Thesis (M.S.)--University of Wisconsin--Madison, 1944. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [22]-24).

Thesis (M.S.)--University of Wisconsin--Madison, 1961. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 37-39).

Thesis (M.S.)--University of Wisconsin--Madison, 1944. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaf 21).

Thesis (M.S.)--University of Wisconsin--Madison, 1962. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 38-41).

Thesis (Ph. D.)--Lehigh University, 1896. / Manuscript. Includes bibliographical references (leaves 1-4). Also available online.

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

The purpose of this investigation was the development of a direct method of investigating the electrical conductance of ampholytic solutions in high electric fields. This was achieved with the use of a cathode ray oscilloscope, which recorded the conductance as a function of the field strength over a wide range. A maximum field of 180,000 volts per centimeter was applied. It was shown that aliphatic ampholytes, such as glycine, valine, etc., in aqueous solution, exhibit a considerable increase of conductance with rising field strength. Further investigations, especially with polypeptides, are planned. The consequences of this investigation for the biophysical problem of permeability in living matter are discussed. / Science, Faculty of / Physics and Astronomy, Department of / Graduate

Stereoselective 9-step conversions of the ketone 17 into the tricyclic ketone 31 via two similar synthetic pathways are described. The highly stereoselective steps involved in the preparation of 31 were: a) the Lewis acid-catalyzed reaction of the silyl enol ethers 50 and 60 with the ethylene ketal of 3-buten-2-one to produce the diketones 37 and 39, respectively, b) the hydrogenation of the enone 32 to give the ketone 56, and c) the Birch reduction of the enone 61 to give 31. Compound 31 contains the correct relative configuration at each of the corresponding chiral centers present in the target molecule 16 and, therefore, appears to be an ideal intermediate for a projected total synthesis of (±)-16 (initially believed to be the sesterterpenoid suvanine). A 15-step total synthesis of the antimicrobial diterpene (±)-8-isocyano-10(14)-amphlilectadiene (23) from the intermediate 24 is described. The key steps in the synthesis of (±)-23 involved the stereoselective Birch reduction of 24, the epimerization of the aldehyde 91 to the corresponding a-formyl isomer, and the degradation of the carboxylic acid function of 95 to an isonitrile group. The last part of the synthetic work described in this thesis resulted in a 15-step conversion of the ketone 17 into the tricyclic ketone 164. Of particular note in this sequence of reactions are: a) the palladium(0)-catalyzed coupling reaction of the enol triflate 143 with lithium cyanide to produce the nitrile 144, b) the stereoselective alkylation of 144 to give compound 148, and c) the stereoselective Birch reduction of the enone 163 to produce 164. The methodology employed in the construction of the tricyclic intermediate 164 contributes to the development of a general route towards the synthesis of the spongiane diterpenes 18-22. [formula omitted] / Science, Faculty of / Chemistry, Department of / Graduate