Higher organisms can regulate gene expression through changes in epigenetic marks present on the genome. However, how this regulation takes place in organisms with highly repetitive/complex genomes is not well understood. The acquisition of de novo DNA methylation in plants is mediated by siRNAs through the RNAdirected DNA methylation (RdDM) pathway. The targeted deposition of DNA methylation by this pathway allows for the transcriptional silencing of transposable elements and repeat sequences within the genome, as well as regulating gene expression. In addition, it has been hypothesized that mobile siRNAs may be involved in the epigenetic communication between different seed components. Thus the mobility of non-coding RNAs from extra-embryonic tissues could contribute to epigenetic modifications that could be transmitted to the offspring. The aim of my thesis is to characterise the mechanisms involved in epigenetic gene silencing in maize through the use of a novel transgenic reporter. My work has identified components of the RdDM pathway to be involved in maintenance of gene silencing and show that imprinting and paramutation could be recapitulated using synthetic transgenes. In addition, I developed a novel grafting technique to demonstrate that epigenetic gene silencing could be efficiently transmitted between different seed components. Collectively, this work provides an insight into the complex mechanisms that regulate gene expression in the highly repetitive/complex genome of maize.

Understanding the regulatory DNA sequences are becoming increasingly important in understanding the way plants integrate signalling cues mediated through the actions of the transcription factors (TFs). This thesis presents an interdisciplinary investigations into regulatory elements found in the promoter regions of a model organism Arabidopsis thaliana. The intergenic DNA sequences are studied between sets of orthologous genes in A. thaliana and 3 other related species to uncover hundreds of evolutionary conserved noncoding sequences (CNSs). The CNSs are found to be more skewed towards the annotated transcription start sites (TSSs) and enriched in previously identified transcription factors binding motifs. Furthermore, the nucleosomes are predicted to have strong presence in the uncovered CNS than random intergenic sequences alone. Altogether the evidence presented in the thesis points to the functional nature of the CNSs. Then, the promoters of genes thought to be co-regulated together and transcriptionally active during infection with fungal pathogen Botrytis cinerea are experimentally tested for direct protein-DNA interaction using high-throughput Yeast One-Hybrid (Y1H) library screens against the TFs found in A. thaliana. The resulting predictions were further validated using pairwise Y1H screen to suggest potential common regulation by ORA59, PIF7, ESE1, At4g38900 and ERF14, and uncovering a complex gene regulatory network (GRN) associated with the tested genes. The promoter fragments together with the predictions from the Y1H screens were used in the computational analysis to establish transcription factor specific binding motifs. Some of the newly predicted motifs were mutated and tested again for altered binding of the associated TFs. Furthermore, in planta mutations of the TFs predicted to be interacting with the promoters of the genes in the Y1H screens were found to have significant impact on the susceptibility of A. thaliana to infection with B. cinerea, further informing gene regulatory network active in response to biotic stress.

Phytopathogens have developed methods to suppress, manipulate and avoid host defences though the production of toxins and proteins that act on the exterior and interior of host cells. Pathogenic proteins that promote susceptibility to the pathogen are termed effectors, and can function through the suppression of host defences and the diversion of host nutrients. Plants are able to detect select effectors, and thus regain resistance. This multilayered defence response and diverse array of pathogenic effectors can cause the outcome of infection to be decided by a single protein from either organism. Current characterised effectors are able to block pathogen-associated molecular pattern recognition, downstream signalling, up-regulate susceptibility genes including nutrient transporters, modify chromatin structure, interfere with RNA metabolism, suppress or prevent effector-triggered immunity and block the defensive and culminating plant cell death. Research conducted here aims to investigate the in uence of effectors from two pathogens upon transcription in their common host, Arabidopsis thaliana. Over 30 Pseudomonas syringae pv. tomato (Pst) DC3000 effectors, which are delivered directly into the host cell, have been at least partially characterised with several more predicted from the sequenced genome. The research presented here analyses a high resolution microarray time series dataset, comparing the transcriptional events that take place during virulent Pst DC3000 infection to infection with an avirulent mutant that is unable to deliver effectors into the host cell; Pst HrpA

To maintain genetic integrity, eukaryotic cells must faithfully segregate their chromosomes to daughter cells during mitosis. Errors during bi-orientation lead to aneuploidy, a hallmark of human cancers. A highly conserved mechanism termed the Spindle Assembly Checkpoint (SAC) delays the onset of anaphase until all chromosomes have correctly aligned on the metaphase plate. The proteins responsible for the SAC signal include Mad1, Mad2, BubR1 (Mad3 in fission yeast), Bub1, Bub3 and Mph1 kinase. The SAC monitors tension across the spindle and the attachment status of kinetochores; large, proteinaceous structures that assemble on centromeres during mitosis, and is maintained until all kinetochores are properly attached to microtubules. Phosphorylation and dephosphorylation at the kinetochore also regulate chromosome biorientation. Although the SAC has been intensively studied, the exact kinetochore binding site of some SAC components and regulatory mechanisms surrounding the SAC are poorly understood. I have shown, subsequent to previous work which implicated Protein Phosphatase 1 (PP1) in SAC silencing, that two PP1-binding motifs within the N-terminus of kinetochore protein Spc7 (KNL-1 in human cells) bind Dis2, a fission yeast homologue of PP1, in vitro. This interaction is essential for viability and efficient SAC silencing. I also present data implicating kinesin motor proteins Klp5 and Klp6 in PP1-binding and SAC silencing. Furthermore, I have established a role for the Spc7 MELT motifs in recruiting Bub1, Bub3 and Mad3 to the kinetochores using phospho-null (spc7-9TA) and phosphomimetic (spc7-9TE) mutants, and present evidence that this interaction is required for the recruitment of other SAC components. Additionally, the MELT motifs have a role in SAC maintenance and chromosome segregation, and spc7-9TE cells are able to silence the SAC more efficiently than wild type cells. Results presented in this thesis highlight KNL-1 as a major platform for SAC silencing at the kinetochore and will likely form the basis of future studies within the field of mitosis.

This dissertation situates eighteenth-century botany within the contexts of contemporary commercial culture and international networks of knowledge formation. I assess the connections between scholars, merchants and consumers in London and Paris between c. 1760 and c. 1815. I ask how individuals who made a commercial profit from selling science understood and related to the notion of a community of scientific practitioners. My aim is to expose the diversity of socio-intellectual configurations that existed in the late eighteenth century. I focus on the histories of two plant nurseries, one based in London and the other in Paris. Their commercial successes rested on their proprietors’ abilities both to serve the growing consumer demand for plants and to actively participate in the international scientific community. The first three chapters address how each participated in scientific and commercial networks, examining which groups composed these networks, the types of social relationships they formed, and how knowledge circulated between them. I highlight the role played by ‘gardener-botanists’ who acted as intermediaries between each of these groups. The final two chapters focus on the people who purchased and exchanged plants. I assess who comprised the ‘public’ that collected specimens and studied botany, and I examine how gardens in London and Paris formed part of an expanding space for science. I emphasise in particular the significance of the culture of connoisseurship to the history of botany, and discuss the range of different publics who collected plants and studied their science. My research is concerned with the interplay between knowledge, commerce and culture. Drawing from the notion that scientific knowledge is always socially and culturally situated, I aim to connect the history of the plant trade to the development of the science of botany, and to place these within a wider cultural context.

A large proportion of the genomes of species belonging to the genus Allium comprises repetitive sequence DNA, a component implicated as a cause of the large variation in C-values between even closely related species. The work presented here represents part of the first phase in the characterisation of some of these repetitive sequences in a number of Allium species. One repetitive DNA sequence family, BIOOO, isolated from the genome of A. sativum, has been characterised with respect to the genomic organisation, reiteration frequency and sequence divergence of its members within A. sativum. Sequences sharing homology with a cloned representative member of the B1OOO family have been detected in the genomes of a number of other Allium species; such sequences display quantitative and qualitative modulations in their organisation. In addition, and by contrast, the distribution and organisation of a satellite DNA family present In a number of Allium species has been investigated; the characteristics of this family differ from those of the B1OOO family in many respects. Data relating to the evolution and maintenance, functions and effects of repetitive-sequence DNA in eukaryotic genomes are reviewed and where possible the data pertaining to Allium are discussed in context with such information from other species.

The work within this thesis is concerned with the creation of a temperature-sensitive Schizosaccharomyces pombe marker protein, and the regulation of the pheromone communication system of Sz. pombe reporter strains by RGS proteins. There are a limited number of marker proteins available for use in the genetic manipulation of Sz. pombe, and the generation of a temperature-sensitive Ura4p was envisaged to expand the scope of carrying out sequential gene disruptions in the fission yeast. PCR-based mutagenesis was used to introduce mutations in the ura4 cassette, and a leucine to proline mutation identified at residue 261 in the ura4 open reading frame conferred a temperature-sensitive requirement for uracil. To demonstrate the use of the Ura4sp marker in gene disruption, the Sz. pombe irpl gene was disrupted with the ura4u cassette, and subsequently, the prkl gene was disrupted with the wild-type ura4 cassette. RGS proteins are a recently discovered family of proteins that negatively regulate G protein-coupled signalling pathways. This thesis describes the ability of mammalian RGS proteins to regulate the pheromone communication system of Sz. pombe reporter strains. Human RGS 1 and human RGS4 displayed the greatest ability to negatively regulate the Sz. pombe pheromone signalling pathway when expressed from multicopy expression vectors. Human RGS2, human RGS3, human RGS9-2 and murine RGS2 displayed lesser, varying abilities. Expression of human RGS 1 from single copy reduced signalling at low pheromone concentrations. Expression of human RGS4 from single copy was incapable of reducing pheromone-independent and pheromone-dependent signalling. This thesis also describes the search for gain-of-function RGS proteins. Two potential gain-of-function szRgslp mutants were previously identified, and these mutants were recreated. The two mutations identified (histidine to arginine at szRgslp residue 171 and valine to isoleucine at szRgslp residue 305) conferred gain-of-function szRgslp phenotypes in an sxa2:: ura4 reporter strain. Hydroxylamine treatment of the human RGS4 open reading frame resulted in the identification of a potential gain-of-function RGS4 mutant. The lysine to arginine mutation at huRGS4p residue 20 conferred a gain-of-function huRGS4p phenotype in an sxa2:: ura4 reporter strain.

There is a growing need to engineer an increased yield in food crops coupled with greater resistance to environmental stresses, such as drought, to meet the requirement of a growing population. As plant responses to drought (and other abiotic stress) are complex, many studies have attempted to understand the drought response in a holistic manner, for example, by analysing transcriptomics data obtained from plants subjected to drought. Preliminary work of obtaining time-series microarray data from a slow-drying experiment of Arabidopsis was used to construct Gene Regulatory Networks using Variational Bayesian State Space Modelling. This led to the identification of a number of transcription factors RAP2.12, FD, BHLH038, ANL2, and two unknown genes, UKTF and POZ, as possibly being important regulatory genes in the drought response. Loss- and gain-of-function mutants of these genes, as well as those of AGL22 identified by Bechtold et al. (2016), were phenotyped under drought conditions. Only the flowering time AGL22 showed a drought phenotype. Network connections in the gene network of AGL22 were tested by qPCR. Drought responsive transcription factors, such as DREB1A and WRKY20 were found to be induced by AGL22.

The brewing properties of an industrial strain of Saccharomyces cerevisiae were investigated by laboratory scale brewing trials in the presence or absence of an uncoupler of oxidative phosphorylation (SX-1). When SX-1 was added the change in specific gravity of brewing wort with respect to time was less, yeast produced less biomass and more ethanol per unit drop in specific gravity than the control. Similar fermentation properties were observed for C2, a haploid laboratory yeast strain with the ability to ferment maltose. Recombinant DNA technology was used to generate a C2 pet mutant, specifically in the ATP12 gene, which encodes a protein essential for mitochondrial ATP synthesis. In brewing trials a comparison of C2 and C2:AATP 12 shows similar results to C2 fermentations in the presence or absence of SX-1 although the effects of SX-1 are more dramatic than with C2:AA TP 12. Together, the results of chemical studies and gene disruption mutagenesis suggest that mitochondrial ATP synthesis affects nuclear functions, although the possibility that results obtained are a consequence of changes to the yeast mitochondrial DNA cannot be eliminated. Problems experienced with yeast DNA transformation protocols lead to the development of a new transformation method that is quicker and more efficient than the standard protocol. Initial studies revealed that DMSO could enhance yeast transformation efficiency, and that the optimal concentration of DMSO used is strain specific. The point at which DMSO was added was found to be important, with maximal transformation efficiency achieved when DMSO was added just before heat shocking. The optimised protocol for S. cerevisiae JRY188 routinely enhanced transformation IS- to 25-fold compared with a control transformation protocol. The osmotic condition was found to be important for DNA uptake as transformation was inhibited if yeast were washed in 1M sorbitol and selected on plates containing the same. Significantly, DMSO enhanced transformation even in the absence of captions, therefore this method may prove useful for yeasts which transform poorly by existing cationic-based yeast transformation methods.

The work presented in this thesis involves the isolation and characterization chaperonin 60 (cpn60) proteins from Rhodobactar sphaaroidas and the cytosol of Pisum sativum. Non-denaturing-PAGE analysis of R. sphaaroidas cell-free extracts fractionated in sucrose density gradients revealed the presence of a Mr 670,000 protein that was considered to be a cpn60 candidate protein. This protein was purified by a combination of ion exchange and size exclusion chromatography. The Mr 670,000 multimer was shown to consist of a single polypeptide of Mr 58,000. Anti-sera raised against the Escharichia coli cpn60 (GroEL) and the pea plastid cpn60 did not cross-react with this protein. However, anti-sera raised against the R. sphaaroidas protein was shown to cross-react with the E coli GroEL protein. Subsequent N- terminal amino acid sequence analysis demonstrated that the R. sphaaroidas polypeptide was identical to GroEL at 15 of the 19 residues determined and showed significant similarity to other known cpn60 sequences. Like other chaperonins the native protein tended to dissociate in the presence of MgATP, in this case into what appears to be a dimeric form. Examination of the R. sphaaroidas protein in the electron microscope revealed 'top' views with seven-fold symmetry and rectangular 'side' views with four equally spaced, approximately equally dense, transverse striations. The protein appeared to be a cylinder of diameter 12 nm and height 9 nm. This structure is characteristic of the majority of the cpn60s examined so far. Rotational symmetry analysis revealed structural details extending beyond (20 A)-|. The image of the chromosome-encoded R. sphaaroidas chaperonin (Rs cpn60) differed from similarly generated images of plasmid-encoded GroEL. The possible significance of these differences is discussed. The effects of various environmental stresses on protein synthesis in R. sphaaroidas was investigated by pulse-labelling cells. Particular attention was paid to the expression of Rs cpn60. Overall the stress responses observed were similar to those reported for other prokaryotes. Of particular note are the quite different views of the stress responses presented by non-denaturing- and SDS-PAGE analyses. Attempts were made to determine the range of polypeptides that might be bound to Rs cpn60 before and after heat shock. These studies possibly indicate that there is a size constraint on polypeptides that interact with Rs cpn60 and that the flux of polypeptides through Rs cpn60 alters during heat shock. The second part of this thesis concerns attempts to identify a cytosolic chaperonin in P. sativum Until very recently no chaperonins had been identified in the cytosol of eukaryotes. Sequence analysis had raised the possibility that the TCP-1 protein from mice might be a cytosolic chaperonin (van der Vies, 1989). Monoclonal antibodies raised against the mouse TCP-1 protein cross-reacted with a Mr 57,000 polypeptide with a possible cytosolic location in P. sativum leaf extracts. Subsequent attempts to purify and determine the sub-cellular distribution of this polypeptide were unsuccessful. However, the sedimentation behaviour of the native protein indicated that the polypeptide was part of a multimeric complex of similar size to other chaperonins. The behaviour on ion exchange columns was also similar to other known chaperonins. The main obstacles to purification and localization of the native protein were: a) the monoclonal antibody used did not recognise the native protein and b) the multimeric complex appeared to dissociate during purification.