Visualization of chromatin substructure: upsilon bodies.

Abstract

Spread chromatin fibers, from isolated eucaryotic nuclei, reveal linear arrays of spherical particles (o bodies), about 70 A in diameter, connected by thin filaments about 15 A wide. These particles have been observed in freshly isolated nuclei from rat thymus, rat liver, and chicken erythrocytes. In addition, v bodies can be visualized in preparations of isolated sheared chromatin, and in chromatin reconstructed from dissociating solvent conditions (i.e., high urea-NaCl concentration). As a criterion for perturbation of native chromatin structure low-angle X-ray diffraction patterns were obtained from nuclear pellets at different stages in the preparation of nuclei for electron microscopy. These results suggest that the particulate (v body) structures observed by electron microscopy may be closely related to the native configuration of chromatin.The DNA of eucaryotic chromosomes exists in a highly folded condition largely as a consequence of its interactions with the histone proteins (Huberman, 1973;Pardon and Richards, 1973;Hnilica, 1972;DuPraw, 1970). Numerous models of this folded state have been presented based upon data from low-angle X-ray diffraction and from electron microscopy. The models which appear to be favored, at present, consist of superhelical coiling of a fundamental nucleohistone strand (Pardon and Wilkins, 1972;Bram and Ris, 1971). Previous studies from our laboratory (Olins and Olins, 1974) have presented evidence that chromatin fibers consist of chains of spheroid particles about 70 A in diameter (v bodies) which could contain DNA folded by association with a small number of histone molecules. This paper extends these observations to soluble chromatin and to reconstructed chromatin. In addition, parallel, low-angle X-ray diffraction studies are included which indicate that the particulate structures observed by electron microscopy are closely related to the native state of chromatin. MATERIALS AND METHODS ReagentsFormaldehyde (10%) was prepared fresh by dilution from a stock of 37% analytical reagent grade formaldehyde. Chemicals were reagent grade or better. Buffers and solutions were made from glass-distilled water. Preparation of NucleiRat thymus, rat liver, and chicken erythrocyte nuclei were prepared as described previously Olins, 1974, 1972;Olins and Wright, 1973). Nuclei, recovered after pelleting from high sucrose concentrations, were washed and centrifuged twice in 0.05 M sodium cacodylate buffer (pH 7.5), 0.025 M KCI, 5 mM MgCI2 (CKM), and washed and centrifuged once with 0.2 M KCI before the swelling procedures.Swelling of the nuclei was generally accomplished by suspending the KCI-washed nuclear pellet in 0.2 M KC1 to a concentration of approximately 108 nuclei/ml, followed by a dilution of about 200-fold with distilled water. Nuclei were allowed to swell for 10-15 rain, then the suspension was made approximately I% in formaldehyde by the addition of 0.1 vol of 10% formaldehyde (pH 57,8

Studies on the elementary fibril of chromatin in pollen mother cells.

“…3, arrows). The granules are the same sizes as the v-bodies re reported by Olins and Olins (1974), Woodcock et al (1976) and Howze et al (1976), but the tiny filaments connecting v-bodies as reported by Olins and Olins (1974) and Howze et al (1976) could not be observed.…”

“…2). Olins and Olins (1974) and Wood cock et al (1976) concluded that the extended chromatin threads have a "beads on-a-string" substructure, with particles called "v-bodies" which are about 70 A in diameter and are connected by DNA fibers of about 15 A diameter . Howze et al (1976) observed fine fibrils of about 89+11 A length between particles in the chromosomes of Chinese hamsters.…”

“…In electron microscopic studies Olins and Olins (1974), Olins et al (1975), Se nior et al (1975) and Howze et al (1976) observed particles of 100 A diameter aligned in tandem along the elementary chromatin fibrils. These particles have been called "u-bodies" (or "nucleosomes" by Oudet et al 1975).…”

The SV40 transcription complex. II. Non-dissociation of protein from SV40 chromatin during transcription

“…110A (4)(5)(6). Nucleosomes are comprised of about 200 base pairs of DNA wound around a complex containing two each of the four major histones (11)(12)(13)(14)(15)(16)(17). Transcriptionally active and inactive regions of cellular chromatin are both organized in this basic manner (18)(19)(20), although there is some structural difference between them, as evidenced by a selective deoxyribonuclease I sensitivity of active genes (21,22).…”

Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei.

“…One basic structural component of the chromosome results from the organization of histone and DNA into a regular array of nucleosomes (1)(2)(3)(4)(5)(6)(7). This basic unit of structure involves about 190 base pairs of DNA complexed with two each of the four major histones and encompasses virtually all genomic sequences.…”