Ultrastructural and biochemical studies on the isolation of nucleolar chromatin from Novikoff hepatoma cell nucleoli

Daskal, Yerach; Ballal, N.R.; Busch, Harris

doi:10.1016/0014-4827(78)90245-8

Cited by 9 publications

(7 citation statements)

References 16 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…DNA was isolated from nuclei and from nucleolar subfractions, nick translated, and hybridized to a 100-fold excess of mouse L-cell total cytoplasmic RNA (18 Under the hybridization conditions used (16), mature rRNA and any other relatively frequent species of mouse L-cell cytoplasmic RNA would be expected to hybridize. Compared with total nuclear DNA, there was an enrichment for transcribed sequences in nucleoli prepared by conventional procedures (4,6). The proportion of transcribed sequences in these nucleoli was 12.0 ± 2.1%, whereas DNA from core nucleoli contained no comparable proportion of transcribed sequences (<0.15%; Fig.…”

Section: Resultsmentioning

confidence: 94%

“…L-cell nucleoli prepared by traditional procedures (see Materials and Methods) retained their characteristic in vivo morphology (4,6).…”

Section: Resultsmentioning

confidence: 99%

“…When we have digested non-nucleolar and core nucleolar chromatin from the same cells with micrococcal nuclease, only the non-nucleolar fractions give standard nucleosomal patterns (as in references 6 and 11, for example). In contrast, the extracted core nucleolar DNA shows bands of fragment size 20 to 200 bp differing by about 25 bp between bands (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The extensive studies of Busch and co-workers have characterized DNA fragments probably unique to nucleoli (10), preribosomes, and the nucleolar-specific U3 small nuclear RNA (snRNA) (8). However, nucleoli prepared by conventional methods (4,6,23) also include up to 50% of the nuclear DNA and its associated proteins (4,6,23). Since only about 0.1 to 0.3% of the nuclear DNA is rDNA, these preparations contain a great deal of other chromatin and nuclear material.…”

mentioning

confidence: 99%

See 3 more Smart Citations

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Bolla¹,

Braaten²,

Shiomi³

et al. 1985

Mol. Cell. Biol.

View full text Add to dashboard Cite

Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse sateilite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.Because nucleoli show a structural arrangement which can be cytologically correlated with events in ribosome formation (9), their molecular organization is of great interest. The extensive studies of Busch and co-workers have characterized DNA fragments probably unique to nucleoli (10), preribosomes, and the nucleolar-specific U3 small nuclear RNA (snRNA) (8). However, nucleoli prepared by conventional methods (4, 6, 23) also include up to 50% of the nuclear DNA and its associated proteins (4, 6, 23). Since only about 0.1 to 0.3% of the nuclear DNA is rDNA, these preparations contain a great deal of other chromatin and nuclear material. Thus nucleolar-specific DNA, RNA, or protein species are seen against a very high background.We have used extensive treatment of nucleoli with DNase I or micrococcal nuclease to try to remove perinuclear or adventitiously associated chromatin and nucleoplasm selectively. We will report here that the resulting "core" fraction retains the preribosomes and snRNAs characteristic of nucleoli but now contains only a small fraction of the cellular DNA in a form highly protected against nuclease action. The DNA shows a characteristic size distribution and a restricted sequence content, including only spacer sequences of rDNA. Nuclei were collected by centrifugation through 0.5 M sucrose in buffer B (10 mM Tris-chloride [pH 7.4], 10 mM MgC12, 150 mM NaCl) at 800 x g for 10 min. They were then resuspended in buffer B (107/ml) and disrupted by sonication (50 W, 4 x 30 s, with a Branson Sonifier). A 20-,ug portion of DNase I per 107 nuclei (55 U/107 nuclei per ml) was added, and the extract was incubated at 37°C as indicated in the text. Nucleoli were isolated by centrifugation through a 10-ml sucrose step gradient containing 5 ml each of 0.25 and 0.88 M sucrose at 800 x g for 15 min and suspended in 1 mM Tris-chloride (pH 8.0)-i mM EDTA on ice for 15 min with occasional agitatio...

show abstract

Section: Resultsmentioning

confidence: 94%

“…L-cell nucleoli prepared by traditional procedures (see Materials and Methods) retained their characteristic in vivo morphology (4,6).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Bolla¹,

Braaten²,

Shiomi³

et al. 1985

Mol. Cell. Biol.

View full text Add to dashboard Cite

show abstract

“…There are several hundred proteins in the nucleolus which include the proteins of the preribosomal RNP particles and those involved in processing and transport of pre-rRNA (6,7). Most of these proteins were removed when nucleoli were extracted with low ionic strength solutions to isolate chromatin (8,9). Although the isolated chromatin was only about 20% as active as intact nucleoli in its ability to synthesize RNA (8), the transcripts were essentially the same as those of intact nucleoli and closely resemble pre-rRNA by the homochromatography fingerprinting technique (2).…”

Section: Introductionmentioning

confidence: 99%

Studies on the specificity of preribosomal RNA transcription in nucleoli after selective deproteinization

et al. 1979

Self Cite

View full text Add to dashboard Cite

Fidelity of preribosomal RNA transcription in vitro was studied after selective deproteinization of nucleoli using either sequential salt extraction or sodium deoxycholate treatment. Homochromatography fingerprinting and identification of marker oligonucleotides from a T1 ribonuclease digest of the transcripts were used to evaluate the RNA products. These studies indicated that: (1) nucleoli retained their endogenous RNA polymerase I activity and the specificity of transcription up to 0.6 M NaCl extraction; (2) exogenous RNA polymerase I transcribed nucleolar chromatin only after 1.0 M NaCl extraction and the transcription pattern, like that of totally deproteinized DNA, was completely random; (3) extraction of nucleoli with deoxycholate resulted in a DNP complex in which the endogenous RNA polymerase I transcribed pre-rRNA specifically; however, it also initiated random transcription, producing a "mixed" fingerprint pattern on the homochromatogram. The random transcription was selectively inhibited either by deoxycholate or rifampicin AF/013. These studies indicate that the selectivity of pre-rRNA transcription is due both to the endogenous RNA polymerase I molecules that were involved in transcription in vivo and are tightly bound to the template and to factors in intact nucleoli which prevent random transcription by the released RNA polymerase I molecules.

show abstract

Scanning electron microscopy of amphibian lampbrush chromosomes

Angelier¹,

Paintrand²,

Lavaud³

et al. 1984

Chromosoma

View full text Add to dashboard Cite

Ultrastructural and biochemical studies on the isolation of nucleolar chromatin from Novikoff hepatoma cell nucleoli

Cited by 9 publications

References 16 publications

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Localization of specific rDNA spacer sequences to the mouse L-cell nucleolar matrix.

Studies on the specificity of preribosomal RNA transcription in nucleoli after selective deproteinization

Scanning electron microscopy of amphibian lampbrush chromosomes

Contact Info

Product

Resources

About