xix, 246 leaves : photos., graphs, tables, diagr. (fold. in end pocket) ; 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, 1979

xxi, 278 leaves : photos, tables, graphs ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.1978) from the Dept. of Biochemistry, University of Adelaide

Bibliography: leaves 120-138. / xi, 138, [41] leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Characterises the 186 coliphage site-specific integration reaction in vitro with the expectation that it could be used in mammalian systems to insert genes or modify existing ones, to provide an alternative method for the production of transgenic livestock species. The analyzed system is the temperate bacteriophage 186. The in-vitro requirements for 186 integrative site-specific recombination were investigated. In vivo investigations were conducted whereby active 186-intasomes were microinjected into fertilised mouse eggs containing genomic copies of 186-attB. The 186 system has not been shown to work in vivo. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry 1999?

Clostridium difficile is an important hospital-acquired pathogen causing C. difficile-associated diarrhoea (CDAD) in patients exposed to antibiotics. The lack of information on bacteriophages of C. difficile, and the potential of phages as therapeutic agents for the treatment of CDAD, prompted the isolation and characterisation of phages active against clinical isolates of C. difficile in order to determine the prevalence and significance of phages of this anaerobe. Three (5.4 %) of 56 clinical C. difficile isolates induced by mitomycin C yielded dsDNA phages C2, C5, C6 and C8. The four phages differed from previously described C. difficile phages in particle morphology, burst size and host range. C2, C5 and C8 particles were members of the family Myoviridae, while C6 belonged to Siphoviridae. The burst sizes were 5 for C2, 7 for C5, 19 for C6 and 33 for C8. C8 had the broadest host range, lysing 27 out of 56 (48 %) C. difficile isolates, followed by C6 (43 %), C5 (20 %) and C2 (20 %). Superinfection experiments, restriction enzyme analysis and Southern hybridisation showed C2 and C5 to be closely related with C8 somewhat related to them, however, C6 was distantly related to the other three phages. C2 was further characterised as a representative phage. Its genome did not possess cohesive ends, and was shown to integrate chromosomally via an attP site identified within a 1.9 kb HindIII fragment. However, an integrase gene, which is typically close to the attP region, was not located. Nine of 16 HindIII fragments of C2, including the 1.9 kb fragment, were cloned into pUC18. Approximately 9 kb of the estimated 43 kb genome of C2 was sequenced and analysed. Seven of the nine translated sequences were homologous to phage structural proteins, two sequences were not homologous to any relevant protein in the Genbank and EMBL databases, and one was homologous to proteins of Clostridium species. Nucleotide homology between the C2 sequences and the recently sequenced C. difficile strain CD630 was found in three regions within CD630 genome. Seven of the nine sequences, including the 1.9 kb fragment, were clustered in one region. These data suggest that the genes constitute a phage structural gene module. The presence of C2-like sequences in CD630, and Southern hybridisation of C. difficile strains using phage probes, suggested related prophage sequences may be commonly present in this bacterial species. An investigation was carried out to determine the presence of toxin genes tcdA and tcdB, and PaLoc-associated gene tcdE, in phage DNA. In addition, the effect of phage infection on toxin production of toxigenic C. difficile strains was studied. Of the three genes, tcdE only was detected in phages C2, C5 and C8, but not in C6. Strains that maintained phages in a stable manner (lysogens) were isolated and used in toxin studies. The amount of toxin B produced was measured by cytotoxic assays using Vero cells, and toxin A production was measured by ELISA. Although phages did not encode toxin A or B genes, there was a significant increase in toxin B production in some lysogens. There was no increase in toxin A production. Transcriptional analyses of tcdA and tcdB in lysogens and parental strains was performed by real-time RT-PCR and Northern hybridisation to determine whether phage was affecting regulation of toxin transcription. Phage did not appear to affect toxin gene transcription, although results from real-time RT-PCR and Northern hybridisation were conflicting. A phage induced from the highly toxigenic reference strain VPI 10463 was also briefly characterised and investigated for its effect on toxin production in VPI 10463. The phage, ΦCV, had similar particle morphology to C2, C5 and C8, and had some HindIII bands in common with C2 and C5. Two cured variant strains produced significantly less toxin B compared to VPI 10463. In conclusion, several important properties of C. difficile phages were characterised. It appears these temperate phages may play a role in toxin production making them unsuitable as therapeutic agents for the treatment of CDAD. However, C2 phage may have potential as the basis for an integrative vector that will add to the genetic tools available for clostridia.

The initial aim of this project was to characterise the integrative recombination mechanism of bacteriophage ÖAR29 , to provide a better understanding for development of the shuttle plasmid pBA as a site-specific Bacteroides integration vector. RT-PCR showed that the previously identified ÖAR29 recombination genes, integrase (Int) and excisionase (Xis), were transcribed from pBA in E. coli SCS110, B. thetaiotaomicron AR29 and B. uniformis AR20. In silico derived amino acid sequences from both genes showed only very low levels of similarity to other known Int and Xis in GenBank. To improve understanding of the phage recombination system, the ÖAR29 genome was sequenced. This revealed a 35,558 bp double-stranded DNA genome with GC content of 39.11%. Bioinformatic analysis identified 53 open reading frames (>30 codons) and gene promoters and terminators that allowed the genome arrangement to be compared with other phages. Comparison of deduced gene products with proteins from other phages identified 6 reading frames, allowed tentative identification of 7 others, but left 40 ORFs unidentified. Those with strong homology to known genes were: large terminase subunit (44.66 kDa), dnaC (27.94 kDa), helix-turn-helix (HTH) transcription regulator (14.69 kDa), cI repressor (26.48 kDa), amidase (18.42kDa) and a novel integrase (54.22 kDa). The integrase gene is located 162 base-pairs downstream of the phage attachment (attP) core site, rather than the previously suggested location upstream of the integration site. The ÖAR29 attP was shown to include a 16-bp att core region, 117 bp upstream of the previously suggested location. Integration of ÖAR29 was found to occur at the 3’end of an arg-tRNA gene on the AR29 genome (attB). Imperfect direct repeats with a consensus sequence (ANGTTGTGCAA) were found surrounding the attP core. A review of pBA sequence showed that only the 5’ end (435 bp) of the newly identified Int gene was cloned in pBA. Despite this, PCR analysis revealed integration of pBA into the AR29 genome. Serial subculturing of pBA transformed AR29 was able to cure AR29 of the ÖAR29 prophage, providing an improved host for integrative plasmids, and for detailed studies of AR29 physiology and ÖAR29 life cycles.

Thesis (Ph. D.)--University of Adelaide, Dept. of Biochemistry, 1984. / Includes bibliographical references (p. 90-96).

Thesis (Ph. D.)--University of Adelaide, Dept of Biochemistry, 1987. / Includes bibliographical references.

Thesis (Ph. D.)--University of Adelaide, 1986. / Includes bibliographical references.

Thesis (M.S. in veterinary science)--Washington State University, May 2008. / Includes bibliographical references (p. 13-19).

Diss. (sammanfattning) Stockholm : Tekniska högskolan, 1999. / Härtill 5 uppsatser.