Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Cell ghosts have been prepared from mature chicken erythrocytes using 0.05% saponin. Such preparations provide a system, where the nucleus is permeable to nucleotides and macromolecules, for studying the low level RNA synthesis characteristic of these cells. Although several fold lower than that in HeLa cell nuclei, the ('3)HUTP incorporation in the erythrocyte can be detected using high specific activity isotope and assaying on DEAE filters with extensive washing to reduce background. RNase A (50 (mu)g/ml) and KOH (0.3M) eliminate all radioactivity binding to filters, indicating the product is RNA. Preincubation with DNase I (10 (mu)g/ml) or actinomycin D (10 (mu)g/ml) inhibits UTP incorporation by about 70%, suggesting that the synthesis is DNA dependent. Maximum synthesis occurs in the presence of high salt (0.175 M KCl) and Mn('++) (0.5 mM), and is inhibited 90% by 0.1 (mu)g/ml of (alpha)-amanitin. Polyacrylamide gel electrophoresis indicates that the newly synthesized RNA is heterogeneous in size, having a distribution from 5-60 S. Approximately 20% of the total RNA is bound by an oligo(dT)-cellulose column suggesting that some RNA processing is occurring, although attempts to detect the incorporation of label from (alpha)-('32)PGTP into a 5'-cap structure were unsuccessful. Hybridization of radiolabeled RNA to excess chicken DNA indicates that the majority (80%) of the in vitro transcripts which hybridize are complementary to unique sequence DNA (C(,o)t(, 1/2) = 5 x 10('3)). When RNA synthesized by either erythrocyte or reticulocyte nuclei is hybridized to globin cDNA, about 10% of the reticulocyte RNA but less than 1% of the erythrocyte RNA is resistant to RNase A digestion. Taken together, these data suggest that nuclei, prepared by saponin lysis of chicken erythrocytes, synthesize messenger-like RNA via endogenous polymerase II activity. A fraction of this RNA is also polyadenylated, but contains few, if any, globin sequences. The erythrocyte system was further developed by determining conditions for the optimal uptake of proteins and the effect of various extracts on reactivation of transcription. Uptake is optimal at Mg('++) or Mn('++) concentration greater than 2.5 mM, at a pH of around 5, and in the absence of KCl. No stimulation of transcription was observed for a number of extracts tested.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Paul Allen Wiersma
Wiersma, Paul Allen, "Synthesis of messenger-like RNA by isolated avian erythrocyte nuclei " (1984). Retrospective Theses and Dissertations. 7805.