Thymidine kinase is a key enzyme in the nucleotide salvage pathway, catalyzing the production of dTMP from thymidine and ATP. In order to identify the structural features of the TK protein and/or primary amino acid sequences which contribute to the catalytic and regulatory activities of this enzyme, an in vitro transcription and translation system has been used in concert with protein engineering techniques for the production and phenotypic characterization of mutant and wild-type TK enzymes. Because of discrepancies in the literature regarding the quaternary structure of the VVTK, the native molecular weight and quaternary structure was determined to be an 80kDa homotetrameric enzyme by glycerol gradient fractionation, gel filtration and glutaraldehyde cross-linking analyses. Computer-assisted alignment of the predicted amino acid sequences derived from cellular and poxvirus TK genes identified seven highly-conserved domains distributed throughout the VVTK polypeptide (domains I-VII). Domain I (amino acid residues 11-18 ) exhibits a high degree of similarity to both ATP and GTP binding site consensus sequences, although the VVTK utilizes only ATP as a phosphate donor. Site directed mutagenesis and ATP-agarose affinity chromatography techniques were employed to confirm that this region was responsible for ATP binding and to determine which amino acids were essential for efficient binding. The TK gene (tdk) from E. coli was isolated and sequenced to serve as a prokaryotic enzyme with which to compare VVTK. The alignment revealed only 23% shared identity with VVTK and, interestingly, the identical and similar residues were clustered into three of the seven domains identified previously (domains I, III and VII). Preliminary evidence supports domain III (residues 78-90) as a putative magnesium binding site and that a highly conserved cysteine residue (cysteine 170) within domain VII (residues 168-171) may be a component of the catalytic site. Secondary structure alignment between Herpes Simplex Virus (HSV) TK and monkeypox TK (a close relative of VVTK) revealed that the putative nucleoside binding site of HSVTK aligns with residues within domain IV. Replacement of a VVTK residue (Q114) with the corresponding residue of HSVTK (an aspartic acid) greatly alters the substrate specificity and dTTP sensitivity of VVTK. / Graduation date: 1991

No description available.

Thesis (Ph.D.) -- University of Adelaide, Dept. of Applied and Molecular Ecology, 2001. / Corrigendum inserted at the back. Includes bibliographical references (leaves 133-158).

Corrigendum inserted at the back. Includes bibliographical references (leaves 133-158). Ch. 1. General introduction -- ch. 2. General materials and methods -- ch. 3. Biological characterisation of Australian PSbMV isolates -- ch. 4. Developing nucleic acid based diagnostics for PSbMV -- ch. 5. Detection of PSbMV isolates by RT-PCR and RFLP analysis -- ch. 6. Developing an internal control for PSbMV RT-PCR -- ch. 7. Molecular analysis of the PSbMV VPG -- ch. 8. PSbMV sequence and phylogenetic analysis -- ch. 9. General discussion Sixteen pea seed borne mosaic virus (PSbMV) isolates were collected between 1995 and 1998. These isolates were biologically distinct yet serologically indistinguishable. The conclusion is that PSbMV is widespread and occurs at a low incidence in Australia. Reports sequence information on new isolates of PSbMV which has allowed genomic regions to be identified which distinguish PSbMV pathotypes and isolates; and, to the development of PSbMV nucleic acid hybridisation and RT-PCR assays.

Corrigendum inserted at the back. / Includes bibliographical references (leaves 133-158). / xvi, 158 leaves : ill., col. map ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Sixteen pea seed borne mosaic virus (PSbMV) isolates were collected between 1995 and 1998. These isolates were biologically distinct yet serologically indistinguishable. The conclusion is that PSbMV is widespread and occurs at a low incidence in Australia. Reports sequence information on new isolates of PSbMV which has allowed genomic regions to be identified which distinguish PSbMV pathotypes and isolates; and, to the development of PSbMV nucleic acid hybridisation and RT-PCR assays. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 2001

vii, 121 leaves : ill., graphs, tables ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1981

Thesis (Ph. D.)--University of Adelaide, 1983. / Includes bibliographical references (leaves 116-117).

Thesis (Ph.D.) -- University of Adelaide, Dept. of Biochemistry, 1981.

Thesis (Ph. D.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 160-180). Also available on the World Wide Web.

Bibliography: leaves 116-117.