The Prp2 protein of <I>Saccharomyces cerevisiae </I>is a 100kDa non-snRNP splicing factor with amino acids common to the DEAD/H-box family of ATP-dependant RNA helicases. Prp2, Prp16 and Prp22 proteins have extensive sequence similarity, and function at sequential steps in the splicing pathway. Prp2 and Prp16 form transient reactions with the spliceosome, Prp2 at step 1 and Prp16 at step 2. In addition to the conserved motifs of the DEAD/H-box family, Prp2 also contains a sequence resembling a zinc finger motif. This sequence has been extensively mutagenised, and two mutations were found by <I>in vivo </I>complementation assays to affect the function of Prp2. In order to study the molecular interactions of Prp2 at step 1, several dominant negative <I>PRP2</I> alleles had been generated previously in this laboratory by site-directed mutagenesis. When overexpressed from an inducible promoter, these cause accumulation of pre-mRNA in the presence of wild-type <I>PRP2. </I>These dominant negative alleles and wild-type <I>PRP2 </I>were subjected to deletion analyses. In the case of one dominant negative mutant which has a GKT-AKT change in the ATP-binding motif, some dominant activity was retained despite the removal of 19kDa from the protein. It has been suggested that the Prp16 protein plays a proof-reading role in splicing, as a mutant of <I>PRP16, prp16-1,</I> suppresses an A-C change of the branch nucleotide. A mutation analogous to the <I>prp16-1</I> allele was created in <I>PRP2 </I>to determine if Prp2 plays a role in influencing the fidelity of splicing. Although no effect was found for this and other mutants of <I>PRP2, </I>it was discovered that overexpression of wild-type <I>PRP16</I> had an effect similar to that of the <I>prp16-1 </I>mutant on the fidelity of splicing. A new hypothesis is proposed for the mechanism of suppression of branchpoint mutants by Prp16 whereby the Prp16 protein might mediate the binding of an unknown proof-reading factor to the branchpoint.

BACKGROUND: FtsZ is an essential cell division protein in bacteria. It is involved in the initiation, timing, and frequency of cell divisions. Consequently it is subject to strict regulation, including transcriptional and post-transcriptional control and protein inhibition. Regulation of <i>ftsZ</i> expression by an antisense RNA has also been proposed. Dewar and Donachie suggested that there is a gene <i>stfZ</i> encoded by the antisense strand of DNA at the <i>ftsA-ftsA </i>gene junction in the <i>mra </i>gene cluster of the <i>Escherichia coli</i> chromosome. Multiple copies of the DNA fragment containing the <i>stfZ</i> gene block cell division. Since an RNA transcribed from <i>stfZ</i> would be complementary to the ribosomal binding site of <i>ftsZ</i>, it has been proposed that the product of <i>stfZ</i> is an antisense RNA that controls translation of <i>ftsZ</i>. RESULTS: To see if there are other antisense genes in the <i>mra</i> gene cluster of the <i>E. coli</i> chromosome, we looked for antisense promoters and terminators in the region. While no promoters have been identified, all the DNA fragments that have been tested had strong termination activity. Northern blotting, RT-PCR, and primer extension experiments confirm that there is an RNA produced from the antisense strand of DNA at the <i>ftsA-ftsZ</i> gene junction of <i>E. coli</i> cells. RT-PCR experiments indicate that the StfZ RNA is at least 423 nt long. Overexpression of an artificial RNA containing a part of the StfZ sequence lowers FtsZ concentration in cells, prevents FtsZ ring formation, and inhibits cell division. Experiments with fusion constructs show that the overexpressed RNA acts on the ribosomal binding site (RBS) of <i>ftsZ</i>. Other experiments using the fusion constructs indicate that the expression from the RBS of <i>ftsZ</i> is strongly dependent on the growth phase of cells. CONCLUSIONS: The antisense RNA StfZ is produced in <i>E. coli</i> cells. It is very likely that StfZ controls translation of <i>ftsZ</i>, and that it does this by binding to the RBS of the <i>ftsZ</i> mRNA.

This thesis has focused on the regulation of the gene encoding the essential DNA replication protein Proliferating Cell Nuclear Antigen (PCNA). PCNA expression in <I>Plasmodium falciparum </I>had earlier been shown to be stage specific during development in the intraerythrocytic cycle. Here its expression was examined in more detail. In addition, a preliminary analysis of the regulation of the gene encoding DNA polymerase δ, of which PCNA is an auxiliary factor, was made. Antisera raised against <I>P. falciparum </I>PCNA (PfPCNA) and DNA polymerase δ (PfPolδ) have been used against extracts from synchronised parasites to show that both proteins accumulate in trophozoites and persist in schizonts. The steady-state transcripts from both PfPCNA and PfPolδ also accumulate at the trophozoite stage. However, nuclear run on analysis shows that, whereas PfPolδ promoter activity is absent in rings but present in trophozoites and schizonts, the PfPCNA promoter is active throughout the intraerythrocytic cycle. This suggests that mechanisms regulating the expression of these two genes may be different although their coordinated activity is required for DNA replication. A major transcription start site 960bp upstream of the translational start of the PfPCNA coding sequence was identified. A second, minor, site is situated a further 40bp upstream. These results were developed from a number of methods to reduce the possibility of ambiguities which may arise due to the extreme AT-richness of <I>P. falciparum </I>non-coding DNA. Consensus TATA boxes and an OCT-1 box were identified upstream of the putative transcription start sites. Analysis of cDNA clones identified a putative transcription stop site 250bp downstream of the stop codon. Intraerythrocytic stages were successfully transfected with constructs containing a firefly luciferase reporter gene under the transcriptional control of variously modified elements of the PfPCNA 5' flanking sequence to test the effect on promoter activity.

As part of a larger study to identify genes and gene products expressed <I>in vivo</I> by <I>Mycobacterium avium </I>subsp. <I>paratuberculosis, </I>two immunogenic clones designated S4 (1.6 kb) and S8 (3 kb) were extracted from a λgt11 genomic expression library of <I>M.a. paratubeculosis </I>by screening with serum from a sheep with clinical Paratuberculosis (Johne's disease). The clones were shown by antibody elution and DNA sequencing to contain fragments of the same 1.1 kb open reading frame (ORF), which encoded a native protein of approximately 34 kDa in <I>M.a. paratuberculosis. </I>The ORF also encoded a possible signal peptide from amino acids 1-39, suggesting the native protein is secretory. Database searches using the deduced amino acid sequence of the ORF identified a motif 'GDSGG' which displayed 100% homology to the residues surrounding the active serine in a trypsin-like serine protease. An overall homology of approximately 30% was detected with the HtrA proteins of <I>Escherichia coli, Salmonella typhimurium, Bruncella abortus, Rochalimea henselae, </I>and <I>Campylobacter jejuni, </I>which are also thought to be serine proteases. The S8 DNA insert contained the entire 1.1 kb ORF, but due to the presence of the signal peptide, the recombinant could not be expressed as a fusion protein. However the insert was successfully expressed as a free protein by translational coupling, and was found to be secreted into the <I>E. coli</I> periplasm, confirming the presence of a signal sequence. When several species of mycobacteria were screened with the 1.1. kb ORF only members of the <I>M. avium </I>complex (<I>M. avium, M.a. paratuberculosis, M.a. silvaticum, M. intracellulare</I> and <I>M. scrofulaceum </I>were found to contain the gene, although <I>M. malmoense </I>and <I>M. marinum </I>reacted weakly and may therefore contain homologous genes. The native 34 kDa protein encoded by these clones therefore appears to be a novel secretory serine protease specific to the <I>M. avium </I>complex of mycobacteria. However, no proteolytic activity has been demonstrated by either native or recombinant proteins.

Green fluorescent protein (GFP) was used as a tool to examine the interactions between pairs of bacterial species. A plasmid encoding GFP from <I>Aequorea victoria</I> was transformed into strains of <I>Enterobacter agglomerans (EntGFP) </I>and <I>E. coli </I>ATCC 11229 (<I>E. coliGFP</I>). UV illumination of plates enabled species to be identified in a mixture; their spatial distribution was examined by UV microscopy; and fluorescence measurements were used to quantify adhesion and inhibition of adhesion of the strains to other cells or cell components. Cooperation between <I>EntGFP </I>and <I>Klebsiella pneumoniae G1 (KlebG1) </I>resulted in enhanced biofilm formation and alteration of dual species biofilm properties. <I>E. coliGFP</I> and <I>Serratia marcescens 87b (Serr87b) </I>stably coexisted in biofilms but did not affect the growth of each other. The other bacterial partnerships examined were competitive, with the end result that one species dominated the biofilm. Microscopic examination of <I>EntGFP</I> and <I>KlebGI</I> dual species biofilms showed that the two species were often closely juxtaposed in microcolonies, suggesting the interactions involved surface associated macromolecules. They appeared to directly interact through adhesin/receptor interactions and proteins were isolated which could form the basis of their specific interactions. In addition, EPS affected coadhesion non-specifically. Compared to single species biofilms, both species in cooperative dual species biofilms had increased resistance to disinfectants and antibiotics. Neutral dual species biofilms of <I>E. coliGFP </I>and <I>Serr87b </I>did not show increased resistance to disinfection. Therefore, the enhanced resistance was related to the specific interactions and disruption of the cooperative partnership though the integration of a third species, <I>Serr87b, </I>led to a decrease in resistance. The methods developed provide a convenient technique for the examination of mixed species biofilm communities where the unique interactions between species determine the true properties and resistance of the resultant biofilms.

No description available.

PRP8 protein is a component of the nuclear pre-mRNA splicing machinery. The Prp8p had previously been identified as a component of the U5 snRNP, U4/U6, U5 tri-snRNPs of the spliceosome, which contacts the catalytic centre of the spliceosome, as detected by photochemical crosslinks of Prp8p with functional important region of the U5 and U6 snRNAs, and with the 5’ splice site, 3’ splice site, and branchpoint of pre-mRNA. In this thesis, I present further work on protein interactions between Prp8p and other splicing factors using Prp8p partitioned strains. The Prp8p partitioned constructs allowed investigation of factors that associate with the N- or C-terminal regions of Prp8p by isolation of the sub-complexes associated with different Prp8 protein fragments. Thus, the U5 snRNP protein Snu114p associates with Prp8p in the region 437-770 whereas fragmenting Prp8p at residue 2173 destabilises its association with Aar2p. The role of Prp8p on U5 snRNP assembly was investigated. Upon the identification of nuclear localisation signal of Prp8p, I further investigated the role of Prp8p in U5 snRNP assembly, and showed that Prp8p-Snu114p-Aar2p-U5 complex formation happened in cytoplasm, and Prp8p-Snu114p-Brr2p-U5 association of the complex took place in the nucleus. Despite the fact that Prp8p is nuclear localised, the association between Prp8p and Brr2p is disrupted by Prp8p C-terminal mutations that resemble human Prp8p mutations in result of retinitis pigmentosa type 13. In addition, I also identified the importin, Kap95p, is needed for Snu114p, and snRNAs nuclear import. In this thesis I also characterised a Prp8p co-purified protein, Spp382p, and results indicate that Sp382p is needed in the late-stage of splicing.

RNase MRP is a ribonucleoprotein particle (RNP) with endoribonucleolytic activity which is involved in the processing of pre-rRNA at site A<SUB>3</SUB>. A genetic screen for mutants which are synthetic lethal (sl) with a temperature sensitive (ts) mutation in the RNA component of RNase MRP (<I>rrp2-1</I>) has been performed in order to identify new gene products which physically and/or functionally interact with RNase MRP. Analysis of the obtained sl mutant strains led to the identification of a new and essential gene, <I>POP3</I>. Depletion of Pop3p <I>in vivo</I> results in a phenotype characteristic of the loss of RNase MRP activity; A<SUB>3</SUB> cleavage is inhibited, leading to under-accumulation of the short form of the 5.8S rRNA (5.8S<SUB>S</SUB>) and formation of an aberrant 5.8S rRNA precursor which is 5' extended to site A<SUB>2</SUB>. Pop3p depletion also inhibits pre-tRNA processing. Primary tRNA transcripts accumulate, as well as spliced but 5' and 3' unprocessed pre-tRNAs. The Pop3p depletion phenotypes resemble those previously described for mutations in components of RNase MRP and RNase P (<I>rrp2-1, rpr1-1 </I>and <I>pop1-1</I>). Immunoprecipitation of epitope tagged Pop3p efficiently co-precipitates the RNA components of both RNase MRP and RNase P. Thus, Pop3p is a common component of both RNPs and is required for the function of both enzymes <I>in vivo</I>. Strain SL158 carried a mutation in <I>HAL2</I> which is sl in combination with <I>rrp2-1. </I>Hal2p is an enzyme that converts pAp (adenosine 3', 5' bisphosphate), a product of sulfate assimilation, into 5' AMP and P<SUB>i</SUB>.

Sigma-70 is the subunit of <i>Escherichia coli</i> RNA polymerase which confers specificity for initiation of transcription at most promoters in actively growing cells. The protein shares four conserved regions with major and many alternative sigmas within all studied prokaryotes. However, a stretch of 245 amino acids lying between conserved regions 1 and 2, is absent from all minor sigmas and even from the similar and functionally homologous sigma-43 of <i>Bacillus subtilis</i>. Deletion of these 245 residues from sigma-70 generates a protein which retains significant <i>in vitro</i> and partial <i>in vivo</i> function. The three natural cysteine residues of sigma-70 are confined to the deleted region. Plasmids allowing overproduction of the internally deleted protein and of a number of single-cysteine substituted mutant derivatives were constructed, and these were tested for function <i>in vivo</i> using a chimaeric sigma/promoter system. The overproduced proteins were isolated employing a novel purification method, and their activities analysed <i>in vitro</i> at l_PR and T7A1 promoters. An extraordinary tendency towards reversible aggregation and hydrophobicity were observed. A possible role of the 245 amino acids domain is discussed. The <i>scrP</i> gene encodes sigma cross-reacting protein, originally identified during a search for alternative sigma factors, but was later shown not to be a sigma protein. As part of the effort to elucidate its possible function, the regulation of expression of the <i>scrP</i> gene was studied. Transcriptional fusions of <i>lacZ</i> to <i>scrP</i> and <i>arcB</i> and <i>mtqA</i>, the genes adjacent to it, were produced and utilised to analyse the expression of ScrP. <i>scrP</i> belongs to the same operon as <i>arcB</i> and <i>mtgA</i>, and is transcribed both from its own promoter and from the promoter upstream from <i>arcB</i>. Further analysis was used to demonstrate that ScrP is localised in the periplasm of <i>E. coli</i>.

The chemical compositions and physical characteristics of a range of bacterial polymers were analysed. The polymer elaborated by <i>Rhizobium meliloti</i> appeared to be a succinoglycan containing acetyl groups whereas the <i>Zoogloea ramigera</i> polymer contained glucose and galactose in the ratio 2:1 and contained acetate and pyruvate. In the presence of cations, the succinoglycan existed in an ordered state, wheras the <i>Zoogloea</i> polymer appeared to exist in a gel like state, rather than a true polymer solution. The bacterial alginates contained various amounts of mannuronic acid (M) and guluronic acid (G) residues. As expected, the <i>Pseudomonas aeruginosa</i> alginate lack contiguous G residues. The alginate from <i>Azotobacter vinelandii</i> 206 was unusual in that it contained a very low amount of G residues. Metal uptake by a range of polymers was carried out by dialysis and samples were analysed using ion chromatography. The alginate samples showed that uptake of the ions was dependent on the ratio of mannuronic acid L: guluronic acid and a binding selectivity of SR > Ca > Mg > K > Na > Li was found. The algal alginates showed greatest uptake and selectivity, although values obtained were low in comparison to published results for alginates in the gel phase. The bacterial alginates showed limited uptake and selectivity. Once the bacterial alginates were deacetylated the uptake and selectivity increased significantly. It is thought that the acetyl groups sterically inhibit the binding of cations. The uptake of cations by polysaccharides isolated from four other bacteria was also exmained. The polysaccharides which contained acetyl groups showed a selectivity of Ca > Mg > monovalent cations, however when the polymers were deacetylated, the selectivity of uptake of monovalent ions was > Mg > Ca. The polymer and ion concentrations were too low to allow gelatin of certain polymers, but even at low concentrations, the acetyl group appeared to play a significant role in ion binding. The reason for the altered selectivity of ion binding following deacetylation is unclear. The <i>Zoogloea</i> polymer had a low capacity to remove cations compared to A3(sl) and XM6 which had the highest uptake capacities. The selectivity coefficients were all close to unity, perhaps suggesting similar ion binding.