VEZF1 is a ubiquitously expressed transcription factor with epigenetic roles at gene regulatory elements, including protection from DNA methylation. Genetic ablation of Vezf1 in murine embryos is lethal due to haemorrhages localised in head and neck mainly. Interestingly, Vezf1 seems to be important for the generation of erythroid cells. The role of human VEZF1 in erythroid gene regulation was the primary focus of this study. Previous work found that VEZF1 unexpectedly interacts with the majority of erythroid-specific enhancer elements, including those at the α- and β-globin gene clusters, in the K562 cell line, a long- standing model for erythroid progenitor cells. The co-occupancy of VEZF1 with well characterised erythroid transcription factors and histone marks associated with active enhancers indicated that VEZF1 may support the regulation of the erythroid gene regulatory programme. To understand the role of VEZF1 in erythroid gene regulation and differentiation, we used CRISPR technology to knock out VEZF1 in K562 and hESCs. RNA-seq analysis revealed that VEZF1 does have regulatory roles at several erythroid genes, but it is not essential for the erythroid gene regulatory programme. VEZF1 null K562 cells display increased levels of haemoglobin biosynthesis. Intriguingly, we find that VEZF1 protein levels are progressively downregulated during the in vitro erythroid differentiation of human ESCs. VEZF1 null ESC cultures commit more readily to the erythroid lineage at the onset of erythropoiesis, which then proceeds as normal. VEZF1 is therefore not required for human erythropoiesis and appears to be an early regulatory block to erythropoiesis proceeding effectively. Careful inspection of the cultures at a stage prior to the onset of erythroid differentiation revealed that a sub-population of endothelial precursors was absent in VEZF1 null ESC cultures. We demonstrate that the absence of VEZF1 prevents the efficient differentiation of endothelial cells. The role of VEZF1 in vascular endothelial development is therefore conserved in mammals, although the impact on human endothelial differentiation appears to be more profound. VEZF1 is anticipated to be a key regulator of the vascular endothelial gene regulatory programme.

Type 1 diabetes (DMT1) is a chronic autoimmune disease that leads to apoptosis and death of pancreatic islet beta cells. These beta cells are influenced by and contribute to a network of gut derived hormones that regulate glucose homeostasis. Our research explores the effect that DMT1 has on this network of gut hormones. A series of clinical research studies was established to collect samples from both healthy participants and individuals with DMT1. Methods to measure gut hormones were explored and refined prior to sample analysis. Results from this analysis revealed that the homeostasis of glucagon, active-ghrelin and peptide YY are affected by DMT1 and that glucagon secretion may be controlled by a glucose independent signal derived from the gut. Important lifestyle markers in DMT1 were identified and included a correlation between leptin and body mass index and gastric inhibitory polypeptide and sedentary behaviour. Histopathological analysis of pancreatic slides taken from donors with DMT1 suggest that the islet area stained for glucagon does not correlate with duration of diabetes, but does correlate with age. Together these suggest that type 1 diabetes does affect other gut hormones involved in glucose homeostasis and this can be influenced by lifestyle factors.

RK2 is a low copy number plasmid responsible for spread and maintenance of important properties (including antibiotic resistance and degradation functions) among bacteria. Gene expression in RK2 is controlled by cooperativity among four repressors (i.e. KorA, KorB, KorC and TrbA) to tightly regulate replication, stable inheritance and conjugative transfer functions. KorB (358 aa) has dual roles as a global regulator and as an active partitioning protein. This study focuses on its role as a global regulatory protein and its interaction with DNA, RNAP and other repressor proteins (e.g KorA and TrbA) of RK2. It is shown for the first time that DNA binding by negatively charged protein KorB (-21) is modulated via a balance of charge in the internal region from aa 235 to 245. KorB binds O\(_B\) and silences the genes around, showing that KorB can spread. TrbA and KorA bound to DNA adjacent to KorB do not block gene silencing by KorB and indeed potentiate its repression, suggesting that KorB can spread past DNA binding proteins and thus that they do not act as road blocks. The fact that KorB E237A, which is defective in silencing, cannot repress at a distance when alone but can do so in presence of TrbA, provides strong evidence of looping. The fact that KorA and TrbA do not potentiate gene silencing by E237A, but do potentiate its repression, indicates strongly that gene silencing is because of spreading instead of looping. Full length KorB is required for distal repression. However, only the region 225-255 aa is critical for proximal repression by KorB. The results suggest a model in which KorB organises DNA loosely over a long region through a wrapping in a way that can accommodate other regulatory proteins. This nucleoprotein complex may also be critical for plasmid partitioning.

Transcription is the first step in gene expression. Thus, RNA polymerase copies information from template DNA to generate the mRNA template for protein production. Transcription is divided into three steps; each step provides a platform for regulation of gene expression. This work contributes to understanding novel aspects of regulatory processes. For example, complex regulation of expression of the nucleoid-associated protein cbpA was found to be dependent not only on sigma factor specificity, but also on binding of the transcription factor Fis upstream of the cbpA start codon. Binding of Fis to the cbpA regulatory region prevents the action of a strong σ70-dependent promoter found within the coding region of a neighbouring gene, yccE. This work carefully dissects the sequence specificity and orientation specificity of DNA sequences found upstream of the cbpA coding region that allows for the binding of Fis. Binding of Fis to the cbpA regulatory region is a redundant process, whereby in the absence of Fis another factor binds to this region. Furthermore, the regulation of cbpA was also found to be dependent on the interplay between the strong σ70-dependent promoter and a weak but convergent σ32-dependent promoter. Finally, horizontally acquired DNA, which is AT-rich in nature, has been known to be regulated by the transcription factor, H-NS. However, the field only has limited knowledge on the mechanisms behind this regulation. It was previously thought that AT-rich genes are subject to canonical regulation by H-NS. However, this work demonstrates the phenomenon of “pseudo-regulation”. Here, H-NS silences activity coming from intragenic promoters, rather than the genuine promoter. This phenomenon is likely widespread, and is demonstrated by this study in a number of AT-rich genes.

Following the molecular cloning of the PVM genome, the opportunity to the individual genes and proteins of PVM has arisen. This study investigated nucleocapsid (N) gene and the phosphoprotein (P) gene of PVM and attempted to characterise the polypeptide products expressed from the N and P genes both in vitro in PVM-infected cells. The ability of the PVM N and P proteins to interact with other was also investigated. The nucleotide sequence of the PVM P gene was determined to be 903 nucleotides in length and shown to comprise a long open reading frame capable of encoding the 295 amino acid long P protein and also a smaller second ORF with the potential to express a polypeptide 137 amino acids in length. The PVM P protein shows overall amino acid homology of 35.3%, 35.6% and 28.3% to the P proteins of pneumovirus members HRSV, BRSV and TRTV respectively. The PVM P gene contrasts with the P genes of other pneumovirus genus members which do not possess extensive alternative ORFs. Both the N and P genes of PVM were shown to be capable of directing the synthesis of more than one polypeptide product both in vitro and in PVM-infected BSC1 cells. mRNA transcribed from the PVM P gene long ORF directed the in vitro expression of the 39 kDa P protein and four additional polypeptides. By constructing transcription plasmids that contained 5' terminally truncated P gene cDNA insets, these polypeptides were determined to be expressed by translational initiation on internal P gene initiation codons. Western blot analysis determined that in addition to PVM P protein, two of these in vitro expressed P protein species, with molecular weights of 26 kDa and 23 kDa, were expressed in PVM-infected BSC 1 cells and this observation was supported by the results of anti-P protein monoclonal antibody epitope mapping studies. The ability of the PVM P gene to direct the expression of P protein related polypeptides from internal initiation codons is a feature not yet described for any other pneumovirus member. By immunising rats with a synthetic peptide, antiserum specific for the second polypeptide product (P2) was generated. Western blot analysis using this anti-P2 antiserum identified a species thought to represent P2 in PVM-infected BSC 1 cell material. The ability of the PVM P gene to express a polypeptide from an alternative is a feature common to the P genes of most other morbilliviruses and paramyxoviruses. mRNA transcribed from PVM N gene cDNA was able to direct the in vitro translation of the 43 kDa N protein and also a highly abundant polypeptide with a molecular weight of 24 kDa which was shown to be expressed by way of internal initiation on the fifth N gene AUG codon of the N gene sequence. The 24 kDa N protein related polypeptide was expressed in E. coli, purified, and used to immunise a rabbit for the production of anti-24 kDa polypeptide antiserum. Western blot analysis using this antiserum with PVM-infected BSC1 cells detected the 43 kDa N protein, a highly abundant 30 kDa N protein related species, but not the 24 kDa polypeptide. precise identity of the 30 kDa polypeptide was not determined. Possible mechanisms which could account for the expression of the protein products of the N P genes are discussed. By using a protein blotting technique the interaction that occurs between the N P proteins of PVM was investigated. The P protein binding affinities of in vitro expressed truncated N proteins suggested that many regions of the N protein are co- operatively involved in the binding process, although some regions contributed more than others. The N protein of Sendai virus is believed to bind to the Sendai virus P protein in a similar way. It was also determined that both the amino and the carboxyl- terminal regions of the PVM P protein were found to be essential for binding to N protein. This contrasts with the situation determined for Sendai virus in which 344 P protein amino-terminal amino acids were found to be dispensable for binding N protein.

Long lasting calcium (Ca2+) oscillations are necessary and sufficient for mammalian egg activation and early embryological development. In mammals, phospholipase C zeta (PLCζ) has been identified as the likely endogenous trigger of Ca2+ oscillations at fertilization. Some cases of male factor infertility have been associated with the absence / reduced or presence a mutant form of PLCζ. In these cases sperm fails to activate eggs after intra-cytoplasmic sperm injection (ICSI). Artificial egg activation is the potential way to trigger Ca2+ oscillations and egg activation. Strontium (Sr2+) is the main artificial agent for this purpose in rodent eggs. The work in this Thesis aims to examine the mechanism of PLCζ or Sr2+ ions to trigger Ca2+ oscillations in mammalian eggs. It was not clear how Sr2+ causes Ca2+ oscillations and why it is only effective in rodents but not human eggs or domestic animals. My studies show that Sr2+ is effective in causing Ca2+ oscillations in mouse eggs over a range of concentrations, but that its actions are influenced by the osmolarity of the medium. Low osmolarity enhances the ability of low concentrations of Sr2+ to cause Ca2+ oscillations. Further investigation revealed that Sr2+ influx is mainly through the reverse mode of the Na+/Ca2+ exchange protein (NCX) which can be controlled by the membrane potential and Na+ gradient across the plasma membrane. Preliminary studies investigated the ability of a modified Sr2+ media that maximizes reverse mode NCX to trigger Ca2+ changes in human eggs. In other studies, various PLCζ-luciferase cRNAs were injected into mouse and human eggs. PLCζ expression in mouse eggs was measured by imaging light due to luciferase iv activity, and Ca2+-oscillations were monitored with Ca2+ sensitive fluorescent dye. Aspects of the structure of PLCζ and the effects and the recent discovery of PLCζ sequence mutations were investigated. Preliminary studies were also carried out to test the ability of recombinant PLCζ protein to cause Ca2+ oscillations in mammalian eggs. It is hoped that these studies might open up new therapies for some male factor infertility couples that acconts 1-5% of failed ICSI.

The African sleeping sickness parasite Trypanosoma brucei evades the immune system of its mammalian host by periodically switching the variant surface glycoprotein (VSG) that forms its cell-surface coat. This process of antigenic variation utilises a large archive of VSG genes, which are primarily subtelomeric and appear to evolve rapidly. Subtelomeres are the location of multi-member, variable gene families in many organisms, and often seem to have an elevated rate of mutation. The VSG archive is a particularly striking example of an organism taking advantage of this environment to promote hyperevolution. The aim of this project was to investigate the changes that occur in VSG evolution. In collaboration with researchers at the Sanger Institute, genomes from two time-separated isolates of the same trypanosome strain were sequenced and assembled. The quality of the genome assemblies was assessed, and the genomes concluded to be of sufficient quality for further analysis. Chromosome core genes and VSG N-terminal domain (NTD) genes and pseudogenes were annotated in each genome, and mutations between the genomes in each gene were catalogued. VSG NTDs had a significantly higher mutation frequency than core genes, and the specific patterns of mutations differed significantly between the two genome regions. These results together implied that VSG are subject to different mutational processes from core genes. However, mutation frequency did not appear to differ between VSG NTDs and other subtelomeric sequence, indicating that it is the subtelomeres in general that are subject to elevated mutational activity. Further examination of the VSG NTDs within each new genome reinforced published observations in the reference genome strain VSG archive of extensive substructuring and abundance of pseudogenes. Finally, to address the question of which mechanisms may be involved in elevating the mutation rate in subtelomeres, an attempt was made to characterise two members of a gene family predicted to encode error-prone lesion bypass DNA polymerases, a class of enzymes that have been suggested to have a role in the systematic generation of mutations. Such results as were obtained suggested that the genes examined may not encode active polymerases, and the results did not provide any evidence for a role for these polymerases in VSG hyperevolution. Overall, however, the project has uncovered considerable detail of how hypermutation proceeds in this highly variable gene family.

The results presented were concerned with the survival of Streptomyces coelicolor A3(2) (pll673) inoculated into soil microcosms, which were destructively fractionated so that the total propagules and spore counts could be determined in each of the soil fractions. It was found that this microorganism became associated with the smallest soil aggregates at the time of inoculation but with incubation of the soil microcosms the mycelia and spores became attached to the larger soil aggregates. In the sterile soil, the streptomycete growth was much greater than in nonsterile soil, perhaps due to the increased supply of nutrients created by autoclaving the soil, and the lack of competition. Many of the newly formed spores in sterile soil were not attached to the soil aggregates, which may have enabled them to be distributed to new micro sites. When the distribution of indigenous actinomycetes in soil was investigated, it ressembled the distribution of Streptomyces coelicolor in nonsterile soil after the inoculant had been through one life cycle. Actinomycetes were then isolated from each of the soil fractions, as well as the unfractionated soil, and each of these strains were identified to genera, if possible. It was found that many of the micromonosporas and streptosporangia were isolated from the 63-251 μm soil aggregates, probably because this fraction contained low eubacterial and streptomycetes populations caused by the low organic content within this soil fraction. There was a high eubacterial count in the 2-20 μm soil aggregates and although the actinomycetes were outcompeted within this soil fraction, their diversity was greatest within this fraction. This diversity was also reflected by their production of different secondary metabolites. DNA was extracted from each of the isolates and amplified using specifically designed primers for high GC microorganisms. Each of the products were individually run on denaturing gradient gels. It was found that the amplified products from actinomycetes formed bands on the denaturing gels which migrated to 3 positions. Each of these positions corresponded to major groups of actinomycetes of which streptomycetes formed one group. The patterns corresponding to the isolates of each soil fraction would be compared with the amplified products derived from in situ soil DNA extracts. It was found that the results were not comparable but this work is still being investigated.

The purpose of this investigation was to study the heterologous expression of soluble methane monooxygenase (sMMO) genes from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. Using the T7-RNA polymerase expression system, the entire sMMO operon and subclones (constructed using the polymerase chain reaction) were over-expressed in E. coli. Results obtained using the Me. capsulatus (Bath) sMMO operon confirmed previous reports (C. West, G. P. C. Salmond, H. Dalton and 1. C. Murrell (1992). J Gen. Microbial. 138, 1301-1307) that functional expression of protein B and the reductase occurred but the hydroxylase was inactive. Similar results were obtained by expressing the sMMO operon of Ms. trichosporium OB3b in E. coli, using plasmids previously described (D. Jahng and T. K. Wood (1994). Appl. Environ. Microbiol. 60, 2473-2482). Protein B, the reductase and orfY were over-expressed and purified from E. coli using glutathione-Stransferase fusion proteins and affinity chromatography. The expression of sMMO genes from Mc. capsulatus (Bath) and Ms. trichosporium OB3b was studied in Pseudomonas putida. A previous report (D. Jahng and T. K. Wood (1994). Appl. Environ. Microbial. 60, 2473-2482) had suggested that functional expression of sMMO from Ms. trichosporium OB3b was achieved in P. putida Fl. Attempts to repeat this work proved that protein B and the reductase were functionally expressed, but the hydroxylase was inactive. Similar results were obtained for the heterologous expression of the sMMO operon from Mc. capsulatus (Bath) in P. putida. Methanotrophs were used for the heterologous expression of sMMO via two strategies. (1) The expression of sMMO from Mc. capsulatus (Bath) and Ms. trichosporium OB3b was studied in Methylomonas album B08 and Methylocystis parvus OBBP. These are methanotrophs that do not express sMMO, but express particulate MMO (pMMO) only, to utilise methane as a sole carbon and energy source. Functional expression of the sMMO operon of Ms. trichosporium OB3b was achieved in Mm. album BG8, however, recombinant sMMO enzyme activity was poor and problems were encountered with the growth of the sMMO positive transconjugant methanotrophs. (2) sMMO-minus marker exchange mutants of Ms. trichosporium OB3b (H. Martin and 1. C. Murrell (1995). FEMS Microbiol. Letts. 127, 243-248) were complemented with plasmid encoded genes and functional sMMO expression was obtained. Southern hybridisation analysis revealed that the plasmid DNA had integrated into the chromosome of the Ms. trichosporium OB3b sMMO-minus mutant via a single homologous recombination event between the mmoX genes. Protein B from Mc. capsulatus (Bath) is inactivated by proteolysis to give rise to a truncated form designated protein B'. The Met 12-Gly 13 cleavage site was modified by site-directed mutagenesis to Met12-Gln13 which improved the stability of the protein when incubated at room temperature. Only after prolonged incubation was protein B' formed. Recombinant protein B from Ms. trichosporium OB3b also appears to be unstable, and readily degraded when incubated at room temperature. The cleavage of protein B to inactive protein B' may be a general regulatory mechanism that occurs within the cell to regulate sMMO activity.

A nitrous acid-induced, temperature sensitive mutant of cowpea mosaic virus (CPMV) known as 8-14, (Evans 1985, Virology 1985, 141, 275-282), was characterised. The phenotypic defect in 8 -14 was shown not to affect translation of the RNA or the first proteolytic cleavage of the B RNA-encoded polyprotein. The defect is probably at the level of genome replication. The technique of two dimensional RNA fingerprinting showed the mutant genome to be similar to the parental wild-type but did not resolve the genetic alteration(s) specific for the mutation. The mechanism of CPMV translation was investigated by site-directed mutagenesis of a full-length cDNA clone of CPMV M RNA from which infectious RNA could be generated by in vitro transcription. The results obtained confirm the AUG at position 161 is used to direct the synthesis of the 105K protein in vitro. The detection of a 58K protein in infected protoplasts suggests that it is also used in vivo. The synthesis of the 95K protein can be initiated from either of the AUGs at positions 512 and 524. Synthesis of this protein is not essential for CPMV replication in protoplasts. Several deletion mutations were created in the M RNA cDNA clone in order to determine the regions of M RNA essential for replication of M RNA. Analysis of one mutant indicated that sequences between 1446 and 1620 are probably not required for replicase recognition. However, the accumulation of this mutant in protoplasts was reduced, presumably as a result of lack of encapsidation of the RNA as this mutant is thought not to synthesise functional coat protein. Data from several mutants showed that alterations of M RNA around nucleotides 161 and 189 prevent transcript accumulation in protoplasts possibly owing to a severe reduction in replicability of the input RNA.