Nucleosome electron microscopy

Inaga S, Osatake H, Tanaka, K. SEM images of DNA double hehx and nucleosomes observed by ultrahigh-resolution scanning electron microscopy. J Electron Microsc 1991 40 181-186. 

Fig. 3. Gallery of representative nucleosomes reconstituted in the absence (a) or presence of GH5 (b) or H5 (c), and visualized by scanning transmission electron microscopy, (a) and (b) 256 bp 5S rDNA fragment [65]. (c) 357 bp fragment from the 5S series (see text). Samples were diluted in TE buffer supplemented with 50 mM NaCl and 5 mM MgCl2 before adsorption to the grids. Note the nucleosome different positions relative to the DNA ends. Bars 25 nm and 75 bp. (Adapted from Fig. 7,9, and 10 in Ref [34].) Schemes of the corresponding DNA conformations are shown.

B. Effect of the ionic strength on hyperacetylated 208-12 nucleosome arrays as visualized by electron microscopy. The numbers to the left indicate the milimolar NaCl concentration [369]. [Reproduced from Garcia-Ramirez M. et al. (1995) J. Biol. Chem. 270, 17923-17928, with permission from The American Society for Biochemistry and Molecular Biology.]... 

DNA, since proximity eflfects demand that the DNA or nascent RNA closest to the histones at the point of disruption will be the polyanion for which those histones will preferentially reassociate. Ten Heggeler-Bordier et al. [95] have verified these observations. They used immuno-electron microscopy to determine what happens to histones after transcription with T7 RNA polymerase of a multi-nucleosomal template and also observed transfer to the nascent RNA. In contrast, Kirov et al. [96] have reported that no histones displace during transcription with this polymerase. However, as described above, transcriptional efficiency and ultimately histone displacement is not efficient in very low ionic strength conditions. 

Shioda et al. [43,44] visualized by electron microscopy both regions of naked DNA and of DNA covered with particles in the chromosome of Halobacterium salinarium isolated from gently lysed cells. In a control experiment, they did not detect such particles in E. coli. They also reported the existence of nucleosome-like structures in S. acidocaldarius and methanogens (unpublished results cited in ref. [43]). The size of the particles detected in H. salinarium (9.5 nm) is similar to that of eukaryotic nucleosomes (10.3 nm) however, this putative archaebacterial chromatin is not as regular as eukaryotic chromatin, since not all of the DNA is covered with nucleosomes and since the length of the DNA spacer between the particles is not uniform. In contrast to these results, Bohrmann and coworkers [45] did not visualize nucleosome-like structures in isolated chromosome fibers of Thermoplasma acidophilum. These authors also reported that in situ the nucleoid of T. acidophilum appears to be highly dispersed in the cytoplasm. 

Chromatin is made up of repeating units, each containing 200 bp of DNA and two copies each of H2A, H2B, H3, and H4, called the histone octamer. These repeating units are known as nucleosomes. Strong support for this model comes from the results of a variety of experiments, including observations of appropriately prepared samples of chromatin viewed by electron microscopy (Figure 31.19), Chromatin viewed with the electron microscope... 

Yeast RSC chromatin remodeling complex structure determined by electron microscopy. The complex (red) is shown bound to a nucleosome (yellow). [Courtesy of Francisco J. Asturias, 2002, PAMS 99 13477]... 

Folding occurs at several levels, or hierarchies. In the lowest level, DNA is wound around protein cores to form nucleosomes, which are described in more detail below. In the next hierarchy, strings of nucleosomes are coiled into a helix known as the 30-nm fiber because of its apparent width (30 nm) when viewed by electron microscopy. 

Nucleosome cores have been crystallised and studied by X-ray diffraction and electron microscopy. The X-ray diffraction model to 7 A resolution has been published (Richmond et al 1984). The resolution of the X-ray diffraction data has been extended to =5 A using SR (Richmond, Searles and Simpson (1988) see table 10.3). 

In eukaryotic cells, DNA is packaged into a highly compacted and condensed nucleoprotein structure called chromatin. Biochemical studies and electron microscopy indicated that DNA in eukaryotic chromatin is folded as regular units, each of which contains 146 base pairs of DNA and a core of histone proteins. Structurally, DNA makes approximately 1.8 turns around a central histone octamer that consists of two molecules of each of the four core histones H2A, H2B, H3, and H4. The combination of a histone core and associated DNA makes up the nucleosome. Nucleo-somes are linked by 20-100 base pairs (bp) of linker DNA, so as to form a beadlike nucleosomal array. In conjunction with the linker histone, Hl,... 

When rat pancreatic polynucleosomes were poly(ADP-ribosylated) with purified calf thymus poly(ADPR) polymerase and examined by electron microscopy a relaxation of their native zigzag structure was observed, even at high ionic strengths they showed a close resemblance to chromatin depleted of histones HI. The relaxed state of poly(ADP-ribosylated) polynucleosomes was also confirmed by sedimentation velocity analysis [19, 20]. Locally relaxed regions can also be generated within poly-nucleosome chains by the activity of their intrinsic poly(ADPR) polymerase and appeared to be correlated with the formation of hyper(ADP-ribosylated) forms of histone HI and an increase of DNA polymerase activity [21]. The posttranslational transitory modifications of histones are potential modulators of chromatin stmcture. This may be involved in DNA transcription, replication, and repair. 

Preliminary results indicate that the DNA purified after micrococcal nuclease digestion of the enzyme—DNA complex activates more efficiently the DNA-free poly(ADP-ribose) polymerase than the total purified sDNA and much more than the purified nucleosomal core particles DNA (data not shown). Ohgushi et al. [8] and Benjamin and Gill [7] correlate the activation of poly(ADP-ribose) polymerase only to its binding to nicks or ends and not to a specific DNA sequence. Although our observations of poly(ADP-ribose) polymerase-sDNA complexes by electron microscopy and by polyacrylamide gel analysis do not exclude the possibUity that the enzyme is activated preferentially by internal nicks on sDNA fragments, they raise the question whether... 

Fig. 3. Electron microscopic visualization of control and poly(ADP-ribosyl)ated native chromatin, HI-depleted chromatin and core particles. Following poly(ADP-ribosyl)ation at 200 yM NAD as described in the legend of Fig. 1, the samples were diluted to 0.01 OD unit at 260 nm and fixed for 1 h at 20 C in buffer containing 40 mM NaCl, 10 vaM TEACL, 0.2 mM EDTA and 0.1% (v/v) glutaraldehyde and processed for electron microscopy according to Poirier et al. [6]. Control native chromatin, Hl-depleted chromatin and core particles (a-c). Poly(ADP-ribosyl)ated native chromatin, Hl-depleted chromatin and core particles (d-f). Notice the dissociation of the DNA from the nucleosome cores in the poly(ADP-ribosyl)ated core particles. Also notice the relaxation of chromatin structure in the native chromatin. Big arrows indicate the automodified enzyme and small arrows indicate the DNA (145 bp) free after poly(ADP-ribosyl)ation. Th bars indicate 1000 A...

We have recently shown that, in vitro, poly(ADP-ribosyl)ation of nucleosomes either by purified poIy(ADP-ribose) polymerase [8] or by the endogenous chromatin bound enzyme [9] leads to relaxation of the chromatin superstructure throu histone HI modification. To test the reversibility of this phenomenon, we have used partially purified bull testis poly(ADP-ribose) glycohydrolase [10], which splits the ribosyl-ribose bond between two ADP-ribose units and produces ADP-ribose [11,12]. Changes of the nucleosome structure were examined by electron microscopy and by ultra-centrifugation, and the poly(ADP-ribosyl)ated histones were characterized. 

In order to demonstrate that the recondensation effect observed on poly(ADP-ribosyl)-ated nucleosomes was essentially due to the glycohydrolase activity, we decided to add the glycohydrolase preparation to the reaction mixture in combination with 10 xoM ADP-ribose, which is a known inhibitor of this enzyme activity [11]. Figure 3 shows that 70% of the glycohydrolase activity was inhibited under these conditions. At the end of the reaction (120 min), an aliquot was fixed and visualized by electron microscopy (Fig. 3, insert). Similarly, an inhibition of chromatin structure recondensation was observed. 

Fig. 3. Inhibition of glycohydrolase activity by ADP-ribose. Nucleosomes were incubated as described in Fig. 1. After 60 min (arrows), the reaction was stopped with 10 vc M nicotinamide and 10 vcM ADP-ribose was added. Then 20 Mg/OD unit of glycohydrolase were added and the reaction mixture was incubated for 60 min more. At 120 min, an aliquot was taken and visualized by electron microscopy (inset)...

Sedimentation coefficient determinations [4,21] on polynucleosomes ADP-ribosylated in the presence of 200 fjM NAD for different time intervals, gave values of S20 w ing from 51.5 0.4 S for control nucleosomes to 44.9 0.2 S for fully relaxed nucleosomes (Fig. 3). The time course was in good agreement with the relaxation process visualized by electron microscopy. 

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