scholarly journals Transcription of fractionated mammalian chromatin by mammalian ribonucleic acid polymerase. Demonstration of temperature-dependent rifampicin-resistant initiation sites in euchromatin deoxyribonucleic acid

1974 ◽  
Vol 143 (1) ◽  
pp. 73-81 ◽  
Author(s):  
C. James Chesterton ◽  
Barbara E. H. Coupar ◽  
Peter H. W. Butterworth
Keyword(s):  
Rna Polymerase ◽  
Rat Liver ◽  
Deoxyribonucleic Acid ◽  
Temperature Dependent ◽  
Fractionation Method ◽  
Liver Nuclei ◽  
Nascent Rna ◽  
Chromatin Fractionation

The chromatin fractionation method of Frenster et al. (1963) as modified by Leake et al. (1972) was used to prepare fragments of euchromatin from rat liver nuclei. These remain soluble in 5mm-MgCl2, and contain DNA of maximum mol.wt. 1×106–2×106. The fragments were separated from condensable chromatin on a sucrose gradient. Euchromatin contains endogenous DNA-dependent RNA polymerase, and most of the nascent RNA labelled in vivo or in vitro. Euchromatin fragments allow initiation of transcription by added purified rat liver form-B RNA polymerase and contain temperature-dependent rifampicin-resistant initiation sites for the form-B enzyme. These findings indicate that transcription of the euchromatin regions of interphase chromosomes is not initiated in condensed chromatin, but is initiated within the euchromatin stretches. Condensable chromatin also contains most of these activities, but is not associated with nascent RNA.

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

1978 ◽  
Vol 176 (3) ◽  
pp. 715-725 ◽  
Author(s):  
T J C Beebee
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rat Liver ◽  
Dna Sequences ◽  
Acid Synthesis ◽  
Rna Molecules ◽  
Liver Nuclei

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 × 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.

scholarly journals Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

1967 ◽  
Vol 105 (2) ◽  
pp. 779-782 ◽  
Author(s):  
F. Stirpe ◽  
L. Fiume
Keyword(s):  
Rna Polymerase ◽  
Ammonium Sulphate ◽  
Ribonucleic Acid ◽  
Mouse Liver ◽  
Orotic Acid ◽  
Acid Synthesis ◽  
Liver Necrosis ◽  
Liver Nuclei

1. Injection of α-amanitin to mice causes a decreased incorporation of [6−14C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn2+ and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with α-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg2+-activated RNA polymerase is only slightly affected by α-amanitin either administered to mice or added in vitro.

scholarly journals Phosphotriesters in rat liver deoxyribonucleic acid after the administration of the carcinogen NN-dimethylnitrosamine in vivo

1975 ◽  
Vol 145 (3) ◽  
pp. 475-482 ◽  
Author(s):  
P J O'Connor ◽  
G P Marigison ◽  
A W Craig
Keyword(s):  
Rat Liver ◽  
Deoxyribonucleic Acid ◽  
Methylation Pattern ◽  
Rat Livers

After treatment with NN-di[14C]methylnitrosamine, samples of DNA were isolated from rat livers by a conventional phenol procedure and examined for the presence of phosphotriesters. A method of capable of detecting relatively small amounts of 14C-labelled phosphotriesters was developed and used to establish that these products account for 10-12% of the total methylation pattern found after treatment with this agent in vitro. The significance of the presence of phosphotriesters in DNA is discussed.

scholarly journals Inhibition by α-amanitin of ribonucleic acid polymerase solubilized from rat liver nuclei

1970 ◽  
Vol 116 (2) ◽  
pp. 177-180 ◽  
Author(s):  
F. Novello ◽  
L. Fiume ◽  
F. Stirpe
Keyword(s):  
Rna Polymerase ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Ribonucleic Acid ◽  
Isolated Rat Liver ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Isolated Rat

1. α-Amanitin inhibits in vitro the RNA polymerase solubilized from isolated rat liver nuclei. 2. In contrast with previous observations with whole nuclei, the inhibition occurs approximately to the same extent in the presence and in the absence of ammonium sulphate. 3. Evidence is presented that the toxin acts by interacting with the enzyme itself and not with DNA or other components.

scholarly journals Subnuclear fractionation by mild micrococcal-nuclease treatment of nuclei of different transcriptional activities causes a partition of expressed and non-expressed genes

1980 ◽  
Vol 187 (2) ◽  
pp. 467-467 ◽  
Author(s):  
G J Dimitriadis ◽  
J R Tata
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rat Liver ◽  
Nuclear Dna ◽  
Globin Gene ◽  
Micrococcal Nuclease ◽  
Deoxyribonuclease I ◽  
Liver Nuclei ◽  
Albumin Mrna

Extremely mild treatment with micrococcal nuclease of isolated nuclei yields subnuclear fractions in which the majority of RNA polymerase II transcriptional complexes formed in vivo are segregated [Tata & Baker (1978) J. Mol. Biol. 118, 249-272]. We now describe different approaches followed to established whether or not the nuclei are thus resolved into transcribed and non-transcribed DNA. First, we have compared the sensitivity to deoxyribonuclease I, which is known to digest preferably expressed genes as present in nuclei or chromatin, of three micrococcal-nuclease-derived fractions from nuclei of different transcriptional activities. In transcriptionally active nuclei (rat liver, hen liver and oviduct, and Xenopus liver), the DNA in a polynucleosomal fraction comprising 6-15% of DNA and the majority of template-engaged RNA polymerase II (fraction P2) was 10-50 times as sensitive to deoxyribonuclease I as the DNA in the other two fractions (fractions P1 and S, comprising 78-88% of total nuclear DNA as large polynucleosomal aggregates and 2-6% of DNA mostly as mononucleosomes, respectively). In transcriptionally inactive nuclei obtained from hen erythrocytes, micrococcal nuclease did not separate DNA into fractions exhibiting such differential sensitivities. Second, we have monitored the partition of an expressed gene. Hybridization of complementary DNA to Xenopus albumin mRNA revealed a 5-10-fold enrichment of the albumin (but not the globin) gene in the P2 fraction of nuclei from Xenopus liver in which this gene is fully expressed. Third, a large part of the nascent rapidly labelled RNA synthesized in vivo in rat liver nuclei was recovered in the micrococcal-nuclease-derived fraction that is more susceptible to digestion with deoxyribonuclease I. It is concluded that mild micrococcal-nuclease treatment of nuclei causes their separation into transcribed and non-transcribed DNA as determined by a number of very different criteria.

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