Changes of nucleosome frequency in nucleolar and non-nucleolar chromatin as a function of transcription: an electron microscopic study

Abstract

SummaryThe morphology of nucleolar and non-nucleolar (Iampbrush chromosome loops) chromatin was studied in the electron microscope during states of reduced transcriptional activity in amphibian oocytes (Xenopus laevis, Triturus alpestris, T. cristatus). Reduced transcriptional activity was observed in maturing stages of oocyte development and after treatment with an inhibitor, actinomycin D.Strands of nucleolar chromatin appear smooth and thin, and contain only few, if any, nucleosomal particles in the transcribed units. This is true whether they are densely or only sparsely covered with lateral ribonucleoprotein fibrils. This smooth and non-nucleosomal character is also predominant in the interspersed, apparently nontranscribed rDNA spacer regions. During inactivation, however, nucleolar chromatin frequently and progressively assumes a beaded appearance in extended fibril-free-that is, apparently nontranscribed -regions. I n either fUll-grown 00-cytes or late after drug treatment, most of the nucleolar chromatin is no longer smooth and thin, but rather shows a beaded configuration indistinguishable from inactive non -nucleolar chromatin. In many chromatin strands, transitions of fibril-associated regions of smooth character into beaded regions wihout lateral fibrils are seen.Similarly, in the non-nucleolar chromatin of the retracting lampbrush chromosome loops, reduced transcriptional activity is correlated with a change from smooth to beaded morphology. Here, however, beaded regions are also commonly found interspersed between the more or less distant bases of the lateral fibrils, the putative transcriptional complexes.I n both sorts of chromatin, detergents (in particular Sarkosyl) that remove most of the chromatin proteins including histones from the DNA axis but leave the RNA polymerases of the transcriptional complexes attached were used to discriminate between polymerases and nucleosomal particles. The results suggest that nucleosomes are absent in heavily transcribed chromatin regions but are reformed after inactivation. In contrast to the findings with inactivated nucleolar genes, in lampbrush chromosome loops the beaded nucleosomal configuration appears to be assumed also in regions within transcriptional units that, perhaps temporarily, are not involved in transcription.

Mitochondrial DNA arranged into chromatin-like structures after injection into amphibian oocyte nuclei

“…The morphology of the rings was indistinguishable from that of inactive chromatin endogenous to the injected nuclei ( fig. 2d; cf [52]) and was also similar to that of inactive chromatin observed in a diversity of other cells (for refs see [39,[41][42][43][52][53][54]), including non-transcribed chromatin formed from plasmid or SV40 DNA injected to Xenopus laevis oocyte nuclei (cffig. 5e; [3,6]).…”

Microinjection of actin-binding proteins and actin antibodies demonstrates involvement of nuclear actin in transcription of lampbrush chromosomes

“…Typical complete "Christmas tree" structures, characteristic of fully active rRNA genes, were found to the same extent in experimental preparations as in controls (Figure 3c; for correlation of structure and transcriptional activity of rRNA genes see Scheer et aI., 1976;Scheer, 1978). This indicates that RNA polymerase 11 dependent transcription is specifically inhibited by these substances.…”

“…Since transcription units of lampbrush loops from untreated oocytes and oocytes receiving control injections are characterized by an extremely high packing density of transcriptional complexes (reviewed in Miller, 1981;Scheer et aI., 1979b), their reduced number in injected nuclei indicates premature detachment from the loop axes and/or reduced frequency of initiation events. A premature release of transcripts from the template chromatin which in early stages results in intragenic regions without RNP fibrils ("gaps") seems to be a general consequence of reduced transcriptional activity in chromosomal loops (Scheer, 1978;Scheer et aI., 1979aScheer et aI., , 1979bBona et aI., 1981;Kleinschmidt et aI., 1983).…”

“…Ultrathin sections were stained according to standard procedures. Chromatin spread preparations were made essentially according to the method of Miller and Bakken (1972) with some modifications (Scheer, 1978;Scheer et aI. , 1976Scheer et aI.…”

DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

“…When viewed in the electron microscope, active ribosomal genes are invariably extended to an extent which precludes typical nucleosome structure (55,56,(58)(59)(60)(61). In Oncopeltus, the extended state appears before the onset of transcription (56).…”