RNA synthesis in intact rat liver nuclei |
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| Authors: | R R MacGregor H R Mahler |
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| Affiliation: | 1. Materials Science and Engineering, Physical Science and Engineering Division, King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia;1. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02142, USA;2. Department of Intelligent Precision Healthcare Convergence, Sungkyunkwan University, Suwon 16419, Korea;3. Department of Biomedical Engineering, Sungkyunkwan University, Suwon 16419, Korea;4. Division of Gastroenterology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA;5. Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;6. Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA;1. Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education and Wuhan University School of Pharmaceutical Sciences, Wuhan 430071, China;2. Department of Endocrinology, Zhongnan Hospital of Wuhan University, Wuhan 430071, China |
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| Abstract: | A system has been developed for the in vitro synthesis of RNA from nucleoside triphosphates in morphologically intact rat liver nuclei in a medium of low ionic strength. It requires all four nucleoside triphosphates (NTP's), Mg2+ (or possibly Mn2+), and is stimulated by NaF, NH4+, an ATP generating system, and several oligoamines. Spermidine or other oligo-, di-, or monoamines are required for the preservation of nuclear morphology at the incubation temperature; immediate nuclear lysis results from agitation of the assay mixture in their absence. Sucrose, sorbitol, Mg2+, or monovalent ions at higher ionic strength cannot substitute for the amine, while NH4+ shows only a slight stabilizing effect.Incorporation is inhibited by RNase, DNase, actinomycin, Mn2+, deletion of one NTP, Ca2+, and calf thymus histones. Potassium chloride and NaCl have no effect at moderate concentrations but lead to nuclear lysis at high ionic strength. Mercaptoethanol, sonication, trypsinization, or puromycin treatment of the nuclei do not affect the incorporation. The system is highly active (7 mμmoles GMP incorporated/ mg DNA-P in 5 minutes), and functions over a broad pH range from about 7.8 to 9.5.A stimulation of incorporation in vitro is observed if the fluorinated steroid hormone triamcinolone diacetate is injected into the intraperitoneal cavity of normal rats 3–8 hours before sacrifice. This stimulation by hormone is similar in magnitude to one observed by means of an in vivo pulse of 14C-orotate. The optical density profiles obtained from sucrose density gradients of RNA isolated from the nuclei after in vitro incubations and after in vivo pulses are similar, but the radioactivity profiles and double labeling experiments indicate extensive degradatation of RNA with s values greater than 16 during the in vitro incubations. Differences in radioactivity profiles of RNA from control and hormone-treated rats after short pulses of 14C-orotic acid can be interpreted in terms of stimulation of RNA anabolism by the hormone.No stimulation of incorporation has as yet been obtained by in vitro incubation of the nuclei with triamcinolone. |
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