Micrococcal nuclease digestion of Dictyostelium discoideum nuclei from various developmental stages was used to investigate transcription-related changes in the chromatin structure of the coding region of four genes. Gene activity was determined by Northern blotting and nuclear run on experiments. During strong transcription of the developmentally regulated cysteine proteinase I gene, a smear superimposed on a nucleosomal ladder was observed, indicating perturbation of nucleosomal structure was occurring. However, two other developmentally regulated genes, discoidin I and pSC253, showed only slight nucleosome disruption during high levels of transcription. The chromatin structure of a fourth gene (pCZ22) was disrupted throughout development, even at those stages where transcription was greatly reduced. We suggest that although nucleosome structure can be transiently perturbed by the passage of the transcription complex in vivo, the degree of perturbation and the speed with which nucleosomes reassemble is also influenced by the DNA sequence.
We aimed to establish whether there is a matrix structure in the nucleolus to which the ribosomal DNA (rDNA) is strongly attached. To detect artifacts that might occur during the harsh histone extraction procedures frequently used for matrix preparation, we dissociated nucleoli of Dictyostelium discoideum with a range of NaCl or heparin concentrations. With heparin treatment significant amounts of rDNA were solubilized into the dissociating solution. When the residual nucleoli were digested with Eco RI, none of the Eco RI fragments of the rDNA remained preferentially bound to the residual nucleoli, indicating that there is no matrix attached to a specific site on the rDNA. When residual nucleoli were examined by electron microscopy, a correlation was found between the extent of solubilization of rDNA, the loss of nucleosomes, and, in heparin-treated nucleoli, the loss of ribonucleoprotein-bound components. These results suggest that the rDNA is released from the nucleoli as soon as nucleosomes have been dissociated and transcription complexes disrupted. Electron microscopy also showed that the NaCl concentration required for dissociation of nucleosomes was higher when divalent cations (Ca2+, Mg2+, Cu2+) were used during the isolation or the treatment of the nucleoli prior to dissociation in high salt. Furthermore, the residual, high-salt-resistant structures were much larger when nucleoli were pretreated with divalent cations or when they were purified in the presence of Ca2+ than when they were purified in its absence. Hence divalent cations, which induce chromatin condensation, prevented nucleolar dissociation whereas treatment with chelating agents, which loosen chromatin compaction, led to much smaller residual matrixlike structures. Nucleoli could be dissociated with heparin to a larger extent than with NaCl so that in Ca2+-free preparations no residual nucleolar matrixlike structures could be detected. Our results suggest that the nucleolar "matrix" seen in the electron microscope is due to incomplete dissociation of the nucleolar material. We propose that in nucleoli of Dictyostelium the rDNA is not attached to a tightly binding matrix structure, but that nucleoli are stabilized by side-to-side contacts between chromatin fibers and transcription complexes.
We have used methidiumpropyl-EDTA-iron(II) [MPE.Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dictyostelium. Confirming our earlier results with micrococcal nuclease (1,2), MPE.Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE.Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE.Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.
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