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• W2145432810 abstract "The experiments to be described are conicerned with some of the factors regulating DNA template activity in differentiated cells. They deal specifically with methods for modifying the types of ribonucleic acid synthesized and the sites of RNA synthesis in isolated cell nuclei. It is now well established that the patterns of RNA synthesis in intact cells can be varied and that they are subject to physiological and environmental controls which regulate both the kinds of RNA synthesized and their rates of formation. This control over nuclear function is particularly well documented in studies of hormone effects on RNA metabolism (e.g., ref. 1). A capacity for variable gene transcription is also evideiit in liver cells during regeneration2-4 and in cells adapting to changing envirornments in culture.'-7 What determines which portions of the total DNA in the cell nucleus will be active as a template for RNA synthesis? One approach to this problemn is based on a study of the RNA polymerase activity of isolated nuclei. On the premise that a carefully isolated nucleus may nmaintain a native intactness of organization, preserve its differentiated chromnatin stucture, and continue to synthesize the types of RNA characteristic of the cell of origin, we have analyzed the RNA formed by liver nuclei during short-term incubations in vitro. The biochemical findings have been supplemented with a study of the intranuclear sites of RNA synthesis as revealed by high-resolution autoradiography under the electron microscope (EM). A biochemical and cytological comparison can thus be made between RNA synthesis in vitro and the results of earlier experiments on RNA metabolism in liver cells in vivo. Evidence will be presented to show that the isolated liver nucleus appears to maintain the pattern of RNA synthesis typical of the tissue of origin. It will also be shown that the pattern and sites of RNA synthesis in isolated nuclei can be changed. Both the eomposition of the RNA formed and its localization during synthesis in the nucleus can be influenced by specific divalent cations and depend on the salt concentration of the suspending medium. It will be stressed that these changes can be effected at very low salt concentr-ations without disruption of nuclear structure. Methods.-The isolation of nuclei from normal and regenerating rat liver has been described.4 The nuclei were resuspended in 0.25 M sucrose-4 mM MgCl2-0.01 M tris-HCI buffer (pH 8.3) (TMS), washed and incubated as described below. RNA polymerase assay: The procedure is based on the observations of S. Weiss,8 modified to permit studies of specific ion effects. Nuclei from normal and regenerating liver were washed twice with 20 vol of TMS solutioin, or with TMS containing 0.6 mM MnCl2. They were resuspended in 10 vol of TMS, or TMS + MnCl2. To each aliquot (0.2 ml) containing 20-30 ,ug DNA-P was added 0.1 ml of a water solution of the ribonucleoside triphosphates in the concentrations shown in Table l and Figure 2. In parallel experiments the incorporation of each of the four C14-labeled nucleotides was measured in the presence of the other three unlabeled nucleotides. All were present at saturating concentrations for the amount of nuclei tested. I'he mixture was" @default.
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• W2145432810 date "1967-03-01" @default.
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• W2145432810 title "MODIFICATION OF RIBONUCLEIC ACID SYNTHESIS IN NUCLEI ISOLATED FROM NORMAL AND REGENERATING LIVER: SOME EFFECTS OF SALT AND SPECIFIC DIVALENT CATIONS" @default.
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• W2145432810 doi "https://doi.org/10.1073/pnas.57.3.743" @default.
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