Differences of supranucleosomal organization in different kinds of chromatin: cell type-specific globular subunits containing different numbers of nucleosomes.

Abstract

Fractions of homogeneously-sized supranucleosomal particles can be obtained in high yield and purity from various types of cells by brief micrococcal nuclease digestion (10 or 20 s) of condensed chromatin in 100 mM NaCl followed by sucrose gradient centrifugation and agarose gel electrophoresis . These chromatin particles, which contain only DNA and histones, differed according to cell type. Sea urchin spermatozoa (Paracentrotus lividus) gave rise to heavy particles (ca. 260 S) with a mean diameter (48 nm) . These resembled the unit chromatin fibrils fixed in situ, which contain an average of 48 nucleosomes, as determined both by electron microscopy after unraveling in low salt buffer and gel electrophoresis . In contrast, higher order particles from chicken erythrocyte chromatin were smaller (105 S; 36-nm diam) and contained approximately 20 nucleosomes . The smallest type of supranucleosomal particle was obtained from chicken and rat liver (39 S; 32-nm diam ; eight nucleosomes). Oligomeric chains of such granular particles could be recognized in regions of higher sucrose density, indicating that distinct supranucleosomal particles of globular shape are not an artifact of exposure to law salt concentrations but can be obtained at near-physiological ionic strength . The demonstration of different particle sizes in chromatin from different types of nuclei is contrary to the view that such granular particles are produced by artificial breakdown into "detached turns" from a uniform and general solenoid structure of approximately six nucleosomes per turn. Our observations indicate that the higher order packing of the nucleosomal chain can differ greatly in different types of nuclei and the supranucleosomal organization of chromatin differs between cell types and is related to the specific state of cell differentiation .The question of how the nucleosomal chain, the basic structural element of organization of chromatin, is organized at the secondary level of chromatin packing remains unanswered . Two different models of supranucleosomal packing have been presented : (a) Finch and Klug (15) have proposed a continuous "solenoid" in which the nucleosomal chain is helically coiled, with about six nucleosomas per turn and a pitch of -I 1 nm, thus forming a homogeneously thick fibril -30 nm in diameter. Various observations have been interpreted to support this model (2,4,6,7,9,10,30,31,41,53,58). However, the majority of these experiments have been performed upon chromatin that has undergone destabilization through/by preparative "stress," such as lengthy dialysis and/or washes in buffers of drastically reduced ionic strength or containing chelating agents. According to this model, the solenoid should be identical in different kinds of condensed chromatin, irrespective of the cell type examined (31). (b) From electron microscopic observations of spread chromatin preparations and from studies of the products of limited digestion with micrococcal nuclease (16,20,24) other authors have proposed that the nucleosomal c...

EM measurements define the dimensions of the “30-nm” chromatin fiber: Evidence for a compact, interdigitated structure

“…This finding resolves the controversy in the literature, showing that the fiber diameter does not increase linearly with linker length. The diameter of our reconstituted fibers containing 40 bp of linker DNA or less is consistent with previous measurements of chromatin isolated from mudpuppy erythrocytes (28-bp linker) (20)(21)(22)27), rat liver (31-bp linker) (17) and chicken erythrocytes (42-bp linker) (17,19,23). Furthermore, the larger diameter of Ϸ44 nm observed for fibers with linker DNA between 50 and 70 bp is in good agreement with measurements from sea urchin sperm chromatin (67-bp linker) (17,(20)(21)(22)27).…”

Electron microscopy and atomic force microscopy studies of chromatin and metaphase chromosome structure

“…1 (panels C and D). The observation of globular oligonucleosome particles in the electron microscope after nuclease digestion of chromatin led to the suggestion that fibers may be discontinuous (Hozier et al, 1977;Zentgraf and Franke, 1984). Three-dimensional reconstruction of fibers from sea cucumber sperm embedded in araldite-epon ( Fig.…”

Structural elements of bulk chromatin within metaphase chromosomes

“…Some results suggested that superbeads isolated after mild nuclease digestion of chromatin could be ribonucleoprotein complexes (Muyldermans et al 1980) but the detailed studies of Zentgraf & Franke (1984) demonstrated that these particles obtained from different types of cells contain 8À48 nucleosomes with the normal histone stoichiometry. The results obtained in our laboratory (Bartolomé et al 1994, 1995 with small fragments of chicken erythrocyte chromatin (containing 6À36 nucleosomes) have shown that, in the presence of Mg 2+ , these fragments form cylindrical structures of about 35 nm in diameter corresponding to short fibres more compact than the typical solenoid and the other models previously suggested.…”