A structural concept for nucleoli of Dictyostelium discoideum deduced from dissociation studies

Labhart, Paul; Banz, Elizabeth; Ness, Penelope J.; Parish, Roger W.; Koller, Th.

doi:10.1007/bf00292894

Cited by 20 publications

(7 citation statements)

References 36 publications

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“…From the intensity of the two distinct bands, it can be determined that in this X. laevis cell line, the active gene copies represent only about 25% of the total population. When the nuclei are cross-linked in the presence of heparin, which removes the histones from the DNA (15,43), the same ribosomal coding fragment is resolved in a single retarded band (Fig. 2a, lane 3) with mobility similar to that of the s band, indicating that the inactive copies have also been heavily cross-linked.…”

Section: Resultsmentioning

confidence: 99%

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

Lucchini¹,

Sogo²

1992

Mol. Cell. Biol.

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The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer.In the last few years, much progress has been achieved in identifying the different sequence elements located in the intergenic spacers that are involved in the regulation of the rRNA genes. Despite the fact that the DNA sequence of the ribosomal spacers has diverged very rapidly between different organisms, recent studies indicate a general consensus in the function as well as in the arrangement of transcriptional regulatory elements in almost all higher eukaryotes (for reviews, see references 35, 44, and 45). However, some exceptions seem to exist even among closely related species. The most striking difference resides in the function of the spacer elements close to the 3' end of the rRNA precursor coding regions in the two frog species Xenopus laevis and Xenopus borealis. In fact, a series of transcriptional analyses failed to detect a true termination site at the 3' end of the X. laevis rRNA genes (9, 18), whereas the related sites in X. borealis behave like efficient terminators (21) (sites T2, see Fig.

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Section: Resultsmentioning

confidence: 99%

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

Lucchini¹,

Sogo²

1992

Mol. Cell. Biol.

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“…However, their structural and functional stability during isolation requires divalent cations and/or polyamines at concentrations in the mM range, conditions which cannot be readily compared with and may not correspond to those in vivo, and the effect of these cations of increasing the resistance of nucleoli to dissociation by NaCl (Labhart et al, 1984) suggests that they may provide stabilisation by alternative mechanisms. Here, bacterial chromosomes offer Fig.…”

Section: Discussionmentioning

confidence: 96%

A role for macromolecular crowding effects in the assembly and function of compartments in the nucleus

Hancock

2004

Journal of Structural Biology

152

136

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“…According to it, chromatin DNA is not tightly bound, but rather loosely interwoven with the lamina layer. The DNA may be additionally held inside the shell in the form of a huge network by side to side interactions, possibly involving elements of the nuclear ribonucleoprotein structures, as suggested for DNA in the nucleolus [51]. When chromatin is fragmented at low ionic strength in the absence of divalent cations and then dehistonized, DNA is completely released.…”

Section: Dna-nl Association Irreversibly Stabilized By Mg + +mentioning

confidence: 99%

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells

Krachmarov

Iovcheva

Hancock³

et al. 1986

J of Cellular Biochemistry

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We have studied in vitro binding of DNA to nuclear lamina structures isolated from Ehrlich ascites tumor cells. At low ionic strength in the presence of Mg++, they bind considerable amounts of mouse and bacterial DNA, forming complexes stable in 2 M NaCl. Single-stranded DNA and pulse-labeled DNA show higher binding efficiencies than native uniformly labeled DNA. When mixing occurs in 2 M NaCl, complex formation is inhibited. When nuclei are digested with DNAse I under conditions that favor chromatin condensation, DNA associated with matrices subsequently prepared from such nuclei is markedly enriched in satellite DNA. If digestion is carried out with DNAse II while nuclei are decondensed in EDTA, no enrichment in satellite DNA is observed. Preparations of purified, high-molecular weight, double-stranded DNA contain variable amounts of fast-sedimenting aggregates, which are insoluble in 2 M NaCl but are dispersed by DNA fragmentation or denaturation. These results point at some artifacts inherent in studies of DNA bound to residual nuclear structures in vivo and suggest conditions expected to avoid these artifacts. Further, using controlled digestion with DNAse II, we have studied the in vivo association of DNA with nuclear lamina isolated from Ehrlich ascites tumor cells. In the course of DNA fragmentation from above 50 kbp to about 20 kbp average size, the following events were observed. The DNA of high molecular weight (much longer than 50 kbp) behaved as if tightly bound to the nuclear lamina, as judged by sedimentation in sucrose and metrizamide density gradients, electron microscopy, and retention on glass fiber filters. As the size of DNA decreased, it was progressively detached from the nuclear lamina, and at about 20 kbp average length practically all DNA was released. The last 1-4% of DNA, although cosedimenting with the nuclear lamina in sucrose gradients, behaved as free DNA, banding at 1.14 g/cm3 in metrizamide density gradients and showing less than 4% retention on filters. At no stage of digestion did the DNA cosedimenting with nuclear lamina show changes in satellite DNA content relative to that of total DNA or enrichment in newly replicated DNA. It was shown, however, that digestion of nuclear lamina-DNA complex with EcoRI or Hae III led to the formation of DNA-protein aggregates, which banded at 1.35 g/cm3 in high salt containing metrizamide density gradients and which were strongly enriched in satellite DNA.(ABSTRACT TRUNCATED AT 400 WORDS)

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A structural concept for nucleoli of Dictyostelium discoideum deduced from dissociation studies

Cited by 20 publications

References 36 publications

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

A role for macromolecular crowding effects in the assembly and function of compartments in the nucleus

Association of DNA with the nuclear lamina in Ehrlich ascites tumor cells

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