Macromolecular synthesis in mitochondria isolated from different regions of developing rat brain

DNA, RNA, and protein synthesis in mitochondria isolated from cerebral hemispheres, brain stem, and cerebellum of 10- and 30-day-old rats was measured. Synthesis of different macromolecules was affected by the respective mitochondrial specific inhibitors, showing a good level of purity of mitochondrial preparations. DNA and protein synthesis in 10-day-old rats was about 70% higher than in 30-day-old animals. In contrast, RNA synthesis did not decrease with age in all the regions examined.     

Messenger ribonucleic acid of cerebral nuclei

1. RNA was isolated from crude nuclear preparations and from ribosomes derived from rat brain and liver. Nuclear RNA was obtained by lysis of the nuclei with sodium dodecyl sulphate, followed by denaturation and removal of DNA and protein with hot phenol. 2. Base composition analyses indicated that the cerebral nuclear RNA preparation contained a higher proportion of non-ribosomal RNA than the analogous hepatic preparation. 3. Sucrose-density-gradient analyses revealed a heterogeneous profile for each nuclear RNA preparation, with two major peaks possessing the sedimentation properties of ribosomal RNA (18s and 28s). 4. Template activities of both preparations were widely distributed through the sucrose density gradients. 5. The cerebral nuclear RNA preparation was more active than the hepatic nuclear RNA preparation in promoting amino acid incorporation in cell-free systems from Escherichia coli and rat brain. 6. Cerebral nuclear RNA stimulated amino acid incorporation in a cerebral ribosomal system even in the presence of an excess of purified E. coli transfer RNA. 7. It is concluded that a significant proportion of cerebral nuclear RNA has the characteristics of messenger RNA.     

Postnatal ontogeny of uridine kinase in the cerebellum,hypothalamus, and cerebral cortex of the rat

Postnatal developmental patterns of uridine kinase were determined in crude subcellular fractions of the rat cerebellum, hypothalamus and cerebral cortex at ages 3 through 60 days. The highest specific activity and predominant distribution of enzyme was in the 105,000g supernatant of the 3 brain regions. Enzyme activity in hypothalamus and cerebral cortex was maximum at 3 days and decreased with age; in cerebellum it increased through 13 days and decreased thereafter. Thus, the pattern of activity in hypothalamus and cerebral cortex paralleled changes in DNA and RNA synthesis through age 60 days; in cerebellum, it more closely approximated changes in DNA synthesis during early development. Changes inK _m with aging suggest that the brain regions contain more than one form of enzyme. The highest particulate activity was in the microsomal fraction of the cerebellum and hypothalamus at all ages and in the cortex at 35 and 60 days. Relative specific activity for microsomal fractions of the brain regions at 60 days indicate a concentration of the enzyme which may be relevant in the maintenance of RNA activity in adult brain.     

大鼠衰老过程中肝细胞核及染色质的体外转录活性

用同位素掺入法研究不同年龄大鼠的肝细胞核及染色质体外转录活性,所得结果表明:(1)老年大鼠肝细胞核的转录起始能力较断乳鼠及青年鼠分别下降68％及56％。(2)大鼠肝细胞核内与染色质结合的RNA聚合酶所致的转录活性随增龄呈近似线性下降,而不与染色质结合的RNA聚合酶所致的转录活性随增龄则无变化。(3)老年大鼠肝染色质体外转录活性较断乳鼠及青年鼠分别下降52％及35％。这些结果提示。老年大鼠肝染色质功能的改变可能是转录活性改变的主要原因。     

Studies by DNA-RNA hybridization of transcriptional diversity in human brain

Abstract— Purified unique sequences of human DNA (from HeLa cells) were hybridized with various preparations of human brain KNA, to obtain estimates of the fraction of DNA transcribed. Values of 12 per cent were obtained for fetal brain increasing to 24 per cent for specific regions of adult brain. Right and left parietal and temporal cortical RNA gave similar plateau values but left frontal cortical RNA appeared to represent 2–3 times as much of the total genome as that from right frontal cortex. Lower values were obtained for fetal and adult brain stem and for RNA prepared from temporal cortex of a patient exhibiting gross cerebral atrophy. Attempts were made to assess the contributions of glial and neuronal cells by using human astrocytoma and mouse neuroblastoma RNA.     

Effect of RNA synthesis on the binding of 3H-cortisol to nuclear ribonucleoprotein particles from rat liver carrying DNA-like RNA in vivo.

3H-cortisol was found to associate with rat liver nuclear 30S ribonucleoprotein particles carrying D-RNA in vivo. No interaction was detectable when RNA synthesis was inhibited by alpha-amanitine. The association appears to be specifically for RNP carrying RNA synthesized after the administration of cortisol to adrenalectomized rats. The DNA/protein/RNA ratio of rat liver nuclei was not effected by alpha-amanitine under our conditions. However, the drug caused a 5-10 fold decrease in nuclear uptake of cortisol. The results are discussed in relation to a supposed transfer of cortisol-receptor complexes from the chromatin template to the nascent RNA chains.     

Transglutaminase Catalyzes the Formation of Sodium Dodecyl Sulfate-Insoluble, Alz-50-Reactive Polymers of τ

Abstract: The gene for tryptophan 2,3-dioxygenase (TDO) heretofore was believed to be expressed only in liver. The data presented here demonstrate that RNA encoding TDO is present in rodent brain. Oligonucleotide primers based on the rat liver TDO cDNA sequence were synthesized and used to amplify RNA derived from mouse whole brain and liver and rat brain regions by the RNA-PCR. Reaction products were purified and subjected to DNA sequencing. Identical sequences were obtained when mouse whole brain and liver RNAs were amplified, and these sequences were shown to be 96% identical to the published rat liver tryptophan TDO cDNA sequence. In addition, TDO sequences were found in RNA derived from rat brainstem, cerebellum, cortex, hypothalamus, and the remainder of the brain.     

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.     

Majority of RNA which co-purifies with unactivated rat hepatic glucocorticoid-receptor complexes is nonspecific and not receptor-associated.

Molybdate-stabilized, unactivated rat hepatic glucocorticoid-receptor complexes were purified by a three-step procedure which includes affinity chromatography, gel filtration and anion exchange chromatography. Following elution of unactivated steroid-receptor complexes from the final DEAE-cellulose column, RNA which remained bound to the anion exchange resin was eluted with 1 M KCl. This RNA was small and heterogeneous in size. Equivalent amounts of RNA were detected after a mock purification which was devoid of receptors, suggesting that the presence of this RNA is not dependent on that of receptors. Both a [32P]DNA complementary to the RNA eluted from DEAE-cellulose and a [32P]DNA probe synthesized from total rat liver RNA gave similar results when hybridized to total rat liver RNA. These data indicated that the RNA which co-purified with unactivated receptors through the first two steps was very similar to total RNA in overall composition. Virtually identical hybridization patterns were also detected when end-labeled probes generated from the DEAE-cellulose eluted RNA or total liver RNA were hybridized to total genomic rat DNA, suggesting that the RNA eluted from the anion exchange resin is not specific or unique. Although these results do not exclude the possibility that there could be specific RNA species associated with the unactivated glucocorticoid receptor, they do indicate that the majority of the RNA eluted from DEAE-cellulose following elution of receptor complexes appears indistinguishable from total rat liver RNA and can be detected in parallel mock purifications.