Covalent protein-DNA complexes at the 5' strand termini of meiosis-specific double-strand breaks in yeast.

During meiosis in Saccharomyces cerevisiae, the first chemical step in homologous recombination is the occurrence of site-specific DNA double-strand breaks (DSBs). In wild-type cells, these breaks undergo resection of their 5' strand termini to yield molecules with 3' single-stranded tails. We have further characterized the breaks that accumulate in rad50S mutant stains defective in DSB resection. We find that these DSBs are tightly associated with protein via what appears to be a covalent linkage. When genomic DNA is prepared from meiotic rad50S cultures without protease treatment steps, the restriction fragments diagnostic of DSBs selectively partition to the organic-aqueous interphase in phenol extractions and band at lower than normal density in CsCl density gradients. Selective partitioning and decreased buoyant density are abolished if the DNA is treated with proteinase K prior to analysis. Similar results are obtained with sae2-1 mutant strains, which have phenotypes identical to rad50S mutants. The protein is bound specifically to the 5' strand termini of DSBs and is present at both 5' ends in at least a fraction of breaks. The stability of the complex to various protein denaturants and the strand specificity of the attachment are most consistent with a covalent linkage to DSB termini. We propose that the DSB-associated protein is the catalytic subunit of the meiotic recombination initiation nuclease and that it cleaves DNA via a covalent protein-DNA intermediate.

Single-site cleavage in the 5'-untranslated region of Leishmaniavirus RNA is mediated by the viral capsid protein.

Leishmaniavirus (LRV) is a double-stranded RNA virus that persistently infects the protozoan parasite Leishmania. LRV produces a short RNA transcript, corresponding to the 5' end of positive-sense viral RNA, both in vivo and in in vitro polymerase assays. The short transcript is generated by a single site-specific cleavage event in the 5' untranslated region of the 5.3-kb genome. This cleavage event can be reproduced in vitro with purified viral particles and a substrate RNA transcript possessing the viral cleavage site. A region of nucleotides required for cleavage was identified by analyzing the cleavage sites yielding the short transcripts of various LRV isolates. A 6-nt deletion at this cleavage site completely abolished RNA processing. In an in vitro cleavage assay, baculovirus-expressed capsid protein possessed an endonuclease activity identical to that of native virions, showing that the viral capsid protein is the RNA endonuclease. Identification of the LRV capsid protein as an RNA endonuclease is unprecedented among known viral capsid proteins.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted, in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites: (c) 2006 Elsevier Inc. All rights reserved.

Targeted delivery of a novel group of site-directed transglutaminase inhibitors to the liver using liposomes:a new approach for the potential treatment of liver fibrosis

Liver fibrosis and its end-stage disease cirrhosis are a main cause of mortality and morbidity worldwide. Thus far, there is no efficient pharmaceutical intervention for the treatment of liver fibrosis. Liver fibrosis is characterized by excessive accumulation of the extracellular matrix (ECM) proteins. Transglutaminase (TG)-mediated covalent cross-linking has been implicated in the stabilization and accumulation of ECM in a number of fibrotic diseases. Thus, the use of tissue TG2 inhibitors has potential in the treatment of liver fibrosis. Recently, we introduced a novel group of site-directed irreversible specific inhibitors of TGs. Here, we describe the development of a liposome-based drug-delivery system for the site-specific delivery of these TG inhibitors into the liver. By using anionic or neutral-based DSPC liposomes, the TG inhibitor can be successfully incorporated into these liposomes and delivered specifically to the liver. Liposomes can therefore be used as a potential carrier system for site-specific delivery of the TG2 inhibitors into the liver, opening up a potential new avenue for the treatment of liver fibrosis and its end-stage disease cirrhosis.

Residue-specific investigation of the relationship between oxidation and activity of a dual specificity phosphatase by mass spectrometry

Redox regulation of signalling pathways is critical in proliferation and apoptosis; redox imbalance can lead to pathologies such as inflammation and cancer. Vaccinia H1-related protein (VHR; DUSP3) is a dual-specificity phosphatase important in controlling MAP kinase activity during cell cycle. the active-site motif contains a cysteine that acts as a nucleophile during catalysis. We used VHR to investigate the effect of oxidation in vitro on phosphatase activity, with the aim of determining how the profile of site-specific modification related to catalytic activity. Recombinant human VHR was expressed in E. coli and purified using a GST-tag. Protein was subjected to oxidation with various concentrations of SIN-1 or tetranitromethane (TNM) as nitrating agents, or HOCl. the activity was assayed using either 3-O-methylfluorescein phosphate with fluorescence detection or PIP3 by phosphate release with malachite green. the sites of oxidation were mapped using HPLC coupled to tandem mass spectrometry on an ABSciex 5600TripleTOF following in-gel digestion. More than 25 different concentration-dependent oxidative modifications to the protein were detected, including oxidations of methionine, cysteine, histidine, lysine, proline and tyrosine, and the % oxidized peptide (versus unmodified peptide) was determined from the extracted ion chromatograms. Unsurprisingly, methionine residues were very susceptible to oxidation, but there was a significant different in the extent of their oxidation. Similarly, tyrosine residues varied greatly in their modifications: Y85 and Y138 were readily nitrated, whereas Y38, Y78 and Y101 showed little modification. Y138 must be phosphorylated for MAPK phosphatase activity, so this susceptibility impacts on signalling pathways. Di- and tri- oxidations of cysteine residues were observed, but did not correlate directly with loss of activity. Overall, the catalytic activity did not correlate with redox state of any individual residue, but the total oxidative load correlated with treatment concentration and activity. This study provides the first comprehensive analysis of oxidation modifications of VHR, and demonstrates both heterogenous oxidant effects and differential residue susceptibility in a signalling phosphatase.

Site Machines

Thesis (Ph.D.)--University of Washington, 2016-07

Site Specifc Growth of Metal Catalyzed Silica Nanowires for Biological and Chemical Sensing

In this research the integration of nanostructures and micro-scale devices was investigated using silica nanowires to develop a simple yet robust nanomanufacturing technique for improving the detection parameters of chemical and biological sensors. This has been achieved with the use of a dielectric barrier layer, to restrict nanowire growth to site-specific locations which has removed the need for post growth processing, by making it possible to place nanostructures on pre-pattern substrates. Nanowires were synthesized using the Vapor-Liquid-Solid growth method. Process parameters (temperature and time) and manufacturing aspects (structural integrity and biocompatibility) were investigated. Silica nanowires were observed experimentally to determine how their physical and chemical properties could be tuned for integration into existing sensing structures. Growth kinetic experiments performed using gold and palladium catalysts at 1050 ˚C for 60 minutes in an open-tube furnace yielded dense and consistent silica nanowire growth. This consistent growth led to the development of growth model fitting, through use of the Maximum Likelihood Estimation (MLE) and Bayesian hierarchical modeling. Transmission electron microscopy studies revealed the nanowires to be amorphous and X-ray diffraction confirmed the composition to be SiO2 . Silica nanowires were monitored in epithelial breast cancer media using Impedance spectroscopy, to test biocompatibility, due to potential in vivo use as a diagnostic aid. It was found that palladium catalyzed silica nanowires were toxic to breast cancer cells, however, nanowires were inert at 1µg/mL concentrations. Additionally a method for direct nanowire integration was developed that allowed for silica nanowires to be grown directly into interdigitated sensing structures. This technique eliminates the need for physical nanowire transfer thus preserving nanowire structure and performance integrity and further reduces fabrication cost. Successful nanowire integration was physically verified using Scanning electron microscopy and confirmed electrically using Electrochemical Impedance Spectroscopy of immobilized Prostate Specific Antigens (PSA). The experiments performed above serve as a guideline to addressing the metallurgic challenges in nanoscale integration of materials with varying composition and to understanding the effects of nanomaterials on biological structures that come in contact with the human body.

here/there/then/now

here/there/then/now was a practice-led research project that brought together 10 independent artists in dance, music, theatre and visual/media arts to create a site-specific program within the walls of the Brisbane Powerhouse. The purpose was to explore how to best conceive flexible performance platforms, theatricalise site-specific work and engage new audiences through forms of promenade experience that could provide open choices on how and where to view it. The sold out season of 6 performances, which took place 14-19 May 2002, presented three discrete performance installations set in intimate parts of the building, each with their own aesthetic and communicative intention, culminating in a fourth in-theatre installation, where memories of the first three coalesced and were reinterrogated. Each site thereby investigated meaning-making via the moving body and its critical relationship with space and objects, in a dramatic re-contextualisation of traditional solo dance forms, now re-articulated through interdisciplinary practices. The benefit of this approach was the creation of a layered and multimodal experience that could be both shared and subsequently critiqued by performers and audience alike.

Eco-retrofitting with building integrated living systems

Building integrated living systems (BILS), such as green roofs and living walls, could mitigate many of the challenges presented by climate change and biodiversity protection. However, few if any such systems have been constructed, and current tools for evaluating them are limited, especially under Australian subtropical conditions. BILS are difficult to assess, because living systems interact with complex, changing and site-specific social and environmental conditions. Our past research in design for eco-services has confirmed the need for better means of assessing the ecological values of BILS - let alone better models for assessing their thermal and hydrological performance. To address this problem, a research project is being developed jointly by researchers at the Central Queensland University (CQ University) and the Queensland University of Technology (QUT), along with industry collaborators. A mathematical model under development at CQ University will be applied and tested to determine its potential for predicting their complex, dynamic behaviour in different contexts. However, the paper focuses on the work at QUT. The QUT school of design is generating designs for living walls and roofs that provide a range of ecosystem goods and services, or ‘eco-services’, for a variety of micro-climates and functional contexts. The research at QUT aims to develop appropriate designs, virtual prototypes and quantitative methods for assessing the potential multiple benefits of BILS in subtropical climates. It is anticipated that the CQ University model for predicting thermal behaviour of living systems will provide a platform for the integration of ecological criteria and indicators. QUT will also explore means to predict and measure the value of eco-services provided by the systems, which is still largely uncharted territory. This research is ultimately intended to facilitate the eco-retrofitting of cities to increase natural capital and urban resource security - an essential component of sustainability. The talk will present the latest range of multifunctional, eco-productive living walls, roofs and urban space frames and their eco-services.

The moment of exchange : risk, vulnerability, responsibility

Any cycle of production and exchange – be it economic, cultural or aesthetic – involves an element of risk. It involves uncertainty, unpredictability, and a potential for new insight and innovation (the boom) as well as blockages, crises and breakdown (the bust). In performance, the risks are plentiful – economic, political, social, physical and psychological. The risks people are willing to take depend on their position in the exchange (performer, producer, venue manager or spectator), and their aesthetic preferences. This paper considers the often uncertain, confronting or ‘risky’ moment of exchange between performer, spectator and culture in Live Art practices. Encompassing body art, autobiographical art, site-specific art and other sorts of performative intervention in the public sphere, Live Art eschews the artifice of theatre, breaking down barriers between art and life, artist and spectator, to speak back to the public sphere, and challenge assumptions about bodies, identities, memories, relationships and histories. In the process, Live Art frequently privileges an uncertain, confrontational or ‘risky’ mode of exchange between performer, spectator and culture, as a way of challenging power structures. This paper examines the moment of exchange in terms of risk, vulnerability, responsibility and ethics. Why the romance with ‘risky’ behaviours and exchanges? Who is really taking a risk? What risk? With whose permission (or lack thereof)? What potential does a ‘risky’ exchange hold to destabilise aesthetic, social or political norms? Where lies the fine line between subversive intervention in the public sphere and sheer self-indulgence? What are the social and ethical implications of a moment of exchange that puts bodies, beliefs or social boundaries at ‘risk’? In this paper, these questions are addressed with reference to historical and contemporary practices under the broadly defined banner of Live Art, from the early work of Abrovamic and Burden, through to contemporary Australian practitioners like Fiona McGregor.

Can't see the water for the trees

The work was both conceived and constructed in-situ within Gnombup Swamp a seasonal water body at Bremer Bay, Western Australia. The work interacts with site-specific conditions including wind patterns and a datum of seasonal water levels marks. The work is the result of collaboration between soil scientist Paula Deegan and Ian Weir. The installation was documented with a series of 30 still digital photographs, later animated in Microsoft Powerpoint.

H-house

The author's approach to the problems associated with building in bushfire prone landscapes comes from 12 years of study of the biophysical and cultural landscapes in the Great Southern Region of Western Australia - research which resulted in the design and construction of the H-house at Bremer Bay. The house was developed using a 'ground up' approach whereby Dr Weir conducted topographical surveys and worked with a local botanist and a bushfire risk consultant to ascertain the level of threat that fire presented to this particular site. The intention from the outset however, was not to design a bushfire resistant house per se, but to develop a design which would place the owners in close proximity to the highly biodiverse heath vegetation of their site. The research aim was to find ways - through architectural design-to link the patterns of usage of the house with other site specific conditions related to the prevailing winds, solar orientation and seasonal change. The H-house has a number of features which increase the level of bushfire safety. These include: Fire rated roller shutters (tested by the CSIRO for ember attack and radiant heat), Fire resistant double glazing (on windows not protected by the shutters), Fibre-cement sheet cladding of the underside of the elevated timber floor structure, Manually operated high pressure sprinkler system on exposed timber decks, A fire refuge (an enlarged laundry, shower area) within the house with a dedicated cabinet for fire fighting equipment) and A low pressure solar powered domestic water supply system.

Bushfire responsive architecture : strategies for reconciling bushfire and biodiversity with daily life

My perspective on the problems associated with building in bushfire prone landscapes comes from 12 years of study of the biophysical and cultural landscapes in the Great Southern Region of WA which resulted in the design and construction of the ‘Hhouse’ at Bremer Bay. The house was developed using a ‘ground up’ approach whereby I conducted a topographical survey and worked with a local botanist and a bushfire risk consultant to ascertain the level of threat that fire presented to this particular site. My intention from the outset however, was not to design a bushfire resistant house per se, but to develop a design which would place the owners in close proximity to the highly biodiverse heath vegetation of the site. I was also seeking a means—through architectural design—of linking the patterns of usage of the house with other site specific conditions related to the prevailing winds, solar orientation and seasonal change.