Nadjati, Amira (1978) Studies on nuclear RNA polymerases and the role of cyclic nucleotides on transcription. PhD thesis, University of Warwick.

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Abstract

1. An assay for measuring the endogenous RNA polymerase activity in Tetrahymena pyriformis nuclei was developed. The reaction rate was linear for 60 minutes at 25°C. This rate of the reaction was dependent on the presence of four nucleoside triphosphates (ATP, GTP, CTP and UTP), Mn⁺⁺ or Mg⁺⁺ ions, and was optimal at high concentrations of KC1 or (NHO₄)₂80₄, (0.15 M).
2. α-amanitin (a specific inhibitor of RNA polymerase II) was used to differentiate between the different classes of nuclear RNA polymer­ases, RNA polymerase I and RNA polymerase II. 3.6 x 10⁻4 M α-amanitin inhibited approximately 601 of total RNA polymerase activity in nuclei isolated from logarithmically growing cells. Thus about two-thirds of the nuclear RNA polyiserasa activity was due to RNA polymerase II.
3. Cyclic AMP or dibutyryl cyclic AMP stimulated nuclear RNA polymerase activity at physiological concentrations (10⁻⁶ - 10⁻⁸ M). Maximum stimulation was obtained using 10⁻⁷ M cyclic AMP or 10⁻⁷ M dibutyryl cyclic AMP. The increase .in activity stimulated by cyclic AMP in isolated nuclei was dependent on salt concentration. Cyclic AMP stimulated endogenous RNA polymerase I and inhibited endogenous RNA polymerase II activity. Chromatin-bound RNA polymarase activity was also stimulated by this cyclic nucleotide.
4. Cyclic CMP or dibutyryl cyclic GMP in the presence of CaClg stimulated nuclear RNA polymerase activity at physiological concentrations (10⁻⁸8 - 10⁻10 M). Maximum stimulation of nuclear RNA polymerase activity by cyclic GMP in the presence of 2 mM CaCl2 occurred at 10“10 M cyclic nucleotide. 10”8 M dibutyryl cyclic GMP plus 2 mM CaCl2 produced maximum stimulation. The stimulation was CaCl2 dependent.
5 No significant stimulation of nuclear RNA polymerase activity was observed with 5'-AMP, 5'-GMP or 3',5'-cyclic CMP.
6. Chromatin-bound cyclic GMP and cyclic AMP phosphodiesterases (degrading enzymes of cyclic GMP and cyclic AMP respectively) were demonstrated to occur in Tetrahymana pyriformia nuclei.
7. The levels of total SNA polymerase activity were determined during the course of the natural cell cycle. By using α-amanitin, approximately two-thirds of the activity during the S phase of the cell cycle was found to be due to RNA polymerase II. RMA polymerase I activity was predominant in the G2 phase of the cell cycle.
8. An assay for endogenous protein kinase activity in Tetrahymena pyriformia nuclei was developed and validated. The cyclic AMP- dependent phosphorylation of nuclear proteins was found to be a salt- dependent process. Dibutyryl cyclic AMP stimulated the phosphorylation of the endogenous protein (s) and.exogenous substrates in a.partially purified nuclear fraction -containing RNA polymerase activity by 5-fold.

Item Type:	Thesis or Dissertation (PhD)
Subjects:	Q Science > QD Chemistry
Library of Congress Subject Headings (LCSH):	RNA, Tetrahymena pyriformis, RNA polymerases
Official Date:	February 1978
Dates:	Date Event February 1978 Submitted
Institution:	University of Warwick
Theses Department:	Department Chemistry
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Swoboda, Bennett Edward Paul
Extent:	138 leaves
Language:	eng

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