Chicken erythrocytes

The poor resolution of the fine structures of DNA and nucleosomes by conventional SEM was solved by the development of an ultrahigh-resolution SEM (14-15). Inaga et al. (16) observed the DNA double helix and nucleosomes of chicken erythrocytes by using an ultrahigh-resolution SEM. They modified the microspreading technique of Seki et al. (17) and combined it with the carbon plate method devised by Tanaka et al. (18). Briefly, they (procedures were performed at 0-4°C before fixation with the formalin solution) ... 

Seki S, Nakamura T, Oda T. Supranucleosomal fiber loops of chicken erythrocyte chromatin. J Electron Microsc 1984 33 178-181. 

The inverse of the above experiments gave similar results (Whitlock and Stein, 1978). Trypsin-digested histones removed from HeLa core particles can subsequently fold DNA, although DNase I digests the resulting particles more rapidly than the untreated ones. Parallel experiments were performed for chicken erythrocyte core particles (Lilley and Tatchell, 1977). In all cases it could be concluded that it is the trypsin-insensitive carboxy-terminal regions of the histones which are responsible for the folding of the DNA in the nucleosome. 

A 80-100 nm fiber have also been identified in the chromosome by EM (Belmont and Bruce, 1994) and ALM (Yoshimura et al., 2003) (Fig. 2a). The 80 nm fiber exists also in yeast and chicken erythrocyte nuclei (Kobori et al., 2006) (see section 2.4). 

Another group of non-histone proteins have been identified as essential components for the formation of the condensed chromosome (Table 1). Topoisomerase II (topo II) localizes in the scaffold/matrix fraction of the interphase nuclear (Berrios et al., 1985) and the mitotic chromosome (Maeshima and Laemmli, 2003) (see section 3.1). Topo II forms a ring-shaped homodimer (Berger et al, 1996 Nettikadan et al, 1998) and catalyzes the decatenation and relaxation of DNA double strand (Wang, 2002). In fission yeast, chromosomes cannot be condensed without functional topo II (Uemura et al, 1987). In addition, in in vitro experiment, mitotic extracts containing topo II induce chromatin condensation in the isolated nuclei from HeLa and chicken erythrocyte cells (Adachi et al., 1991). 

Figure 5. (Continued) different panels (e and f for HeLa, j and k for chicken erythrocyte, and o and p for yeast). A section profile obtained along X-Y line shows a typical granular structure in die nucleus (e, j, o), and the peak-to-peak distance between the granular structure was distributed from 60 nm to 120 nm (e). The diickness of the chromatin fibers released out of die nucleus varied possibly due to the assembly of diinner fibers (f, k, p). A section profile for the spread fibers was obtained along X-Y line (f, k, p). Isolated HeLa cell nucleus was treated widi (r, s) or without (q) RNase. The treatment releases SOnmfiber from the nucleus. The histogram of die fiber width is shown in an inset of (s). Bars, 250 nm. (See Colour Plate 2.)...

Taquet A, Labarbe R, Houssier C (1998) Calorimetric investigation of ethidium and netropsin binding to chicken erythrocyte chromatin. Biochemistry 37(25) 9119—9126 Temple MD, McEadyen WD, Holmes RJ, Denny WA, Murray V (2000) Interaction of cisplatin and DNA-targeted 9-aminoacridine platinum complexes with DNA. Biochemistry 39(18) 5593-5599 Terasaki T, Iga T, Sugiyama Y, Hanano M (1984) Interaction of doxorubicin with nuclei isolated from rat liver and kidney. J Pharm Sci 73(4) 524—528... 

Nucleosomes isolated and purified from chicken erythrocytes and beef kidney crystallized and diffracted to limits of 5-6 A, but led to structural models of 7-8 A resolution [12,14], In these structures, the path of the DNA around the histone core is clearly seen. With the exception of positions about 1.5 and 4.5 helical turns from the center of the nucleosomal DNA in either direction, the DNA appears uniformly bent. Significant compression of the DNA occurs where the minor grooves face... 

The crystals of NCPs containing a-satellite DNA palindrome and chicken erythrocyte histones diffracted isotropically to 3.0 A using an in-house rotating anode X-ray source and to better than 2.5 A at a moderate intensity synchrotron beamline [30,31]. The crystals used for structure determination were grown in the microgravity environment using a counter-diffusion apparatus [32]. Ground-based... 

Fig. 6. Purified recombinant HMGA proteins bind to four regions of DNA on random sequence nucleosome core particles. Panel A The results of EMSA gel assays in which increasing concentrations of either purified nonhistone HMGN2 (a.k.a., HMG-17, which binds to two sites on nucleosome core particles) or recombinant human HMGAla protein were bound nucleosome core particles isolated from chicken erythrocytes [57]. Panel B Two different views of the nucleosome taken from the X-ray structure of Luger et al. [119] showing the sites of binding of HMGA proteins (dashed circles) determined by DNA foot-printing analyses and other techniques (see text for details).

Hendzel, M.J. and Davie, J.R. (1990) Nucleosomal histones of transcriptionally active/competent chromatin preferentially exchange with newly synthesized histones in quiescent chicken erythrocytes. Biochem. J. 271, 67-73. 

Fig. 4. Amino acid sequence of several histone HI proteins to illustrate the macroheterogeneity of linker histones. Amino acid sequence of two highly specialized development-specific members of the histone HI family. A. Oocyte specific mammalian histone Hlfo (previously Hloo) [116]. B. PL-I (EM-1/6) protein from the sperm of the razor clam Ensis minor [120]. These two sequences are shown in comparison to the highly specialized histone H5 from chicken erythrocytes. The regions corresponding to the trypsin-resistant (winged helix motif [96]) which is characteristic of the protein members of the histone HI family are indicated by a box and have been aligned to show the sequence similarity.

Fig. 10. A. Acetic acid-urea-triton-X-100 polyacrylamide gel electrophoresis [15] of the histones used to reconstitute 208-12 nucleosome arrays consisting of recombinant H2A.Z (lane 2) or recombinant H2A.1 (lane 3). Lanes 1 and 4 respectively are chicken erythrocyte and calf thymus histones used as markers [42]. B. Ionic strength (NaCl concentration) dependence of the average sedimentation coelRcient (s2o,w) of reconstituted 208-12 nucleosome arrays containing either H2A.1 (O) or H2A.Z ( ) [42]. The dotted line represents the behavior of a 208-12 complex reconstituted with chicken erythrocyte histones [406]. [Reproduced from Abbott D.W. et al. (2001) I. Biol. Chem. 276, 41945-41949, with permission from The American Society for Biochemistry and Molecular Biology.]...

A. Effect of the ionic strength (mM NaCl concentration) on the average sedimentation coelRcient (S20,w) of (208-12) oligonucleosome arrays reconstituted with HeLa cell native histone octamers [solid line, ], chicken erythrocyte histone octamers (broken line) [369], or hyperacetylated. HeLa cell histones 208-12 oligonucleosome complexes reconstituted with hyperacetylated HeLa cell histones ( ) [369]. 

The principles whereby a chain of nueleosomes can compact to form a 30 nm chromatin fiber are still not well understood. Nevertheless, important aspects of this process are becoming clear from imaging studies, employing both ECM and SFM. When isolated chicken erythrocyte chromatin or chromatin reconstituted onto six tandem 208 bp nucleosome positioning units were examined by ECM, a linker DNA stem-like architectural motif was observed at the entry-exit sites (Fig. 4) [30]. Particles consistent with an octamer are surrounded with 1.7 turns of DNA, a linker... 

Fig. 4. Images of unfixed and unstained chromatin in a frozen and hydrated state. All samples shown contain linker histone H5. (A) Soluble chromatin prepared from chicken erythrocyte nuclei. Arrow indicates a nucleosome with a linker histone stem conformation. (B-E) Chromatin reconstituted onto an array of the 5S rDNA nucleosome positioning sequence. En face views (B-D) of nucleosomes show the linker DNA entering and exiting the nucleosome tangentially, before interacting and remaining associated for 3-5 nm before separating (arrows). An edge-on view (E) shows the two gyres of DNA (arrow heads) and the apposed linker DNA (arrow) (from Ref. [30]). Scale bar 20 nm (A) and 10 nm (B-E).

Fig. 5. SFM images of chicken erythrocyte chromatin fibers. (A) Untrypsinized, linker histone-containing control fibers, and (B) linker histone-stripped fibers. The stripping of linker histones destroys both the three-dimensional interactions of adjacent nucleosomes and the zig-zag arrangement of consecutive nucleosomes. Trypsinization of the N-terminal histone tails of the linker histones and core histone H3 result in the loss of the three-dimensional association of the consecutive nucleosomes, but does not destroy the zig-zag configuration. Imaging of fibers deposited onto mica was performed in air under conditions of ambient humidity and temperature (from Ref. [32]). Full width of each image corresponds to 500 nm.

Fig. 8. (a) Schematic of the AFM pulling experiments and expected unraveling of an individual nucleosome as a result of pulling on the DNA. (b) Example force-extension curves on isolated chicken erythrocyte chromatin fibers redrawn from Ref [69]. (c) Idealized schematic of a typical force-extension curve obtained on pulling single titin moleeules, as in the experiments of Rief et al. [71]. (d) Explanation of the titin force curve by successive unfolding of individual protein domains (see text). 

Earlier attempts to use the AFM for mechanically stretching chromatin fibers have run into a rather unexpected artifact. Long native chromatin fibers isolated from chicken erythrocytes, or fibers assembled in vitro from purified histones and relatively short, tandemly repeated DNA sequences were deposited on mica or glass surfaces and pulled with the AFM tip [69,70]. In such stretching experiments the scanning of the sample in the x- and y-direction used for imaging was disabled, and the cantilever-mounted tip was allowed to move only in the z-direction, i.e., upwards and downwards, away and towards the surface. When the AFM tip is pushed into the sample, it may attach to the sample by non-specific adsorption upon retraction it stretches the sample and force-extension curves are recorded (see Fig. lb for an explanation of a typical force curve). 

The experimental approach used to mechanically stretch a chromatin fiber with the AFM is depicted schematically in Fig. 8a, and some example curves obtained with native chicken erythrocyte chromatin fibers are presented in Fig. 8b. These curves exhibited a saw-tooth pattern, similar to the patterns obtained upon stretching of multi-domain proteins like titin [71] or tenascin [72] (Fig. 8c). Each of... 

Using optical traps, Cui and Bustamante [76] stretched isolated chicken erythrocyte fibers, and Bennink et al. [77] pulled on fibers directly reconstituted in the flow cell from X-DNA and purified histones with the help of Xenopus extracts (see Fig. 10a for a schematic of the latter experiment). Up to 20 pN, the fibers underwent reversible stretching, but applying stretching forces above 20 pN led to irreversible alterations, interpreted in terms of removal of histone octamers from the fibers with recovery of the mechanical properties of naked DNA. 

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