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PMID: 4348169 Published · ppublish English Journal Article

Fractionation of nuclei from brain by zonal centrifugation and a study of the ribonucleic acid polymerase activity in the various classes of nuclei.

The Biochemical journal ·Vol. 129 ·No. 5 ·1972-10-00 ·Pages 1139-55

Austoker J, Cox D, Mathias AP

Abstract

1. The nuclei of the cells of the whole rat brain have been fractionated in a B-XIV zonal rotor with a discontinuous gradient of sucrose. Five fractions were obtained. Zone (I) contained neuronal nuclei (70%) and astrocytic nuclei (23%). Zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (15%). Zone (III) contained astrocytic nuclei (84%) and oligodendrocytic nuclei (15%). Zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained only oligodendrocytic nuclei. 2. The content of DNA, RNA and protein per nucleus was determined for each zone. Although the amount of DNA per nucleus is constant (7pg) the RNA varies from 4.5 to 2.5pg/nucleus and the protein from 38 to 17.6pg/nucleus. The neuronal nuclei have the greatest amounts of protein. The oligodendrocytic nuclei have the least content of RNA and protein. 3. The effects of pH, ionic strength, and Mg(2+) and Mn(2+) concentration on the activity of the nuclear system for synthesis in vitro of RNA have been investigated for unfractionated nuclei. From these studies a standard set of conditions for the assay of nuclear RNA polymerase has been established. 4. The activity of the RNA polymerase in each of the zonal fractions has been determined in the presence and in the absence of alpha-amanitin. Zone (II) is the most active, followed by zone (I). The nuclei of zones (IV) and (V) have comparable activity, which is 40% of that of zone (II). 5. The extent of incorporation of each of the four labelled nucleoside triphosphates by the nuclei from each zone has been measured. These values have been used to calculate the base composition of the RNA synthesized in vitro in each class of nucleus. 6. The effect of changes in the condition of assay of RNA polymerase in the different classes of nuclei has been investigated. Significant differences in the response to concentrations of metal ions and ammonium sulphate have been observed. 7. Homopolymer formation in each zone of brain nuclei has been determined. The extent of formation of the four homopolymers roughly parallels the RNA polymerase activity.

MeSH Terms
Animals Brain/cytology,enzymology Cell Fractionation Cell Nucleus/enzymology Centrifugation, Zonal DNA/analysis DNA-Directed RNA Polymerases/metabolism Glucose-6-Phosphatase Hydrogen-Ion Concentration Magnesium/pharmacology Male Manganese/pharmacology Microscopy, Electron Microscopy, Phase-Contrast Mycotoxins/pharmacology Neuroglia/enzymology Proteins/analysis RNA/analysis Rats Ribonucleases Tritium
Chemicals
Mycotoxins Proteins Tritium Manganese RNA DNA DNA-Directed RNA Polymerases Ribonucleases Glucose-6-Phosphatase Magnesium
Authors & Affiliations
3 authors, click to expand affiliations / ORCID
Austoker J
Cox D
Mathias A P
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44 references, click to expand
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Article Info
Journal
The Biochemical journal
Abbr.
Biochem J
ISSN
0264-6021
DOI
10.1042/bj1291139
Published
1972-10-00
Pages
1139-55
Language
English
Region
England
NLM ID
2984726R
PMCID
PMC1174273
Subset
IM
External Links
PubMed source DOI: 10.1042/bj1291139 PMC Full Text View journal details
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