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.

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 CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites

2020 ◽  
Vol 28 (3-4) ◽  
pp. 381-393
Author(s):  
Michi Miura ◽  
Honglin Chen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Biological Significance ◽  
Micrococcal Nuclease ◽  
Dna Fragments ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Human Lung Carcinoma Cell

AbstractCUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (> 270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (< 120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.

scholarly journals Effects of glucocorticoid and cycloheximide on the activity and amount of RNA polymerase I in nuclei of rat liver

1986 ◽  
Vol 235 (3) ◽  
pp. 699-705 ◽  
Author(s):  
H Matsui ◽  
H Yazawa ◽  
N Suzuki ◽  
T Hosoya
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rat Liver ◽  
Protein S ◽  
Rna Polymerase I ◽  
Free Form ◽  
Precursor Synthesis ◽  
Liver Nuclei ◽  
Polymerase I ◽  
Adrenalectomized Rats

The activity of the template-engaged form of RNA polymerase I from livers of adrenalectomized rats was about 50-60% of that of normal control rats, and increased about 2-fold at 6 h after the administration of dexamethasone. However, no change was found in the activity of the ‘free’ form of RNA polymerase I or the template-engaged form of RNA polymerase II. Immunochemical studies using guinea-pig anti-(RNA polymerase I) serum disclosed that the total number of RNA polymerase I molecules did not vary during the treatment with dexamethasone. Cycloheximide caused a rapid decrease in the template-engaged form of RNA polymerase I activity in normal rats and in dexamethasone-treated (6 h) adrenalectomized rats, to the value in adrenalectomized rats, but affected it only slightly in adrenalectomized rats. The elongation rate of rRNA-precursor synthesis in liver nuclei was not affected by a change in the concentration of circulating dexamethasone. From these results, it is concluded that about half the rRNA-precursor synthesis in rat liver is regulated by glucocorticoids, probably through the synthesis of short-lived protein(s) which may play a role in conversion of the ‘dormant’ form of RNA polymerase I into the ‘engaged’ form.

scholarly journals CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites

2020 ◽  
Author(s):  
Michi Miura ◽  
Honglin Chen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Immunoprecipitation ◽  
Biological Significance ◽  
Micrococcal Nuclease ◽  
Dna Fragments ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Human Lung Carcinoma Cell

AbstractCUT&RUN is a powerful tool to study protein-DNA interactions in vivo. DNA fragments cleaved by the targeted micrococcal nuclease identify the footprints of DNA-binding proteins on the chromatin. We performed CUT&RUN on human lung carcinoma cell line A549 maintained in a multi-well cell culture plate to profile RNA polymerase II. Long (>270 bp) DNA fragments released by CUT&RUN corresponded to the bimodal peak around the transcription start sites, as previously seen with chromatin immunoprecipitation. However, we found that short (<120 bp) fragments identify a well-defined peak localised at the transcription start sites. This distinct DNA footprint of short fragments, which constituted only about 5% of the total reads, suggests the transient positioning of RNA polymerase II before promoter-proximal pausing, which has not been detected in the physiological settings by standard chromatin immunoprecipitation. We showed that the positioning of the large-size-class DNA footprints around the short-fragment peak was associated with the directionality of transcription, demonstrating the biological significance of distinct CUT&RUN footprints of RNA polymerase II.

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