Fractionation of chromatin by nuclease digestion and sucrose gradient centrifugation : implications for ultrastructure of the nucleus

Recent experimental work underlying the rapid increase in knowledge of chromatin ultrastructure is reviewed. The validity of the evidence for a non-chromatin structural framework within nuclei is discussed.

Preparations of nuclei from mouse spleen were shown to contain contaminating fibers. In the presence of EDTA, nuclei and fibers aggregated into dense clumps. Preparations of liver nuclei did not contain fibers and did not aggregate in the presence of EDTA.

Chromatins prepared by different methods were compared using micrococcal nuclease as a probe for nucleosome integrity. Intranucleosomal DNA was more susceptible to the nuclease in sonicated nuclei than in intact nuclei. Repeated washing of nuclei with chelating agents and buffers of low ionic strength produced a chromatin gel that resisted digestion unless first dispersed by sonication. After sonication, nucleosomes in the washed nuclei were most susceptible to nuclease cleavage of all preparations tested.

After digestion with micrococcal nuclease, mouse liyer nuclei separated into slowly and rapidly sedimenting fractions in 5-65% sucrose gradients. Fractionation was obtained after digestion periods ranging from 1 minute (1% of the DNA acid soluble) to 2 hours (50% of the DNA acid soluble). The slowly sedimenting fraction contained chromatin fragments produced by nuclease cleavage. After brief digestion, this fraction was enriched in a specific nonhistone protein.

Residual nuclear structures were observed with the light microscope in unfixed wet mounts of the rapidly sedimenting fraction. After complete digestion with the nuclease, 15% of the nuclear DNA remained associated with these structures. This DNA was in the form of mononucleosomes and could not be dissociated except by treatments that disrupted the residual nuclei. The presence of these structures in unfixed material prepared under relatively mild conditions is further evidence for the existence of a nuclear matrix in vivo. It is suggested that chromatin is attached to the nuclear structure through individual nucleosomes. . Examination of stained preparations indicated that the attachment sites are uniformly distributed at the level of resolution of the light microscope.

The effects of changes in ion concentrations on the fractionation was investigated. Digestion products were not released from nuclei in the continued presence of divalent cations, suggesting participation of these ions in nucleosome-nucleosome or nucleosome-matrix interactions. In the presence of 0.06 M salt, octanucleosome subunits of a higher order of chromatin structure were detected. The data indicate the possibility that stability of these subunits is not dependent on preservation of the linker DNA between core particles and that close association between higher order subunits may not require continuity of the connecting DNA.

The implications of highly ordered structure for the organization and regulation of nuclear activity and experimental approaches for future investigation of these mechanisms are discussed.