Background Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium... more
Background Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoeba Dictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in the cell, developmental and evolutionary biology. Results We describe REMI-seq, an adaptation of Tn-seq, which allows high throughput, en masse, and quantitative identification of the genomic site of insertion of a drug resistance marker after restriction enzyme-mediated integration. We use REMI-seq to develop tools which greatly enhance the efficiency with which the sequence, transcriptome or proteome variation can be linked to phenotype in D. discoideum. These comprise (1) a near genome-wide resource of individual mutants and (2) a defined pool of ‘barcoded’ mutants to allow large-scale parallel phenotypic analyses. These resources are freely available and easily accessible ...
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Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoebaDictyostelium discoideum, a... more
Genomes can be sequenced with relative ease, but ascribing gene function remains a major challenge. Genetically tractable model systems are crucial to meet this challenge. One powerful model is the social amoebaDictyostelium discoideum, a eukaryotic microbe widely used to study diverse questions in cell, developmental and evolutionary biology. However, its utility is hampered by the inefficiency with which sequence, transcriptome or proteome variation can be linked to phenotype. To address this, we have developed methods (REMI-seq) to (1) generate a near genome-wide resource of individual mutants (2) allow large-scale parallel phenotyping. We demonstrate that integrating these resources allows novel regulators of cell migration, phagocytosis and macropinocytosis to be rapidly identified. Therefore, these methods and resources provide a step change for high throughput gene discovery in a key model system, and the study of genes affecting traits associated with higher eukaryotes.
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Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a... more
Natural compounds often have complex molecular structures and unknown molecular targets. These characteristics make them difficult to analyse using a classical pharmacological approach. Curcumin, the main curcuminoid of turmeric, is a complex molecule possessing wide-ranging biological activities, cellular mechanisms and roles in potential therapeutic treatment including Alzheimer's disease and cancer. Here, we investigate the physiological effects and molecular targets of curcumin in Dictyostelium discoideum We show curcumin causes acute effects on cell behaviour, reduces cell growth, and slows multicellular development. We then employ a range of structurally related compounds to show the distinct role of different structural groups cell behaviour, growth, and development, highlighting active moieties in cell function, and showing that these cellular effects are unrelated to the well-known antioxidant activity of curcumin. Molecular mechanisms underlying the effect of curcumin ...
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Development of chloroplasts and other plastids depends on the import of thousands of nucleus-encoded proteins from the cytosol. Import is initiated by TOC (translocon at the outer envelope of chloroplasts) complexes in the plastid outer... more
Development of chloroplasts and other plastids depends on the import of thousands of nucleus-encoded proteins from the cytosol. Import is initiated by TOC (translocon at the outer envelope of chloroplasts) complexes in the plastid outer membrane that incorporate multiple, client-specific receptors. Modulation of import is thought to control the plastid's proteome, developmental fate, and functions. Using forward genetics, we identified Arabidopsis SP1, which encodes a RING-type ubiquitin E3 ligase of the chloroplast outer membrane. The SP1 protein associated with TOC complexes and mediated ubiquitination of TOC components, promoting their degradation. Mutant sp1 plants performed developmental transitions that involve plastid proteome changes inefficiently, indicating a requirement for reorganization of the TOC machinery. Thus, the ubiquitin-proteasome system acts on plastids to control their development.
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PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for... more
PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, wherea...
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Coupling the activities of normally disparate proteins into one functional unit has significant potential in terms of constructing novel switching components for synthetic biology or as biosensors. It also provides a means of... more
Coupling the activities of normally disparate proteins into one functional unit has significant potential in terms of constructing novel switching components for synthetic biology or as biosensors. It also provides a means of investigating the basis behind transmission of conformation ...
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This chapter introduces a set of transposon-based methods that were developed to sample trinucleotide deletion, trinucleotide replacement, and domain insertion. Each approach has a common initial step that utilizes an engineered version... more
This chapter introduces a set of transposon-based methods that were developed to sample trinucleotide deletion, trinucleotide replacement, and domain insertion. Each approach has a common initial step that utilizes an engineered version of the Mu transposon called MuDel. The inherent low sequence specificity of MuDel results in its random insertion into target DNA during in vitro transposition. Removal of the transposon using a type IIS restriction endonuclease generates blunt-end random breaks at a frequency of one per target gene and the concomitant loss of 3 bp. Self-ligation or insertion of another DNA cassette results in the sampling of trinucleotide deletion or trinucleotide substitution/domain insertion, respectively.
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Research Interests: Membrane Proteins, Molecular Genetics, Protein Translocation, Arabidopsis thaliana, Biological Sciences, and 15 morePhylogeny, Plant Physiology, Mutation, Plant, Arabidopsis, Phenotype, Seeds, Seedling, Amino Acid Sequence, Membrane Protein, Gene Family, Embryos, PISUM SATIVUM, Gene expression profiling, and Molecular Sequence Data
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Research Interests: Molecular Evolution, Directed evolution, Antibiotic Resistance, Protein Structure and Function, Protein structure, and 10 moreEnzyme, Anti-Bacterial Agents, Protein Secondary Structure Prediction, Substrate Specificity, Amino Acid Profile, Ampicillin, Amino Acid Sequence, Structure activity Relationship, Biochemistry and cell biology, and Beta Lactamases
Coupling the activities of normally disparate proteins into one functional unit has significant potential in terms of constructing novel switching components for synthetic biology or as biosensors. It also provides a means of... more
Coupling the activities of normally disparate proteins into one functional unit has significant potential in terms of constructing novel switching components for synthetic biology or as biosensors. It also provides a means of investigating the basis behind transmission of conformation events between remote sites that is integral to many biological processes, including allostery. Here we describe how the structures and functions of two normally unlinked proteins, namely, the heme binding capability of cytochrome b(562) and the antibiotic degrading beta-lactamase activity of TEM, have been coupled using a directed evolution domain insertion approach. The important small biomolecule heme directly modulates in vivo and in vitro the beta-lactamase activity of selected integral fusion proteins. The presence of heme decreased the concentration of ampicillin tolerated by Escherichia coli and the level of in vitro hydrolysis of nitrocefin by up to 2 orders of magnitude. Variants with the largest switching magnitudes contained insertions at second-shell sites that abut key catalytic residues. Spectrophotometry confirmed that heme bound to the integral fusion proteins in a manner similar to that of cytochrome b(562). Circular dichroism suggested that only subtle structural changes rather than gross folding-unfolding events were responsible for modulating beta-lactamase activity, and size exclusion chromatography confirmed that the integral fusion proteins remained monomeric in both the apo and holo forms. Thus, by sampling a variety of insertion positions and linker sequences, we are able to couple the functions of two unrelated proteins by domain insertion.
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Cardiff sciSCREEN is a public engagement programme that brings together local experts and publics to discuss issues raised by contemporary cinema. Since 2010, Cardiff sciSCREEN (short for science on screen) has exhibited more than 50... more
Cardiff sciSCREEN is a public engagement programme that brings together local experts and publics to discuss issues raised by contemporary cinema. Since 2010, Cardiff sciSCREEN (short for science on screen) has exhibited more than 50 films alongside short talks and discussions that draw on a range of disciplinary perspectives to explore the broad repertoire of themes found within different film genres. The aim of Cardiff sciSCREEN is to increase the local community's access to university research, whilst enabling university staff and students to engage a variety of publics with their work. In this paper, we first describe our method of public engagement, then draw on data from a research survey we administered to sciSCREENers to discuss the relationship between the theory and practice of public engagement. Using research from public understanding of science (PUS), public engagement with science and technology (PEST), science and technology studies (STS), and film literacy, we discuss the ways in which our flexible characterisation of science has made the programme inclusive, attracting a wide and varied audience. We consider the benefits of cross-disciplinary perspectives when communicating and engaging contemporary developments in science, where the term science is taken to stand for the breadth of academic research and not merely the natural sciences, as well as discussing the importance of space in public engagement events. Three Key Points • Cardiff sciSCREEN is an effective public engagement initiative with which to engage publics with contemporary scientific and societal issues. The screening of a film creates a shared experience between panellists and attendees. • Attendees enjoy the pan-disciplinary perspective provided my multiple speakers from different disciplinary backgrounds. • Public engagement is more than an activity. It is a process, and engagement programmes should be flexible enough to develop and adapt over time.