Transcription of isolated mouse liver chromatin

Bacheler, Lee T.; Smith, Kirby D.

doi:10.1021/bi00660a018

Cited by 21 publications

(3 citation statements)

References 36 publications

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“…To obtain the chromatin fraction, MgCl, was added to the sonicate at a final concentration of I m M [4] and the precipitate was collected by centrifugation at 10000 x g for I0 min. The chromatin fraction thus obtained showed the same composition and proteolytic activity as that obtained according to Bacheler and Smith [5] as reported previously [6].…”

Section: Chromatin Preparationsupporting

confidence: 85%

Purification and properties of a thiol protease from rat liver nuclei

Motizuki

Tsurugi

Ogata

1984

European Journal of Biochemistry

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A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29 000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30 -40 % of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2-4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.When the synthesis of rRNA in regenerating rat liver was selectively inhibited by the administration of a low dose of actinomycin D in vivo, the synthesis of ribosomal proteins remained almost normal 1 h after the actinomycin D treatment. These newly synthesized and unbound ribosomal proteins were found to be degraded rapidly with a half-life of 20 -40 min [I]. Furthermore, we found that the ribosomal proteins and histones, synthesized in actinomycin-D-treated rat liver under the conditions as described above, accumulated in liver nuclei after injection of E-64, a thiol protease inhibitor [2]. From the results we have concluded that these two kinds of proteins, which are not associated with the nascent rRNA or DNA, are degraded post-translationally by thiol protease(s) in the nuclei of regenerating rat liver. It must be added that the degradation of newly synthesized and unassociated ribosomal proteins and especially that of histones may occur in the regenerating liver even without actinomycin D treatment [2]. More recently we demonstrated the presence of an acidic protease in the pure nuclei of regenerating rat liver. It was extracted with 0.7 M NaCl from the chromatin fraction and partially purified with Sepharose 6B. The acidic protease was endopeptidase of a thiol type [3]. During the course of these experiments the presence of the same kind of acidic protease was shown in normal rat liver. Partially purified enzyme from normal rat liver was also sensitive to -SH reagents and had almost the same molecular weight as the enzyme from regenerating rat liver. In the present report the purification of the thiol protease to near homogeneity from normal rat liver chromatin, and some of its characteristics are described. MATERIALS AND METHODS MaterialsWistar strain rats weighing 200 -300 g were used. Iodo-[l-14C]acetamide (23 Ci/mol) was obtained from New England Nuclear Co. Sepharose 6B, Sephadex G-100, PBE94 and Chromatin preparationThe chromatin fraction was prepared from rat liver nuclei as described previously [3]. Briefly, rat liver homogenate in medium A (0.25 M sucrose, 5 mM MgCl,, 25 mM KCl and 50 mM Tris/HCl buffer, pH 7.6) was centrifuged at 10000 x g for 10 min and the precipitate was suspended in 10 volumes of 2.3 M sucrose containing 5 m M MgC...

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Section: Chromatin Preparationsupporting

confidence: 85%

Purification and properties of a thiol protease from rat liver nuclei

Motizuki

Tsurugi

Ogata

1984

European Journal of Biochemistry

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“…Total cellular RNA was isolated [7] and separated into poly(A) + and poly(A)-fractions by oligo(dT)-cellulose chromatography [8]. Total nuclear RNA was prepared from nuclei [9] by hot phenol extraction [10] followed by DNase digestion [ 11 ]. Hybridizations in 24/ll mixtures containing a constant amount of cDNA (500 cpm) and varying amounts of RNA were assayed by $1 nuclease digestion as in [6].…”

Section: Methodsmentioning

confidence: 99%

Ratios of α‐ to β‐globin RNA sequences in the erythropoietic mouse spleen

et al. 1979

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“…Such studies as have been carried out on these aspects of chromatin transcription have yielded valuable insights into the fidelity of the process in vitro (e.g. Bacheler & Smith, 1976;Coupar & Chesterton, 1977).…”

mentioning

confidence: 99%

The use of rat liver nucleoplasm for the characterization of heterogeneous nuclear ribonucleic acid synthesis in vitro

Beebee¹

1978

Biochemical Journal

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1. A nucleoplasmic fraction rich in endogenous RNA polymerase II activity was isolated from rat liver nuclei and conditions were determined under which elongation of RNA molecules initiated in vivo continued at maximal rates in vitro. 2. Elongation rates in vitro were calculated to be about 0.25 nucleotide/s and there were about 7 X 10(3) RNA molecules in the process of being elongated by form-II RNA polymerase per original nucleus. 3. Evidence was obtained suggesting that transcription-dependent release of RNA polymerase II molecules from the template occurred during the incubations in vitro. 4. The nascent RNA was tightly associated with protein and banded as ribonucleoprotein in caesium salt gradients. 5. RNA molecules labelled in vitro were up to 13000 nucleotides in length, but consisted of long unlabelled chains transcribed in vivo with only short labelled sequences added in vitro, and without significant polyadenylation. 6. Hybridization of transcripts in the presence of a vast excess of DNA demonstrated that both form-II RNA polymerase and another enzyme, resistant to low alpha-amanitin concentrations, were synthesizing RNA molecules complementary to both reiterated and unique DNA sequences in the genome.

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Transcription of isolated mouse liver chromatin

Cited by 21 publications

References 36 publications

Purification and properties of a thiol protease from rat liver nuclei

Purification and properties of a thiol protease from rat liver nuclei

Ratios of α‐ to β‐globin RNA sequences in the erythropoietic mouse spleen

The use of rat liver nucleoplasm for the characterization of heterogeneous nuclear ribonucleic acid synthesis in vitro

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