Thesis (Ph. D.)--University of Wisconsin, 1965. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Adenine phosphoribosyl transferase is a ubiquitous enzyme which salvages endogenous adenine, via the nucleotide AMP, for use by the cell. This activity, in conjunction with other interconnected purine salvage mechanisms is an energy-efficient way for the cell to satisfy its purine requirements. APRT is a target molecule in certain human diseases, for chemotherapeutics, and in vivo mutagenesis studies. There is little known about structure-function relationships in APRT. In the absence of solved three-dimensional crystal structures, we have explored structure-function relationships in APRT by sequence comparison, in vitro mutagenesis and kinetic analysis, protein crosslinking, and in vivo selection of mutant enzymes with altered substrate affinities. Chinese hamster APRT shares identifiable sequence similarities to all other phosphoribosyl transferases, and many other nucleotide binding proteins, in regions which probably serve closely similar functions across diverse protein families. Predicted secondary structures of CHO APRT are very similar to other APRT molecules, and to a lesser degree to other phosphoribosyl transferases. Residues of part of the generalized nucleotide binding motif of APRT were found to have specific roles in binding substrate, which can be extrapolated to the same functional elements in other nucleotide binding proteins. In addition, mutants identified by selection for altered substrate affinities are widely dispersed in the primary sequence. Although APRT is thought to exist as a dimer in its native context, certain mutants of APRT which have impaired ability to form dimers appear to have near-wildtype activity. / Graduate

No description available.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1950. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 66-70).

by Shinichi Watanabe. / Vita. / Thesis (M.S.) - Florida State University. / Bibliography: leaves 87-96.

No description available.

DNA adenine methyltransferase (Dam) plays an important role in different bacterial functions. It has been shown that Dam is required for regulation of bacterial replication initiation and is required for proofreading newly synthesized DNA through methylation directed mismatch repair. Dam is also involved in the regulation of different genes and is required for virulence in several different bacterial genera though its degree of importance depends on the specific bacteria being studied. During this work, a Dam-negative strain (JSM1) was constructed in Klebsiella pneumoniae strain 43816 to ascertain its importance for K. pneumoniae viability and virulence. To test JSM1 for expression of fimbrial virulence factors, agglutinations were used to detect the presence of type three and type one fimbriae, respectively. No differences between 43816 and JSM1 were discernable. Similarly, JSM1 production of capsular material appeared to be unaltered. K. pneumoniae JSM1 virulence in a murine model was examined following intranasal or intraperitoneal inoculation, and it was determined that JSM1 is partially attenuated. Quantitative analysis of 43816 and JSM1 biofilm growth revealed only slight decreases in JSM1 biofilm mass and thickness, but live/dead staining of developed biofilms showed decreased JSM1 biofilm viability over time compared to 43816 biofilms. JSM1 was also examined for alterations in the frequency of spontaneous antibiotic resistance mutations and tested for increased susceptibility to various DNA damaging agents, and statistically significant differences were found for some of the spontaneous antibiotic resistance mutation frequencies tested. Fimbriae in K. pneumoniae are important virulence factors which facilitate respiratory and urinary tract infections in vivo. They also contribute to formation of biofilms which are believed to cause chronic infections and increased antibiotic resistance. Searches for homologous regions within the Klebsiella chromosome using the chaperone and usher components of E. coli type 1 fimbriae revealed five putative fimbrial gene clusters on the Klebsiella chromosome which had not been characterized. Mutations created within select gene clusters did not yield detectable deficiencies in biofilm formation or murine respiratory virulence. However, based on the multiplicity of fimbrial expression observed in Salmonella enterica serovar Typhimurium, combinational mutations may be required prior to detection of a discernable phenotype.

The cytoplasmic enzyme, NAD⁺ kinase (ATP: NAD⁺ 2-phosphotransferase, [E.C. 2.7.1.23}) has been characterized and purified from yeast. A continuous fluorescence assay was developed. A purification procedure was developed utilizing NAD⁺-Agarose affinity column chromatography.

No description available.

No description available.