Small RNA are short, 18-25 nt molecules that regulate gene expression in plants and animals. Two main types, microRNA (miRNA) and short interfering RNA (siRNA) perform this regulation by transcript silencing, translation inhibition, DNA methylation and chromatin remodeling. This thesis is an investigation into small RNA activity in archaeological plant material, specifically barley grain from Qasr Ibrim, a multi-period archaeological site in southern Egypt. It is of particular interest due to its unusual phenotype, suggestive of stunted development that is unexpected in a staple, domesticated cultivar, and the unusual level of DNA and RNA preservation attributable to the extremely arid climate at the site. The research presented here is a comparative analysis of small RNA profiles and epigenetic states of Qasr Ibrim barley and modern, unstressed counterparts. It concludes that differential microRNA and epigenetic profiles are the result of stress response, adaptation, dormancy and / or viral infection unique to the archaeological grain. The primary method of investigation was generation of small RNA sequence data using the Illumina GAIIx platform. This was followed by extensive bioinformatic analysis (RNA diagenesis patterns, miRNA prediction, siRNA target prediction and small genome in silico reconstruction) the results of which were in turn validated experimentally (genomic methylation states, locus-specific methylation analysis and direct miRNA detection). The research represents a twofold contribution to knowledge: first, proof-of-principle that biologically meaningful archaeological RNA can be extracted despite its relative instability to DNA, and second that a unique miRNA profile and epigenetic state is detectable in this particular cultivar of archaeological barley.

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.

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 Ruv ABC and RecBCD protein complexes together can collapse and repair arrested replication forks. With their help a fork structure can be re-established on which replication can be restarted. ruv and recB mutants are therefore quite sensitive to UV light. Their survival is greatly decreased in the absence of the signalling molecules (p)ppGpp and increased when excess (p)ppGpp is present. (p)ppGpp are the effector molecules of the stringent response, regulating adaptation to starvation and other stressful environmental changes. Absence of (p)ppGpp can be compensated for by mutations in RNA polymerase that are called stringent mutations. Some of those, called rpo *, also - like excess (p)ppGpp - increase the survival of UV irradiated ruv and recB cells. A model proposed by McGlynn and Lloyd (Cell, Vol. 101, pp35-45, March 31, 2000) suggests that this is achieved by modulation of RNA polymerase, which decreases the incidence of replication fork blocks. In this work twenty-seven rpo * mutants were isolated, sequenced and mapped on the 3D structure of Thermus aquatic us RNA polymerase. I have found mutants in the ~ and ~' subunits of RNA polymerase. They lie mostly on the inner surface of the protein, well placed to make contact with the DNA substrate or the RNA product. A large number of rifampicin resistant mutations among rpo* mutations is explained by an overlap between the so-called Rif pocket and the "rpo* pocket". rpo * mutations, like stringent mutations, lead to a decrease in cell size, suppress filamentation and increase viability. For in vitro studies I purified wild type and two mutant RNA polymerases with help of a his-tagged a subunit. The experiments confirmed that rpo* mutant RNA polymerases form less stable open complexes than wild type, just like previously investigated stringent RNA polymerases. In addition I have shown here that (p)ppGpp leads to the destabilisation of RNA polymerase complexes stalled by nucleotide starvation or UV-induced lesions, though there is as yet no indication that rpo * mutations act in the same way.

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.

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.

This thesis is an investigation into the function of the drongo, a novel gene with a zinc finger motif, orignally isolated via enhancer trapping from its expresssion in the embryonic neuroectoderm. Drongo has previously been shown to be expressed during oogenesis, neurogenesis and eye development. In this project, sequencing of a Drongo cDNA clone, shows homology to human nucleoporin protein hRIP; and a lesser extent to other proteins including the mammalian ARF-l GTPase activating protein (ARF-l GAP), a protein involved in vesicular transport across the cell; and family of yeast zinc finger genes GTSI/GCSI/GL03, members of which have also recently been shown to have GAP activity. Overexpression of drongo during early oogenesis results in egg chambers with supernumary nurse cells, probably as a result of a delay in follicle cell migration; a phenotype similar to that of braniac mutants. Overexpression during late oogenesis causes a mislocalisation of Oskar, producing embryos which lacked denticle belts and which often had posterior defects, suggesting that ectopic expression of the gene at different times in development can have different consequences. A mutagenesis screen carried out generated a number of potential mutant alleles, although none as yet have identified a mutation in the drongo gene. The use of an peptide antibody to the protein on S2 cells and western blots has identified a possible localisation of the Drongo protein to the endoplasmic reticulum, suggesting a role in vesicle transport. Drongo has also been shown to be developmentally expressed and the gene appears to encode two proteins which may or may not be functionally distinct. The role of Drongo as a possible ARF GAP, are discussed.