Chicken erythrocyte histones

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. 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]. 

Poirier GG, Niedergang C, Champagne M, Mazen A, Mandel P (1982) Adenosine diphosphate ribosylation of chicken-erythrocyte histones HI, H5 and high-mobility group proteins by purified calf thymus polyadenosine diphosphate-ribose polymerase. Eur J Biochem 127 437-442... 

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. 

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). 

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... 

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. 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.

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). 

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. 

One of the very early research tools that were used to study the nucleosomal state of active genes were the nucleases, DNase I and Micrococcal nuclease. With the development of protocols for the isolation of nuclei from cells, it was possible to add these reagents to probe the accessibility of DNA. DNase I makes single nicks in double stranded DNA and when the DNA is associated with histones within the nucleosome, the DNA is extensively protected. Those nicks that are observed are found to occur only after extensive digestion and are limited to the outside surface of the DNA in 10 base increments [7,8]. Weintraub and Groudine in 1976 [9] first used this nuclease and observed that when nuclei from chicken erythrocytes were treated with DNase I, the active /1-globin gene was preferentially... 

Yau, P.M. and Burlingame, A.L. (2002) Identification of acetylation and methylation sites of histone H3 from chicken erythrocytes by high-accuracy matrix-assisted laser desorption ionization-time-of-flight, matrix-assisted laser desorption ionization-postsource decay, and nanoelectrospray ionization tandem mass spectrometry. Analytical Biochemistry, 306, 259-269. 

Interestingly poly(ADP-ribosyl)ation of chicken erythrocyte core particles resulted in the dissociation of nucleosomal DNA from the histone octamer (Fig. 3c,f). The dissociation coincided with the generation of the hyper(ADP-ribosyl)ated forms of histone H2B (Fig. 2). This result is noteworthy because on exposing hepatoma cells to DMS, histone H2B becomes hyper(ADP-ribosyl)ated [18]. The dissociation of nucleosomal DNA from the histone octamer after poly(ADP-ribosyl)ation explains why nucleosomal DNA becomes more accessible to micrococcal nuclease [19]. The increased accessibility of nucleosomal core DNA caused by poly(ADP-ribosyl)ation could explain in part the increased accessibility of DNA repair patches to micrococcal nuclease observed during the early phase of DNA repair [20]. 

Although the histones prepared from various cell types in the chick are very similar, the histones from rooster testes and chicken erythrocytes differ considerably from the histones obtained from other tissues. Minor but consistent differences have been observed in the histone composition at various stages during the development of the chick embryo the significance of these findings remains to be established. The differences between histones of tumor cells and histones of normal tissues are discussed in greater detail in the chapter devoted to cancer. 

On the other hand, slight tissue differences in the ratio between fractions of jS-histones isolated from chicken erythrocytes, liver, and spleen were observed by Bellair and Mauritzen (1967). Gutierrez and Hnilica (1967) studied phosphorylation of various histone fractions in rat tissues (by determining incorporation of -labeled phosphates into histones) and observed definite fraction and tissue specificity in histone phosphorylation activity. 

Bina-Stein, M., Vogel, T., Singer, D. S., and Singer, M. F., 1976, H5 histone and DNA-relaxing enzyme of chicken erythrocytes, /. Biol. Chem. 251 7363. 

Acetylated isoforms of H3 and H4 are often the targets of ongoing methylation [126,150,151,217,218]. In chicken immature erythrocytes, rapidly acetylated and deacetylated H3 and H4 are selectively methylated, while in Hela cells dynamically acetylated H3, but not H4, is methylated [150,219,220]. H4 that is slowly acetylated and deacetylated is methylated in HeLa [150]. Acetylated yeast H3 was preferentially methylated at Lys-4 [138]. These studies suggested that the processes of histone methylation and dynamic acetylation are not directly coupled, with neither modification predisposing H3 or H4 to the other [138,220]. 

---