Effect of hypophysectomy on liver nuclear ribonucleic acid synthesis in aging rats.

Changes in RNA synthesis in liver nuclei were observed at different ages and after hypophysectomy and hormone replacement in female Sprague-Dawley rats. As determined by the incorporation of [3H]UMP into an acid-insoluble product, RNA synthesis decreased by about 75% in intact rats from 6 months to 24 months of age. This decline with age was not observed in liver nuclei from 24-month-old rats that had been hypophysectomized at 12 months and maintained on a minimal hormone-replacement therapy. Thyroid hormones and somatotropin (growth hormone) had an additive effect on RNA synthesis in liver nuclei from these hypophysectomized rats. The same hormones had no significant effect on intact, age-matched rats. With advancing age, nuclei of intact rats had an increase in the pool of free RNA polymerase and an apparent decrease in the enzyme activity bound to nuclear chromatin. There was no change in total enzyme with age. In hypophysectomized, hormone-treated rats, free RNA polymerase activity decreased and chromatin-bound activity increased. There was no difference in total nuclear RNA polymerase activity between operated or intact rats. However, the ratio of the bound to the free activity was different. These results suggest that the ability of RNA polymerase to bind to chromatin may be involved in the age-related decrease in liver nuclear RNA synthesis of intact rats.     

Influence of steroid-hormone-receptor-protein complexes on initiation of ribonucleic acid synthesis in liver nuclei isolated from rats of various ages.

The effect of age on the induction of the initiation of RNA synthesis was investigated in liver nuclei isolated from adrenalectomized rats of various ages after binding of a dexamethasone-receptor-protein complex. Binding of this complex to nuclear chromatin resulted in increased initiation of nuclear RNA synthesis at all ages; however, an age-associated decline in the extent of this induction was observed. This suggests an age-related decrease of total rat liver nuclear RNA synthesis with a decreased response to glucocorticoid hormones.     

Developmental and regional variations in ribonucleic acid synthesis on cerebral chromatin

1. Chromatin was prepared from purified nuclei isolated from liver and cerebral regions of the rat. 2. The capacity of these preparations to promote RNA synthesis in the presence of bacterial RNA polymerase was determined. 3. The rate of RNA synthesis on chromatin was normally 12-21% of the rate observed with native DNA, but was markedly stimulated on addition of 200mm-ammonium sulphate. 4. At physiological concentrations (80mug./ml.), the brain-specific S-100 protein inhibited RNA synthesis on DNA and chromatin. 5. Cerebral chromatin from foetal and newborn animals was more active in RNA synthesis than were the analogous preparations from liver. 6. Cerebellar chromatin maintained a high rate of RNA synthesis during brain maturation. In contrast, RNA synthesis on chromatin from other brain regions and liver declined with age of the rat. 7. RNA synthesized on chromatin stimulated amino acid incorporation in an Escherichia coli ribosomal system and hybridized with homologous DNA. 8. RNA synthesized on chromatin from adult cortex or hindbrain hybridized with DNA to a greater extent than that synthesized on cerebellar chromatin. 9. The proportion of RNA formed on cerebral-cortical chromatin that hybridized with DNA increased with age of the rat. 10. The results indicate that the total amount and the types of RNA synthesized on cerebral chromatin vary regionally and during development.     

Effect of growth hormone on ribonucleic acid metabolism. The template activity of the chromatin and molecular species of ribonucleic acid synthesized after treatment with the hormone

Growth hormone stimulates the synthesis of RNA in hypophysectomized rat liver. The question whether the hormonal stimulation of RNA synthesis is due to the activation of repressed cistrons or to other factors was studied. Nuclear RNA from the livers of adult female hypophysectomized and growth-hormone-treated rats was examined for molecular homology by hybridization techniques: no new species of RNA were detected after hormone treatment. The template activity of the chromatin for RNA synthesis is also not increased by the action of growth hormone. Short- and long-pulse-labelling experiments demonstrate that the hormonal stimulation of RNA synthesis is most marked in experiments where the period of incorporation of radioactive precursors is limited to 1-2hr. It is concluded that the hormone influences essentially the rate of RNA synthesis in these tissues.     

Modification of the template capacity of liver chromatin for form-B ribonucleic acid polymerase by food intake in rats under controlled feeding schedules.

Nuclei from liver of rats accustomed to eating during the first 8h of a daily 12h dark period demonstrate an increased capacity to synthesize RNA 6H after the beginning of the feeding period. 2. This increase is accompanied by a higher yield of extractable form-B DNA-dependent RNA polymerase activity. 3. The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by food intake. Both purified and chromatin-associated form-B enzyme activities exhibit different ionic strength requirements after food intake. 4. The sensitivity of exogenous (added) form-B-enzyme to changes in ionic strength changes after feeding when chromatin is used as template. 5. Chromatin extracted from the liver of fed rats is a better template for form-B-enzyme than chromatin extracted from starved rats.     

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。     

Effect of chromium(III) on nucleolar RNA synthesis

The enhancement of hepatic nucleolar RNA synthesis induced by Cr(III) in partially hepatectomized rats and its mechanisms are described. Cr(III)-administered (0.5 mg Cr/kg, ip) and then partially hepatectomized rats were significantly enhanced in the hepatic nucleolar RNA synthesis at the very early stage of liver regeneration. This enhancement was caused both by the induction of newly found nucleolar Cr-bound protein of 70 kD (Cr-p70) and by the activation of nucleolar chromatin, both of which arose from nuclear accumulation of Cr together with partial hepatectomy. Studies on the mechanism of this enhancement indicated that the Cr-p70 bound to the activated nucleolar chromatin and loosened its higher-order structure, resulting in an increase of the B-form fraction of chromatin DNA. The degree of this loosening well correlated with the amount of Cr-p70 bound to chromatin and also with the extent of elevation of RNA synthesis. Some molecular species of nonhistone proteins in chromatin were found to play an important role in the interaction to Cr-p70. These results suggest a possibility that the action of Cr is involved in cell proliferation process.     

Ribonucleic acid labelling perfused livers of protein-fed and protein-deprived rats

Several observations suggest an increased RNA synthesis in livers of protein-deprived rats, though the RNA/DNA ratio is decreased. A number of hormones may be involved in these changes. Therefore, we studied in RNA metabolism in isolated perfused livers taken from protein-fed and protein-deprived rats. (3H)-orotic acid was given to the rats 2 h before liver explantation, and (14C)-orotic acid was added to the perfusate. Other rats, called controls in vivo, whose livers were not transplanted were also given (3H)-orotic acid followed by (14C)-orotic acid. The livers of these rats, which were not hormone supplemented, were labelled for the same length of times as the livers in vitro. The ratio specific RNA radioactivity/specific nucleotide radioactivity x RNA/DNA was determined and taken as a measure of the RNA synthesis per liver cell. In the controls in vivo, this ratio was significantly higher for protein-deprived than for protein-fed rats. In livers from the protein-fed rats, labelling in vitro increased significantly when growth hormone, hydrocortisone, insulin and tri-iodothyronine were added to the perfusate. Labelling was also significantly higher in these livers than in the controls in vivo. In livers from protein-deprived rats, the ratio in question was the same whether the hormones were added to the perfusate or not, and was significantly lower than in the controls in vivo. Differences in RNA labelling are thus obtained in our in vitro system. Gel electrophoresis of RNA demonstrated normal RNA labelling, showing that the system is suitable for studying liver RNA synthesis. Further refinement can be made by studying the labelling of UTP and CTP. The results might suggest that the liver from a protein-fed rat, explanted in vitro, may increase its RNA synthesis under the influence of the four hormones in question, and that the RNA synthesis of the liver of a protein-deprived rat is high in-vivo and that it might decrease, when it is explanted to in vitro conditions.     

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.     

Nature of hydrocortisone-elicited amplification of template activity of liver chromatin.

The template efficiency of euchromatin region and number of RNA polymerase binding sites on this region of rat liver chromatin were significantly elevated at 3.5 h after administration of hydrocortisone to adrenalectomised rats. The euchromatin from the liver chromatin of horomone-treated rats was also found to have significantly increased levels of nonhistone proteins as compared to those in euchromatin fraction derived from adrenalectomised rats.     

Some peculiarities of gene expression in inbred and hybrid rats under normal conditions and after partial hepatectomy

The peculiarities of gene expression in line and hybrid rats in the normalcy and after partial hepatectomy were studied. It was shown that the relationship between the RNA synthesis rate in chromatin and RNA transport in the cytoplasm as well as the correlation between the RNA transport rate and the activity of the protein-synthesizing mechanism vary in animals with different genotypes. After partial hepatectomy the hybrids have their S-period of the cell cycle earlier than the line animals. The hybrids do not differ from the parental forms in the rate of RNA chromatin synthesis; however, the newly synthesized RNA of the hybrids in transported into the cytoplasm 5.7 and 5 times faster than that of the lines Vistar and August. The rapidly transported RNA of hybrids is likely to have a more prolonged life as compared to the line animals. After partial hepatectomy an activation of the whole gene expression system occurs. The differences in the gene expression system of hepatocytes of inbred and hybrid animals in the regenerating liver become less pronounced.     

CHANGES IN CHROMATIN TEMPLATE ACTIVITY AND THEIR RELATIONSHIP TO DNA SYNTHESIS IN MOUSE PAROTID GLANDS STIMULATED BY ISOPROTERENOL

Chromatin template activity of mouse parotid glands increases after a single injection of isoproterenol (IPR), a procedure that causes, after a lag period of 20 hr, a marked stimulation of DNA synthesis and cell division in salivary glands of rodents. The increase in chromatin template activity occurs as early as 1 hr and peaks between 6 and 10 hr after IPR, paralleling previously reported changes in the incorporation of uridine-³H into total cellular RNA of mouse parotids. Template activity was measured in vitro in a system in which parotid gland chromatin was incubated with an exogenous RNA polymerase isolated from Escherichia coli. Similar results were obtained when template activity of parotid gland chromatin was assayed using an homologous RNA polymerase from mouse liver. Chromatin template activity in mouse parotids was also studied after the administration of drugs capable of inducing in salivary glands both DNA synthesis and secretion or secretion alone. The results indicate that the increased chromatin template activity occurring 6 hr after IPR is related to the subsequent onset of DNA synthesis. Furthermore, the increased chromatin template activity caused by IPR is inhibited by the previous administration of puromycin, an inhibitor of IPR-stimulated DNA synthesis.     

RNA biosynthesis in mitochondria under conditions of prolonged inhibition of protein biosynthesis in rat liver cytoplasm]

When cytoplasmic protein synthesis is inhibited by cycloheximide (CHI) in vivo synthesis of water-soluble mitochondrial proteins and of mitochondrial RNA is decreased. These changes measured in isolated rat liver mitochondria are similar to those observed in vivo and correlate with the changes the synthesis of water-soluble proteins in mitochondria. When the cytoplasmic fraction (30,000 g-supernatant) had been added to the mitochondria showing decreased RNA synthesis, the RNA synthesis increased to the control level (the incubation conditions were favourable for the protein transport from microsomes to mitochondria). RNA synthesis in mitochondria was not stimulated by cytoplasmic fractions from the CHI-pretreated rats. After prolonged dialysis these fraction stimulated RNA synthesis even to a greater extent than cytoplasmic fractions from the untreated animals. Mitochondrial RNA polymerase activity (measured in mitochondrial extracts supplemented with exogenous DNA) was higher in extracts of mitochondria from livers of normal rats than in extracts of mitochondria from livers of animals injected with CHI.