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.

Myxococcus xanthus is a soil-dwelling bacterium which produces carotenoids upon irradiation with blue light. Genetic analysis has allowed elucidation of transduction of the light signal to the carotenogenic machinery within the cell. The primary element within the carotenogenic regulon is the genetic switch manifested by CarR and CarQ. CarR is an integral membrane protein which binds to the sigma factor CarQ and holds it in an inactive state at the cell membrane. Illumination of the cell with blue light excites the photosensitiser protoporphyrin IX (PPIX) within the bacterial membrane, which then excites molecular oxygen to the excited singlet state. Both singlet oxygen and excited triplet state PPIX can cause large amounts of cellular damage. Carotenoids prevent this damage by absorbing the excess energy from these excited species and dissipating it harmlessly as heat. The presence of singlet oxygen within the bacterial membrane causes the inactivation/degradation of CarR. Removal of CarR releases CarQ from the membrane enabling it to mediate transcription from various promoters. CarQ causes transcription of the crtI gene and of the carQRS operon which produces further CarQ and CarS. CarS causes de-repression of the crtEBDC cluster. The carotenogenic enzymes encoded by crtI and the crtEBDC cluster catalyse the production of carotenoids which quench the initial signalling molecules, singlet oxygen and triplet PPIX. This causes down-regulation of the regulon as a whole as CarR is no longer degraded and once again carries nascent CarQ to the membrane in an inactive state. The negative feedback loop described above is an important consideration when assessing mutants which produce carotenoids either constitutively (Car c phenotype), or under no conditions (Car- phenotype). This work investigates the consequences of Carc and Car- mutations on the activity of promoters within the Car regulon in order to clarify the roles of various genetic loci. It is demonstrated that CarA has no regulatory role in expression of crtI or carQRS and that the expression of crtI has no regulatory consequences. Sequencing downstream of crtI revealed a novel gene gufB (gene of unknown function B) which has homologues of no known function. The critical event in the activation of the carotenogenic system is expression of the carQRS operon allowed by the release of CarQ from its complex with CarR at the membrane. Attempts were made to extract information about the interaction of CarQ with its cognate promoter at carQRS through a variety of in vivo and in vitro molecular and genetic techniques. Site-directed mutations within pcarQRS were assessed in vivo through the use of lacZ transcriptional fusions, enabling identification of important regions within the carQRS promoter. In vitro experiments provided information about the possibility of using molecular methods to assess interactions between CarQ and the pcarQRS promoter.

Molecular detection methods were developed to study the distribution of type II polyketide synthase (PKS) genes in Cuban soils. A PCR based detection method targeting the α and β ketosynthase genes was applied to a number of different total community DNA samples. These genes were detected in 43% of samples tested from a number of different locations. A botanical garden site located in Havana, Cuba, was found to show the greatest distribution of type II PKS genes across the sites tested. It was not possible to amplify type II PKS genes from a pristine island site off the coast of Cuba. Further investigation revealed that actinornycetes containing type II PKS were present in the soil community at a level above the detection limit of the PCR protocol. Further total community DNA cleanup steps failed to allow the detection of type II PKS genes within the DNA samples suggesting PCR inhibition was responsible for negative results. The molecular detection of type II PKS genes in total community DNA was compared to the detection of type II PKS genes in actinomycete isolates. A lack of correlation between these two approaches was observed with the molecular detection limit unable to amplify type II PKS genes in <50% of crop soils tested. Actinomycetes containing type II PKS genes could be isolated from all crop soils tested. No difference was seen in the detection of type II PKS genes between rhizosphere and bulk soil samples. Actinomycetes were isolated using a selective isolation procedure at a level of approximately 10(7) cfu g-1 soil compared to 10(8) cfu g-1 for total bacterial counts. Actinomycetes were isolated from Cuban crop soils and screened for the presence of type II PKS genes. Out of 100 isolates 26 were found to contain the genes of interest. Phylogenetic analysis of these isolates based on 16S rDNA and recA sequence data showed them to be closely grouped within the streptomycetes. Sequence data based on KSα genes from Cuban isolates showed them to be representative of both spore pigment and antibiotic polyketide genes. A representative clone library was constructed containing type II PKS genes amplified from total community DNA. Rhizosphere and bulk soil samples were compared from the same site. Sequences obtained from rhizosphere total community DNA appeared to be widely distributed when compared to published sequences and included examples of both spore pigment and antibiotic polyketide genes. A molecular method was developed to amplify near full length α and β KS genes from type II PKS gene clusters. Expression vectors were constructed to allow these genes to be expressed along with an ACP to give a functional minimal PKS for polyketide chain production. This method was used on total community DNA in an attempt to extract diverse genes from as yet uncultured organisms.

Although the starvation or non-growth state is probably the most common physiological state of bacteria, it has been studied in relatively few organisms. In spite of its importance in pathogenesis, bioremediation and several industrial processes, limited research has been performed on Rhodococcus under starvation/stationary phase conditions. The objectives of this study were to analyse the physiological adaptation of Rhodococcus erythropolis SQ1 to starvation/stationary phase, and to generate and screen a bank of mutants to identify genetic elements involved in this adaptation. It was found that R. erythropolis SQ1 can survive for at least 43 days in LB and distilled water, and 65 days in chemically defined medium (CDM) containing high (1 % w/v) or low (0.1 % w/v) glucose concentrations. Early stationary phase R. erythropolis SQ1 cells grown in 0.1 % glucose also exhibited enhanced resistance to heat and oxidative stress compared with exponential phase cells. A mutant bank of 898 R. erythropolis SQ1 mutants was generated and screened; four mutants were of particular interest. The culturability of mutants 4G6 and 10D3 dropped to <0.1 % of the maximum CFU/ml at 27 days incubation, and to <3 % of the maximum CFU/ml for mutants 1B2 and 1H1, when grown in 1 % glucose medium. No drop in culturability was observed when mutants were grown in 0.1 % glucose. Mutant 4G6 had a transposon insertion in uvrB (UvrB, part of the DNA excision repair mechanism), while the insertion for mutant 10D3 was immediately downstream of a putative guaB gene, which, based on bioinformatic analyses, is followed by another putative IMP dehydrogenase (guaB-like) and/or a cholesterol oxidase gene. In mutant 1H1 the transposon inserted 272 nucleotides downstream of a gene encoding a putative phosphoglycerate mutase and upstream of putative thioredoxin and cytochrome c biogenesis genes. In conclusion, R. erythropolis SQ1 was shown to present a classic starvation/stationary phase survival response, with the associated increase in resistance to various external stresses. A mutant bank has been generated which can be used in the future to analyse other phenotypes of interest. Several genes linked to starvation/stationary phase survival were identified. These findings show that a wide variety of genes are involved in starvation/ stationary phase survival. Indeed, over 100 such genes have been identified in Escherichia coli and Mycobacterium tuberculosis.

Nucleosomes comprise the most basic repeating unit of chromatin and provide hubs for the regulation of DNA transcription, replication and repair. ATPase chromatin remodelling complexes establish nucleosome occupancy, positioning and structure in a dynamic fashion to allow fine-tuning of protein-DNA interactions. The ISWI and CHD families of remodelers possess the ability to sample DNA linker length between nucleosomes and space nucleosomes evenly. How these spacing remodelers combine their functions to maintain phasing of nucleosomal arrays, and re-organise these arrays during development remains poorly understood. Furthermore the relationship between nucleosomal array structure and transcriptional regulation is unclear. Dictyostelium discoideum provides a complex chromatin environment and remodeler repertoire, while retaining a compact genome and ease of genetic manipulation. Thus we have utilized this model to generate remodeler null mutants, and double mutants to observe phenotypic effects and interactions. We further compiled comprehensive nucleosome mapping and RNA sequencing data sets for all spacing remodelers in Dictyostelium. Bioinformatic analysis of these data provide novel insights into remodeler functions, and help to establish a paradigm to explain the relationship between remodeler-mediated chromatin organisation and transcriptional regulation.

Hi-Spot are highly re-aggregated neural cultures grown on PTFE membrane at the liquidair interface. The Hi-Spot protocol was developed by scientists at Capsant Neurotechnologies using embryonic neural tissue. We characterised Hi-Spots made using existing protocols to confirm they represented a maturing neural network with molecular, cellular and functional signatures. We have additionally modified existing protocols to allow use of postnatal tissue as the source for dissociation and re-aggregation. Hi-Spots made from postnatal day zero (P0) rats self organised into a complex 3D tissue-like structure containing anatomically synaptically-interconnected neurons, astroglia and microglia. This CNS analogue of brain tissue provides for the emergence of a co-ordinated excitatory and inhibitory network, demonstrating a maturing pattern of activity involving single spikes developing into bursting behaviour driven by intrinsic synaptic activity. This activity can be represented as frequency or averaged amplitude (RMS), increases with time in culture and is blocked by glutamate receptor antagonists and stimulated by the inhibitory receptor antagonist bicuculline. The validity of its more in vivo-like organization, not observed in more conventional dissociated cultures, is evidenced by a glutamate toxicity resistance in the Hi-Spot cultures. Further, the thesis describes the optimisations to the Hi-Spot protocol to allow viable high density re-aggregated cultures to be made from individual rather than pooled brain dissociations. Modifications to the protocol included dissociation of the mouse tissue using a protease papain kit and cell plating at an increased cell density. Using this as a platform we have gone on to investigate the neuronal dysfunction occurring in Cysteine String Protein (CSP)α-/- mice. CSPα is a presynaptic protein thought to have co-chaperone like functions, mice lacking CSPα are born alive but show progressive weakness and neuronal degeneration soon after birth (Fernandez-Chacon, Wolfel et al. 2004). Hi-Spots formed from CSPα -/- tissue did not show overt neurodegenerative characteristics compared to +/+ controls and functional analysis demonstrated that at~DIV14 CSPα +/+ and -/- mice displayed equal levels of basal spontaneous network activity. Addition of bicuculline (50μM) to +/+ cultures lead to a significantly increasedfrequency and RMS value. However, in -/- cultures there was no increase induced by bicuculline. This may be due to an inability of CSPα -/- cultures to sustain high frequency synaptic transmission that is associated with bursting activity, or a selective degeneration of a sub-population of inhibitory neurons and a homeostatic network plasticity. The data suggest CSPα may act to protect the ability of neurons for high frequency synaptic transmission and/ or protect inhibitory neurons from degeneration.

Click chemistry has the potential to be employed for the assembly of large DNA fragments, by purely chemical methods. However to enable this, the bio-compatibility of the resulting click-linked DNA must be examined. Click DNA linkers were incorporated into a plasmid within the gene encoding for an ampicillin resistance marker. The plasmid was transformed into E. coli and resulting colonies found to survive on LB agar plates supplemented with ampicillin. This indicated that the click DNA linker was replicated and transcribed correctly by the cellular machinery of E. coli. The observed bio-compatibility was further probed by demonstrating the functionality of the click-linked DNA in nucleotide excision repair deficient cells line. The bio-compatibility of the click DNA linker was then investigated in a non-essential gene by constructing a click-linked variant of the gene encoding for the fluorescent mCherry protein. Experiments carried out using a plasmid containing two click DNA linkers in the region of the gene encoding for the mCherry fluorophore provided further evidence that the click DNA linker was functional in E. coli. Moreover, using a coupled in vitro transcription/translation system the yield and fluorescence of the mCherry protein expressed from the plasmid containing the click DNA linker was similar to that from canonical DNA. Investigation of the bio-compatibility of the click DNA linker in mammalian cells showed that a plasmid containing the click-linked DNA, had the same viability of a plasmid containing canonical DNA. Finally, the use of click ligation for the assembly of the first 229 bp of the mCherry gene was investigated. Two different approaches referred as “templated click assembly” and “one-pot click assembly” were employed and the bio-compatibility of the click DNA linker was confirmed in both cases.

Predictive Adaptive Responses are changes in development made in the perinatal period in response to maternally transmitted information, and a mismatch between the diet selected during human evolution and the contemporary Western diet can produce an adult phenotype characterised by weight gain, cardiovascular disease, hypertension and diabetes. In humans, most evidence is epidemiological. Using Drosophila melanogaster, the problem can be approached from an adaptive phenotypic plasticity perspective. Health effects in humans stem from predictive adaptations made to enhance fitness and so it must first be shown that D. melanogaster make these responses. To model the human dietary transition, two equivalent fly diets were designed, one a human Palaeolithic diet and the other a contemporary Western diet. Using isofemale lines, flies were swapped between diets over three generations and fitness indicators measured in the offspring generation. Fitness indicator responses to a range of diets differing in protein: carbohydrate ratio and total macronutrient content were also investigated. There were adaptive, compensatory effects on survival rate and male thorax size from parental diet, and development time from grandparental diets, but also non-adaptive effects on development time and female thorax size from the parental diets. Higher dietary protein: carbohydrate ratios reduced development time and increased thorax size and survival rate, while increased macronutrient content increased weight, lipid content and survival. Diet had no effect on ommatidia number relative to fly size. Whether a response to diet is predictive and adaptive depends not only on diet composition, but whether offspring, parents or grandparents consumed the diet, the phenotypic character measured and the genotype of the fly. The variety of responses in relation to parental and grandparental diets show that intergenerational effects are complex, and D. melanogaster is a suitable model to help unravel the causes of human diseases.

DNA is regularly subjected to endogenous and exogenous reagents that cause mutations that can be detrimental to a cell if they are not repaired. One class of enzymes responsible for DNA repair is the family of DNA glycosylases and their role is to remove damaged bases. Uracil DNA Glycosylase (UDG) is a member of this family and is highly specific, removing only uracil, an RNA base, from DNA. Uracil arises in DNA through misincorporation of deoxyuridine monophosphate (dUMP) creating an A.U base pair, or through deamination of cytosine resulting in a G.U base pair. Though UDG acts on A.U pairs, this is not it’s primarily role as A.U pairings are not mutagenic. However the G.U mispair is highly mutagenic and leads to a G.C to A.T transition on subsequent rounds of replication. UDG only reacts with uracil and has no activity at thymine since the 5-methyl group on the base is excluded from the active site. This thesis examines mutants of UDG that can cleave cytosine but not 5-methylcytosine. Methylation of cytosine at CpG sites leads to gene silencing and is an important epigenetic signal. Knowing the methylation state of cytosines will therefore be important for understanding gene control and may be beneficial for treating many diseases. The most common method for detecting cytosine methylation uses a bisulphite reaction followed by normal DNA sequencing methods. This process has several drawbacks and the aim of this work is to create an enzyme that is capable of distinguishing between5-methylcytosine and cytosine. It has been reported that mutation of a critical asparagine in UDG to an aspartate allows the enzyme to accommodate cytosine into its active site; generating a cytosine DNA glycosylase (CDG). Using the natural ability of UDG to distinguish between uracil and thymine due to the presence of the 5-methyl group, we hypothesised that the mutant enzyme should be able to discriminate between5-methylcytosine and cytosine, which differ by the presence or absence of a methyl group in the same position. E. coli and human CDGs were prepared and their ability to remove cytosine or 5-methylcytosine examined when placed in different sequence contexts. hCDG was generated through complete gene synthesis of hUDG followed by the N204D mutation. The corresponding mutation in E.coli (N123D) generates a highly cytotoxic enzyme that cannot even be cloned in pUC19. As L191 aids base flipping, mutation to alanine (L191A) renders the enzyme inactive; activity can then be rescued using a bulky synthetic nucleoside that occupies the base pair and forces the target base into an extrahelical conformation. The L191A mutation was followed by N123D to generate an expressible and functional eCDG, denoted eCYDG. We demonstrate that these mutants have cytosine glycosylase activity when the cytosine is mispaired or unpaired, but not when paired with guanine, and show no activity against5-methylcytosine in any context. The activity of these CDGs varies with the stability of the base pair, with the fastest cleavage rates being obtained with the least stable base pairs, and also varies with the local sequence context. As CDGs are able to discriminate between cytosine and 5-methylcytosine we began development of a real-time PCR assay for detection of 5-methylcytosine. This employed a hexaethylene glycol (HEG) linker opposite the target cytosine, as this produces one of the fastest cleavage rates and cannot be read by a DNA polymerase.

Chromatin is the complex of DNA, histones and non-histone proteins that make up chromosomes and the region of dispersed chromosomes during the interphases and S phase of the cell cycle. It is found inside cell nuclei in eukaryotic cells. There are many important proteins in the nuclei which are involved in DNA replication, gene expression, DNA repair, etc. Core histones, linker histones and HMGs are the most important proteins in this group. In this thesis, a new, quick and efficient method is developed, to extract and purify proteins from cell nuclei, which is named "forward technology". By using the "forward technology", ultra-pure native core histone octamers, dimers and tetramers were extracted from chick erythrocytes. By using the pure histone dimers and tetramers, the complexes of NAP1-dimer and NAP1-tetramer were obtained in collaboration with others. Crystals of pure histone octamers with a higher resolution than before were produced (Chapter 2). A low KCI/phosphate wash of chick erythrocyte nuclei removed up to 1 g of proteins without nuclei lysis (cePNE1 proteins). The high KCI/phosphate soluble fraction of the cePNE1 is rich in HMG proteins, peptidyl-prolyl isomerases (FKBP3) and heatshock protein 70 which were fractionated by cation-exchange chromatography and anion-exchange chromatography (Chapter 3). Valuable (in terms of function) high-molecular-weight proteins were enriched, and another group of nucleoproteins called cePNE5 is fractionated by the "forward technology" from chick erythrocyte nuclei. It was confirmed that HMGB proteins prefer to bind to core histone H2A-H2B dimers (Chapter 4). It has proved possible to fractionate HMG-rich PNE1 proteins separate from a linker- histone rich nucleoprotein extract, separate from pure core histones (not as oetamers) from human tissue culture cells. The method has been applied to human leukocytes obtained from the National Blood Service. This enrichment of particular groups of proteins will be useful for proteomic studies (Chapter 5).