This thesis is a comparative study of gene arrangements in Pseudomonas species, and is organized into three major sections. The first section compares gene arrangements for different pathways in Pseudomonas aeruginosa PAO1 to determine if the gene arrangements are similar to previous studies. It also serves as a reference for pyrimidine gene arrangements in P. aeruginosa. The second part compares the physical, and genetic maps of P. aeruginosa PAO1 with the genome sequence. The final section compares pyrimidine gene arrangements in three species of Pseudomonas. Pyrimidine biosynthesis and salvage genes will be aligned for P. aeruginosa PAO1, P. putida KT2440, and P. syringae DC3000. The whole study will gives insight into gene patterns in Pseudomonas, with a focus on pyrimidine genes.

The objective of this research was to study the pyrimidine salvage pathways of Rhizobium. Three approaches were used to define the pyrimidine salvage pathways operative in two species of Rhizobium, R. meliloti and R. leguminosarum . The first approach was to ascertain the pyrimidine bases and nucleosides that could satisfy the pyrimidine requirement of pyrimidine auxotrophs. Uracil, cytosine, uridine or cytidine all satisfied the absolute pyrimidine requirement. The second approach was to select for mutants resistant to 5-fluoropyrimidine analogues which block known steps in the interconversion of the pyrimidine bases and nucleosides. Mutants resistant to 5-fluorouracil lacked the enzyme uracil phosphoribosyltransferase (upp ) and could no longer use uracil to satisfy their pyrimidine requirement. Mutants resistant to 5-fluorocytosine, while remaining sensitive to 5- fluorouracil, lacked cytosine deaminase (cod) and thus could no longer use cytosine to satisfy their pyrimidine auxotrophy. The third approach used a reversed phase HPLC column to identify the products that accumulated when cytidine, uridine or cytosine was incubated with cell extracts of wild type and analogue resistant mutants of Rhizobium. When cytidine was incubated with cell extracts of Rhizobium wild type, uridine, uracil and cytosine were produced. This Indicated that Rhizobium had an active cytidine deaminase (cdd) and either uridine phosphorylase or uridine hydrolase. By dialyzing the extract and reincubating it with cytidine, uridine and uracil still appeared. This proved that it was a hydrolase ( nuh ) rather than a phosphorylase that degraded the nucleoside. Thus, Rhizobium was found to contain an active cytidine deaminase and cytosine deaminase with no uridine phosphorylase present. The nucleoside hydrolase was active with cytidine, uridine and to a far lesser extent with purines, adenosine and inosine. When high concentrations of cytidine were added to mutants devoid of hydrolase, cytosine was produced from cytidine - 5-monophosphate by the sequential action of uridine ( cytidine ) kinase and nucleoside monophosphate glycosylase. Both ft meliloti and ft leguminosarum had identical salvage pathways.

Cells of Clostridium oroticum, an anaerobic bacterium, were grown on orotate as a carbon and energy source. Ureidosuccinase, an inducible enzyme in the pathway for pyrimidine degradation has been shown to convert ureidosuccinate to aspartate, C02 and NH3 as reported by Liebenmm and Kornberg (7). Aspartate and C02 were formed in approximately a 1: 1 ratio from ureidosuccinase activity. Ureidosuccinase was found to be a Mn+2 requiring enzyme with a pH optimum of approximately pH 6.5. Enzyme activity is labile to 02, temperature, pH, dilution and high ionic strength. The optimum conditions for storage of ureidosuccinase were found to be mixtures which contained Mn+2, PO4-3 and ureidosuccinate (the substrate of the enzyme) at -20°C in argon gassed serum vials. Hydantoinase, an enzyme which converts ureidosuccinate to 5'-carboxymethylhydantoin in a ratio of two 5'-carboxymethylhydantoin to one ureidosuccinate, was present in cells grown on orotate. Hydantoinase was able to convert both the D and the L isomers of ureidosuccinate to carboxymethylhydantoin. High levels of a Mg+2 dependent carbamyl phosphate kinase were found in extracts from cells grown on orotate. The activity was dependent upon addition of ADP or AMP to the reaction mixture. The pH optimum of the carbamyl phosphate kinase was approximately pH 6.5. Cells grown on orotate contained high levels of an induced system for the degradation of pyrimidines whereas cells grown on glucose contained a constitutive level of enzymes for pyrimidine biosyntheses. Low levels of ureidosuccinase, carbamyl phosphate kinase and hydantoinase were found in glucose grown cells. Cells grown on glucose showed a high level of a Mn+2 dependent aspartate transcarbamylase (the analogous enzyme to ureidosuccinase which is operative during pyrimidine biosynthesis) which forms ureidosuccinate from aspartate and carbamyl phosphate. A consideration of the energy and reducing power supply and demand was made for cells grown both on glucose and orotate as carbon and energy source.

Pyrazolopyrimidines are a general class of compounds which exhibit Aj adenosine receptor affmity. A number of pyrazolo(3,4-d)pyrimidine analogues of isoguanosine and i-methylisoguanosine has been synthesised. All compounds were tested forAi adenosine receptor affinity using a (311) R-PIA competitive binding assay. The N-i and N-5 positions were substituted with a number of different ailcyl and aryi groups. 3-Chiorophenyl substitution of the N-i position and butyl substitution of the N-5 position greatly enhanced the overall adenosine receptor affinity. Substitution by a methyl group at the N-7 position fixed the C-4 position in the imino tautomeric form. This resulted in a marked reduction in activity. The substitution of the N-2 position with a phenyl group produced an analogue with a similar structure to i,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX). A 2-phenyl substituent was favourable for interaction with the adenosine receptor. A number of pyrazolo(3,4-d)pyrirnidine analogues of 4,6-bis-a-carbamoylethylthio-i-phenylthiopyrazolo(3,4-d)pyrinhidine (DJB-KK) has also been synthesised and tested for Aj adenosine receptor affinity. 4,6-Bis-alkylthio-1-phenylpyrazolo(3,4-d)pyrimidines with a-carbamoylethyl and u-carbamoylpropyi groups were compared. The additional methyiene of the a-carbamoylpropyl group produced increased adenosine receptor affinity. 6-a-Carbamoylethylthio-4-mercapto-1-phenylpyrazolo(3,4-d)pyrimidine and 4-cc-carbamoylethylthio- i-phenylpyrazolo(3,4-dlpyrimidine were compared. Substitution of the C-6 position maintained activity, while substitution of the C-4 reduced activity.

Thesis (PhD)--University of South Australia, 2003.

Thesis (Ph. D. in Chemistry)--Vanderbilt University, Dec. 2007. / Title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Accompany CD-ROM contains html files that illustrate in 3-D the structures discussed in Chapter 1. Typescript. Vita. Includes bibliographical references. Also available on the Internet but lacking CD-ROM contents.

Thesis (Ph. D.)--University of Missouri-Columbia, 2003. / Accompany CD-ROM contains html files that illustrate in 3-D the structures discussed in Chapter 1. Typescript. Vita. Includes bibliographical references. Also available on the Internet but lacking CD-ROM contents.

No description available.

To further understand the ATCase/DHOase bifunctional complex formed in Streptomyces, the genes encoding these and other pyrimidine enzymes were identified and characterized. Polymerase chain reaction (PCR) was utilized in this effort. Primers were constructed by selecting conserved regions of pyrimidine genes from known gene and protein sequences of a wide variety of organisms. These sequences were then optimized to Streptomyces codon usage. PCR products were obtained from internal sites within pyrimidine genes and also from primer combinations of different genes. The size, orientation, and partial sequence of the resulting products shows that Streptomyces has a gene organization of pyrR followed by pyrB, pyrC, carA, carB, and pyrF in an operon similar to that found in other Gram-positive bacteria.