Localization of highly repeated <i>Hin</i>dIII fragments of novikoff rat DNA to the nucleolus

Fuke, Motohiro; Davis, Frances M.; Busch, Harris

doi:10.1016/0014-5793(79)80925-4

Cited by 9 publications

(5 citation statements)

References 7 publications

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“…The 141 GC pairs found in the 370 bp repeat unit correspond to a GC content of 38.1 %. We have no explanation for the disagreement with a previous estimate (12,13).…”

Section: Resultscontrasting

confidence: 95%

“…A similar value was obtained on the basis of fluorescence intensities of fragments in agarose gels stained with ethidium bromide. Our value is comparable to the one of 2.9 % found for the HindIII monomer (13). Satellite DNA I was purified on a preparative scale by gel filtration of a Sau3AI digest on Sepharose 2B columns ( Fig.…”

Section: Resultssupporting

confidence: 84%

“…These results were recently confirmed by Fuke and Busch (12) and extended to include the HaeIII and HinfI sites. Moreover in fractionation experiments these authors found most of the DNA component to occur in the nucleolar fraction (13).…”

Section: Introductionmentioning

confidence: 98%

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Nucleotide sequence of a highly repetitive component of rat DNA

1979

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A highly repetitive component of rat DNA which could not yet be enriched by density gradient centrifugation was isolated with the help of the restriction nuclease Sau3AI. This nuclease converted the bulk of the DNA to small fragments and left a repetitive DNA component as large fragments which were subsequently purified by gel filtration and electrophoresis. This DNA component which was termed rat satellite DNA I is composed of tandemly repeated 370 bp blocks. According to sequence analysis the 370 bp repeats consist of alternating 92 and 93 bp units with homologous but not identical sequences. Methylation of CpG residues was correlated to the rate of cleavage by restriction nucleases. Significant homologies exist between the sequences of rat satellite DNA I and satellite DNAs of several other organisms. The divergence of the sequence of rat satellite DNA I was discussed with respect to evolutionary considerations.

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“…The 141 GC pairs found in the 370 bp repeat unit correspond to a GC content of 38.1 %. We have no explanation for the disagreement with a previous estimate (12,13).…”

Section: Resultscontrasting

confidence: 95%

Section: Resultssupporting

confidence: 84%

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Nucleotide sequence of a highly repetitive component of rat DNA

1979

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“…First, medium was removed and the cells were washed three times with phosphate-buffered saline, pH 7.4. The cells were suspended in homogenizing buffer (buffer A: 10 mM Trischloride [pH 7.4], 150 mM NaCI, 10 mM MgCl2, 0.35% Triton X-100) at a density of 107 cells per ml and were then briefly homogenized, using a Dounce homogenizer. Sodium * Corresponding author.…”

Section: Methodsmentioning

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).…”

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confidence: 99%

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

Bolla¹,

Braaten²,

Shiomi³

et al. 1985

Mol. Cell. Biol.

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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...

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