RNA POLYMERASE ACTIVITY IN VARIOUS CLASSES OF NUCLEI FROM DIFFERENT REGIONS OF RAT BRAIN DURING POSTNATAL DEVELOPMENT

—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.     

THE RELATIONS BETWEEN DNA, RNA, AND PROTEIN IN NORMAL EMBRYONIC CELL NUCLEI AND SPONTANEOUS TUMOUR CELL NUCLEI

Interferometric and photometric measurements have been made successively on individual cell nuclei derived from normal embryonic tissues and spontaneous tumour tissues of the mouse grown in vivo. From the measurements, the relations between nucleic acid and dry mass content have been studied in the two types of nuclei and the results shown to be consistent with differences in cell metabolism previously reported to exist in vitro. In the nuclei of normal embryonic cells, the syntheses of DNA, nuclear RNA, and protein appear to be closely associated, whereas in the tumour cell nuclei an appreciable fraction of the chromatin RNA and protein synthesis is dissociated from the replication of DNA.     

THE ISOLATION OF CELL NUCLEI FROM RAT BRAIN

A method for preparing highly pure cell nuclei from adult rat brain, using both differential and isopycnic centrifugation in sucrose media, is described. The morphology of these preparations was examined by both phase contrast and electron microscopy. The isolated nuclei retained many aspects of their in situ morphology; in particular, the nuclear envelope was double layered and interrupted by pore-like discontinuities, and the nucleoli consisted of irregular masses of densely packed granules. Analyses of these nuclear preparations for cytochrome oxidase and cholinesterase activity, as well as RNA/DNA ratio, indicated minimal contamination with mitochondria and microsomes. Problems involving the homogenization technique, choice of ionic conditions in the homogenization medium, and choice of optimal density of the sucrose solution used for the final purification of nuclei are discussed. Results of application of the technique to isolation of adult rat liver nuclei are also reported.     

THE RELATIONSHIP BETWEEN DNA SYNTHESIS AND NUCLEAR PROTEINS IN THE CEREBRUM AND CEREBELLUM OF 8-DAY-OLD RATS

In-8-day-old rats the higher rate of DNA replication in cerebellum than in cerebrum is accompanied by an enhanced synthesis of nuclear proteins. The greatest difference between the incorporation of tritiated leucine into proteins of cerebral and cerebellar cell nuclei occurs in the acid-soluble deoxyribonucleoproteins. However, the specific radioactivity of the acidic deoxyribonucleoproteins is similar in both tissues. The relative content of these proteins and the activity of the RNA polymerase is higher in cerebrum than in cerebellum. The results suggest that in the cerebrum of young rats these proteins are mainly concerned with the regulation of RNA synthesis.     

NUCLEAR-CYTOSOL INTERACTIONS THAT FACILITATE RELEASE OF RNA FROM MOUSE BRAIN NUCLEI

Abstract— Mouse brain nuclei were incubated in vitro under conditions that primarily lead to the synthesis of radioactive polydisperse and messengerlike nuclear RNA. After incubation the effects of Mg² concentrations, nucleoside triphosphate levels and brain cytosol were examined with regard to their ability to influence the release of RNA from brain nuclei. The presence of 8 mM -MgCl₂ and a total of 0.3 mM-nuclcoside triphosphates during the labelling procedure allowed only a minimal amount of RNA to be released. However, when the MgCl₂ was decreased to 2 mM and the nucleoside triphosphates were increased to 1 mM, a stimulation of RNA release was observed. The addition of unfractionated brain cytosol under these conditions resulted in an inhibition of RNA release.
G-100 Sephadex filtration removed detectable RNase activity from the cytosol preparations and allowed the identification of fractions that were able to facilitate nuclear RNA release by 3-fold. The fractions that stimulated release did not have detectable levels of RNase, protease or DNA-dependenl RNA polymerase. Under conditions that provided maximum nuclear RNA release by both labelled mouse brain and neuroblastoma nuclei, no release of DNA could be measured. The cytosol fractions that facilitated RNA release did not have a high affinity for nuclear RNA or an ability to stimulate nuclear RNA synthesis. However, other components in the cytosol were shown to stimulate RNA metabolism in isolated mouse brain nuclei and to have a relatively high binding affinity to nuclear RNA. Further purification of the RNA release components in the brain cytosol by DEAF. Sephadex chromatography allowed an increase in specific activity of at least 40-fold. The thermal lability, effective filtration size, and solubility in phenol suggested that the cytosol factors that facilitiated nuclear RNA release were associated with cellular proteins.     

STABILITY OF DNA IN PURKINJE CELL NUCLEI OF THE MOUSE : An Autoradiographic Study

Neurons of the mouse were labeled with [³H]thymidine during their prenatal period of proliferation. The ³H activity of the Purkinje cell nuclei was then studied autoradiographically 8, 25, 55, and 90 days after birth. The measured grain number per nucleus decreased by about 14% between the 8th and 25th postnatal days and then remained constant up to 90 days. There was no significant decrease of the ³H activity of the Purkinje cell nuclei after correction of the measured grain number per nucleus for increasing nuclear volume of the growing Purkinje cells and for the influence of [³H]β self-absorption in the material of the sections. Injection of a high dose of [³H]thymidine into young adult mice did not result in ³H labeling of either Purkinje or other neurons in other brain regions. The results agree with the concept of metabolic stability of nuclear DNA. "Metabolic" DNA could not be observed in these experiments.     

PROTEIN SYNTHESIS IN FRACTIONS FROM ISOLATED BRAIN CELL NUCLEI

Abstract— 1. The incorporation in vivo and in vitro of isotopically labelled leucine into fractions of nuclear proteins from young and adult rat brain was investigated.
2. During post-natal cerebral maturation, the ability of nuclei from brain cells to synthesize proteins decreased. The specific activities of all the fractions of nuclear protein were highest in 3-day-old rats and declined thereafter. Nuclei from adult brain cells exhibited only 10 per cent of the activity found in nuclei from brain cells of 3-day-old rats.
3. The 'residual protein' fraction was most rapidly labelled, peak activity being reached within 30 min after injection. In vitro , the 'residual protein' fraction attained maximum activity within 40 min.
4. The specific activity of the chromatin acidic proteins (HCl-insoluble) was considerably higher than that of the histones both in vivo and in vitro. Histones were the most inert of all the nuclear protein fractions studied.
The possible functional significance of the various protein fractions during the process of cerebral maturation and in the adult brain is discussed.     

EFFECTS OF CORTICOSTEROIDS ON THE BIOCHEMICAL MATURATION OF RAT BRAIN: POSTNATAL CELL FORMATION

(1) Treatment with cortisol acetate (0.2 mg daily during the first 4 days after birth) reduced the rate of growth in the rat: at 35 days of age the body weight was reduced by 50 per cent and the brain weight, depending on the region, by up to 30 per cent. (2) In the brain the normal increase in cell number was severely inhibited during the period of cortisol treatment; this resulted in a final deficit in cell number of about 20 per cent in the cerebrum and 30 per cent in the cerebellum. (3) To determine whether cortisol affected primarily cell formation or cell destruction the labelling of brain DNA was studied 1 h after a subcutaneous injection of 20 Ci/100 g [2-¹⁴C]thymidine. In the controls the amount of labelled DNA increased by a factor of two in the cerebrum and seven in the cerebellum during the period 2-13 days, and it decreased to 40 and 27 per cent of the peak values in the cerebrum and cerebellum respectively in the following 7 days. The results indicated that mitotic activity is higher in the cerebellum than in the cerebrum in the 2nd week of life. It would appear that in the cerebrum appreciable cell death accompanies new cell formation, especially during the period 13-35 days of age. (4) Cortisol treatment affected cell division rather than cell destruction in the brain since it strongly inhibited the incorporation of [2-¹⁴C]thymidine into DNA. The inhibition was severe during the period of treatment but it did not result in a lasting fall in mitotic activity. At the age of 13 days the amount of labelled DNA formed approached the normal level and it was twice that in controls at 20 days, indicating a tendency for compensating cell deficit by an accelerated mitotic activity. Nevertheless, massive cell proliferation ceased at about the same age as in normals; the labelling of DNA decreased markedly between 13 and 20 days after birth, and the DNA content did not increase after the age of 20 days. (5) In contrast to the marked effect on cell number, cortisol treatment did not influence significantly the maturational changes related to average cell size (DNA concentration) or the chemical composition of cells (RNA/DNA and protein/DNA).     

Diurnal variations in endogenous RNA polymerase activity and amounts of nuclear non-histone protein, DNA and cytoplasmic protein in rat liver.

From rats fed ad libitum and kept under a 12 + 12 h light/dark regimen, the DNA dependent RNA polymerase activity of liver cell nuclei was determined avery four hours. From identical rats, nuclear non-histone protein and DNA, and cytoplasmic protein was determined by Feulgen-Naphthol Yellow S cytophotometry of isolated liver cells. The minimum: maximum ratio of the RNA polymerase activity is 0.77; the min:max ratio of nuclear non-histone protein is 0.84. These two parameters have identical time courses with a gradual decline during the light period and a sharp rise after the onset of the dark period. The variations in nuclear DNA content, estimated as the amount of Feulgen stain bound, closely parallel those of the RNA polymerase activity and nuclear non-histone protein content (min:max = 0.96). The amount of cytoplasmic protein per cell also varies throughout the day, but its time curve lags behind those of nuclear non-histone content and RNA polymerase activity. These results are consistent with the concept of nuclear non-histone proteins as de-repressors of the DNA template in differentiated, non-proliferating cells, and support the validity of using Feulgen-Naphthol Yellow S cytophotometry of nuclear non-histone proteins as an estimate of gene expression in such cells.     

Diurnal variations in endogenous RNA polymerase activity and amounts of nuclear non-histone protein,DNA and cytoplasmic protein in rat liver

Summary From rats fed ad libitum and kept under a 12+12 h light/dark regimen, the DNA dependent RNA polymerase activity of liver cell nuclei was determined every four hours. From identical rats, nuclear non-histone protein and DNA, and cytoplasmic protein was determined by Feulgen-Naphtol Yellow S cytophotometry of isolated liver cells. The minimum: maximum ratio of the RNA polymerase activity is 0.77; the min:max ratio of nuclear non-histone protein is 0.84. These two parameters have identical time courses with a gradual decline during the light period and a sharp rise after the onset of the dark period. The variations in nuclear DNA content, estimated as the amount of Feulgen stain bound, closely parallel those of the RNA polymerase activity and nuclear non-histone protein content (min:max=0.96). The amount of cytoplasmic protein per cell also varies throughout the day, but its time curve lags behind those of nuclear nonhistone content and RNA polymerase activity. These results are consistent with the concept of nuclear non-histone proteins as de-repressors of the DNA template in differentiated, non-proliferating cells, and support the validity of using Feulgen-Naphthol Yellow S cytophotometry of nuclear non-histone proteins as an estimate of gene expression in such cells.     

BIOCHEMICAL EFFECTS OF THYROID DEFICIENCY ON THE DEVELOPING BRAIN

Abstract— The effects of neonatal thyroidectomy on some constituents of the cerebrum, cerebellum and liver of the rat have been studied during the first 7 weeks of life. In the normal rat between the 6th and 14th post-natal days the RNA content per unit of DNA in the brain increased by 70 per cent. Although the brain continued to grow from the 14th to the 35th day, the amount of RNA relative to DNA decreased by about 20 per cent. The ratio of protein to DNA increased during the whole period studied and in the cerebral cortex it was more than trebled between the age of 6 and 35 days. The growth of the cerebellum extended over a longer period than that of the cerebrum, its weight increasing by 88 per cent between the ages of 14 and 35 days as compared with a cerebral increase of 34 per cent. The DNA content showed a 50 per cent increase during this period. Qualitatively these maturational changes were not affected by neonatal thyroidectomy. Quantitative changes, which applied equally to the cerebral cortex and brain as a whole, were observed. At the age of 35 days, the weights of the cerebral hemispheres and cerebellum were reduced by thyroidectomy by 20 per cent; the overall DNA content per organ did not change, but the amounts of protein and RNA relative to DNA decreased significantly. It is therefore inferred that thyroid deficiency affects the size of the cells in brain and cerebellum rather than their total number. Conversely, the cell population of the liver was only a quarter of that in the control. There was a small but significant decrease in the hepatic protein and RNA content in the hypothyroid animal. The activities of the following enzymes which served as markers for subcellular fractions in homogenates of cerebral cortex were determined: lactate dehydrogenase for the supernatant, glutamate dehydrogenase for the mitochondrial and glutamate decarboxylase for the synaptosomal fractions. When the activities were expressed on a fresh weight basis a significant decrease by comparison with the control values was observed only in the case of glutamate decarboxylase (—15 per cent at the age of 17–32 days); when the activities were based on DNA content all values were reduced, probably as a result of the general decrease in cell size. Pyrimidine metabolism of brain and liver, studied after the administration of [6-¹⁴C]-orotic acid, was not affected in either tissue by neonatal thyroidectomy. A small but significant reduction in the incorporation of labelled pyrimidine nucleotides in liver RNA was observed, but no significant decrease in the incorporation in cerebral RNA was found in the hypothyroid rats.     

EFFECT OF UNDERNUTRITION ON CELL FORMATION IN THE RAT BRAIN

Abstract— Rats were undernourished by approximately halving the normal food given from the 6th day of gestation throughout lactation. Growth of the foetuses was nearly normal, in marked contrast to the severe retardation caused by undernutrition during the suckling period. In comparison with controls the size and the DNA content of the brain were permanently reduced by undernutrition during the suckling period: this effect was relatively small, approx. 15 per cent decrease at 21 and 35 days. The rate of ¹⁴C incorporation into brain DNA at 30 min after administration of [2-¹⁴C] thymidine was taken as an index of mitotic activity; compared with controls there was severe reduction in mitotic activity (maximal decrease by about 80 per cent at 6 days in the cerebrum and by 70 per cent at 10 days in the cerebellum). The rate of acquisition of cells was calculated from the slopes of the logistic curves fitted to the estimated DNA contents. In normal animals the maximal slope was attained at 2·7 days and at 12·8 days after birth in cerebrum and cerebellum respectively; the daily acquisition of cells at these times was 4·8 × 10⁶ and 18 × 10⁶ cells respectively. The fractional increase in cell number at the maximum was 5·4 percent per day in the cerebrum and 15·2 per cent per day in the cerebellum. The rate of acquisition of cells relative to the rate of mitotic activity was higher in the brains of undernourished animals than in controls. One of the compensatory mechanisms for the severe depression of mitotic activity in the brain of undernourished animals Seems to involve a reduction in the normal rate of cell loss.     

THE CYTOPLASMIC CONTROL OF NUCLEAR ACTIVITY IN ANIMAL DEVELOPMENT

1.This article reviews the occurrence, mechanism, and functional significance of the cytoplasmic regulation of nuclear activity during cell differentiation and especially during early animal development. 2.Nuclei from brain, and from other kinds of adult cell normally inactive in DNA synthesis, are rapidly induced to commence DNA synthesis by components or properties of intact egg cytoplasm. The components of egg cytoplasm which induce DNA synthesis are not species-specific and they are likely to include DNA polymerase. It is known that DNA polymerase exists in egg cytoplasm before it becomes associated with nuclei in which it is effective. The induction of DNA synthesis in brain nuclei by living egg cytoplasm is always preceded by a pronounced nuclear swelling, a dispersion of chromosomes or chromatin, and the entry of cytoplasmic protein into the nucleus. 3.RNA synthesis can be experimentally induced or repressed by living cytoplasm. The cytoplasm of unfertilized and fertilized eggs appears to contain components which can reversibly and independently repress the synthesis of ribosomal RNA, transfer RNA, and heterogeneous RNA. RNA synthesis can be induced by introducing nuclei inactive in this respect into the cytoplasm of cells very active in RNA synthesis. The induction and repression of RNA synthesis is preceded by a marked swelling of the nucleus and the dispersion of its chromosome material. 4.The cytoplasmic control of chromosome condensation before division has been demonstrated by introducing sperm or adult brain nuclei into the cytoplasm of oocytes undergoing meiotic maturation. 5.The evidence that regional differences in the composition of eggs and other cells are associated with changes in nuclear and gene activity is reviewed in Section 111. While it is certain that these regional differences are of great importance in cell differentiation, evidence that they have a direct effect on nuclear activity has been obtained in a few instances only. In some species it has been shown that the cytoplasmic components related to germ-cell differentiation include RNA and, frequently, granules. 6.It is concluded that whenever nuclei are introduced experimentally into the cytoplasm of another cell, they very quickly assume, in nearly every respect, the nuclear activity characteristic of the host cell. In many instances, altered function has been demonstrated in nuclei which subsequently support normal development. The induced nuclear changes are therefore regarded as normal and it is believed that they are achieved through the same mechanism as that by which the host cell nucleus originally came to function in its characteristic way. Examples are cited to show that changes in gene activity very frequently arise immediately after mitosis. The changes induced experimentally in transplanted nuclei resemble in very many respects those undergone by nuclei which are naturally reconstituted after mitosis, and it is argued that the two processes are functionally equivalent, It is suggested that during telophase of mitosis, chromosomes are reprogrammed in respect of potential gene activity by association with cytoplasmic proteins. Inter-phase nuclei seem not to show changes of gene activity except when they undergo a pronounced enlargement after entering a new cytoplasmic environment.     

Transcription in Isolated Wheat Nuclei: I. ISOLATION OF NUCLEI AND ELIMINATION OF ENDOGENOUS RIBONUCLEASE ACTIVITY

Nuclei isolated from embryos of wheat (var. Yamhill) incorporated [(3)H]UTP into a trichloroacetic acid-insoluble product linearly for 60 minutes. When the RNA synthesized in vitro was analyzed on a sucrose gradient, the amount of RNA in the 4S region increased with longer incubation times. These data and the absence of higher molecular weight RNA of specific size classes in our work (and previously published reports) suggested that nuclear fractions from plant tissue contained active nucleases. This was confirmed when wheat nuclei were mixed with [(3)H]yeast RNA (4, 18, 26S). All of the radioactive yeast RNA was degraded within 30 minutes to species sedimenting between 4 and 10S. The inclusion of high salt (125 millimolar (NH(4))(2)SO(4), 100 millimolar KCl), EGTA, and exogenous RNA or DNA reduced but did not eliminate endogenous RNase activity. Wheat embryo nuclei were further purified by centrifugation on a gradient of a polyvinylpyrrolidone-coated colloidal silica suspension (Percoll). These nuclei were ellipsoidal, free of cytoplasmic material, and lacked endogenous nuclease activity when assayed with [(3)H]yeast RNA. Sucrose gradients were not as effective as Percoll gradients in purifying nuclei free of RNase activity. The Percoll method of isolating nuclei and the RNase assay reported here will be useful in isolating plant nuclei that are capable of synthesizing distinct RNA species in vitro.     

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

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.