Small Nuclear RNA Sythesis in Isolated Mouse Erythroleukemia Cell Nuclei
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A set of small nuclear RNA.sp~cies (snRNA) in eukaryotic cells have been described. Their cellular function and method of formation is unknown. SnRNAs are found predominately in the nucleus and have been - shown to consist of 9-14 discrete species (1). An in vitro isolated nuclear system capable of synthesizing this class of RNA would greatly facilitate the study of l) the RNA polymerase(s) responsible for their. synthesis and pos$ible derivation from other classes of nuclear RNA, 2) transport from the nucleus and 3) post transcriptional modifications such as methylation and capping. Mouse erythroleukemia cell nuclei were capable of synthesizing RNA linearly for 70 minutes. A labeled ribonucleoside triphosphate was . ' incorporated into· RNA at a rate of 7 pmols/~gnuclear DNA/50 minutes. . " Maximum synthetic activity required the presence of high concentrations of all four ribonucleoside triphosphates (1 mM), a low incubation temperature (25°C), ~nd 5 x 107 nuclei/ml. The effect of a-amanitin on total RNA synthesis was examined. RNA polymerases A, B, and C were functioning in nuclei isolated from MEL cells in mid-log phase. The RNA synthesized .:!.!!_ vitro sedimented in sucrose gradients at approximately 4 to 20S. The synthesis of two species of rapid turn-over snRNA (C and A) was· detected, one with the same electrophoretic mobility as 5S rRNA and one with that of 4.5S pre-tRNA. Indications of the synthesis qf snRNA species D, E, arid F were observed as was the synth~sis ·of snRNA species· ~~, which unlike D, E; and F can be recovered from both cytoplasmic and nuclear cell fractions .
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Dissertation