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P Labhart

Publications and source records attributed to P Labhart.

32 records · Page 2Linked to original sources
PubMed1987

Heat shock stabilizes highly unstable transcripts of the Xenopus ribosomal gene spacer.

We have shown recently that, in Xenopus laevis oocytes, the 3' end of the longest detectable ribosomal precursor RNA is not formed by transcription termination but by RNA processing and that RNA polymerase I continues to transcribe through the intergenic spacer region. In oocytes, these spacer transcripts are turned over rapidly, and the only apparent transcription termination site is located 215 base pairs upstream of the 5' end of the next transcription unit. In this paper we show that, at heat shock temperature (34 degrees C), processing at the 3' end of the precursor, rapid turnover of spacer transcripts, and termination are all severely impaired. In contrast, transcription initiation and chain elongation are not significantly affected by heat shock. This results in the appearance of large RNA in the range of 10-20 kilobases and longer.

Animals↗
PubMed1987

A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis.

rRNA transcription in Xenopus laevis terminates near a 7-base-pair (bp) conserved sequence (T3 box) located 200 bp upstream of the site of transcription initiation for the adjacent gene promoter. We present evidence here that a 12-bp element containing the T3 box is an essential part of the terminator. Using an oocyte injection assay, we found that the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself and that the T3 box within the 12-bp element was required to specify the formation of correct 3' ends. This requirement for the 12-bp element was also seen in pulse-label experiments by using a homogenate of oocyte nuclei, but the present data did not allow us to determine the exact mechanism by which the 12-bp element acts. Removal of the T3 region from its normal location allowed a significant amount of readthrough transcripts to accumulate, indicating that additional sequences may be required for complete terminator function.

Animals↗
PubMed1986

Characterization of three sites of RNA 3' end formation in the Xenopus ribosomal gene spacer.

We have studied three sites of 3' end formation on the Xenopus laevis ribosomal genes, designated T1, T2, and T3. Site T1 coincides with the unique HindIII site at the 3' end of the 28S sequence and is shown to be a site of rapid RNA processing. Transcription continues another 235 bp past T1 to site T2, which marks a boundary between relatively stable and highly unstable transcripts. However, T2 does not cause polymerase release. In nuclear run-off experiments transcription is detected across the entire spacer until site T3, 215 bp upstream of the gene promoter. T3 shares some sequence homology with T2 but appears to cause polymerase release and is presently the best candidate for a true terminator of transcription on these genes.

Animals↗
PubMed1985

Xenopus ribosomal gene enhancers function when inserted inside the gene they enhance.

The ribosomal DNA of Xenopus laevis contains repeated sequence elements in the intergenic spacer region that enhance transcription from the adjacent gene promoter (1,2). Previous work has shown that these RNA polymerase I enhancers influence the target promoter when they are in either orientation, at a distance of several kilobases, and only when they are in cis (3-5). In this work, we further show that enhancer activity is unaffected by inserting the enhancers within the transcription unit whose promoter is being enhanced. In addition, enhancer activity does not interfere with transcription through its sequences. The results suggest that the enhancers act at a point prior to the initiation of transcription and that they are likely to be dispensable once transcription has begun.

Animals↗
PubMed1984

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

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.

Cell Fractionation↗
PubMed1984

Enhancer-like properties of the 60/81 bp elements in the ribosomal gene spacer of Xenopus laevis.

The spacer region of the Xenopus laevis ribosomal gene contains blocks of repetitive sequence elements that are 60 or 81 bp long. These 60/81 bp elements function as enhancer elements for the RNA polymerase I promoter at the 5' end of the gene. An RNA polymerase I promoter adjacent to a block of 60/81 bp elements is always dominant over a promoter on a second plasmid when both are coinjected into oocyte nuclei. If two promoters are placed on the same plasmid containing enhancers, both promoters come under their influence and are codominant. The influence of the enhancers can be transmitted through several kilobases of plasmid sequence, through a potentially active promoter, and is independent of the orientation of the enhancers. The enhancers appear to compete with promoters for the same transcription factor(s); however, the enhancers can only compete when they are on a circular plasmid.

Animals↗
PubMed1983

Chromatin structure along the ribosomal DNA of Dictyostelium. Regional differences and changes accompanying cell differentiation.

The ribosomal genes of Dictyostelium discoideum are extrachromosomal palindromic DNA molecules situated in the nucleolus. Each molecule comprises ribosomal RNA coding regions and non-transcribed spacer regions. We used both biochemical and electron microscopic approaches to investigate the structure of transcribing and non-transcribing chromatin. Nucleoli from exponentially growing cells were digested with micrococcal nuclease, and the resulting DNA fragments were separated by gel electrophoresis and transferred to DBM paper. They were hybridized with cloned EcoRI fragments derived from different parts of the ribosomal gene. Probes of the coding region showed a smear, while probes of the non-transcribed regions gave pronounced banding patterns more complex than typical nucleosome repeats, but not due solely to sequence-specific cutting by micrococcal nuclease. The DNA of the coding region was digested more quickly than that of the non-transcribed ones. When nucleoli were digested with restriction enzymes, sites within the coding region were accessible and sites in the non-transcribed region were protected. The structure of ribosomal chromatin in differentiating cells, in which the rate of ribosomal RNA synthesis is reduced, was examined using essentially the same methods. The coding region, probed by hybridization to micrococcal digests, then showed a typical DNA repeat pattern indicating that this region had become condensed into nucleosomes, and its accessibility to restriction enzymes was very much reduced. On electron micrographs of lysed nucleoli from exponentially growing cells, two types of chromatin were observed, one with a beaded nucleosomal appearance, the other with putative RNA polymerase molecules attached to fibres indistinguishable from free DNA adsorbed to the same grid. The combined results suggest that whereas regions that are not transcribed are packaged with proteins that protect them from nuclease digestion, actively transcribing ribosomal genes are associated with few macromolecular constituents apart from those required for transcription and its regulation.

Cell Nucleolus↗
PubMed1983

Comparative studies on the structural organization of membrane-depleted nuclei and metaphase chromosomes.

Interphase membrane-depleted nuclei and metaphase chromosomes were prepared in parallel with a nonionic detergent lysis procedure at low ionic strength. By flow microfluorometry we showed for the first time that cell lysates contain all stages of the cell cycle in the same proportions as the starting cell population. Morphologically intact membrane-depleted nuclei and metaphase chromosomes were isolated as non-aggregated structures on sucrose gradients. When analysed in the electron microscope, membrane-depleted nuclei that had been treated with 2M NaCl appeared as residual structures containing the pore complex-lamina layer attached to a halo of DNA filaments. In contrast, no distinct high salt-resistant structure was found with metaphase chromosomes. They formed a highly fragile network which disintegrated easily into small complexes connected with DNA filaments. High salt-resistant DNA-protein complexes were purified by Metrizamide density gradient centrifugation. The main difference in the protein composition of interphase and metaphase residual complexes was the presence in interphase of a protein triplet in the 60-75 kilodalton molecular weight range and its absence in metaphase. This protein triplet most likely corresponds to the lamins A, B, and C of the nuclear lamina. The combined results suggest that the main difference in the structural organization of interphase nuclei and metaphase chromosomes is the presence or absence of the pore complex-lamina layer.

Animals↗
PubMed1982

Involvement of higher order chromatin structures in metaphase chromosome organization.

Using electron microscopy we show that the metaphase chromatin fibers of Chinese hamster ovary cells form the same ionic strength-dependent higher order structures as the corresponding interphase chromatin fibers. We present evidence that such intact chromatin fibers are a prerequisite for the maintenance of the characteristic shape of metaphase chromosomes. The evidence is based on the finding that treatment of chromosomes with 0.5 M NaCl, a condition which is known to remove histone H1 and which destroys the higher order structure of chromatin fibers, also leads to a disintegration of the metaphase chromosome structure, whereas treatment with 0.3 M (or less) NaCl has no effect on the integrity of the chromosomes and their chromatin fibers. These data support a model in which the metaphase chromosome is maintained by a tight assembly of the 25-30 nm thick chromatin fibers containing all the histones.

Animals↗
PubMed1982

Structure of the active nucleolar chromatin of Xenopus laevis Oocytes.

Active nucleolar chromatin of Xenopus laevis oocytes was prepared for electron microscopy by a step gradient method, which separates the chromatin from proteins and other constituents that might nonspecifically bind at low ionic strength. Between putative RNA polymerases and within the nontranscribed spacer region, the chromatin appears as smooth, thin filaments. For the first time, it is shown here that these filaments are indistinguishable from pure DNA absorbed to the same specimen, even when the ionic strength is raised up to 100 mM NaCl. Bulk rat liver chromatin, however, which was coprepared as a biochemically well-characterized standard with the active nucleolar chromatin, shows nucleosomes containing fibers, which condense into supranucleosomal structures with increasing ionic strength. Since the appearance and the behavior of active nucleolar chromatin at different ionic strengths and pHs resembles tht of pure DNA, but not of any known type of chromatin, it is suggested that, except for the transcription apparatus, very few macromolecular constituents are associated with ribosomal DNA during transcription. The observations described in this paper explain most of the published and partly conflicting results obtained by electron microscopy of nucleolar chromatin.

Animals↗
PubMed1982

Structure of the active nucleolar chromatin of Xenopus laevis oocytes.

Active nucleolar chromatin of Xenopus laevis oocytes was prepared for electron microscopy by a step gradient method, which separates the chromatin from proteins and other constituents which might unspecifically bind at low ionic strength. Between putative RNA polymerases and within the non-transcribed spacer region, the chromatin appears as smooth, thin filaments, indistinguishable from pure DNA adsorbed to the same specimen. These filaments are found under all conditions tested, even in the presence of 100 mM NaCl. On the other hand, bulk rat liver chromatin, which was co-prepared with the active nucleolar chromatin, shows nucleosomes containing fibers, which condense into supranucleosomal structures with increasing ionic strength. Since the appearance and the behaviour of active nucleolar chromatin at different ionic strength and pH resembles that of pure DNA, but not that of any known type of chromatin, it is suggested that, except for the transcription apparatus, few macromolecular constituents are associated with ribosomal DNA during transcription.

Animals↗
PubMed1981

Electron microscope specimen preparation of rat liver chromatin by a modified Miller spreading technique.

We describe a modification of the spreading method of Miller and Bakken [17]. Prior to mounting the samples, the carbon supports used for electron microscopy are coated with Alcian blue. By this method the chromatin fibres stick firmly to the supporting film such that the shape of the fibres is independent of the washing and drying procedures used during specimen preparation. Using this technique we show an ionic strength-dependent condensation of rat liver chromatin protruding from lysed nuclei which is fully compatible with that reported previously [34] for soluble chromatin.

Adhesiveness↗
PubMed1981

Structural changes of soluble rat liver chromatin induced by the shift in pH from 7 to 9.

Soluble rat liver chromatin was studied at pH 7 and at pH 9. In order to remove selectively non-histone components or non-histone components and histone H1, fractionation of chromatin was performed at pH 7 and pH 9 at different ionic strengths. The salt-dependent condensation of the fractionated chromatin was analysed in the electron microscope. There is no difference between the appearance of H1-depleted chromatin at poH 7 and pH 9. In H1-containing chromatin the shift from pH 7 to pH 9 leads to the following morphological changes: a) at very low ionic strength the nucleosomes unravel partially or totally and the zigzag-shaped fibres disappear in favour of beads-on-a-string; b) with increasing ionic strength the filaments condense into fibres, however, these fibres appear distorted and clearly less ordered than at pH 7. There is no indication of a release or displacement of histone H1. The pH-effect is completely reversible. The data suggest a pH-induced change in the mode of action of histone H1 in the formation of nucleosome beads and higher order chromatin structures.

Animals↗