No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Bacteriophage T4 encodes three polynucleotide ligases that seal phosphodiester backbones during infection of E. coli, referred to as T4Dnl, T4Rnll and T4Rn12. A detailed biochemical examination of the nucleic acid substrate specificity of each ligase was performed using recombinant proteins and a variety of double stranded substrates comprised of a mixture of DNA and RNA, with a single nick. The RNA ligases sealed a broad range of substrates, with the activity of T4Rnll being 50-1000 fold less than that of T4Rn12. Mutagenesis identified regions of the RNA ligases that are important for nick-joining activity. T4Dni had a greater specificity than both RNA ligases, since it was not active on some substrates containing RNA. All proteins joined a double stranded substrate with the 3'-hydroxyl group at the nick being RNA, but with the 5'phosphate group at the nick and the complementary strand being DNA. The conserved use of this substrate questions whether it may be important during nucleic acid metabolism. In assays under identical conditions, the rates of nick-joining by all three recombinant ligases were best at 37°C. For all substrates, optimal1igation was at pH 8.0 for T4Dni and T4Rnll, and pH 7.0 for T4Rn12, therefore showing that the pH optima of ligation is determined by the protein. Changes to buffer conditions showed that all three proteins are dependent upon ATP and Mi+ in order for ligation to occur, although Mn2+ can provide an alternative to Mg2+. Furthermore, Ml+ and ATP along with DTT influenced intrinsic fluorescence emission by the proteins, suggesting that these constituents influence the structure of the proteins. The genome of Streptomyces avermitilis encodes a possible RNA ligase, which was cloned and purified for biochemical analysis. The recombinant protein exhibited weak nick joining activity, although further study is required to provide a better insight into this novel nucleic acid repair process in bacteria.

No description available.

The M13 bacteriophage is ~1 μm long, with a capsid comprised of five coat proteins. Small peptides can be displayed upon the coat proteins. The aim of this work was to use the phage display of small peptides for the purpose of fabricating a self-assembling molecular transistor with the M13 bacteriophage acting as the scaffold. Each aspect of this fabrication was considered. Firstly, a 50 nm long microphage particle was re-created and used to produce a 215 nm long phage particle. These smaller phage particles could be used to make the transistor 100 nm in size. To create the scaffold, selenocysteine was displayed on the coat protein pIII and used to self-assemble two to four phage particles around 10 nm gold nanoparticles or quantum dots. These higher order structures were then purified using a linear sucrose gradient. So that the transistor could be directed to a specific area of an existing electronic circuit, the coiled-coil pair ACID:BASE was used. ACID was displayed on the coat protein pIX whilst a BASE peptide containing a Cvterminal cysteine was immobilised onto a gold surface. It was shown that ACID and BASE form a coiled-coil when the BASE is immobilised on a surface and that the M13 bacteriophage, displaying ACID, can be anchored to the surface via coiled-coil formation. Finally, tyrosine was displayed on the major coat protein pVlIl so that gold could be specifically reduced onto the M13 phage particle scaffold to create the gold electrodes of the transistor. Although within solution, and on a carbon surface, gold was reduced onto the phage particles, when the particles were adsorbed onto an Si02 surface there was no gold deposition. Therefore, although much progress was made towards the goal of a self assembling transistor, the aspects devised within this study need to be combined.

Members of the genus Campylobacter are a major cause of food-borne disease worldwide. They can colonize the intestinal mucosa of poultry, to high levels leading to contamination of meat, at slaughter. Their numbers can be reduced in different ways including chicken treatment with bacteriophages. For such treatments to be successful, in depth understanding of the bacteriophage that infects and kills campylobacters is vital. The work in this thesis describes: isolation and comprehensive characterisation of bacteriophage candidates for future therapy applications. In order to increase the available stocks of characterized candidate bacteriophage, a number of attempts were made to isolate bacteriophages from poultry excreta. The new isolates together with some uncharacterized phages from our laboratory stocks were characterized with respect to their host range and genomic size. Some bacteriophages preparations in previous studies showed genomes of different sizes and a number of attempts were done for their separation. This raised questions about the relationship between the two different sized genomes. Prior to this work, a co isolate pair had been successfully separated and the sequence of the larger genome, CP220, was determined. Part of the work here, was performed to extend this study by obtaining the sequence of the smaller co isolate, CPX and compare it to CP220. They did not appear to have any identifiable relationship at the genetic level, but the availability of the CPX sequence will further extend our knowledge of bacteriophage genetics and this phage has clear therapeutic potential. Attempts were also made to separate and characterize a second co-isolate pair but these were unsuccessful. The availability of the DNA sequence of CP220 allowed a much closer molecular characterisation and comparison of Campylobacter phage genomes, than had previously been possible. One area that was investigated in this study was the presence of repeat regions identified in the CP220 genome, which were amplified by PCR, but could not be cloned in E. coli. Furthermore, genes encoding potential lysins were identified in the CP220 genome and they were amplified, cloned and attempts were made to express the proteins, which may have potential therapeutic value. One gene product was successfully expressed and showed evidence of lytic activity on Campylobacter and other bacterial genera. In summary, this thesis describes a much closer examination of molecular biology of Campylobacter bacteriophage than had previously been possible, including the determination of the sequence CPX phage.