The number of flagella in Salmonella enterica serovar Typhimurium cells is closely related with the activity of FlhD4C2, a transcriptional regulator that controls the synthesis of a flagellum. In multiflagelated cells of S. enterica, FlhD4C2 activity is negatively controlled by the Type 3 Secretion chaperone, FliT. FliT interacts with the flagellar filament cap protein FliD. FliD is thus indicated as an anti-regulator in the same aspect describing FlgM inhibition of σ28 activity. Protein interactions between FliT, FliD and FlhD4C2 were explored in detail. Two independent studies, Native gel electrophoresis and gel filtration, revealed the stoichiometry of FliT:FliD interaction to be 1 to 1. In addition, Bacterial Two Hybrid (B2H) analysis showed FliT interacting with FliD In vivo. FliT was also shown to interact with FlhD4C2 by destroying the FlhD4C2 complex. FlhD4C2 activity reduced in the presence of FliT but not all FlhD4C2 activity was inhibited. Biochemical analysis of the interaction between FlhD4C2 and DNA showed that FlhD4C2 bound to DNA was insensitive to FliT regulation. Using a FliT variant, FliT94, that had the last α-helix deleted, helped show that the binding site of FliT to both FliD and FlhD4C2 possibly overlap. The negative regulation of FliT is proposed to be a flagellar specific regulatory mechanism that effectively decreases FlhD4C2 activity to a minimum level rather than inhibiting all flagellar production. FliD counteracts FliT activity to increase FlhD4C2 activity by forming the FliT:FliD complex. Importantly, FlhD4C2 bound to DNA is insensitive to FliT regulation and permits a low number of cells in a population to build a low number of flagella, even during the action of FliT. This study highlighted the importance of including the regulatory protein:DNA interaction in the working model of the regulatory circuit. This model unveils a mechanism to allow bacterial cells to remain prepared to respond quickly and efficiently to the decision to control the activity of flagellation to multiple signals generated by the external environment, internal cues and flagellar specific feedback mechanisms.

The experimental measurement of gene expression levels has produced preliminary results on the regulation, pathways and networks of genes in cells. Furthermore, the number of available three-dimensional folded structures of proteins increases on a daily basis. The ultimate aim of both genomics and proteomics froni the perspective of bioinformatics, is to map out all the circuits of energy and information processing in life by terms of molecular interactions in a system~tic way with minimal human intervention. In this thesis we propose a new rule mining framework for ill silico inference of protein-protein interactions and an effective class of techniques that identify domain-domain interactions from multiple domain protein structures. The above two approaches allow molecular biologists to use both gene expression data and three dimensional folded protein structures to efficiently predict or validate potential protein-protein interactions. A novel temporal rule mining technique is used to infer rules that relate local expression patterns across a set of genes. The set of these rules is associated to a set of potential interacting pairs of proteins. . I Probable protein-protein interactions can be validated against a network of protein domain interactions that are computed by considering interacting domains in known multiple domain protein structures. We introduce efficient algorithms that can effectively identify protein domain-domain interactions in multiple protein domain structures which are solved and publicly available. To analyse the vast amount of interactions between all the protein domains classified to superf~milies we propose a new graphtheoretic measure which is able to rank protein superfamilies by incorporating information about the topology of the whole network. To summarise, the work in this thesis consists of a new temporal rule mining framework applied .' to gene expression data analysis and furthermore, a novel class of algorithms that effectively identify ' domain-domain interactions from known protein structures.

The polymerase chain reaction (PCR) is one of the most widely used techniques in the biosciences, and has found extensive use in a variety of processes including gene cloning and mutagenesis. The PCR requires the use of a thermostable DNA polymerase that is able to tolerate the multiple heat/cool steps that occur during each cycle of the reaction. Archaeal family B DNA polymerases have found extensive use in this process, as in addition to their high thermostability they also contain a 3’-5’ exonuclease or proofreading activity, which increases the fidelity of replication. A polymerase that exhibits high processivity, defined as the number of nucleotides added per association with the DNA, is also desirable from a commercial perspective as it will reduce the amount of time taken to replicate any given amplicon. In this thesis, the processivity of a variety of commercially available archaeal Pol B enzymes is determined, which reveals significant differences in the processivity of polymerases closely related in sequence. The PCR performance of Pfu-Pol and Tkod-Pol, representing poorly and highly processive enzymes respectively is investigated, which reveals that Tkod-Pol is less efficient at replicating long amplicons (> 1000 bp) than Pfu-Pol, attributed to the increased thermostability of the latter. Based on this observation, an attempt is made to enhance the processivity of Pfu-Pol to improve the PCR performance of this enzyme.

No description available.

Fluorinated heterocyclic compounds have drug like properties and possess a valuable biological activity due to their rigid chemical structures and the high solubility profile. Novel fluorinated heteroarenes have been synthesised by SNAr reaction of a range of fluorinated arenes including pentafluoropyridine, hexafluorobenzene and pentafluorotoluene to introduce a range of groups specially nitriles, benzimidazole, carbazole and benzimidazole. A number of cyclization reactions have been investigated with the aim of forming polycyclic structures that could act as DNA intercalators. The synthesised compounds have been characterized by elemental analysis, IR, 1H and 19F NMR spectroscopy and single crystal analysis. These compounds have been screened for their biological activities including DNA thermal denaturation assay, UV-Visible spectroscopy, fluorescence spectroscopy, X-ray co-crystallization and antimicrobial activity study. Some of the compounds showed potential DNA bonding activity in particular the carbazole derivatives.

Bacterial conjugation is the transport of a DNA molecule from a donor cell to a recipient. Since bacteria do not reproduce sexually, conjugation is a major contributor to prokaryotic genome plasticity and the spread of antibiotic resistance genes. A Type IV Secretion System (T4SS) mediates the DNA transport during conjugation. T4SSs are large macromolecular assemblies embedded in the membrane of bacteria, and are associated with pathogenesis, bacterial conjugation and natural transformation. They are a versatile family of secretion systems, who transport a wide variety of substrates, such as virulence proteins, DNA––protein complexes as well as only DNA. Here, we investigate the minimal requirements for conjugation, and the T4SS’s localisation within the cell. We show that the conjugative T4SS of the plasmid pR388 requires a total of 14 genes to efficiently mobilise DNA from a donor cell to a recipient cell and is arranged around the cell circumfence in a helical array. Our study of two reconstituted and fully functional conjugative T4SSs opens doors for further structural and functional analysis.

Kaposi’s sarcoma-associated herpesvirus, is a double-stranded DNA γ - herpesvirus and the main causative agent of Kaposi’s sarcoma (KS). γ - herpesviruses undergo both lytic and latent replication cycles; and encode proteins that modulate host transcription at the RNA level, by inducing decay of certain mRNAs. Here we describe a mechanism that allows the viral endo-/exonuclease SOX to recognise mRNA targets on the basis of an RNA motif and fold. To induce rapid RNA degradation by subverting the main host mRNA degradation pathway SOX was shown to directly bind Xrn1. This may shed light as to how some viruses evade the host antiviral response and how mRNA degradation processes in the eukaryotic cell are involves in this.

Nance-Horan Syndrome (NHS) is an X-linked developmental syndrome characterised by congenital cataract, dental anomalies, and dysmorphological features often associated with mental retardation. The NHS locus on Xp22.13 is encompassed by the disease locus for X- linked congenital cataract (CXN). Analysis of microsatellites within the CXN family resulted in refinement of the CXN disease interval, reducing the region of overlap between the CXN and NHS disease loci. Candidate genes in the overlapping intervals were identified bioinformatically and their genomic structures evaluated. Patient DNA was screened by direct sequencing, resulting in the identification of mutations within a novel gene in four British families with NHS, but not the CXN family. This novel gene, named NHS, is encoded by at least 10 exons transcribed into at least five mRNA isoforms A, B, C, D, and E (encoding a putative 1,630 a.a., 1,335 a.a., 1,474 a.a., 1,453 a.a., and 1,473 a.a. protein, respectively). All mutations identified are truncating and three mutations have been identified in exon 1, which are only expressed in isoform A. This implies that mutations in isoform A are sufficient to cause disease in families with NHS. Functional clues for the NHS protein were investigated resulting in identification of three new genes with significant homology to NHS {NHS-Like 1 (NHSL1), NHSL2 and NHSL3). All four genes share a conserved genomic structure. Fetal expression analysis of NHS, NHSL1 and NHSL2 suggests that NHSL1 and NHSL2 are more ubiquitous than NHS. Analysis of the NHS family of proteins revealed significant homology to members of the WASP family, which consists of WASP, N-WASP and WAVE 1-3. The WASP protein family play a crucial role in regulating actin dynamics, directly linking small GTPase signalling to actin polymerisation through activation of the Arp2/3 complex. An anti-peptide antibody to the C-terminus of NHS, completely conserved across species, was raised and characterised. A major NHS isoform (approximately 170 kDa) was detected in several cell lines. Subcellular localisation studies in MTLn3 cells showed localization of endogenous NHS to the leading edge of lamellipodia, a localisation pattern reminiscent of the Arp2/3 complex. Endogenous NHS also localised to some actin stress fibres. Homology to the WASP protein family and localisation of endogenous NHS to the leading edge of lamellipods strongly supports a role for NHS in actin cytoskeletal dynamics during development.

In response to DNA damage, eukaryotic cells activate a checkpoint signalling cascade, resulting in cell cycle arrest, stabilisation of replication forks and activation of repair. While many players in these pathways have been identified, little is known about the original sensors, or of the DNA structures involved. Because it is present in all checkpoint-inducing lesions, single-stranded DNA (ssDNA) is a good candidate for a common structure recognised by the DNA damage response. The role of ssDNA in checkpoint activation in the yeast Saccharomyces cerevisiae was investigated using three different approaches. Firstly, an attempt was made to produce ssDNA independently of strand breaks by inducing replication-independent plasmid unwinding. Secondly, the effects of depleting the major ssDNA-binding complex, replication protein A (RPA) were analysed. Lastly, an assay to quantify ssDNA generated at a defined DNA double-strand break (DSB) was developed. Despite extensive efforts, the first approach proved unsuccessful, as the method used did not generate unwound plasmid. Using the second approach, it was found that depletion of RPA did not inhibit checkpoint activation during replication stress. Furthermore, replication with limiting amounts of RPA led to rapid cell death and checkpoint activation that was mediated independently of the response to stalled replication forks. Lastly, at a defined DSB it was found that less ssDNA was being generated than had previously been estimated from results based on non-quantitative methods. Additionally, an element of dose dependency was observed in the checkpoint response to DSBs, with stronger and more rapid responses being generated by higher numbers of breaks. Formation of four DSBs resulted in checkpoint activation even in G1 arrested cells. Together, these results raise the possibility of a DNA damage checkpoint pathway largely independent of long tracts of RPA-coated ssDNA and show that checkpoint activation to DSB-damage is possible in G1.

In the human genome, cytosine is exclusively methylated at CpG sequences to give 5-methylcytosine. The C4-amino group of 5-methylcytosine is susceptible to spontaneous hydrolysis, generating thymine in a G T mismatch. Thymine DNA glycosylase (TDG) detects the mismatch and cleaves the glycosidic bond of thymine. Then apyrimidinic/apurinic endonuclease 1 (APEX1) displaces TDG from the abasic site and cuts the phosphodiester bond 5' to the abasic site in base excision repair. The kinetic parameters, K<i and k2, for TDG acting on thymine and ethenocytosine substrates, were measured. The excision of both substrates was very dependent upon the base 5' to the mismatched guanine. TDG has a strong preference for both thymine and ethenocytosine in a CpG sequence. This is understandable for thymine since G T mismatches arise in this context. However, ethenocytosine is not exclusively formed at CpG sequences. The catalytic step (k2) for CpG-T was six-fold faster than CpG-eC, but was bound, as shown by Kd, approximately 800-fold less tightly by TDG. This large difference in Kd is probably due to the unstable structure of G sC, which allows TDG to easily flip ethenocytosine out of the DNA. Thus, the reaction of TDG with ethenocytosine in vitro is misleading. This, together with the sequence dependence of ethenocytosine excision by TDG, means that TDG cannot be the main ethenocytosine-DNA glycosylase in vivo. The mechanism of TDG displacement by APEX1 was examined by looking at the involvement of the DNA and by looking for protein-protein interactions between TDG and APEX1. Although the DNA is not absolutely required for the displacement of TDG by APEX1, a weak/transient protein-protein interaction was detected between TDG and APEX1 using a pull-down assay. These results suggest that APEX1 transiently interacts with TDG bound to abasic DNA to induce a conformational change in TDG that dissociates it from the abasic site.