etc.) (Figure 4). Next, the protein-DNA complex is immunoprecipitated. After reversal of the cross-link, ChIP-enriched DNA and control DNA are amplified by PCR and labeled with fluorescent dyes (Cy3 and Cy5). Finally, the samples are hybridized onto a specific microarray. The ratio of the Cy5 to Cy3 intensities measured for each DNA sequence in the array is a measure of the amount of a specific histone bound to the DNA. 

Chromatin fragmentation (fluid-filled structures) Mitochondrial swelling... 

Fragmentation of DNA into intemucleosomal fragments chromatin condensation chromatin fragmentation... 

Step El DNA in the immunoprecipitated chromatin fragments is released by reversing the cross-link and then is quantitated using a sensitive PCR method. The method can be used to analyze the in vivo association of any protein with a specific sequence of DNA by using an antibody against the protein of interest in step B- [See S. E. Rundlett et al., 1998, Nature 392 831]... 

Scattering Patterns from Chromatin Fragments in Solution.219... 

Fairly uniform fibres or threads of nucleosomes, 10 nm nucleofilament , have also been reported for the low ionic strength chromatin structure In interphase nuclei, metaphase chromosomes or in solutions of isolated chromatin fragments at ionic strength above 40 mM monovalent salt, the 30 nm fibre is observed. This structure is generally described as resulting from the condensation of... 

Most of the results given in the following sections were obtained using nuclei and chromatin fragments isolated from chicken erythrocytes (CE). Rat liver nuclei and chromatin, calf thymus and yeast nuclei were also used in some experiments for... 

Patterns from (partially) oriented condensed diromatin fibres allow to identify the origin of some of the bands seen in the patterns of intact nuclei. Some insight into the behaviour of chromatin in nuclei under different ionic conditions can also be gained by studies on gels obtained by concentrating isolated chromatin fragments. 

Fig. 9. Changes in the scattering pattern of rat liver chromatin fragments (3.5 mg DNA/ml) as a function of NaQ concentration and corresponding values of R, and relative M/L. As the fibres condense the 0.05 nm band shifts to larger s-values and R and 1(0) progressively increase (after figure 5 from Koch et al., 1987a)...

Observed in nuclei and chromatin fragments in solution at ionic strength below 20 mM. 

Observed in nuclei and chromatin fragments in solution at high ionic strength (100 mM salt) or in the presence of multivalent cations. ... 

Arises from intranucleosomal DNA and protein. Observed in solutions of isolated nucleosomes, nuclei and chromatin fragments. 

Uncondensed and condensed CE chromatin fragments. Uncondensed rat liver chromatin fragments. ... 

If the average size of chromatin fragments is greater than several kilobytes, repeat sonication as described previously. After dialysis and reversal of crosslinks, the average fragment size is generally 0.5 to 1 kb. 

Save 50 jL of chromatin (supernatant above concentrated beads when in MPC) from antibody negative control tube and transfer to a new Eppendorf. This sample will be valuable later to estimate final chromatin fragment size. Add 50 jL of TE and put on ice. 

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