Lengths and patterns of transcriptional units in the amplified nucleoli of oocytes of Xenopus laevis

Scheer, Ulrich; Trendelenburg, Michael F.; Krohne, Georg; Franke, Werner W.

doi:10.1007/bf00288462

Cited by 86 publications

(64 citation statements)

References 49 publications

(59 reference statements)

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“…Another outcome of the model is that the binding of polymerase I to the NTS might produce a degree of initiation at a 240 bp repeat as observed in in vitro studies (3). This is consistent also with the finding of NTS transcription in Xenopus (24), initiated at the promoter-like regions (T. Moss; pers. comm.).…”

Section: Construction Of Clonessupporting

confidence: 85%

Multiple Pol I initiation sequences in rDNA spacers ofDrosophila melanogaster

1982

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Section: Construction Of Clonessupporting

confidence: 85%

Multiple Pol I initiation sequences in rDNA spacers ofDrosophila melanogaster

1982

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“…This differential stability is illustrated in spread preparations of the amplified nucleoli from mature oocytes of urodelan Triturus cristatus and anuran Xenopus laevis amphibia (Fig-ures 7a and 7b RNA poly me rases stand out as very electron dense particles (Figure 7a; compare Scheer et al ., 1977). These residual matrix units are seen next to , and often in structural continuity with , rDNA-containing fibrils free of transcriptional complexes which are uniformly thin (20-40 A) and do not reveal a beaded configuration of nucleosomal size .…”

Section: Resultsmentioning

confidence: 88%

“…, 1976;Franke et aI., 1976a;Laird et al, 1976 ;Trendelenburg et aI. , 1976;Woodcock et al, 1976b;McKnight, Bustin and Miller, 1977 ;Scheer et al, 1977) . For non-nucleolar genes , the situation observed with this method appears to be somewhat different.…”

Section: Introductionmentioning

confidence: 99%

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

Scheer

1978

Cell

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152

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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.

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“…The material was fixed with gluteraldehyde, placed directly on glow-discharged, carbon-coated grids, washed, and stained with uranyl acetate as described by Muller et al (14). The grids were rotory shadowed at an 80 angle with 2 cm of Pt/Pd (80%:20%) wire wound around a tungsten filament (15). The current passing through the tungsten was sufficient to vaporize some of the tungsten.…”

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confidence: 99%

Isolation of a condensed, intracellular form of the 2-micrometer DNA plasmid of Saccharomyces cerevisiae.

Livingston

Hahne

1979

Proc. Natl. Acad. Sci. U.S.A.

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We have initiated an investigation of the proteins bound in vivo to the 2-m DNA plasmid ound in the yeast Saccharomyces cerevisiae by examining its intracellular form.To isolate 2-#m DNA without disturbing proteins bound to the plasmid, an extract was prepared from a strain lacking mitochondrial DNA and the nuc ear chromatin was removed from the extract by centrifugation. When the DNA in this extract was sedimented through a sucrose gradient containing 0.15 M NaCl, plasmid DNA (5) supplemented with glucose and the necessary nutrients. To radioactively label the DNA, the concentration of uracil in the minimal medium was decreased to 2 Ag/ml and either [6-3H]uracil was added to 1 ,gCi/ml (1 Ci = 3.7 X 1010 becquerels) or [2-14C]uracil was added to 0.5 MACi/ml. Cells were harvested in late logarithmic phase, 5-7 X 107 cells per ml for rich medium and 1-2 X 107 cells per ml for minimal medium.Preparation of Extracts. Unless otherwise noted all steps were carried out at 00C. Cells were harvested by centrifugation at 1500 X g for 5 min. The cell pellet was suspended in 1 M sorbitol/20 mM K P04 buffer, pH 7.5/20 mM EDTA and subjected to centrifugation at 12,000 X g for 5 min. The cells were suspended in buffer A [20 mM Na Hepes, pH 7.5/0.15 M NaCl/5 mM KCl/1 mM EDTA/1 mM dithiothreitol/l mM phenylmethylsulfonyl fluoride/0.2% (wt/vol) Triton X-106J containing 1 M sorbitol such that 1 ml of buffer was added for every 1 X 1010 cells. Zymolyase 60,000 (Kirin Breweries, Takasaki, Japan) was added to 2 mg/ml, and the mixture was incubated at 37°C for 1 hr at which time microscopic eximination revealed that most cells had lysed. The lysate was subjected to centrifugation at 27,000( X g for 30 min. The supernatant solution (fraction I) containing 2-Mum DNA was decanted, and up to 2 ml of this fraction was passed through a Sepharose 2B column (0.9 X 23 cm) equilibrated with buffer A. Fractions were collected, the 2-,um DNA was located by agarose gel electrophoresis in the turbid fractions appearing at the void volume, and the peak fractions were pooled. The pooled fractions (totaling 3 ml) were subjected to centrifugation at 27,000 X g for 30 min, the supernatant solution (fraction II) was saved, and the pellet was discarded. In some experiments fraction II was passed through the Sepharose 2B column a second time.Nuclear Chromatin Preparation. To recover nuclear chromatin from the extracts, the pellet remaining after centrifugation of the lysate yielding fraction I was suspended in buffer A (1 ml for every 1 X 1010 cells in the initial lysate) and subjected to centrifugation at 3000 X g for 5 min. This washing was repeated once more. The pellet was suspended in 1 ml of buffer A, layered on 15 ml of buffer A containing 2.6 M sorbitol in a 5/8 X 3 1/2 inch centrifuge tube, and sedimented for 1 hr at 27,000 rpm in a Beckman SW27 rotor. The pellet containing nuclear chromatin was suspended in buffer A and used for either micrococcal nuclease digestion or histone extraction.Sedimentation Analysis. Up to 2 ml of fraction I...

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Lengths and patterns of transcriptional units in the amplified nucleoli of oocytes of Xenopus laevis

Cited by 86 publications

References 49 publications

Multiple Pol I initiation sequences in rDNA spacers ofDrosophila melanogaster

Multiple Pol I initiation sequences in rDNA spacers ofDrosophila melanogaster

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

Isolation of a condensed, intracellular form of the 2-micrometer DNA plasmid of Saccharomyces cerevisiae.

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