Tetrahymena ribosomal RNA gene chromatin is digested by micrococcal nuclease at sites which have the same regular spacing on the DNA as corresponding sites in the bulk nuclear chromatin.

Piper, Peter W.; Celis, Julio; Kaltoft, Keld; Leer, Johan C.; Nielsen, Ole F.; Westergaard, Ole

doi:10.1093/nar/3.2.493

Cited by 54 publications

(25 citation statements)

References 20 publications

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“…Our results clearly indicate that the preferential digestion by DNase I of DNA which has been labeled during refeeding of starved Tetrahymena is similar in intact nuclei, isolated nuclesomes and purified nucleosome cores. It has been demonstrated that this defined starvation-refeeding scheme preferentially labels the ribosomal genes (10,11,29,42; see Fig. 1).…”

Section: Discussionmentioning

confidence: 89%

“…There is general agreement that prestunably active ribosomal (10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21) and non-ribosomal (19,(22)(23)(24)(25)(26) genes show both periodic and particulate structure after digestion with staphylococcal nuclease. The periodicity of the presumably active genes is indistinguishable from that of bulk chromatin (10,11,19,26). There is some controversy over whether active genes show the same susceptibility as bulk chromatin to staphylococcal nuclease.…”

Section: Introductionmentioning

confidence: 99%

“…Further digestion with staphylococcal nuclease produces a third, metastable intermediate containing about 160-168 bp of DNA (5,9) recently named a chromatosome (5). Continued [10][11][12][13][14][15][16][17][18][19][20] bp.…”

Section: Introductionmentioning

confidence: 99%

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DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

Giri¹,

Gorovsky²

1980

Nucl Acids Res

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The level of chromatin structure at which DNase I recognizes conformationial differences between inert and activated genes has been investigated. Bulk and ribosomal DNA's of Tetrahymena pyriformis were differentially labeled in vivo with [14C]-and [5H]-thymidineI respectively, utilizing a defined starvation-refeeding protocol. The H-labeled ribosomal genes were shown to be preferentially digested by DNase I in isolated nuclei. Staphylococcal nuclease digested the ribosomal genes more slowly than bulk DNA, probably owing to the higher GC content of rDNA. DNase I and staphylococcal nuclease digestions of purified nucleosomes and of nucleosome core particles isolated from dual-labeled, starved-refed nuclei were indistinguishable from those of intact nuclei. We conclude from these studies that DNase I recognizes an alteration in the internal nucleosome core structure of activated ribosomal genes.

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Section: Discussionmentioning

confidence: 89%

Section: Introductionmentioning

confidence: 99%

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DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

Giri¹,

Gorovsky²

1980

Nucl Acids Res

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“…Furthermore, data on cross-linking in vivo of r-chromatin from Tetrahymena with trimethylpsoralen support these results [27]. Previous results using differential labelling of rDNA in vivo [28,29] indicated that part of the total r-chromatin in a presumably active state has a nucleosomal structure. However, the observed repeat pattern of DNA fragments generated by micrococcal nuclease digestion might originate from the spacer region and not from the transcribed regions.…”

Section: Discussionmentioning

confidence: 61%

Nuclease‐Sensitive Regions on the Extrachromosomal r‐Chromatin from Tetrahymena pyriformis

Borchsenius¹,

Bonven²,

Leer³

et al. 1981

European Journal of Biochemistry

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The extrachromosomal D N A coding for the ribosomal R N A precursor in Tetrahymenu contains a transcribed region with a size of 6 x lo3 base pairs plus non-transcribed central and distal spacers. In the present study the chromatin structure of the transcribed region and the terminal spacer have been compared. Micrococcal nuclease and DNase I were used to investigate the nucleosomal and the higher order structures. The specific D N A fragments were visualized by gel electrophoresis, Southern blotting onto nitrocellulose sheets and hybridization with specific 32P-labelled RNA probes. Investigations of the cleavage patterns demonstrate the presence of a defined nucleosomal structure in the non-transcribed region, while there is no indication of a nucleosomal pattern in the transcribed region. Specific regions on the r-chromatin are hypersensitive to DNase I. The first cleavage occurs in the non-transcribed central spacer region, while the second cleavage takes place in a region near the 3' end. The hypersensitivity of the central part ofr-chromatin is also found by autodigestion in isolated nucleoli.An understanding of gene expression in eukaryotes requires a detailed description of the structural organization of the chromosome. The basic structural units of chromatin consist of D N A folded around a histone matrix creating the so-called nucleosomes (see e.g. review by McGhee and Felsenfeld [I]). Preferential susceptibility of active sequences to DNase I has been reported in several cases suggesting a modified nucleosomal structure of active loci [2 -81. Evidence has also been presented for the organization of chromatin in higher-order structures above the nucleosomal level. Thus nucleases cleave the chromatin of Drosuphila at specific positions, creating discrete DNA fragments with sizes ranging from a few thousand base pairs to more than twenty thousand base pairs [9]. Activation of heat shock loci in Drosophila results in partial disruption of both the nucleosomal and higher-order organization. Recent investigations of two heatshock protein genes have revealed that sites hypersensitive to DNase I are located near the 5' end of the coding sequences [lo].Hybridization experiments have also revealed that DNaseI introduces specific double-stranded cuts into both the a-globin introduced by DNase I are due to structural modjfications of the chromatin and that the sensitive sites are associated with origins for replication or promotors for transcription.In the present study the structure of active and inactive sequences of the extrachromosomal r-chromatin [13,14] from exponentially growing cultures of Tetrahymenu has been investigated. Digestions with micrococcal nuclease demonstrate a clear difference in the nucleosomal structure of the active regions versus the inactive region. Two regions on the r-chromatin are hypersensitive to DNase I. One region is located near the centre of the molecule (less than lo3 base pairs from the 5' end of the coding sequences), while the second lies near the 3' end of the coding sequenc...

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“…The concept of an absence of nucleosomal globules in transcribed genes of ribosomal RNAs seems to be in conflict with reports of the protection of ca. 200 base pair units of rDNA to digestion with endonuclease in macronuclei of the ciliate, Tetrahymena pyriformis Mathis & Gorovsky 1976;Piper et al 1976), and in somatic cells of Xenopus laevis (Reeves 1976;Reeves & Jones 1976). In both systems, however, the number of transcribed genes has to be regarded as unknown.…”

Section: Discussionmentioning

confidence: 99%

Morphology of transcriptional units at different states of activity

Franke¹,

Scheer

1978

Phil. Trans. R. Soc. Lond. B

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The morphology of two forms of transcriptionally active chromatin, the nucleoli and the loops of lampbrush chromosomes, has been examined after fixation in situ or after isolation and dispersion of the material in media of low ionic strengths, using a variety of electron microscopic preparation techniques (e.g. spread preparations with positive or negative staining or without any staining at all, with bright and dark field illumination, with autoradiography, after pretreatment of the chromatin with specific detergents such as Sarkosyl NL-30; transmission and scanning transmission electron microscopy of ultrathin sections). Nucleolar chromatin and chromosomes from oocytes of various amphibia and insects as well as from green algae of the family of the Dasycladaceae were studied in particular detail. The morphology of transcriptional units that are densely packed with lateral ribonucleoprotein fibrils, indicative of great transcriptional activity, was compared with that of chromatin of reduced lateral fibril density, including stages of drug-induced inhibition. The micrographs showed that under conditions which preserve the nucleosomal organization in condensed chromatin studied in parallel, nucleosomes are not recognized in transcriptionally active chromatin. This holds for the transcribed regions as well as for apparently untranscribed (i.e. fibril-free) regions interspersed between (‘spacer’) and/or adjacent to transcribed genes and for the fibril-free regions within transcriptional units of reduced fibril density. In addition, comparison of lengths of repeating units of isolated rDNA with those observed in spread nucleolar chromatin indicated that this DNA is not foreshortened and packed into nucleosomal structures. Granular particles which were observed, at irregular frequencies and in variable patterns, in some spacer regions, did not result in a proportional shortening of the spacer axis, and were found to be resistant to detergent treatment effective in removing most of the chromatin associated proteins including histones. Thus, these particles behave like RNA polymerases rather than nucleosomes. It is suggested that structural changes from nucleosomal packing to an extended form of DNA are involved in the transcriptional activation of chromatin.

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Tetrahymena ribosomal RNA gene chromatin is digested by micrococcal nuclease at sites which have the same regular spacing on the DNA as corresponding sites in the bulk nuclear chromatin.

Cited by 54 publications

References 20 publications

DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

DNase I sensitivity of ribosomal genes in isolated nucleosome core particles

Nuclease‐Sensitive Regions on the Extrachromosomal r‐Chromatin from Tetrahymena pyriformis

Morphology of transcriptional units at different states of activity

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