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UBC Theses and Dissertations

Use of benzoylated deae-cellulose for the isolation of glycine transfer ribonucleic acids of yeast and for the development of new methods for sequence determination of nucleic acids Warrington, Robert Charles

Abstract

Part I of this thesis describes a method for the isolation of a specific family of aminoacyl-tRNAs. Reaction of the aminoacyl-tRNAs with the N-hydroxysuccinimide ester of 2-naphthoxyacetic acid produces N-2-naphthoxyacetylaminoacy1-tRNAs which possess sufficient affinity for BD-cellulose to allow their separation from unmodified tRNA by simple chromatographic steps. The N-2-naphthoxyacetyl amino acid is removed by mild alkaline hydrolysis and the iso-acceptor tRNAs are separated by chromatography on BD-cellulose. tRNA₁Gly and tRNA₂Gly have been purified from brewer's yeast (Saccharomyces cerevisiae) by this method and a simplified procedure for the large scale isolation of these tRNAs was developed. The object of Part II of this thesis was to develop a new method of nucleotide sequence analysis based on the following steps: (i) specific introduction of hydrophobic groups onto the 3'- or 5'-terminals of polynucleotides, (ii) subsequent cleavage of the appropriately derivatized polymer under conditions which favor rupture of only one bond per molecule of polymer, (iii) separation of fragments bearing the derivative from those not bearing the derivative, (iv) resolution of derivatized fragments according to chain length, (v) complete degradation of the purified fragments from step (iv) with the reagent used to generate these fragments followed by identification of the components so liberated from each fragment, and finally, (vi) assembly of the data obtained from step (v) to order the composite fragments from 5' and 3' ends to derive an unambiguous primary sequence. It was reasoned that N-2-naphthoxyacetylglycyl-tRNA₁Gly and tRNA₂Gly could be used as a model polymer bearing an appropriate 3’-derivative, that BD-cel1u1ose chromatography could effect step (iii), that DEAE-cel1ulose-7 M urea column chromatography could effect step (iv) and that either DEAE-cellulose chromatography or combined chromatographic-electrophoretic techniques could effect step (v). A method for placing a hydrophobic derivative onto the 5’-terminus of polynucleotides needed to be developed. Further, it was necessary to discover if available endonuc1eases could degrade the model polymer in a manner approaching random, single-hit kinetics. To ascertain the feasibility of the proposed method, two studies were undertaken. First, the 3'-terminal fragments released by exhaustive digestion of N-2-naphthoxyacetylglycyl-tRNA₁Gly and tRNA₂Gly with RNase T₁ were isolated by the procedure to verify the efficiency of a number of the required steps. The fragments (from the results of the nucleostide-analysis and on the assumption that the 3'-sequence cytidy1y1(3’-5’)cytidy1y1 (3’5’adenosine is common to all functional tRNAs) are (Cp, Ap)CpCpA and (Up, Ap, Cp, Cp) CpCpA. The relative yields of the two fragments suggested they were derived from tRNA₁Gly and tRNA₂Gly, respectively. Second, a study was undertaken of the degradation products liberated from crude and purified glycyl-tRNAs as well as from crude and purified N-2-naphthoxyacety1g1ycy1-tRNAs by RNase T₁ (and, to a lesser extent, by pancreatic RNase). RNase T₁ was found to degrade crude glycyl-tRNA with a product distribution suitable for testing the feasibility of the method. It was not possible, however, to label specifically the glycyl-oligonucleotides with a hydrophobic group in the presence of other degradation products. Fragmentation patterns suitable for execution of the proposed procedure were not obtained with crude or purified N-2-naphthoxyacetylglycyl-tRNAs with either RNase T₁ or with pancreatic RNase. Studies on the pH-dependent activity of RNase T₁ degradation of tRNA showed, in addition to the expected optimum at pH 7-5, a second pH.optimum at pH 4.5. The latter activity was dramatically augmented by the presence of 7 M. urea. Studies on the specificities of several RNase T₁ preparations under various conditions showed the presence of substantial levels of adenosine as an end-group of tRNA degradation products. Preliminary results of a search for reagents capable of adding specifically an aromatic residue onto the 5'-terminus of polynucleotides demonstrated that reacting either 2-naphthyl-phosphoromorpholidate or 2, 4-dinitrof1uorobenzene with tRNA resulted in increased binding between the modified tRNAs and BD-cel1ulose.

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