Partial characterization of RNA polymerase II complex released by micrococcal nuclease digestion of rat liver nuclei

1981 ◽  
Vol 652 (2) ◽  
pp. 245-255 ◽  
Author(s):  
Takumi Hatayama ◽  
Koichiro Omori ◽  
Akira Inoue ◽  
Munehiko Yukioka
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rat Liver ◽  
Micrococcal Nuclease ◽  
Partial Characterization ◽  
Rat Liver Nuclei ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion ◽  
Liver Nuclei

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.

Ultrastructural studies on chromatin digestion by micrococcal nuclease in the presence of polyamines

1981 ◽  
Vol 48 (1) ◽  
pp. 171-179
Author(s):  
C. Rowlatt ◽  
G.J. Smith
Keyword(s):  
Electron Microscopy ◽  
Salivary Gland ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Micrococcal Nuclease ◽  
Submandibular Salivary Gland ◽  
C57bl Mouse ◽  
Ultrastructural Studies ◽  
Micrococcal Nuclease Digestion ◽  
Nuclease Digestion

Nuclei purified from C57BL mouse submandibular salivary gland were treated with a range of micrococcal nuclease concentrations and times of treatment (from 0.5 unit for 2.5 min to 50 units for 30 min) in the presence of polyamines. About 50% of the chromatin was solubilized initially but with prolonged digestion this chromatin became insoluble again. Electron microscopy showed destruction of the finely dispersed chromatin with mild digestion, followed by aggregation of chromatin with more vigorous digestion. The early disappearance of finely dispersed chromatin filaments was not accompanied by preferential solubilization of chromatin associated with RNA polymerase II (euchromatin). These data suggest that the polyamines markedly reduce the susceptibility of euchromatin to micrococcal nuclease digestion.

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.

Immunochemical characterization of protein kinase C in rat liver nuclei and subnuclear fractions

1987 ◽  
Vol 142 (2) ◽  
pp. 367-375 ◽  
Author(s):  
Silvano Capitani ◽  
Peggy R. Girard ◽  
Gonzalo J. Mazzei ◽  
J.F. Kuo ◽  
Ronald Berezney ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Rat Liver ◽  
Immunochemical Characterization ◽  
Rat Liver Nuclei ◽  
Kinase C ◽  
Liver Nuclei

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.

Sign in / Sign up

Export Citation Format

Share Document