When Aspergillus nidulans grows with ammonium or glutamine combined with an alternative nitrogen source, the organism preferentially utilises the ammonium or glutamine. The pathways for utilising alternative nitrogen source remains inactive in the presence of ammonium or glutamine and this phenomenon is called nitrogen metabolite repression (NMR). The regulation of NMR in A. nidulans is mediated by multiple mechanisms which include transcription, mRNA stability, translation, posttranslational modification and direct protein-protein interactions. A key feature of NMR in A. nidulans is the interaction between the GATA type transcription activator AreA and the transcription repressor protein NmrA. The transcription repressor NmrA of A. nidulans discriminates between oxidised and reduced dinucleotides; however dinucleotide binding has no effect on its interaction with the zinc finger in the transcription activator AreA. The role of specific cleavage of transcription repressor proteins by proteases and how this may be related to the emerging theme of dinucleotides as cellular signalling molecules is poorly characterised. Protease activity in A. nidulans was assayed using NmrA as the substrate, and was absent in mycelia grown under nitrogen sufficient conditions but abundant in mycelia starved of nitrogen. Three proteases were identified and two were purified and identified by mass spectroscopy as serine proteases: Q5BGU2_EMENI and Q5BAR4_EMENI, encoded by the genes AN0238.2 and AN2366.2, respectively. Production of the third protease was absent in strain deleted for the areA gene. Proteolysis of NmrA occurred in an ordered manner by preferential digestion within a C-terminal surface exposed loop and subsequent digestion at other sites. The two proteolytic fragments of NmrA produced by digestion at the C-terminal site remained associated; however the digested NmrA was unable to bind to the AreA zinc finger but retained the ability to bind NAD+. These data reveal a potential new layer of control of nitrogen metabolite repression by the ordered proteolytic cleavage of NmrA. NmrA digested at the C-terminal site showed a resistance to further digestion that was enhanced by the presence of NAD+ and to a lesser extent by NADP+. This is the first time that an effect of dinucleotide binding to NmrA has been demonstrated. The in vitro ordered proteolysis of NmrA reveals a potential new level of regulation relating to nitrogen metabolite repression. The dynamic interplay between the production of NmrA and its subsequent ordered proteolysis facilitates a rapid and finely tuned response to changes in the concentration and nature of the nitrogen source supporting growth.
During the last decades our knowledge about the human mitochondrial translation system has been expanded. However, our understanding of this unique system is far from complete. Mitochondria contain a minimal genome, whose expression is dependent on factors encoded by the nuclear genome. Derived from a bacterial ancestor it is very close to the translation system found in bacteria, but there are also a lot of differences especially the translating ribosome, which differs in a number of features including the sedimentation coefficient, protein-RNA ratio and number of tRNA sites. Available cryo-EM structures of the mitochondrial ribosome are limited, making it difficult to understand the system completely. There are still a lot of open questions concerning the composition, assembly, translation initiation or the recycling of stalled ribosomes in mammalian mitochondria. The study presented in this thesis contributes to our understanding of this unique system by the characterisation of two mitochondrial proteins, found in association with the mitochondrial ribosome, ICT1 and mtRbfA. These proteins are found in association with the mitochondrial ribosome, and were immunoprecipitated together with the mitochondrial ribosome recycling factor (mtRRF). ICT1 as a member of the mitochondrial release factor family has been identified as a peptidyl-tRNA hydrolase. Data shown here indicates that ICT1 has been recruited into the mitochondrial ribosome and further, suggests the involvement of ICT1 in the rescue of stalled ribosomal complexes with immobilised peptidyl-tRNA. In contrast mtRbfA was identified as a potential ribosome assembly factor rather than a permanent component. The function of this protein is still elusive, but data generated for this thesis shows that this protein is preferentially associated with the mitochondrial ribosomal small subunit at a late assembly point, suggesting possible roles of mtRbfA in quality control or in translation initiation.
Ward, C. P.
<i>Plasmodium falciparum var</i> genes encode the PfEMP1 family of variant antigens expressed on the surface of infected erythrocytes. PfEMP1 mediates the adhesion of the parasitized erythrocyte to the venular endothelium and to uninfected erythrocytes. PfEMP1 variants use a range of different host molecules as receptors in these adhesive interactions. The expression of different PfEMP1 variants may directly affect the clinical course of malaria infection by defining the distribution and intensity of parasite adhesion in the microvasculature of various organs within the host. PfEMP1 is a major focus of the host immune response, and the slow onset of clinical immunity in endemic areas may be explained by the gradual accumulation of effective responses to a wide range of PfEMP1 variants present in the local parasite population. It has been hypothesised that the immune response to PfEMP1 may act to stratify the parasite population into co-circulating 'strains' defined by discrete, non-overlapping repertoires of <i>var </i>genes. Here, the DBL1 region of 56 <i>var</i> gene variants from 6 genetically distinct co-circulating Sudanese parasites have been cloned and sequenced. Sequence comparisons suggest that recombination and gene duplication are important mechanisms in the generation of new <i>var </i>variants. A model of the basic structural framework of DBL1 sequences is described and "sequence subtypes" identified within variable regions of sequence. Phylogenetic analysis of the Sudanese and other <i>var </i>sequences from GenBank fail to support the 'strain' model for Sudanese <i>P. falciparum</i> and suggests that the global pool of <i>var</i> genes is finite. 40 Sudanese variants have been expressed as GST-fusion proteins with yields of varying quantity and degree of degradation. 10 of the recombinant proteins have been tested against a cohort of sera in a pilot ELISA, and the role of anti-PfEMP1 immune responses in the development of clinical immunity is discussed.
Duffield, S. J.
<i>Rhodococcus aetherivorans </i>I24 is a saprophytic actinomycete bacteria originally isolated from a toluene-contaminated aquifer. Here, two alcohol dehydrogenases capable of enantioselectively reducing halomethyl ketones were isolated by chromatographic techniques, and their corresponding DNA sequences identified by comparison with experimentally determined amino acid sequence data. Attempts were made to overexpress these genes in the standard cloning host <i>Escherichia coli</i>. One enzyme, Adh1, expressed well, and was characterised successfully in a recombinant form. The second, Adh2, was difficult to express recombinantly in an active form in this system. Concurrently, tools for rhodococcal gene expression were developed, including characterisation of the ohp promoter region of <i>Rhodococcus ruber </i>V49, and creation of a chromosomal expression system. Use of the <i>ohp</i> promoter for expression of recombinant proteins in <i>Rhodococcus </i>was investigated, using the luciferase genes from the marine bacterium <i>Vibrio fischeri </i>as a model. In concert, the mode of action of OhpR, the regulator of the operon was elucidated, and its binding site identified. Protein folding of highly expressed gene products is frequently problematic. To address this potential bottleneck, the GroE chaperone system of <i>R. aetherivorans </i>I24 was investigated through a bioinformatics approach coupled with mutational analyses. Of the three GroEL homologs present in <i>R. aetherivorans </i>I24, two were found to respond to heat shocks by increased levels of transcription. This response was observed to correlate with the presence of binding sites for the heat shock regulatory protein, HrcA, upstream of the homologs.
Bowden, S. D.
<i>Erwinia carotovora</i> subspecies<i> carotovora (Ecc) </i>is a phytopathogenic member of the Enterobacteriaceae and causes soft rot in potato tubers. <i>Ecc</i> strain ATTn10 also produces the β-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid (Car). In order to select for mutants with increased production of Car, a β-lactamase translational fusion was engineered into <i>carH</i>, the last gene in the Car biosynthetic and auto-resistance operon. The <i>carH::blaM</i> reporter was shown to encode for a functional β-lactamase and by selecting in the presence of ampicillin (Ap), spontaneous <i>Ecc</i> mutants with increased Ap<sup>R</sup> were isolated. The mechanism of increased Ap<sup>R</sup> in the spontaneous mutants was shown to be due to IS<i>10</i> and Tn<i>10</i> mobile genetic elements that had been introduced during the previous construction of ATTn10 from its parent strain, ATCC 39048. Subsequently, <i>carH::blaM</i> was introduced <i>via</i> a series of generalised transductions into ATCC 39048 in order to employ the positive selection system in a strain that did not possess IS<i>10</i> or Tn<i>10</i> elements. By using a combined mini-transposon approach, the <i>carH::blaM</i> positive selection system enabled the isolation of a mutant disrupted in a porin gene with high homology to <i>ompF </i>from <i>Escherichia coli.</i> An alternative approach to isolate mutants that were affected in Car production relied on the observation that many ATTn10 spontaneous caseinase mutants were also influenced in Car production. A mini-transposon mutagenesis screen of mutants with altered caseinase activity identified five mutants that were also reduced in Car production.
Rowan, Andrew Stephen
Nucleoside triphosphates (NTPs) are substrates for numerous NTP-dependent enzymes within the body, and thus are crucial for a wide variety of biological processes. NTP mimetics would therefore be ideal candidates for chemical genetics programmes, to perturb intracellular processes with exquisite precision by the inhibition of selected proteins. We believe that replacement of the ~,y-pyrophosphate moiety of an NTP with a monosaccharide may still lead to favourable enzyme binding but lead to inhibition. Replacement of the a-phosphate group with a triazole linker would facilitate cell permeability by removing any remaining charge, as well as increase stability of the mimetic. Triazoles can be formed via the Cu(I)-catalysed Huisgen 1,3-dipolar cycloaddition reaction of azides and terminal alkynes. A series of propargyl glycosides of various monosaccharides have been synthesised, in most cases possessing anomeric purity, and reliable protocols developed. A number of 5'azido nucleosides have also been synthesised, and coupling reactions carried out to obtain a library of 45 novel sugar-triazole-nucleosides. These compounds have been tested as potential inhibitors of the type III pantothenate kinase from Bacillus anthracis and one potent inhibitor discovered with a Kj of 164 p.M. This shows that the inhibitor binds approximately three times more tightly in the active site than the natural substrate ATP (Km = 475 M).
To facilitate the isolation and characterisation of polymorphic markers for the Poultry Genome Mapping project, and to define potential transcriptional factors or disease candidate genes, the following studies were carried out as the purpose of this thesis. Procedures were developed for the construction of specific microsatellite-enriched DNA libraries. [CA/TG]<SUB>n</SUB> microsatellite-enriched genomic DNA libraries were constructed through marker selection and magnetic particle selection respectively; whereas magnetic particle selection was used in the construction of [CA/TG]<SUB>n</SUB> and [CAG/CTG]<SUB>n </SUB>microsatellite enriched liver cDNA libraries. Positive clones in the DNA libraries constructed ranged from 0.5% to 5% depending on the type of microsatellite repeat. Two hundred microsatellite positive clones from genomic DNA library and a hundred from liver cDNA library were identified by screening once with an oligo probe and further characterised by sequencing. Polymorphisms of these microsatellites were studied by polymerase chain reaction and mapped using the EAST LANSING and COMPTON mapping cross. A nonredundant database search using the available expressed sequence information revealed chicken homologous sequences of human transcriptional factor gene, MEF2 and human Fragile X gene, FMR1 as well as a substantial number of new genes. Chromosome mapping and study of differential expression of these genes are underway. The isolation of these microsatellites will facilitate the mapping of important chicken quantitative traits, e.g. production traits. The mapping of expressed sequences will help to define candidate genes of these and other traits.
It is now commonly agreed that Rett Syndrome is a monogenic neurological disease caused by mutations in <i>MECP2 </i>gene. Rett Syndrome mainly occurs in girls and it is characterised by a period of normal development until around 618 months, followed by a rapid regression. After the regression, symptoms persist as severe mental retardation, reduced head size, seizures, ataxia, hyperventilation and repetitive hand wringing movements. The phenotype of mice with a deleted <i>Mecp2 </i>gene mimics some Rett Syndrome symptoms. The <i>Mecp2</i>null mouse develops normally until about 6 weeks of age after which tremors, irregular breathing, lack of mobility and hindlimb clasping develop. To understand how the lack of MeCP2 causes Rett Syndrome, the search for MeCP2 regulated genes was initiated in <i>Mecp2-null </i>mouse brain. Examination of candidate genes revealed that <i>Bdnf is </i>down-regulated and <i>Hes1 is </i>up-regulated in pre, early and late symptomatic <i>Mecp2-null </i>mice. Further, global analysis of gene expression was examined by ADDER differential display. Some mis-regulated genes were identified, two of which are involved in mitochondrial respiration. Oxygen electrode measurements revealed defects in brain mitochondrial respiration, which commenced coincident with symptom onset in <i>Mecp2-null </i>mice. This finding suggests mitochondrial involvement in the pathogenesis of Rett Syndrome symptoms. In the course of these studies, the structure of the <i>Mecp2 </i>gene was re-investigated, leading to the identification of a new MeCP2 isoform. Data in this thesis demonstrates that the new isoform is the major form of MeCP2 in both mouse and human brain.
Modulation and bioavailability via tissue-specific induction of metabolising enzymes, transporters and nuclear receptorsMartin, Philip John January 2008 (has links)
Investigating the mechanisms of regulation of drug disposition is important in understanding pharmacokinetic (PK) variability and rationalising drug-drug interactions. Ultimately, drug absorption in the intestine and drug elimination from the liver are the major determinants of PK. Key proteins involved in these processes are cytochrome P450 enzymes (CYPs) and ABC-transporters. These proteins are transcriptionally regulated by a complex network of nuclear receptor (NR) type transcription factors and many of these NRs are activated by endogenous or exogenous chemicals. The study of these mechanisms in rodent . models has been complicated by the reported species differences in activation of NRs. The aim of this thesis was to assess the spectrum of induction of CYPs, transporters and NRs in response to paradigm NR-activators in a battery of human and rat in-vitro systems. The aim of chapter two was to validate robust methodology for quantitatively assessing mRNA and protein expression of CYP2B6, CYP3A4, ABCB1, ABCC1, ABCC2, CAR, FXR, PXR and their rat homologues. In addition, since there are many conflicting reports in the literature, the impact of using dexamethasone (OEX) as a media supplement was assessed. Previous studies have illustrated species differences with pregnenalone-16a-carbonitrile (PCN) activating rodent but not human PXR, and rifampicin (RIF) activating human but not rodent PXR. The data presented in this chapter illustrate that OEX induces human PXR, CYP3A4, ABCB1 and rat orthologues. Furthermore, in the absence of OEX, RIF and PCN increased expression of human CYP3A4, ABCB1 and rat orthologues to a similar extent. These data suggest that there is a maximal induction and the addition of OEX effectively increases the baseline. However, since the maximum is fixed a reduction in the observed effect is evident. Therefore, although there are species differences in activation of PXR, the impact on target gene expression appears to be similar. For this reason, OEX was not incorporated into the culture media in SUbsequent chapters. The aim of chapter three was to investigate the impact of Phenobarbital (PB; CAR activator), PCN (rodent PXR activator), chenodeoxycholic acid (COCA; FXR activator), RIF (human PXR activator) and 9-cis-retinoic acid (9-cisRA; RXR activator) on expression of CYPs, transporters and NRs in human (Caco-2) and rat (iec-6) intestinal cell lines. Significant induction of transcripts in both human and rat intestinal cell lines were observed. However, some differences were seen in the induction profiles in Caco-2 versus lec-6 cells. In general, logarithmic correlations between the changes in mRNA and protein were also observed. Lower changes in mRNA elicited linear increases in protein but there appeared to be a threshold beyond which no additional increase in protein was observed. In chapter four, these experiments were repeated in the human (HepG2) and rat (H411e) hepatic cell lines. Again, induction of transcripts in both human and rat hepatic cell lines was observed. In hepatic and intestinal cell lines concentration-dependent responses were observed in most cases. However, i~ some cases, bell shaped concentration-response profiles were observed. In most cases these were related to toxicity but in some cases sub-maximal induction was observed at concentrations that were non-toxic. These data indicate that using a single concentration may result in gross underestimation of induction and may also explain some of the discrepancies in the literature. Logarithmic correlations between the changes in mRNA and protein in HepG2 and H411 e cells were also observed. Similar to intestinal cell lines, lower changes in mRNA elicited linear increases in protein. This is most likely due to the time which analysis was conducted; mRNA induction being more rapid than that of protein. The aim of chapter five was to investigate these observations in human (cryopreserved from two donors) and rat (fresh) hepatocYtes. In these studies, a sub·toxic concentration (1IJM) causing maximal induction of most transcripts in cell lines was chosen and a time course was conducted to circumvent problems associated with a decrease in viability over time. Protein was not investigated because insufficient material was observed after incubation with typical NR activators. Generally, maximal induction was observed at 6 hours in human and at 4 hours in rat hepatocytes, and in most instances time-dependent induction was observed. Interestingly, there was variation in the observed induction between the human donors. These data were in broad agreement with data generated in cell lines, and where overlap existed were also in agreement with previous studies in the literature. The aim of chapter six was to investigate activation of a range of human NRsby the activators utilised in previous chapters. This was particularly important given that similar induction of target genes was observed, even where species differences have been documented. In order to probe the mechanisms that underpin these observations, mammalian 1 hybrid ligand binding assays were employed for PXR, VOR, PPARy, LXRa, FXRa and RXRa in Caco-2 and CHO-K1 cells. All NRs showed some degree of promiscuity, with LXRa appearing the most promiscuous. PPARy was activated by PCN, which is particularly interesting in light of previously reported PPAR response elements within the PXR proximal promoter. COCA significantly activated FXRa and LXRa in CHO-K1 cells. In addition, LXRa, FXRa and VOR were all activated by COCA in Caco-2 cells. In summary, variation in induction was observed between model systems for both tissue and species. However, in the absence of OEX, both rat and human PXR and PXR target genes were activated by PCN and RIF to a similar extent. In addition, a number of novel observations were made. For example upregulation of NRs by their purported activators was observed, which raises the possibility of self regulation. Further studies, which utilise reporter constructs from the target gene promoters coupled with NR expression vectors, are now required to investigate which NRs are involved. Work has now also begun to utilise targeted knockdown of NRs (with SiRNA), in order to determine the relative contribution of individual NRs in the reported induction.
Hastings, Robert Kevin
The human genome contains vast numbers of sequences that have copied themselves to new genomic locations by retrotransposition. Long Interspersed Nuclear Element-1 (LINE-1 or L1) is the only sequence in the human genome still capable of autonomous retrotransposition. L1 elements have contributed to the evolution of the human genome via insertional mutagenesis, pseudogene formation, sequence transduction, and recombination events (producing insertions, deletions and inversions). Currently general and L1- specific sequence databases do not reflect the true level of Full Length Human Specific L1 (FL-L1HS) variation, due to the polymorphic nature of these elements and the way the databases were compiled. Methods to identify FL-L1HS were applied to three sequence assemblies (Reference, Celera and HuRef) and the nucleotide accession database from NCBI. A non-redundant set of 533 FL-L1HS was discovered in these four sources, of which 164 resided in genes. The trace archives from Ensembl were also searched and a further 48 potential FL-L1HS were found. Computational analyses showed 154 FL-L1HS were potentially capable of retrotransposition, including 54 that resided in genes. Alongside these analyses a Target Site Duplication (TSD) detection and analysis tool, TSDmapper was developed to automatically detect TSDs in FL-L1HS sequences and provide annotation on sequence transduction. TSDmapper was used to predict the pre-insertion sequence of all 533 unique L1s, which facilitated in-silico genotyping. A new informatic resource, baseLINE (http://baseline.gene.le.ac.uk), was created to display and enable searching of all the L1 annotation information generated. Data can be viewed in a genomic context in chromosome ideograms or can be exported via the Distributed Annotation Service (DAS) on to the Ensembl genome browser. TSDmapper is also provided as a web application at baseline for users to perform TSD annotation of their sequences of interest.
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