The microflora of two acidic environments was investigated using analysis of 16S rDNA amplified by the polymerase chain reaction (PCR) from environmental DNA. These environments had different chemical characteristics from most of the acidic environments studied by others. The first sample site, a coal spoil (Birch Coppice, Warwickshire), might have been expected to produce niches enriched in humic matter. The second, comprising geothermal vents on the Island of Vulcano, was unusual for natural acidic environments since it was saline. Three vent regions of different temperatures (30°C, 45°C and 80°C) were examined. Prior to the 16S rDNA analysis of the sites, a brief investigation into selection of a suitable method of DNA extraction was carried out. A bead-beating method and a chemical lysis/freeze-thaw method were compared. With regard to clone types found via each method, there was little qualitative difference. DNA was extracted from the two sites and 16S rRNA genes were amplified by PCR. PCR products were ligated and competent E. coli cells were transformed to produce clone libraries. Restriction fragment length polymorphisms (RFLPs) were examined and representatives of each RFLP type were sequenced and analysed with reference to RNA gene sequence data bases. The coal spoil clone library was dominated by sequences related to those from uncultured actinobacteria, particularly those found previously in peat bogs and various soils. Representatives of some well-known acidophiles were also found (e.g. Leptospirillum species). The clone bank from the saline, geothermal site DNA comprised sequences from acidophiles capable of growth at the respective temperatures of different samples. The lowest temperature samples produced sequences from a novel Acidithiobacillus species and also indicated a novel species probably related to Thiobacillus prosperus (which was isolated previously from Vulcano). A high temperature sample gave sequences from archaeal acidophiles, Acidianus brierleyi and, previously isolated from Vulcano, Acidianus infernus and Thermoplasma volcanium. Where the clone banks revealed the presence of novel organisms, attempts were made to isolate and characterise them. The novel actinobacteria did not appear to grow in laboratory enrichment cultures. The novel Acidithiobacillus species and two novel Thiobacillus prosperus-like species were characterised.

The rotaviruses are a recently defined ubiquitous group of viruses responsible for causing acute-gastroenteritis in human infants and young animals. Biochemical studies have shown that the rotavirus genome consists of 11 segments of double-stranded RNA (dsRNA). This thesis concerns an investigation of the nature and extent of genomic variation in rotaviruses. A rapid and sensitive method for analyzing the genome profiles of rotavirus field isolates was developed. This is based on the direct extraction of dsRNA from faecal samples followed by radiolabelling with [³²P] pCp using T4 RNA ligase. This procedure has been further developed to produce a method for generating diagnostic fingerprints from individual species of dsRNA. A detailed structural study making use of this fingerprinting method has been undertaken on bovine, porcine and human rotavirus isolates. These analyses show that genome segment mobility variations are always associated with detectable changes in nucleotide sequence. They also show that corresponding genome segments with no mobility variation can have sequence-changes at least as great as those found in segments showing electrophoretic mobility variation. These results also revealed evidence for genome segment specific regions of terminal sequence conservation. Evidence for the occurrence of genome segment reassortment between viruses in the field was obtained. Finally evidence for the existence of a 'new' porcine rotavirus which is antigenically unrelated to previously described rotaviruses and has an unusual pattern for its 11 genome segments is presented.

‘Cutting out’ HIV-1 proviral DNA could potentially cure a person of the infection. Genome editing approaches have been proffered for eradicating the provirus in infected persons by activating all latent viral reservoirs for further antiretroviral therapy or for the excision of the proviral DNA from memory T- cells. Previous approaches to do this have used nuclease-based tools or reprogrammed tyrosine recombinases; the former presenting unpredictable therapeutic outcomes and the latter, lengthy design time for newer tool variants if viral mutability erodes their effectiveness. Unlike nuclease-based tools that only cut DNA and rely on host-mediated repair mechanisms, chimaeric recombinases (CRs) cut DNA and carry the inherent ability to re-ligate cut ends at the cleavage site. The modular domain architecture of small serine recombinases can be redesigned to mediate site-specific recombination on non-cognate sites, by replacing the C-terminal DNA binding domains (DBDs) of serine recombinases with programmable DBDs such as Zinc Finger (ZF) proteins, TAL effector proteins and CRISPR-dCas9. For HIV-1 proviral DNA excision, CR requirement for the interaction of two recombinase-bound sites, and the lack of necessity for host cell-encoded factors should maximize the fidelity and efficiency of provirus removal. In this work, the engineering and characterization of CRs with the specificity to recognize and promote site-specific recombination at highly conserved regions within the HIV-1 proviral DNA is explored. This research provides a solid proof-of-concept for the use of CRs to target divergent novel target sequences, expanding their applicability for applied genome editing and wider biotechnological applications.

Replication of the genome is a central process in cellular life, which must be tightly regulated at the risk of genomic instability. DNA replication has been extensively studied in bacteria and eukaryotes, and recently, in archaea. Universally, DNA replication is started at specific genomic sites termed origins of replication, which are recognised by an initiator factor. While in both bacteria and archaea the initiator factor is a single protein, it is assumed that this role is performed in eukaryotes by a highly conserved six-subunit origin recognition complex (ORC). Recent phylogenetic studies, however, suggest that the presence of a six-subunit ORC might not be as conserved as initially believed. Trypanosoma brucei is a protozoan parasite in which little is known about nuclear DNA replication. To date, initiation of T. brucei DNA replication has been associated with a single factor, TbORC1/CDC6, though highly diverged interacting partners have been identified. To elucidate whether T. brucei possesses a diverged ORC-like complex, TbORC1/CDC6 and its known interacting partners, TbORC1B, TbORC4, Tb7980, Tb3120, and a novel factor, Tb1120, were analysed. First, the protein sequences of these factors were re-analysed, revealing varying degrees of conservation and divergence with other eukaryotes’ ORC proteins. Second, expression silencing by inducible-RNA interference (RNAi) of TbORC1/CDC6, TbORC1B, TbORC4, and Tb3120, in procyclic form (PCF) and bloodstream form (BSF) cells, confirmed their involvement in DNA replication. Third, subcellular localisation and dynamics of TbORC1/CDC6 and its interacting partners during the cell cycle of PCF and BSF cells was investigated by immunofluorescence, revealing TbORC1B to be the sole factor to display an apparent cell cycle-dependent localisation pattern, perhaps suggesting that it might be a DNA replication regulatory factor. Finally, immunoprecipitation and gel filtration assays support the existence of an ORC-like complex, apparently large enough to be composed of TbORC1/CDC6 and known interacting partners, and potentially, additional factors. TbORC1/CDC6-binding sites and origins of replication in T. brucei have been mapped in PCF cells. Like most eukaryotes, no specific sequence elements were found to define TbORC1/CDC6-binding sites or origins of replication, and the TbORC1/CDC6-binding sites outnumbered the mapped origins, which appeared to be activated at different times during S phase. It has been reported in other eukaryotes that different cell types activate different origins or the activation timing differs. Here, origins of replication were mapped in T. brucei PCF and BSF cells, revealing a pronounced inflexibility in origin usage in these two life cycle stages. Only one, notable genome-wide difference was found: in BSF cells, the single active variant surface glycoprotein (VSG) expression site was found to be early replicating, whereas all other silent VSG expression sites were late replicating; in PCF cells however, where all VSG expression sites are silenced, these were all late replicating. These data reveal a locus-specific link between DNA replication and transcription in T. brucei, which may relate to immune evasion. The genomes of T. brucei and related kinetoplastids are highly syntenic. Since most eukaryotic origins of replication are not defined by consensus DNA sequences, but appear instead to be defined by, among other features, chromatin context and status, origins were here mapped in both L. major and L. mexicana promastigotes in order to ask if common features could be found relative to T. brucei. Surprisingly, only a single origin could be found per Leishmania chromosome, in contrast with all eukaryotes examined to date, where each chromosome is replicated from multiple detectable origins. Origin-active loci in Leishmania were found to be distinguishable from related non-origin loci in terms of size, a characteristic not observed in T. brucei, although around 40% of the mapped origins are conserved in location relative to T. brucei. These data reveal pronounced differences in replication dynamics between the two genera, despite the considerable overlap in genome organisation.

Alternative splicing occurs in more than 90% of human genes and is particularly abundant in the nervous system. It has been recognized that toxicity can be caused at the level of pre-mRNA processing and potentially lead to age-dependent neurodegeneration upon low-dose chronic exposure. ELAV (Embryonic Lethal Abnormal Visual system)/Hu family proteins are prototype RNA binding protein and gene specific regulators of alternative mRNA splicing in the nervous system. Analysis of mutants in ELAV family proteins shows overlapping and distinct functions during development and age-dependent neurodegeneration. Overexpression of ELAV family proteins further revealed that cytoplasmic localization of ELAV family proteins in associated with enhanced neurotoxicity. Intriguingly all Drosophila ELAV family proteins and mammalian Hu proteins can regulate neuron-specific alternative splicing of Drosophila neuroglian gene- a known ELAV target. The blood brain barrier (BBB) and efficient excretion are protective mechanisms making delivery of many drugs to the brain difficult in vivo. Therefore, I analyzed the roles of a number of key Organic Anion Transporter Protein (OATP) and Multi- Drug Resistance (MDR) proteins and established a sensitized genetic background for CNS drug delivery. To assess if xenobiotics can interfere with ELAV function leading to neurodevelopmental/neurodegenerative defects, I assessed ELAV regulation of its major target erect wing (ewg) using an ewg fluorescent reporter, which recapitulates endogenous ELAV-mediated splicing and allows rapid visualization of potential modulators. From a compound screen in a sensitized genetic background, I identified a number of xenobiotics that cause changes in ewg splicing, indicating interference with ELAV function. Importantly, these compounds also phenocopy specific characteristics of ELAV mutants. My approach demonstrates the potential for using Drosophila in drug screening and neurotoxicity assessments.

This project examined whether it was possible to create functional hybrid serine integrases – proteins responsible for recombining DNA in a site-specific manner. Creating hybrid recognition sites, specifically engineered to be recognised by the new integrases, was examined concurrently. Ultimately, new serine integrases and recognition sites were created with the intention of increasing the repertoire of serine integrases available for use as independently functioning modules in synthetic biology assemblies. Experiments were carried out primarily on two groups of hybrid integrases – BxbI integrase and ΦC31 integrase, and the smaller recombinase Tn3 resolvase and ΦC31 integrase. It was determined that either the BxbI integrase/ΦC31 integrase hybrids were not active on hybrid or parental recognition sites, or that the proteins themselves were not expressed at a high enough level to exhibit any activity. However, one ΦC31 integrase/BxbI integrase hybrid did exhibit activity on ΦC31 integrase recognition sites in vivo, though not on hybrid sites. However, Tn3 resolvase/ΦC31 integrase hybrid proteins proved far more promising. The two hybrids exhibited recombination on sites created for them, whilst exhibiting no activity on any parental recognition sites. When both Tn3 resolvase and either hybrid integrase were present in vitro, recombination on combination substrate plasmids containing one copy of the Tn3 resolvase recognition site res site I and one copy of a hybrid recognition site was much higher than for either hybrid against hybrid sites on its own. Additionally, throughout this investigation, it was discovered that ΦC31 integrase cleaved and recombined several sites very dissimilar to its natural attP and attB sites.

Trypanosoma brucei, the eukaryotic parasite that causes human African trypanosomiasis in humans, evades the immune system through antigenic variation. T. brucei antigenic variation involves the periodic switching of the variant surface glycoprotein (VSG) coat to an antigenically distinct variant. A single VSG is expressed on the cell surface at any one time, but the T. brucei genome contains a vast number of silent VSGs. To be expressed, a VSG must be located in a specialised VSG blood stream form expression site (VSG BES). Silent VSGs are copied into VSG BES by homologous recombination. Several proteins have been demonstrated to be involved in this process but how VSG switching is initiated remains unclear. Four putative DNA repair factors were identified in T. brucei, whose eukaryotic homologues play a range of roles in DNA repair and other aspects of genome maintenance. These were two RecQ-like helicases, a Mus81 endonuclease and a Pif1 family helicase (PIF6). To examine whether these factors play a role in DNA repair and VSG switching, mutants were generated in blood stream form T. brucei cells. Analysis of RecQ1 by RNAi knockdown revealed it to be an essential gene in bloodstream form T. brucei, possibly involved in nuclear DNA replication. Phenotypic analysis of recq2 mutants suggests that RECQ2 is involved in the repair of a range of DNA damaging agents. Furthermore, analysis of survival following DSB induction suggests RECQ2 is involved in the repair of DNA DSBs, including those in the active VSG BES. VSG switching analysis showed that recq2-/- mutants have an elevated VSG switching rate and increase in recombination events upstream of the active VSG. These analyses suggest that RECQ2 suppresses VSG switching in T. brucei by suppressing recombination events near the active VSG. Analysis of mus81 mutants showed mus81-/- mutants to be sensitive to agents inducing replication stalling and DNA breaks, and that MUS81 is important in the repair of DSBs. PIF6 appears to be a complicated DNA repair factor, different from MUS81 and RECQ2. pif6+/- and pif6-/- mutants appear to be more resistant to MMS than wild type cells, though more sensitive to the replication stalling agent hydroxyurea. pif6 mutants do not appear to be more sensitive to DSBs than wild type cells and may even be more resistant. It is unclear whether PIF6 is involved in VSG switching and more work is required on this factor to attempt to understand its DNA repair and VSG switching function in T. brucei. These analyses shed light on the DNA repair functions of four previously uncharacterised T. brucei proteins. In particular, observations that RECQ2 is deficient in repairing DSBs upstream of the active VSG and mutants exhibit an elevated VSG switching rate cannot be reconciled with current thinking that direct formation of DSBs in this location initiates VSG switching. This suggests that the initiation of VSG switching is more complex than currently thought and requires careful further study and consideration of the relevance of using direct DSBs in this location to model VSG switching.

Many parasites of mammals avoid elimination by varying their exposed antigens. African trypanosomes—deadly parasites of humans and livestock in tropical Africa—possess a comprehensive system of antigenic variation (AV). Trypanosoma brucei undergo frequent, stochastic changes to their variant surface glycoprotein (VSG) coats, and therefore a developing immune response will be only partially effective against the trypanosome population as some trypanosomes will have already switched to a different VSG coat. The source of VSG variability is an archive of ~2000, mostly pseudogenic, silent VSG genes, of which only one is expressed. VSG genes can also be segmentally recombined: ‘mosaic’ VSGs, constructed from more than one silent VSG donor, allow both the reparation of pseudogenes and potentially generation of additional VSG variability. The aim of this research was to investigate the patterns of segmental VSG gene conversion in T. brucei, and assess its contribution to AV. Multiple, longitudinal samples were taken from chronic infections to follow the course of AV, and VSG cDNA sequences were analysed, building a detailed portrait of VSG expression across infection. VSG variability during an infection was extensive, and segmental gene conversion was found to be a frequent occurrence from approximately week three. Two main patterns were found: (i) expressed VSGs readily acquired a 3’ end different from their silent copy, a pattern that probably represents the 3’ boundary of gene conversion occurring within the coding sequence; (ii) expressed VSGs often appeared in sets of related ‘mosaics’, whereby more than one donor gene had contributed to the putative epitope-encoding part of the VSG. To test whether varying donor contributions represents an additional source of antigenic variability available to trypanosomes, a set of five mosaic VSGs retrieved from a single infection was expressed in non-switching trypanosomes and used to raise antibody responses. Indirect immuno-fluorescence, complement-mediated lysis, and agglutination assays using both polyclonal and monoclonal antibodies showed that although 4/5 mosaics were cross-reactive, one variant was completely antigenically distinct. Segmental gene conversion was therefore found to be both prominent in chronic African trypanosome antigenic variation, and capable of bringing antigenic novelty to an infection, with important consequences for the dynamics of AV, and the nature of selection pressure on the silent VSG archive.

Under genotoxic stress conditions, the genome of any organism may become compromised thus undermining cellular functions and the high fidelity transmission of the genome. Should integrity become compromised, cells have evolved a plethora of pathways to monitor, assess and direct the removal or bypass of genomic lesions. Collectively, this response is known as the DNA damage response (DDR). At the forefront of the DDR are specialised enzymes known as protein kinases (PKs), which act to co-ordinate many aspects of this response. In the kinetoplastid parasite Trypanosoma brucei, the role of PKs in other processes, such as the control of the cell cycle and during differentiation between the mammalian and insect lifecycle stages, have already been investigated using mutant cells lines, RNA interference (RNAi) and genome wide and kinome focused screens. However, virtually no work has examined the role of PKs in the context of genome repair. To this end, two RNAi targeted screens (RITseqs), one to examine the genome as a whole and the other to focus directly on the kinome compliment, were performed by others in the mammalian infection stage of T. brucei, searching for genes whose loss sensitises the cells to the presence of the DNA alkylating agent Methyl methanesulfonate (MMS). To validate both screens here, putative DNA damage associated PKs were examined by RNA interference (RNAi). Across both screens, a total of eleven PKs, whose loss sensitised cells to MMS, were validated in this study by monitoring their proliferation. Amongst these novel PKs were a pseudokinase (Tb6560) and an aurora kinase (TbAUK2), whose functions were investigated using disruption mutants generated in BSF T. brucei cells combined with immunolocalisaton. Analysis of the Tb6560 mutants revealed the pseudokinase to be non-essential in vitro and uncovered a potential role for Tb6560 during endocytosis or internal trafficking. Analysis of the TbAUK2 mutants also revealed this PK to be non-essential in vitro. However, when exposed to a variety of genotoxic agents, the growth of the mutants was significantly reduced suggesting TbAUK2 is required for parasite survival under DNA damage conditions. Further phenotypic analysis revealed a potential role for TbAUK2 in the maintenance of the parasite’s nuclear genome. Additionally, the roles of two PKs central to the DDR in other organisms were examined by RNAi. The atypical PKs Ataxia Telangiectasia Mutated (ATM) and the related PK ATM and Rad3 related (ATR) are activated in response to double stranded break (DSB) lesions or replicative lesions, respectively, in other eukaryotes. RNAi of TbATR was associated with perturbation of proliferation, extensive nuclear defects and sensitivity to genotoxic agents. Further analysis also revealed a potential role for this kinase in maintaining transcriptional silencing of bloodstream VSG expression sites (BESs). Conversely, RNAi of TbATM did not disrupt cellular proliferation in vitro and nor were TbATM deficient cells sensitised to alkylating or replication blocking agents, suggesting TbATM is not required for T. brucei survival or for maintenance of growth in these conditions. Collectively, these data reveal a diverse array of PKs required for genome maintainance, in BSF parasites, under genotoxic stress conditions. An indepth characterisation of three DNA damage associated PKs uncovered a variety of putative roles ranging from nuclear and chromosomal segregation to cell cycle regulation, proliferation and endocytosis. These data in particular highlight the complexity underpinning a cell’s response when threatened with genomic instability. Furthermore, depletion of one kinase, TbATR, was also associated with loss of transcriptional silencing within BESs suggesting that PK activity could operate during antigenic variation, a process vital for the persistence of this parasite within a host.

Single nucleotide polymorphisms (SNPs) are one of the forensic markers used to resolve the problem of DNA typing from degraded samples. It has been found in previous studies that when profiling heavily degraded forensic samples the small amplicon required for SNP analysis has an advantage over the larger STR loci, which are routinely used in forensic case work. A total of 66 SNPs from the non-coding region of the 22 pairs of autosomal chromosomes were identified and SNP assays developed. Instead of selecting the SNPs from the available GenBank® sites, SNPs were typed from Arab individuals from Kuwait and United Arab Emirates (UAE) to identify polymorphic SNPs. In order to obtain SNP data from Arab populations, a total of 10 unrelated Arab individuals from Kuwait and UAE were typed. The Affymetrix GeneChip® Mapping 250 K Array Sty І was employed to generate profiles for approximately 238,000 SNPs. Only autosomal SNPs were selected from the data. Following selection, allele frequencies were estimated using the SNaPshot™ technique (Applied Biosystems) with 25 UAE individuals. For this technique, PCR forward and reverse primers were designed to generate PCR products less than 150 bp. The single base extension primers were designed to hybridise 1 bp upstream from the target SNP. SNP characterization, including Hardy¬Weinberg equilibrium and pair wise linkage disequilibrium, was carried out using the software package Arlequin v 3.1. Allele frequencies were calculated using Excel spreadsheets. PowerStats v.12 software used for discrimination power and match probability estimation. All the 66 SNPs were polymorphic with average heterozygosity levels of 47%. A high heterozygosity level is very valuable for forensic application improving the individualization of forensic samples (Vallone et al. 2005). The probability that two individuals having identical genotype profile was found to be very low, 3.058 x 10-25. The combined power of discrimination was found to be 0.999999999. This indicated that the selected SNPs met the parameters needed for forensic application. The SNPs genotype sensitivity gave profiles from minute amounts of DNA template as little as 100 pico grams (pg) and optimal and reproducible results at 300 pg of DNA template. The profiling of DNA from forensic samples is not always possible. This can be due to insufficient amount of samples being recovered and in many cases, DNA degradation. Biological materials that are recovered from the scene of the crime have often been exposed to sub-optimal environmental conditions such as high temperature and humidity. SNPs performance on degraded samples was tested on artificially degraded saliva and semen samples. Controlled temperature and humidity experiments were performed to study the effect of these environmental factors on the samples. Also uncontrolled experiments on samples being subjected to different weather conditions (UK summer and UAE winter and summer) was performed in order to study and compare both weather effects on saliva samples. The triplex sets of SNPs that were developed for such study showed full allele profiles when compared to STRs, the current method used in forensic labs. In addition, SNPs produced a higher success rate than STRs when tested with samples obtained from human teeth remains and on samples subjected to DNase 1 digestion. The small size of SNPs, between 90 and 147 base pair (bp), showed more resistance to degradation than the STRs size ranging between 100 and 360 bp. This study demonstrated that the 66 SNPs selected are useful markers when the typing of degraded samples by STRs fails to produce complete or partial profiles.