1. Non-histone chromosomal proteins were isolated from the cell nuclei of whole rat brain and nuclei from different types of brain cells. 2. Brain nuclei were fractionated by zonal centrifugation into five zones deriving from five main categories of brain cells. These are the neuronals, astrocytes I, astrocytes II, oligodendrocytes I and oligodendrocytes II. 3. The non-histone chromosomal proteins were analysed by (a) sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, (b) electrofocusing electrophoresis and (c) two-dimensional electrophoresis. The results of this analysis showed a limited specific pattern of non-histone chromosomal proteins from the different classes of nuclei. Differences were found to exist between the proteins from neuronal and glial nuclei. In particular one polypeptide band with mol.wt. 10000 and pI8.5 was found to be present in the non-histone protein fractions of neuronal nuclei, and absent from the corresponding fractions of nearly all the other classes of nuclei. 4. Two other classes of nuclear proteins, buffered-saline-soluble and 0.35m-NaCl-soluble, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis along with the non-histone chromosomal. The similarities and differences among these groups of proteins are discussed. 5. The patterns of non-histone chromosomal proteins during development were investigated by studying them in two age groups of animals: in infant rats (10 days old) and adult rats. The polypeptide that was found to be specific for the proteins of neuronal nuclei of adult rats is present in all the classes of nuclei of infant rats.

The effect of convulsions, induced by flurothyl, on RNA synthesis in purified unfractionated nuclei and the cytoplasm of rat cerebral cortex was studied by using a double-label technique involving injection of [3H]- and [14C]-orotate intracisternally. 2. Intact RNA was extracted in 80% yield by an enzymic method by using a proteinase in the presence of sodium dodecyl sulphate followed by deoxyribonuclease. Electrophoresis on 1.5% polyacrylamide-0.5% agarose gels revealed the presence of giant nuclear RNA of size up to approx. 300X 10(6) daltons and mRNA of maximal mol.wt. 9 X 10(6)-16 X 10(6). 3. Nuclear RNA synthesis was decreased to 27% in the first 15 min after convulsions but rapidly increased, so that at 1 1/2 h it was 124% of the control, and at 6 h 147%. 4. Labelling of cytoplasmic RNA was decreased to 15% at 15 min after convulsions but had not recovered to control values by 6 h. 5. Analysis of radioactive gel patterns and the 3H/14C ratio at six time-points (15 min-6h) showed that the major effect was inhibition of the processing of heterogeneous nuclear RNA resulting in a sharp decline in the export of newly synthesized RNA from the nucleus. 6. Cytoplasmic RNA patterns indicated that specific messengers were synthesized at different times during the recovery of the cell after convulsions.

—The changes in the wet weight and the numbers of cell nuclei recovered from the cerebral hemispheres, the cerebellum and the brain stem of rats from the period of 5–30 days after birth have been determined. In parallel a study has been made of the RNA polymerase activity, both in the unfractionated nuclei from these regions and in the nuclei separated by zonal centrifugation. In general there is a considerable decline in activity during this period, which occurs in all class of nuclei although not to the same extent. The most dense nuclei from the cerebellum retain relatively high activity at 20 days after birth, possibly due to the contribution of the microneuronal nuclei.

1. The influence of exogenous or activated DNA template on the DNA polymerase activity in the different types of intact nuclei from rat brain tissue was determined. The different amounts or physical state of the DNA template did not produce significant differences in the relative distribution of the DNA polymerase activity between the separate groups of nuclei. 2. The DNA polymerase activities, fractionated by sucrose gradient centrifugation into enzyme A and enzyme B, were found to be present in the extracts of all types of rat brain nuclei. The distribution of these two activities in the ;particulate' and ;soluble' fractions of the separate groups of nuclei from 10-day-old and adult rats was studied. The findings are related to the DNA-synthetic activity in vivo of the intact nuclei and the possible biological functions of the DNA polymerase activities are discussed.

1. Radioactive orotic acid, uridine and adenosine were administered to rats by intracisternal injection. The effects of the size of the dose, the specific radioactivity and time on the incorporation into the RNA of unfractionated nuclei of brain tissue were examined to establish appropriate conditions for studies of the relative activities in vivo of the various sorts of brain nuclei fractionated by zonal centrifugation. Uridine is incorporated more efficiently than either orotic acid or adenosine. 2. With [3H]uridine as precursor the astrocytes in zone (II) contain the highest radioactivity except at the beginning of the experiment when the neuronal nuclei of zone (I) are more highly labelled. This fraction utilizes [14C]orotic acid more readily than the nuclei of zone (II). The astrocytic nuclei of zone (III) show a general resemblance to those of zone (II). Considerable differences between the incorporations into the two types of oligodendrocyte nuclei in zones (IV) and (V) are observed. 3. The relative synthetic activities of the major types of brain nuclei in vitro and in vivo are discussed.

1. The effects of the inclusion of thioacetamide in the diet on the properties of rat liver nuclei were studied both in adolescent rats, in which the parenchymal cells contain diploid nuclei, and in young adult rats, with a high proportion of tetraploid nuclei. 2. These investigations included a survey of the sedimentation properties of the nuclei, the nuclear volumes, content of DNA, RNA and protein, the incorporation in vivo of [(3)H]thymidine into DNA and [(14)C]orotate into RNA, and measurements of the activity of RNA polymerase and ribonuclease. These studies were conducted on nuclei fractionated by zonal centrifugation. 3. In both groups of animals, exposure to thioacetamide produced large numbers of nuclei that were abnormal in their chemical composition and enzymic activity. The changes were complex as regards both the types of nuclei that were affected and in their variation with time. 4. In adolescent rats two waves of synthesis of DNA and RNA were observed, one at 3 days and the other after 2 weeks of treatment. The first decline in the incorporations into both DNA and RNA coincided with a decrease in the pool sizes of some of the precursors. The activity of RNA polymerase was not substantially altered. A marked increase in the content of protein was observed before the first wave of synthesis. The normal progressive increase in tetraploid nuclei was prevented. 5. In young adult rats two waves of DNA synthesis were detected. Each was preceded by a large increase in the amount of protein per nucleus but was not accompanied by increased RNA synthesis. After 4 weeks of treatment, the diploid stromal nuclei appeared mainly unaffected and large numbers of tetraploid nuclei with a greatly increased quantity of protein were observed.

FEBS LettersVolume 33, Issue 1 p. 18-22 Full-length articleFree Access The uses of metrizamide in the fractionation of nuclei from brain and liver tissue by zonal centrifugation A.P. Mathias, A.P. Mathias Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6B7, EnglandSearch for more papers by this authorCoral V.A. Wynter, Coral V.A. Wynter Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6B7, EnglandSearch for more papers by this author A.P. Mathias, A.P. Mathias Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6B7, EnglandSearch for more papers by this authorCoral V.A. Wynter, Coral V.A. Wynter Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6B7, EnglandSearch for more papers by this author First published: June 15, 1973 https://doi.org/10.1016/0014-5793(73)80149-8Citations: 28AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 J. Austoker, D. Cox, A.P. Mathias, Biochem. J., 129, (1972), 1139– 2 I.R. Johnston, A.P. Mathias, F. Pennington, R. Ridge, Biochem. J., 109, (1968), 127– 3 G.A. Davis, R.J. Santen, B.W. Agranoff, Anal. Biochem., 11, (1965), 153– 4 S.E. Kerr, K. Seraidarian, J. Biol. Chem., 159, (1945), 211– 5 K. Burton, Biochem. J., 62, (1956), 315– 6 O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 7 I.R. Johnston, A.P. Mathias, F. Pennington, D. Ridge, Nature, 220, (1968), 668– 8 I.R. Johnson, A.P. Mathias, G.D. Birnie Subcellular components: preparation fractionation (1972), Butterworths and Co London 53– Citing Literature Volume33, Issue1June 15, 1973Pages 18-22 ReferencesRelatedInformation

1. The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg(2+), glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [(3)H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [(3)H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V).

1. In normal rats the amounts of each of the main types of nuclear protein, i.e. soluble proteins, histones, non-histone chromosomal proteins and residual proteins, vary within the different classes of rat liver nuclei fractionated by zonal centrifugation. 2. Heterogeneity is observed in the non-histone chromosomal proteins prepared from different classes of liver nuclei. These differences were observed by analysis of the proteins both by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis and electrofocusing electrophoresis. They are most evident between the non-histone chromosomal proteins obtained from stromal and parenchymal nuclei. However, some differences are also found for the parenchymal nuclei, between the diploid parenchymal and the tetraploid parenchymal, and between them and the nuclei involved in the synthesis of DNA respectively. 3. Drastic alterations in the nuclear proteins are found after the administration of thioacetamide. The changes observed are complex and not uniform. They vary with the age of the animal and the type of nucleus. In general an increase in the soluble proteins and non-histone chromosomal proteins and a decrease in the residual proteins is observed. There is a decrease in the specific radioactivity of soluble and residual proteins. 4. Electrophoretic analysis of the non-histone chromosomal proteins showed that specific changes occurred after administration of thioacetamide, which are different in adolescent and young adult rats.

Conference Article| May 01 1973 The Activities of Deoxyribonucleic Acid Polymerase and other Enzymes in the Various Types of Nuclei found in Liver and Brain Tissue A. P. MATHIAS; A. P. MATHIAS 1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar M. STAMBOLOVA M. STAMBOLOVA 1Department of Biochemistry, University College London, Gower Street, London WC1E 6BT, U.K. Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 1973 Biochemical Society1973 Biochem Soc Trans (1973) 1 (3): 624–626. https://doi.org/10.1042/bst0010624 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation A. P. MATHIAS, M. STAMBOLOVA; The Activities of Deoxyribonucleic Acid Polymerase and other Enzymes in the Various Types of Nuclei found in Liver and Brain Tissue. Biochem Soc Trans 1 May 1973; 1 (3): 624–626. doi: https://doi.org/10.1042/bst0010624 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1973 Biochemical Society1973 Article PDF first page preview Close Modal You do not currently have access to this content.

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 Mg2+and Mn2+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 α-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.

This chapter discusses the biochemical properties of nuclei fractionated by zonal centrifugation. The function of the nucleus in the determination of heredity and its role in protein synthesis explain the intense interest in this organelle. Many biochemists in their studies of the nucleus have used rat liver, and often they have glossed over the complex cellular structure of this organ. It consists of a continuous mass of parenchymal cells through which tunnel a network of capillaries. These convey predominantly venous blood from the gastrointestinal tract to the heart. The capillaries are formed chiefly of flattened overlapping littoral cells, the Kupffer cells, active in phagocytosis, and some other cells including fat storage cells. Connective tissue cells are located in the pericapillary space.

1. The thermally induced change in conformation of ribonuclease A in solution was investigated by differential thermal analysis and the susceptibility of the enzyme to proteolytic digestion by ficin. 2. A transition with a mid-point of 60.5 degrees C at pH4.2 was observed directly by differential thermal analysis and shown to be a property of the native structure. 3. At pH4.2 ribonuclease A is susceptible to ficin digestion at 60 degrees C but not at 18 degrees C. 4. Chromatographic analysis of the digestion products reveals that transient active intermediates are produced during the digestion. 5. Three of these intermediates were purified and partially characterized. 6. The nature of those sections of the ribonuclease molecule that are involved in the thermal transition is discussed.

1. Extrusion from a French Press at low pressure was used to prepare extracts of Escherichia coli containing about half their ribosomes as polysomes. A fraction enriched in polysomes was obtained from these extracts by centrifugation through two sucrose layers.

FEBS LettersVolume 10, Issue 2 p. 113-116 Full-length articleFree Access The role of rat liver nuclear DNA polymerase and its distribution in various classes of liver nuclei M.E. Haines, M.E. Haines Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6 BT EnglandSearch for more papers by this authorI.R. Johnston, I.R. Johnston Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6 BT EnglandSearch for more papers by this authorA.P. Mathias, A.P. Mathias Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6 BT EnglandSearch for more papers by this author M.E. Haines, M.E. Haines Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6 BT EnglandSearch for more papers by this authorI.R. Johnston, I.R. Johnston Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6 BT EnglandSearch for more papers by this authorA.P. Mathias, A.P. Mathias Wellcome Laboratories, Department of Biochemistry, University College London, Gower Street, London WC1E 6 BT EnglandSearch for more papers by this author First published: September 24, 1970 https://doi.org/10.1016/0014-5793(70)80429-XCitations: 17AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 I.R. Johnston, A.P. Mathias, F.A. Pennington, D. Ridge, Biochem, J., 109, (1968), 127– 2 I.R. Johnston, A.P. Mathias, F.A. Pennington, D. Ridge, Nature, 220, (1968), 668– 3 M.E. Haines, I.R. Johnston, A.P. Mathias, D. Ridge, Biochem. J., 115, (1969), 881– 4 F.J. Bollum, V.R. Potter, J. Biol. Chem., 233, (1958), 478– 5 R.M. Behki, W.C. Schneider, Biochim. Biophys. Acta, 68, (1963), 34– 6 H.M. Keir, R.M.S. Smellie, G. Siebert, Nature, 196, (1962), 752– 7 D.L. Friedman, Biochem. Biophys. Res. Commun., 39, (1970), 100– 8 R. Howk, T.Y. Wang, Arch. Biochem. Biophys., 133, (1969), 238– 9 P. Ove, O.E. Brown, J. Laszlo, Cancer Res., 29, (1969), 1562– 10 R.S. Gilmour, J. Paul, J. Mol. Biol., 34, (1968), 305– 11 Y. Iwamura, T. Ono, H.P. Morris, Cancer Res., 28, (1968), 2466– 12 P. Ove, J. Laszlo, M.D. Jenkins, H.P. Morris, Cancer Res., 29, (1969), 1557– Citing Literature Volume10, Issue2September 24, 1970Pages 113-116 ReferencesRelatedInformation

1. Several substances of high molecular weight were examined for their suitability as suspension media in the formation of density gradients for the zonal centrifugation of avian erythrocytes. None proved satisfactory. 2. The behaviour of pigeon erythrocytes in rate-sedimentation experiments in a type A zonal rotor with density gradients of sucrose was examined. The mature cells sediment more rapidly than the younger cells and have a lower RNA/DNA ratio. Maturation is accompanied by a greater loss of RNA from the nucleus than from the cytoplasm. 3. The base composition of the nuclear RNA and of the two species of cytoplasmic ribosomal RNA is reported. 4. The RNA of erythrocytes may be labelled in vivo by injection of inorganic [(32)P]phosphate. The cells most active in the synthesis of RNA sediment less rapidly than the bulk of the cells. 5. Reticulocyte nuclei sediment more slowly than those from erythrocytes. Reticulocyte nuclei have a mean volume of 35mu(3) and are isopycnic with sucrose of density 1.2871 (measured at 20 degrees ). Maturation of the nuclei causes them to shrink to a volume of 25mu(3) and the density to increase to 1.2944.