This Ph.D. work represents the study of nucleic acid interactions of two zinc finger proteins: mammalian Wilms' tumor suppressor (WT1) and Xenopus transcription factor IIIA proteins (TFIIIA). WT1 is a putative transcriptional regulatory protein which is inactivated in a subtype of Wilm s' tumours. Using selection and amplification binding assay (SAAB) we determined that the highest affinity binding sites for WT1[-KTS] consist of a 12 base pair sequence GCG-TGG-GCG-(T/G)(G/A/T)(T/G). These sequences have a four-fold higher affinity for the protein than the nonselected sequence GCG-TGG -GCG-CCC , as measured by a quantitative nitrocellulose filter binding assay. The effects of Denys-Drash syndrome (DOS) point mutations on the DNA binding activity of WT1 were determined. SAAB assay revealed that none of the DDS mutant proteins give rise to a new sequence specificity. One mutation, R394W abolishes specific binding of the protein. The remaining mutations result in reduced DNA- binding activity, ranging from 1.4 to 14-fold, which suggests that even small changes in DNA-binding activity may precipitate the clinical phenotype of Denys-Drash syndrome. Comparative analysis of the DNA binding characteristics of Wilms' tumour and Early growth response proteins was conducted . The stoichiometry of the DNA-protein complexes, their stability to dissociation, and the effects of pH, temperature and salt concentration on the equilibrium binding of these proteins to their cognate DNA sequences have been determined. Under the conditions of 0.1 M salt, p H 7.5, and 22 * C WT1-ZF has an apparent dissociation constant (Kd) of 1.14± 0.2 X 10⁻⁰⁹ M, and EGR-1 protein has a Kd of 3.55 ± 0.4 x 10⁻⁰⁹ M. In addition, we tested relative contribution of each base pair in the consensus binding site to the high affinity binding by point mutational analysis, and identified important differences that exist in the binding modes of the two proteins. Transcription factor IIIA controls the expression of the 5S ribosomal RN A genes during development of Xenopus laevis., and specifically interacts with both 5S DNA and 5S rRNA molecules. The present study assesses contributions of the central zinc fingers four through seven to specific DNA and RNA binding activities of the protein. The results demonstrate that each zinc finger in the zf 4-7 region contributes to both the high affinity DNA and RNA interactions: the largest effect on TFlllA-DNA binding (10-fold) wa s produced when zinc finger 5 of TFIIIA was replaced with the donor sequences of either p43 or WT1. However, while all the zinc fingers 4-7 contribute to the high affinity 5S rRNA binding, substitution of an α- helical portion of zinc finger 6 with the equivalent sequences from WT1 abolished RNA-binding activity of TFIIIA, suggesting that zIinc finger 6 p lay s a particularly im portant role in binding to RNA. / Graduate

Nucleic acid metabolism has been studied in vitro with suspensions of Novikoff rat hepatoma cells. The formation of acid soluble and nucleic acid purines from formate-C¹⁴ and adenine-8-C¹⁴ has been measured. In all cases, adenine had a higher specific activity than guanine. The acid soluble purines were much more radioactive than the nucleic acid purines. Adenine-8-C¹⁴ gave a higher value for the ratio of the specific activities of adenine to guanine nucleotides than that obtained from formate-C¹⁴. Both ribonucleic acid and deoxyribonucleic acid incorporated radioactivity in this system. A standardized set of conditions suitable for testing the activity of possible chemotherapeutic and inhibitory agents was established, and 4 such compounds were examined. Azaserine in low doses was extremely effective in inhibiting de novo purine biosynthesis. 6-Mereaptopurine also blocked de novo synthesis of the purines. A new possible antimetabolic compound; N-benzoylglycinamidine was tested. In low amounts, N-benzoylglycinamidine stimulated purine biosynthesis, but a large dose decreased both purine synthesis and respiration. The Novikoff tumour cell suspension was used to gain some knowledge of the mode of action of actinomycin D. This antibiotic inhibited cellular respiration, but not anaerobic glycolysis. Actinomycin D decreased nucleic acid biosynthesis and acid soluble guanine synthesis, but did not affect the formation of acid soluble adenine. Large amounts either of calcium pantothenate or of coenzyme A were able to reverse the inhibition of nucleic acid metabolism, and to reverse partially the inhibition of respiration. Coenzyme A was more effective than pantothenate. These findings appear to support the suggestion that actinomycin D interferes with coenzyme A-dependent reactions. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Isotopic adenine, labeled with C¹⁴ in position 2, has been prepared in three steps; (a) formylation of 4-amino - 5-imidazolecarboxamidine in aqueous C¹⁴-formic acid, (b) ring closure of the resulting formamido compound to form 2-C¹⁴-adenine, (c) purification by sublimation in vacuo. The overall yield for the three operations was 60 percent. Proof of identity of the adenine prepared in this manner was obtained from the preparation of a derivative, combustion analysis, paper chromatography and ultraviolet spectrophotometry. The metabolism of 2-C¹⁴-adenine was studied in the adult male rat. The labeled compound was administered to the experimental animals by intraperitoneal injection. The isotope of the administered adenine was found distributed in the purines of the visceral nucleic acids, the expired carbon dioxide and urine, where part of the activity was found in both urea and allantoin. Nucleic acid adenine and guanine were synthesized to the extent of 7.7 percent and 5.5 percent respectively from administered 2-C¹⁴-adenine. The adenine renewal is lower than similar values derived from 1,3-N¹⁵-adenine as reported in the literature. Expired carbon dioxide was found to contain over 8 percent of the administered isotope. Combustion analyses of whole urine indicated that 28 percent of the administered isotope was contained therein. Urea and allantoin together accounted for 16-29 percent of the total radioactivity in urine. The presence of radioactive carbon dioxide in the expired air of the experimental animals, when considered in the light of other evidence, is regarded as being indicative of a biological lability in position 2 of the purines. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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