http://www.dspace.cam.ac.uk:80/handle/1810/224358 Wed, 19 Jun 2013 12:13:37 GMT 2013-06-19T12:13:37Z http://www.dspace.cam.ac.uk:80/handle/1810/243616 Title: The role of Dichaete in transcriptional regulation during Drosophila embryonic development Authors: Aleksic, Jelena Abstract: Sox domain genes encode a family of developmentally important transcription factors conserved throughout the Metazoa. The subgroup B, which includes the mammalian Sox1, 2 and 3 proteins and their Drosophila counterparts Dichaete and SoxNeuro, are particularly important for the development of the nervous system where they appear to play conserved roles in neuronal specification and differentiation. Despite years of detailed study we still have a relatively poor idea of how Sox proteins function on a genome wide scale and the aim of my PhD work was to explore this aspect using the fly group B protein, Dichaete. A number of studies have shown that Dichaete performs a variety of critical functions during development and a few individual regulatory targets have been defined, however, at the start of my work no genome-wide data on Dichaete action were available. While such data emerged from large scale initiatives during my work, a systematic analysis of Dichaete action was lacking. Here I describe the first detailed genomic analysis of Dichaete activity, with a particular focus on three areas: finding the locations of Dichaete binding in the genome, a prediction of potential Dichaete cofactors and an analysis of Dichaete effects on gene expression. To address the issue of where Dichaete binds in the genome, I generated whole genome DamID data for embryos and followed this with a detailed comparative analysis, combining my data with three newly published ChIP-chip datasets. The combined studies identify thousands of binding regions, mostly in the vicinity of developmentally important genes. The binding profiles were found to be consistent with Dichaete acting on enhancer regions and also suggest a role in facilitating RNA Polymerase II pausing. The analysis also identified a Dichaete binding motif closely matching that found with in vitro studies. By combined ChIP and DamID datasets I generated a very high confidence core Dichaete binding dataset, which should be of considerable use in future studies. To identify potential Dichaete cofactors, I compiled the available embryonic transcription factor binding data from the Berkeley Drosophila Transcription Network and mod- ENCODE projects, and identified significant overlaps with the core Dichaete binding data. A number of the proteins highlighted in this analysis have known roles during neuroblast development, including Hunchback and Krüppel, transcription factors involved in temporal specification of neuroblast division, and Prospero, which plays a key role in neuroblast differentiation. The analysis suggests that Dichaete has a role during early neuroblast divisions, where it likely interacts with Hb and Kr to maintain neuroblast pluripotency. This is a role consistent with previous studies in Drosophila larval neuroblasts and is analogous to neural functions of Sox2 in mammals. My analysis suggests that Dichaete acts on the same target genes as Prospero but in an antagonistic role, with Dichaete preventing stem cell differentiation and Prospero promoting it. To examine the effects of Dichaete on gene expression, a number of microarray transcript profiling studies were performed, including a global study with Dichaete null mutants, and tissue specific studies in the CNS midline and neuroblasts via the use of dominant negative constructs. Whole transcriptome expression profiling data was combined with the binding data to establish a set of high confidence potential Dichaete targets, both for specific tissues and more globally during neurogenesis. Specific high confidence targets were found, including bancal during nervous system development. It was also concluded that Dichaete is likely to prevent cell cycle exit by repressing the apoptosis genes grim, hid and reaper, as well as the differentiation genes prospero and miranda. An extensive list of potential Dichaete direct targets was generated and can be used for validation and future research. Mon, 02 Jul 2012 23:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/243616 2012-07-02T23:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/241719 Title: Replication and stability of the linear plasmid pBSSB2 Authors: Ahsan, Sunjukta Abstract: Plasmid pBSSB1 is a 27 kb linear DNA with proteins attached at the 5’ termini. It encodes the H: z66 flagellar antigen in Salmonella enterica serovar Typhi (S. Typhi) isolated from Indonesia. Together with the H: j or H: d flagellar antigen encoded by the host chromosome, pBSSB1 renders expression of the flagellar antigen biphasic in S. Typhi. Following the discovery of pBSSB1, initial bioinformatic analyses were carried out. However, no genetic analysis of replication and stability functions was conducted. Such studies form the basis of the present work. Plasmid pBSSB2, that contains a kanamycin cassette inserted at position 1295 bp of pBSSB1, was used in the present investigation. The first objective of the work was to develop a method of purification for the linear plasmid. Conventional plasmid extraction methods which had been used previously were found to produce a very poor yield of plasmid DNA. It was shown in the present study that a proteinase-K treatment was essential for the removal of the linear plasmid terminal proteins to avoid loss of the plasmid in the phenol-chloroform-isoamylalcohol treatment which removes cellular proteins from the plasmid DNA. The region containing the basic replicon of pBSSB2 was identified by screening for a region that was able to support replication in E. coli of a ColE1-like plasmid in a polA host (in which it would not normally replicate). This identified a 2831 bp fragment encompassing nucleotides 12820 to 15649 of pBSSB2. It was expected that this would encode an initiator of replication such as a Rep protein. However, mutagenesis studies showed that none of the annotated ORFs in this fragment was essential for replication. Candidate ORFs, not identified in the original annotation, have been suggested that remain to be tested as possible candidates for the rep encoding gene. The possibility of an alternative RNA primed initiation of replication has also been hypothesized. An adjacent region was found to exert strong incompatibility against pBSSB2, suggesting that it might encode a repressor of replication. The minimum region conferring incompatibility was 179 bp, encompassing nucleotides 10840 bp to 11018 bp of pBSSB2. A six base pair imperfect repeat, (G/T) (G/A) TGTT was found within this sequence. It is hypothesized that these imperfect repeats may function as iterons that titrate a Rep protein and regulate pBSSB2 replication. A 1023 bp region (nucleotides 7236 to 8258 of pBSSB2) was found to confer stability in E. coli upon an otherwise unstable circular plasmid. Mutational analysis showed that an annotated ORF within this region (ORF09) was required for plasmid stabilisation. When expressed independently from an expression vector ORF09 killed host cells. It is proposed that the stability function acts as a toxin-antitoxin system, although the antitoxin has not yet been identified. A candidate promoter for a putative countertranscript and two potential ORFs as candidates for encoding the antitoxin have been suggested for future work to identify the antitoxin. The preliminary functional characterization of pBSSB2 has contributed to our general understanding of the replication and stability functions of linear plasmids. However, further work will be required to achieve a complete understanding of the molecular basis of these functions in pBSSB2. Tue, 07 Feb 2012 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/241719 2012-02-07T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/237378 Title: Mathematical and experimental approaches to the dimer catastrophe theory Authors: Field, Christopher Martyn Abstract: Multicopy plasmids rely on random distribution for stable inheritance by daughter cells at division. Threats to plasmid copy number increase the probability of plasmid loss, which can be detrimental to both plasmid and host. Plasmid dimers emerge through homologous recombination. Dimers have two independent origins of replication and thus have a replicative advantage and reduced copy number. Models of plasmid behaviour suggest that dimers would overtake a cell population, but that this can be prevented if they impose a small metabolic load, which has been observed in vivo. Plasmid ColE1 also contains a cer site, which allows for dimer resolution by XerCD site-specific recombination. A small RNA, Rcd, is expressed from the cer site in dimers and interacts with tryptophanase to increase the concentration of indole in the cell. It is proposed that, as indole inhibits cell division, Rcd imposes a checkpoint on the cell until plasmid dimers are resolved. In this work, plasmid behaviour in a growing cell population was modelled stochastically in more detail than previous work. A plasmid replication model suggested that dimers replicate to more than half the average copy number of monomers, perhaps accounting for their increased metabolic load. A cell population model suggested that the presence of dimer-only cells decreased the average plasmid stability by less than in previous models, which used a fixed plasmid copy number. The rate of dimer resolution required to affect plasmid stability was unreasonably high, indicating the necessity of the Rcd checkpoint. The model thus suggested that the checkpoint may be an escape route for dimer-only cells rather than an immediate response to the emergence of an initial dimer. The Rcd checkpoint itself was also subject to critical analysis. It was realised that neither inhibition of cell division nor cell growth were sufficient to assist dimer resolution; inhibition of plasmid replication was required. Experiments in vivo found that indole inhibited plasmid replication at a concentration that may be achievable endogenously. DNA gyrase was investigated as a component of the mechanism of this inhibition, and indole was found to inhibit its supercoiling activity in vitro. Tue, 08 Feb 2011 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/237378 2011-02-08T00:00:00Z