Heterogeneity of chromatin: Fractionation of sonicated rat liver chromatin by partial precipitation with Mg2+

Arnold, Eugene A.; Young, Keith E.

doi:10.1016/0003-9861(74)90009-5

Cited by 40 publications

(7 citation statements)

References 43 publications

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“…Preparation of Chromatin: Rat liver chromatin was prepared from adult Sprague-Dawley rats by extracting purified liver nuclei with saline-EDTA as described previously (9). The chromatin was purified by washing in buffers of low ionic strength and sonicated briefly as described (9). Isolation of Accessible DNA: Accessible DNA was isolated from chromatin by utilizing poly-D-lysine binding as described previously (14).…”

Section: Methodsmentioning

confidence: 99%

Transcription of the nonrepeated fraction of "accessible" DNA in rat liver chromatin.

Arnold

Young

1976

Nucleic Acids Research

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The relationship between structure and function in eukaryotic chromatin has been studied in rat liver cells. To elucidate the functional significance of "accessible" DNA, the transcription of this DNA (prepared by titration of liver chromatin with poly-D-lysine) has been examined by RNA-DNA hybridization. The maximum extent to which nuclear RNA will hybridize to the nonrepeated fraction of "accessible" DNA has been measured and compared to the extent that whole chromatin DNA will hybridize. The results show that "accessible" DNA has the same number of sequences complementary to nuclear RNA as does total DNA. In addition DNA-DNA reassociation experiments indicate that there is only a small difference between the total unique sequence populations of "accessible" and total DNA. These results indicate that nonrepeated "accessible" DNA is not preferentially transcribed in the cell as is predicted by some models of chromatin structure.

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Section: Methodsmentioning

confidence: 99%

Transcription of the nonrepeated fraction of "accessible" DNA in rat liver chromatin.

Arnold

Young

1976

Nucleic Acids Research

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Section: Methodsmentioning

confidence: 99%

“…Preparation of Chromatin and Polylysine Complexes: Rat liver chromatin was prepared from adult male Sprague-Dawley or Buffalo inbred rats by extracting purified liver nuclei with saline-EDTA as described previously (9). The chromatin was purified by washing in buffers of low ionic strength (9). Chromatin-polylysine complexes were prepared by the slow addition of stock polylysine solutions to dilute and rapidly stirred solutions of chromatin until the chromatin was completely precipitated from the solution.…”

Section: Methodsmentioning

confidence: 99%

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Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine

1975

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The structure of eukaryotic chromatin has been investigated by isolating and analyzing the "accessible" DNA fraction of rat liver chromatin. This DNA fraction has been isolated by titrating the chromatin with the protese-resistant D isomer of polylysine to bind the "accessible" DNA sites. After removal of chromosomal proteins by digestion with pronase, all DNA not protected from attack by bound polylysine was removed by digestion with DNase. Even after exhaustive treatment with pronase and DNase approximately 30% of the chromatin DNA remains resistant to nuclease attack. Analysis of the isolated DNA shows it to be mainly double-stranded with an average size of 200-250 base pairs. The DNA is slightly A-T rich and contains both repetitive and "single-copy" nuleotide sequences. The results suggest that there are extensive regions in chromatin where the DNA is not tightly complexed with protein. Furthermore, the DNA of these regions is similar in gross properties to the DNA of the total genome.

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“…Various chromatin properties have been utilized for fractionation. These include differences in sedimentation behavior (2,4,5,19), melting behavior (17), solubility in saline (15), low concentration of magnesium (1,10) and adsorption on an ECTHAMcellulose column (20). Ribonucleoprotein particles (RNP) containing precursors of rRNA accumulate in the nucleoli, so it was necessary to extract RNP before fractionation of the nucleolar chromatin.…”

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Ribosomal Genes of Fractionated Chromatin from Nucleoli of Ehrlich Ascites Tumor Cells

Higashi

Hanasaki

Shikichi

et al. 1975

Cell Struct. Funct.

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Chromatin from isolated Ehrlich ascites tumor cell nucleoli was fractionated on a sucrose gradient to examine the distribution of ribosomal genes. The isolated nucleoli were treated with polyvinylsulphate, and then the ribonucleoprotein particles were extracted with 20 mM dithiothreitol in buffer solution. The chromatin was then solubilized by sonication in 10 mM Tris-HCI buffer (pH 8.0). Centrifugation of solubilized chromatin fraction on a sucrose gradient gave two peaks of chromatin containing approximately equal amounts of DNA, regardless of whether the sonication period was 20 sec or 40 sec. Peak I (which sedimented slower) had less RNA than peak II. Ribonucleoprotein particles appeared to merely cosediment with peak II because the profiles of DNA component differed from RNA. Therefore, the presence of ribonucleoprotein particles was probably not the primary cause of the rapid sedimentation of peak II. Examination of the distribution of ribosomal genes in the fractionated nucleolar chromatin by hybridization of DNA with "Plabeled ribosomal RNA showed that peak I contained three times more ribosomal genes than peak II. Fractionation of DNA in the chromatin fractions by CsC1 equilibrium density gradient centrifugation showed that the profiles of DNA complimentary to ribosomal RNA in the two fractions were essentially similar, but that peak I contained more ribosomal genes than peak II.

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Heterogeneity of chromatin: Fractionation of sonicated rat liver chromatin by partial precipitation with Mg2+

Cited by 40 publications

References 43 publications

Transcription of the nonrepeated fraction of "accessible" DNA in rat liver chromatin.

Transcription of the nonrepeated fraction of "accessible" DNA in rat liver chromatin.

Isolation and characterization of the DNA fraction of rat liver chromatin which binds polylysine

Ribosomal Genes of Fractionated Chromatin from Nucleoli of Ehrlich Ascites Tumor Cells

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