In this study, 6 bacterial SSBs are investigated which have been obtained from 4 different <i>Shewanella</i> strains, from <i>Aquifex aeolicus</i> and from <i>E. coli.</i> The 4 <i>Shewanella</i> SSBs have been taken from strains isolated at different depths of the ocean, from sea-level down to 8600m. These organisms therefore differ in their growth pressure optima. By comparing the characteristics of each of these proteins, differences will lead to clues which relate to the pressure differences. In order to highlight the different adaptations of these proteins, the thermophilic SSB from <i>Aquifex aeolicus</i> and the mesophilic SSB from <i>E. coli </i>were used as benchmarks to the piezophilic <i>Shewanella </i>SSBs. Circular dichroism was used to determine proportions of secondary structure present in each SSB and these were compared to the values obtained from previous crystallography work on the <i>E. coli, </i>in order to get some preliminary details about each structure. Further biophysical work was carried out using ITC and DSC which provided thermodynamic data regarding the binding between ssDNA and SSB, and also probed the denaturation temperatures of each protein. Exhaustive crystallisation trials were carried out on each <i>Shewanella</i> SSB but unfortunately did not produce any crystals of sufficient quality. As AqSSB had previously been crystallised, the structure determination is described in this study. To complement the binding data, a crystal of AqSSB in complex with ssDNA was produced and its structure determined. The structure showed great similarities with the several previously published structures of EcoSSB. Therefore this study is focussed on SSB proteins from bacteria isolated form very different habitats. By comparing their various structural and biophysical properties, further clues as to how piezophilic proteins are able to survive extreme pressures may be gained.

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<i>Campylobacter jejuni</i> is the most commonly recognized cause of bacterial gastroenteritis in man and also infects cattle, sheep and poultry. Publishing of the genome sequence of <i>Campylobacter jejuni 11168</i> (Parkhill, 2000) revealed the presence of only one cytochrome P450. Its coding sequence (Cj411c) is located in an operon involved in sugar and cell surface biosynthesis. The gene name is Cj1411c, is 1359 bp long and encodes 453 aa. The sequence is strictly conserved in <i>Campylobacter jejuni</i> RM221. Recombinant P450 was expressed in <i>E. coli</i> and showed the 450 nm peak in the presence of CO indicating the correct folding. The protein was partially purified to about 70% purity. By deleting the P450 gene from the <i>Campylobacter jejuni 11168</i> genome clear changes in cell morphology were identified, cells becoming wider and shorter. The capsular sugar profile of the NC1 knockout strain reveals the presence of arabinose which was not found in the wild type strain. The arabinose was identified by both HPLC and NMR. The phenotype studies showed clear differences between NC1 and WT cells: NC1 cells are less resistant to starvation in the stationary phase; by exposure to the atmospheric oxygen 36.47% of the wild type cells survived after 24 hours and only 16.61% of the NC1 cells survived; by growing the NC1 cells in competition with the WT cells the growth rate of NC1 cells approximately 10x lower than the WT; NC1 cells were proved to be less resistant to high temperature, more resistant to low temperatures and pH. By analysing the results obtained with NaC1 and glycerol we have determined that it is not the osmotic pressure that is affecting the growth of <i>Campylobacter </i>and the differences between the WT and NC1 strain might be related with changes in cell surface components.

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p21-activated kinase (paklp) is essential in fission yeast and plays roles in cell polarity and mating. The <i>pakl-34 </i>mutant has a specific mutation that does not affect essential functions but causes highly penetrant defects in cell polarity and morphology. The <i>pakl-34 </i>strain has a specific defect in bipolar growth and the potential to help dissect the role of paklp in mediating cell polarity. Tagging experiments demonstrated that both wild type and pakl-34 proteins localize to cell tips and septa. I hypothesised that pak-34p may be deficient in kinase activity. I adapted a two-dimensional gel electophoresis approach, called Difference Gel Electrophoresis (DIGE), to screen for paklp substrates. Wild type and <i>pak1 </i>mutant strains were compared in this manner and differential proteins identified by mass spectrometry. General results showed very few differences between wild type and <i>pakl-34 </i>strains and huge differences between wild type and <i>pak1 </i>kinase-dead strains, indicating that the kinase-dead strain may not be suitable for dissecting the paklp mechanism. Specific results identified hxklp as a potential substrate but it was generally concluded that the DIGE approach may not have sufficient sensitivity and/or scope for the screening of paklp substrates. In parallel to the hypothesis-driven DIGE approach I attempted to find paklp substrates by a candidate approach, investigating the phosphorylation state of a paklp regulator, ral3p. I found differences in ral3p phosphorylation, between wild type and <i>pak1</i>mutants, by SDS­-polyacrylamide gel electrophoresis and lambda phosphatase treatment. I also looked for differences in the sizes of paklp and pakl-34p complexes using sucrose gradients. This study (1) describes a new way to screen for novel protein kinase substrates in fission yeast and (2) suggests that hxklp and ral3p are substrates for paklp.

The structural gene encoding cytochrome c<SUB>5</SUB> has been cloned and sequenced, along with some surrounding sequence. The inferred amino acid sequence of the cloned gene, <I>scyA</I>, corresponds to a mature protein of 78 amino acids with a single haem attachment motif situated toward the N-terminal end of the protein; a methionine residue near the C-terminus serves as the sixth haem ligand. The <I>scyA </I>open reading frame contains a 21 amino acid N-terminal extension which is absent in purified cytochrome <I>c<SUB>5</SUB></I>. This sequence conforms to the format of a typical periplasmic signal sequence. Two additional open reading frames were identified on analysis of the regions flanking the structural gene, neither of which is functionally related to cytochrome <I>c<SUB>5</SUB></I>. Northern blott analysis confirmed that <I>scyA </I>is transcriptionally isolated. A null mutant which lacked the gene coding for cytochrome <I>c<SUB>5</SUB></I> was constructed. The anaerobic respiratory capacity of the resultant strain was assessed and compared to wild-type. No obvious mutant phenotype was identified. Gene disruption experiments were also used to characterise cytochrome <I>c<SUB>3</SUB></I>. Deletion strains lacking the gene coding for cytochrome <I>c<SUB>3</SUB></I> (<I>cctA</I>) and also strains lacking both cytochrome <I>c<SUB>3</SUB></I> and flavocytochrome <I>c<SUB>3</SUB></I> were constructed. Comparison of the growth characteristics of the mutant strains with wild-type suggest the involvement of cytochrome <I>c<SUB>3</SUB></I> with respiratory iron (III) reduction. Ferrozine extraction experiments similarily demonstrated a decrease in iron (III) reduction activity by strains lacking the cytochrome <I>c<SUB>3</SUB></I> gene. In order to facilitate further study of cytochrome <I>c<SUB>5</SUB></I>, and production of recombinant forms of the protein, an expression system was developed. Cytochrome <I>c<SUB>5</SUB></I> was successfully expressed in <I>Shewanella frigidimarina</I> NCIMB400 by using the expression vector pMMB503 which is inducible with isopropylthio-β-D-galactoside.

RFC plays an essential role in DNA replication by loading the sliding clamp PCNA onto DNA in order to tether DNA polymerase δ to DNA. RFC consists of five subunits, one large subunit and four small subunits. The large subunit of RFC contains an extended C-­terminal domain that is not present in the small subunits and whose function remains unknown. In addition to RFC, eukaryotic cells contain two more putative PCNA loaders known as RLCs. These other PCNA loaders have similar structures to RFC and contains the RFC small subunits, however the large subunit is replaced with a different protein, either Elg1 or Ctf18. The function of the three PCNA loaders is not clear. In this work the function of the Rfcl C-terminal domain (CTD) was examined. The analysis of an Rfcl CTD deletion mutant showed that the domain is essential for cell viability. <i>rfcl-44, </i>a temperature-sensitive mutant with a mutation in the C-terminal domain, displayed sensitivity to DNA damaging agents, abnormal chromosome structure and a synthetic lethal phenotype when combined with DNA replication mutants. <i>rfc5 </i>mutants were isolated as suppressors of <i>rfcl-44 </i>suggesting that the defect in <i>rfcl-44 </i>may be in the Rfcl-Rfc5 interaction. Ctf18, Dccl and Ctf8, components of Ctf18-RLC, were required for the viability of <i>rfc1­-</i>44 whilst Elg1 was not. Deletion of Elg1 restored the viability of <i>rfc1-44 ctf18</i><i>Δ</i><i> </i>double mutant cells, suggesting that Elg1 plays a negative role. The negative role of Elg1 was confirmed by over-expression of Elg1 in <i>rfc1-44 </i>cells showing a lethal phenotype at permissive temperature. These results suggest that RFC plays a key role in DNA replication and that Elg1-RLC and Ctf18-RLC can play negative and positive roles respectively when RFC function is impaired.

The <I>SDB23</I> gene of <I>Saccharomyces cerevisiae</I> was isolated in a search for high copy-number suppressors of mutations in a cell cycle gene, <I>DBF2. SDB23</I> encodes a 21,276 Da protein with significant sequence similarity to characterised mammalian and yeast snRNP core proteins. Examination of multiple sequence alignments of snRNP core proteins with Sdb23p indicates that the amino-termini of all these proteins share a number of highly conserved residues, and identifies a novel motif characteristic of snRNP core proteins. In contrast, the carboxy-termini of these proteins diverge considerably. An exceptionally hydrophilic, asparagine-rich carboxy-terminus distinguishes Sdb23p from the characterised snRNP core proteins, which tend to have an abundance of arginine and glycine residues. Sdb23p is essential for cell growth and is required for nuclear pre-mRNA splicing both <I>in vivo</I> and <I>in vitro</I>. Extracts prepared from Sdb23p-depleted cells are unable to support splicing and have vastly reduced levels of U6 snRNA. The stability of U1, U2, U4 and U5 spliceosomal snRNAs is not affected by the loss of Sdb23p. Overexpression of U6 snRNA can partially compensate for the loss of Sdb23p, indicating that one role of this protein is to stabilise free U6. However, the partial nature of this suppression would suggest that Uss1p has an additional, as yet uncharacterised role. Based on <I>in vitro</I> and <I>in vivo</I> characterisation of <I>SDB23</I>, this gene encodes a novel U6 snRNA-associated polypeptide, related to the mammalian snRNP core proteins, that is essential for nuclear pre-mRNA splicing and U6 snRNA stability. Sdb23p appears to represent a previously unidentified class of U6 snRNP-associated core-like proteins. For this reason, <I>SDB23</I> has been given the more logical name <I>USS1</I> (<I>U-S</I>ix <I>S</I>nRNP), as its original name was based on its uncharacterised suppressor activity.