Methylococcus capsulatus (Bath) is a Gram-negative, spherical-shaped bacterium that gains its needs of carbon and energy via oxidation of methane, a potent greenhouse gas, thus alleviating global warming. This bacterium oxidises methane to methanol using a membrane-bound particulate methane monooxygenase (pMMO) or a soluble, cytoplasmic methane monooxygenases (sMMO). Copper-tobiomass- ratios significantly affect the expression and activity of both enzymes; the biosynthesis of sMMO is switched on when copper-to-biomass ratios are low, while pMMO is up-regulated when they are high. The exact mechanisms by which copper regulates the switching between sMMO and pMMO are not fully elucidated. Therefore, the main aim of this study was to shed some light on this copper switch, taking the advantage of the availability of the genome sequence of this organism, together with mutagenesis and transcriptional regulation studies. Three potential copper transport Mc. capsulatus mutants; ΔcopA1, ΔcopA2 and ΔcopA3 were generated. The genes inactivated encode three different P-type ATPase homologs. This revealed that CopA1, CopA2 and CopA3 have roles in copper homeostasis, although disruption of genes encoding these proteins individually did not result in constitutive sMMO expression. In addition, three mutants; ΔnrpS-1, ΔnrpS-2 and pkS were constructed. pkS encodes for a polyketide synthase, nrpS-1 and nrpS-2 encode for two non-ribosomal peptide synthetases. The products of these genes were proposed to be involved in biosynthesis of methanobactin, a short peptide that scavenges copper when it is limited. Results suggested that nrpS-2 and pkS might be involved in production of a functional methanobactin. Putative coding sequences predicted to be involved in methanobactin biosynthesis in another methane-oxidising bacterium, Methylosinus trichosporium (OB3b), were also mutated. The mutant was unable to produce methanobactin, could not express sMMO, and was copper resistant compared to the wild-type organism. Therefore, methanobactin is ribosomally- produced in Ms. trichosporium. Corresponding genes were not identified in the genome of Mc. capsulatus. A microarray-based comparative expression profiling study of whole-genome transcriptomics of Mc. capsulatus expressing sMMO versus pMMO was carried out to identify genes involved in regulation of MMO by copper. This identified 53 genes that were differentially expressed and hence promising candidate genes for future studies of MMO regulation. For example, tetR, a down-regulated gene, encodes a putative transcriptional regulator and tonB, an up-regulated gene, which encodes a protein that is a part of a membrane transporter. Interestingly, a cluster of six genes 5’ of sMMO was up-regulated; five of them were found to be co-transcribed. A mutant was made in an up-regulated gene encoding ScO protein (synthesis of cytochrome c). The mutant could tolerate high concentrations of copper compared to the wild-type strain. The work presented in this study is considered a step forward towards understanding the regulatory mechanisms of the copper switch in methanotrophs and provided the basis for new lines of future research to fully understand this phenomenon.

The CHD family of ATP-dependent chromatin remodellers, which are present in all eukaryotes, utilise energy from the hydrolysis of ATP to alter nucleosome structure. The multiple CHD proteins have previously been implicated in transcription and developmental regulation, although the relationship between different family members is not well understood. The CHD family in H.sapiens contains nine proteins divided into three subfamilies. Whereas S.cerevisiae possess a single CHD, the Dictyostelium discoideum genome contains three CHD genes: one subfamily I member, related to human CHD1/2, and two subfamily III members, related to human CHD7/8. Dictyostelium fills a gap in the complexity scale, providing a smaller complement of CHD family members than mammalian models whilst still possessing a multicellular development stage and retaining a compact manipulatable genome. I have studied the expression patterns and created knockout mutants for all three Chd family members. The chd mutants have different developmental phenotypes that correlate with their expression profiles. RNA-seq analysis of the mutants showed that each Chd protein is responsible for regulating the expression of distinct classes of genes. Mapping nucleosomes genome wide in wild-type cells revealed that nucleosomes are structured similarly to higher eukaryotes. Mapping of nucleosomes in chdC-null suggested that ChdC regulates nucleosome repeat length in a subset of genes in a developmentally regulated manner. This thesis presents a comprehensive study of the Chd family in Dictyostelium and, to my knowledge, the first in vivo study of the nucleosome remodelling activity of a Chd subfamily III member.

The renin angiotensin system is often maninpulated clinically for the treatment of hypertension and heart failure. This pathway is of major clinical importance and it is thus a major target for therapy. The incidence of cardiovascular diseases continues to increase worldwide, highlighting the need for new therapies to treat these conditions. Gene therapy for the treatment of cardiovascular diseases is currently being developed. Gene therapy is by definition the treatment or prevention of disease by means of gene transfer. The efficiency of gene transfer will determine how successful the gene therapy application will be. Before the full potential of gene therapy can be reached, many limitations common to all methods of gene delivery must be overcome. The current lack of suitable vectors capable of transducing cells of the vasculature or of the myocardium is a major rate-limiting step, but may be overcome by increasing the specifity of gene therapy vectors. This may be achieved through the isolation of new viral serotypes that can be developed into vectors, or the creation of new vectors by the alteration of the tropism of existing ones. This thesis aimed to assess the effect of ACE2 overexpression in vivo on heart function and blood pressure. In order to achieve cardiac gene transfer, we first had to identify an efficient cardiac gene delivery vector. This was approached by the application of the two main techniques; (1) the use of phage-display identified peptides to retarget viral vectors and (2) the comparison and optimisation of rAAV6 and rAAV9 mediated gene delivery to myocardium in vivo in a rat disease model.

No description available.

The stroke-prone spontaneously hypertensive rat (SHRSP) is an inbred model of hypertension. Renal microarrays and functional genomic strategies investigated chromosome 2 candidate hypertension genes, focussing on the oxidative-stress defence gene, glutathione s-transferse mu type 1 (Gstm1). Ingenuity pathway analysis of renal microarrays in 5 and 16-week SHRSP, normotensive Wistar Kyoto (WKY) and chromosome 2 congenic rats identified differential expression of several glutathione cycling genes. The Gstm1 promoter was investigated by luciferase and Transfac bioinformatic analysis, implicating two polymorphism clusters and several transcription factors in reduced SHRSP Gstm1 expression. Recombinant adenoviruses expressing Gstm1 and short-hairpin RNA-interference sequences to reduce Gstm family expression were produced. In-vivo overexpression of Gstm1 did not improve endothelial nitric-oxide bioavailability in SHRSP carotid arteries. Bacterial artificial chromosome and linear expression constructs were purified for production of Gstm1 transgenic rats, putative transgenic rats were screened by PCR. The strategies developed in this project are an example of thorough functional genomic analysis in experimental hypertension research.

Prior to the work described in this thesis, a great deal of the oxytetracycline cluster (otc) had been sequenced and the putative functions of deduced gene products assigned, based on similarity to known gene products in the databases. The 3.0 kb KpnI23-MluI26b fragment located between the otcD and otcX loci was sequenced. Three open reading frames with the same direction of transcription were found, otcD-ORF3, -ORF4 and -ORF5. OtcD-ORF3 has good similarity to oxidoreductases reported previously in other polyketide gene clusters. The biosynthesis of OTC requires three oxidoreductase steps, one at position C9 which removes the keto function, one at position C6 in the conversion of 4-keto-ATC to ATC and another at the last step [OTC dehydrogenase]. A ketoreductase has been reported previously within the otcY locus that shows stronger similarity to actIII of S.coelicolor. OtcD-ORF3 has been tentatively assigned as catalysing the last step of the pathway. OtcD-ORF4 shows good end-to-end similarity to a coding region of unknown function within the gene cluster for daunorubicin biosynthesis in S.griseus. OtcD-ORF5 is clearly defined as a potential protein coding region that shows good similarity to a small region of an open reading frame within the B.subtilis genome which again has unknown function. The B.subtilis coding region is very hydrophobic and carries a series of repeat sequences, the significance of which is not yet understood. Preliminary transcriptional analysis of this 3.0 kb KpnI23-MluI26b fragment using low-resolution S1 mapping has revealed a single transcript which encompasses OtcD-ORF3 and OtcD-ORF4. A stable stem-loop immediately after the translational stop codon of OtcD-ORF4 was located with high-resolution mapping. A potential promoter sequence was located immediately preceding OtcD-ORF5, however no transcript was detected with low-resolution mapping at the time point when the RNA was isolated.

The insertion sequence ISY100 is a member of the IS630/Tc1/mariner superfamily of transposable elements. An in vivo transposition assay was set up in this study, confirming the TA target preference of ISY100 and showing that 30 bp from each end of ISY100 is sufficient for efficient transposition. Purified His-tagged transposase bound the transposon ends, protecting approximately 26 bp from cleavage by DNase I at each end. Two helix-turn-helix DNA binding motifs linked by an ‘AT-hook’-like sequence were predicted in the N-terminal domain of ISY100 transposase. Supercoiled plasmid containing ISY100 ends, and synthetic linear transposon ends were tested for cleavage by transposase in vitro. Cleavage products were observed and the cleavage sites were mapped. Linear DNA fragments containing single ISY100 ends were cleaved mainly one nucleotide inside the transposon end to produce a 3’ OH and one nucleotide outside the transposon end to produce a 5’ phosphate. Changes in the flanking TA dinucleotides at either one end or both ends of ISY100, reducing the efficiency of transposition in vivo. These changes also reduced the efficiency of cleavage in vitro. Changes at only one end inhibited cleavage at both ends implying communication between the two transposon ends. Synthetic pre-cut transposon ends were tested in an in vitro integration assay, and transposase catalysed the insertion of transposon 3’-OH ends into a target plasmid. Transposase mediated efficient integration of a mini-ISY100, pre-excised by transposase or restriction enzyme, into TA targets in vitro, confirming that excised transposon fragments are intermediates in the reaction. Target sequences of ISY100 from published data and this study were analyzed, yielding the consensus target sequence ADWTAWHT, in which the central TA is the duplicated target dinucleotide. When the Zif268 DNA-binding domain of Tn3 resolvase, transposition occurred into TA dinucleotides to one side of a Zif268 binding site with elevated frequency. This could be developed into a genetic tool for target-specific integration.

Duchenne Muscular Dystrophy (DMD) is a genetic disorder which is caused by mutations in the dystrophin gene.  One of the functions of this protein is to anchor the cell membrane to the sacrolemma.  In DMD, the absence of this protein leads to muscle damage during contractions, resulting in continuous regeneration and repair of the muscle until the replicative ability of the muscle is exhausted. There have been many attempts to cure this disorder including the strategy of gene and cell therapy.  Although these approaches have had some success in animal models there has been no improvement in human trials.  At present the treatment of DMD consists of steroid therapy and supportive treatment. The aim of my work was to test the hypothesis that the transduction of telomerase in to muscle cells could produce a non transformed muscle cells with unlimited growth potential that could in principal, could serve as a target cell population for human dystrophin gene therapy. The expression of the catalytic subunit of telomerase (hTERT) has been successfully used to extend the lifespan of a number of different cell types without malignant transformation.  However previous attempts to immortalise muscle cells in this manner had failed.  Here I report the successful immortalisation of normal human skeletal muscle cells by retroviral infection of hTERT.  The telomerase positive cells display an extended lifespan, with 4/5 clones exceeding 120 MPD.  The clones show no feature of transformation in vitro, retain a stable diploid karyotype, have wild type unmethylated CDKN2A genes and do not express muscle specific markers desmin and spectrin.  In vitro, they can repair and reconstitute muscle in immunosuppressed RAG-1 mice.  These results suggest that telomerase expression can extend lifespan of human muscle cells and could aid attempts at gene therapy for muscle diseases.

The combination of physiology and genetics affords opportunities to perform experiments with greater precision, and with less risk of artefact, than heretofore possible. This is illustrated here by demonstrating the selective manipulation of the second messengers cyclic GMP (cGMP), cyclic AMP (cAMP) and calcium (Ca2+) in the renal (Malpighian) tubule of the genetic model organism, Drosophila melanogaster. The importance of the second messengers cGMP, cAMP and Ca2+ in control of fluid transport makes the Malpighian tubule an ideal testbed for such technology. The actions of known hormones can be compared with the results obtained by manipulating levels of the second messenger through which these are thought to act. Flies were constructed or obtained transgenic for the rat atrial natriuretic peptide receptor, GC-A, the Drosophila 5HT7Dro receptor, and the Drosophila 5HT1ADro receptor, under control of the UAS or heat-shock promoter. Tubules dissected from such flies were then demonstrated to show diuresis induced by application of rANP or 5HT, whereas controls showed no response. Second messenger measurements showed that the rat GC-A receptor acted to raise cGMP levels, the 5HT7Dro receptor to raise cAMP levels, and the 5HT1ADro receptor to raise intracellular calcium. In addition, modulation of both cell-specific cell nucleotide phosphodiesterase activity and cell-specific cyclic nucleotide-dependent kinase activity of tubules with elevated cyclic nucleotide levels was observed, implicating these enzymes as key regulators/effectors of signalling in tubules. Cross-talk between cGMP, cAMP and Ca2+ was also assessed and shown to be variable, depending on the particular cell type in which the signal was generated. This reveals further complexity in the control of tubules and suggests a distinct role for cyclic nucleotide-gated channels in principal cells. This study has validated ectopic transgene expression as a generic technology with potential beyond Malpighian tubules: in principle, such transgenes can be expressed specifically in any population of cells that can be delineated by a GAL4 driver line.

Many genes and cellular pathways have been implicated in the initiation and regulation of testicular differentiation, development and function. The genes generally have a temporal expression profile. This study aimed to characterise several of the key genes involved in the androgen synthetic pathway, in both normal mice and also mutant strains. LH receptor and 5 a reductase were the two major genes of interest. Using molecular biology techniques it was established that the LH receptor exists in several forms throughout development, with smaller, alternate-spliced forms being the predominant transcripts expressed in testis during fetal life. It is inconclusive from this study whether these spliced forms encode functional proteins, but the consistency of their expression patterns suggests that they do. Expression levels of the two isoforms of 5 a reductase were found to be very low in mouse testis throughout development, and in addition androgen may be involved in regulation of expression of the type 1 isoform, as levels were significantly reduced in the AR-null mouse testis both at puberty and adulthood. Previously unpublished sequence from both type 1 and type 2 5 a reductase transcripts was obtained using PCR primers designed from rat sequence and mouse EST sequence. The results from this study highlight the importance specific genes play in regulation and expression of others and the down stream signalling pathways which may be involved in development and regulation of a functional testis.