The bacteriophage PRD1 is a lipid bearing phage that infects a wide variety of gram-negative bacteria, including Escherichia coli and Salmonella typhimurium when they harbor the appropriate plasmid. It contains a linear duplex DNA molecule that is covalently bound by its 5' ends to a terminal protein. Like adenovirus and the Bacillus phage φ29, PRD1 specifies its own DNA polymerase which is able to utilize the phage encoded terminal protein to prime DNA synthesis. In addition to these two proteins, PRD1 also specifies an additional replication protein (p12) of unknown function. We have sequenced the origins of replication (termini of the genome) as well as the right most 1700 bp of the bacteriophage PRD1 genome. The right most 1700 bp encompasses the right early region and completes the sequence of all PRD1 early functions. We report here that the PRD1 genome contains a perfect 111 bp inverted terminal repeat. Furthermore, statistical analyses of the right 1700 bp, as well as the examination of transcription and translation signals has allowed us to assign gene XII to an open reading frame and to infer the direction of both early and late transcription. Gene XII, which has been implicated in the replication process and the regulation of gene expression is predicted to encode a 16.7 Kdal protein. Data base searches have revealed a possible evolutionary relationship between this protein and the ε-subunit of E. coli DNA polymerase III. We have also mapped right, early transcription of the PRD1 genome. This has corroborated our inference concerning the direction of right early transcription and confirmed our assignment of gene XII to an open reading frame. It has also revealed that two putative rho-independent terminators are functional in vitro and that the putative right early promoter is utilized in vivo and in vitro. The data presented here have permitted us to ascertain the general genomic and transcriptional organization of PRD1 and to predict the primary structure of the product of gene XII. These results, in turn, have allowed us to develop hypotheses concerning the evolution of linear, protein primed DNA's and the function of gene XII.

Graduation date: 1992

The development of new and effective vaccines and immunodiagnostic reagents requires the characterisation of antigenically relevant proteins and their interactions with the products of the immune system. Phage display technology was investigated as a means of elucidating some of the antigenic properties of the rickettsial parasite, Cowdria ruminantium (Cowdria). Randomly fragmented gene-derived libraries have been useful in elucidating viral and other epitopes, but only limited work has been done with entire genomes. A phage display library expressing a repertoire of Cowdria peptides was constructed. It was sufficiently large to represent the organism's genome, but lacked phages displaying peptides coded for by genes containing a Pvu II restriction enzyme site, including the one coding for the major antigenic protein 1 (MAP1). This was considered advantageous since MAP1 is immunodominant and has already been well characterised. Affinity selection with antibodies against Cowdria proteins other than MAP1 allowed several antibody-reactive peptides to be isolated. These selected sequences were placed in the context of the genome by screening a lambda bacteriophage library and by comparison with Cowdria DNA sequences. Apart from showing that antigenic mimics were present in the phage display library, six open reading frames encoding putative Cowdria proteins were identified. All had similarities to, or motifs in common with, membrane proteins and are thus likely to be exposed to the host's humoral immune system. Some of the proteins identified were larger than the antigens used to elicit the antibodies used for selection, probably as a result of the presence of cross-reactive epitopes. Despite limitations experienced when extending a fragmented-gene approach for epitope location to genomes, it was possible to identify an antigenic region on MAP1 by comparison with selected mimics. In addition, binding peptide sequences were identified with two monoclonal antibodies that had been raised against non-Cowdria antigens. An epitope on the VP7 protein of bluetongue virus was identified and peptides were found that reacted with a monoclonal antibody directed against malignant catarrhal fever virus. Thus, apart from being able to identify several potentially important Cowdria epitopes and genes, the fragmented-genome library holds promise as a universal reagent for identifying useful mimics.

Includes bibliography / 219 leaves, [22] leaves of plates : ill ; 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, 1987

Bibliography: leaves 94-104

Graduation date: 2000

Includes bibliographical references / 183, [111] leaves, [6] leaves of plates : ill ; 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, 1990

Bibliography: p. 90-96 / v, 96 p. [70] leaves [26] leaves of plates : ill ; 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, 1984

The nucleotide sequence of the PRD1 terminal protein gene has been determined. The coding region for PRD1 terminal protein is 777 base pairs long and encodes 259 amino acid residues (29,326 daltons). The deduced amino acid sequence of PRD1 terminal protein reveals no overall homology with other known terminal proteins or related proteins. A closer examination revealed a highly conserved amino acid sequence, YSRLRT, exist among all identified DNA terminal proteins including PRD1, PZA, Nf, φ29 and adenovirus. This is the first conserved amino acid sequence that has been found in all identified DNA terminal proteins. Not only is the YSRLRT sequence conserved, but its spatial location is similar as well. Therefore, the significance of the YSRLRT conserved sequence is suggested by both its conservative spatial location and high degree of homology across species. To study the structure-function relationship of the YSRLRT sequence of PRD1 terminal protein, in vitro site-directed mutagenesis was performed to determine the role of each amino acid in this conserved region. The PRD1 terminal protein and DNA polymerase genes were cloned into phagemid pEMBLex3, and the recombinant plasmid used for constructing mutants. Eleven PRD1 terminal protein mutant clones were examined for their priming complex formation activities. Our results have strongly demonstrated that the positive charge residue of arginine-174 plays an important role for PRD1 terminal protein function. There are 13 tyrosine residues in the predicted PRD1 terminal protein. It was of interest to known which tyrosine is actually linked to terminal nucleotide of the PRD1 DNA. We used a new approach involving replacing the tyrosine residues with phenylalanine residues in the carboxyl terminal portion of the protein. From analyses, the tyrosine-190 has been determined to be the most likely linkage site between terminal protein and PRD1 DNA.

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy