A Genetic and Structural Analysis of P22 Lysozyme: A Thesis

P22 lysozyme, encoded by gene 19, is an essential phage protein responsible for hydrolyzing the bacterial cell wall during lytic infection. P22 lysozyme is related to T4 lysozymein its mode of action, substrate specificities, and in its structure. Gene 19 was located on the phage genome, subcloned, and then sequenced. lysozyme was produced in large quantities and purified for biochemical characterization and for crystallograpic studies. Gene 19consists of 146 codons, and encodes a protein with a molecular weight of 16,117. Amber mutations were created in gene 19 by in vitro primer-directed mutagenesis. The mutations were crossed by homologous recombination onto the phage genome. The phages bearing the amber mutations in gene 19 were screened for the ability to grow on six different amber suppressor strains. Amino acid substitutions that resulted in nonfunctional or less functional lysozyme were determined. Of 60 possible amino acid substitutions at 11 different sites in P22 lysozyme, 20 are deleterious. The phage bearing amber mutations in gene 19that failed to grow on given suppressor strains were reverted and second site intragenic revertants were obtained. The mutations were sequenced. A substitution of serine for glutamine at residue 82 is compensated for by changing residue 46 from serine to leucine. This single change enables the phage to form a plaque at 300C but not at 400C. When the triple change asn42-\u3elys; ser46-\u3eleu; and ser43-\u3epro is present the lysozyme produced is no longer temperature sensitive. The crystal structure of P22 lysozyme is not yet solved. Assuming that the structures of T4 lysozyme and P22 lysozyme are similar, one can examine the positions of equivalent residues in the T4 lysozyme structure. The spatial arrangement of the residues changed by the secondary site mutations and the original substitution can then be visualized. The mutations discussed above all map far from the original mutation on the T4 three dimensional model. A substitution of leucine for tyrosine at position 22 is compensated for by the double mutation of arg18-\u3eser and ser23-\u3elys. When the equivalent residues are mapped on the T4 three dimensional model the changes map in close proximity to the original mutation

Exploring places and landscapes of everyday experience in the Outer Hebridean Iron Age: a study of theory, method and application in experiential landscape archaeology

This thesis explores aspects of everyday experience and the creation of place within the Iron Age island landscapes of the Outer Hebrides. While investigations of place and landscape, as experiential phenomena, are well developed in the context of Neolithic and Bronze Age research such approaches have been largely neglected within British Iron Age studies and in the study of the Outer Hebridean Iron Age more specifically. A hitherto focus upon ritual landscapes partly explains the lack of uptake within British Iron Age contexts more frequently defined by concepts of domesticity. The experience of place and landscape, however, are not only of significance within 'ritual' contexts but play an important role in the shaping of human action in the realm of the everyday. Instead, the principal barrier appears to be methodological - how does one go about investigating everyday experiences within prehistoric landscapes? A major component of this research has therefore been to explore and develop a methodology for this research. Current archaeological practice provides two contrasting methods for the study of landscape experience - one rooted in the analysis of field observations, inspired more directly by phenomenology, and the other via the application of GIS as a means of modeling landscapes from the perspective of human engagement. Despite much shared theoretical ground there remains little dialogue between practitioners of these respective approaches. It is proposed, however, that both approaches can make valued contributions to our understanding of the past and this thesis aims to contribute to an emerging discourse between what are commonly conceived as contradictory methods of enquiry. By exploring the character and diversity of island landscape settlement locales and the everyday experiences of Iron Age places this research offers an alternative framework for understanding the Iron Age societies of the Outer Hebrides

Chronostratigraphy of an eroding complex Atlantic Round House, Baile Sear, Scotland

The excavation team would like to thank Historic Scotland (now Historic Environment Scotland) and the University of St Andrews for providing funding.A high-resolution chronostratigraphy has been established for an eroding Atlantic round house at Sloc Sàbhaidh (North Uist, Scotland), combining detailed OSL profiling and dating of sediments encompassing the main bracketing events associated with the monument, radiocarbon AMS dates on bone recovered from excavated features and fills within it, and TL dates on pottery and burnt clay. Concordant OSL and radiocarbon evidence place construction of the wheelhouse in the first to second centuries AD, contemporary with dates from the primary occupation. Beneath the wheelhouse, clay deposits containing burnt material, attest to cultural activity in vicinity to the monument in the preceding second to first centuries BC. At a later date, the southern wall collapsed, was rebuilt, and the interior spaces to the monument re-structured. The chronology for the later horizons identified from the sediment luminescence dates extends to the second half of the first millennium AD, which goes beyond the range of the radiocarbon dates obtained. The data from ceramics encompass both periods. The juxtaposition of the dating evidence is discussed relative to short and longer chronologies for this Iron Age monument. Corollaries of this research are the implications that based on the long chronology, some of the ecofacts (bone) appear to be residual, and that the temporal duration of Hebridean Coarse Ware may extend into the second half of the first millennium AD.Publisher PDFPeer reviewe

Thermodynamic Prediction of Protein Neutrality

We present a simple theory that uses thermodynamic parameters to predict the probability that a protein retains the wildtype structure after one or more random amino acid substitutions. Our theory predicts that for large numbers of substitutions the probability that a protein retains its structure will decline exponentially with the number of substitutions, with the severity of this decline determined by properties of the structure. Our theory also predicts that a protein can gain extra robustness to the first few substitutions by increasing its thermodynamic stability. We validate our theory with simulations on lattice protein models and by showing that it quantitatively predicts previously published experimental measurements on subtilisin and our own measurements on variants of TEM1 beta-lactamase. Our work unifies observations about the clustering of functional proteins in sequence space, and provides a basis for interpreting the response of proteins to substitutions in protein engineering applications

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made in vivo. Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods

In the light of directed evolution: Pathways of adaptive protein evolution

Directed evolution is a widely-used engineering strategy for improving the stabilities or biochemical functions of proteins by repeated rounds of mutation and selection. These experiments offer empirical lessons about how proteins evolve in the face of clearly-defined laboratory selection pressures. Directed evolution has revealed that single amino acid mutations can enhance properties such as catalytic activity or stability and that adaptation can often occur through pathways consisting of sequential beneficial mutations. When there are no single mutations that improve a particular protein property experiments always find a wealth of mutations that are neutral with respect to the laboratory-defined measure of fitness. These neutral mutations can open new adaptive pathways by at least 2 different mechanisms. Functionally-neutral mutations can enhance a protein's stability, thereby increasing its tolerance for subsequent functionally beneficial but destabilizing mutations. They can also lead to changes in “promiscuous” functions that are not currently under selective pressure, but can subsequently become the starting points for the adaptive evolution of new functions. These lessons about the coupling between adaptive and neutral protein evolution in the laboratory offer insight into the evolution of proteins in nature

“Till the muddle in my mind have cleared awa”: can we help shape policy using systems modelling?

This paper considers how some well-documented deficiencies of mental models make it difficult to create effective policies, and suggests that systems modelling can begin to address this issue. To illustrate the argument three short cases are presented. These relate to specific domains but demonstrate how systems modelling can illuminate different general phenomena: effects on labour costs (unintended consequences and feedback); fishery management (accumulation and non-linearity) and child protection (worldviews and sense-making). Six levers for increasing the use of systems modelling in the policy arena are then discussed. The paper closes by emphasising the opportunities for systems modellers in the Anthropocene Era

On the history of meridional overturning circulation schematic diagrams

Author Posting. © Elsevier B.V., 2008. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Progress In Oceanography 76 (2008): 466-486, doi:10.1016/j.pocean.2008.01.005.Recent global warming caused by humans and the prediction of a reduced Atlantic Ocean meridional overturning circulation in the future has increased interest in the role of the overturning circulation in climate change. A schematic diagram of the overturning circulation called the “Great Ocean Conveyor Belt,” published by Wallace Broecker in 1987, has become a popular image that emphasizes the inter-connected ocean circulation and the northward flux of heat in the Atlantic. This would appear to be a good time to review the development of the conveyor belt concept and summarize the history of overturning circulation schematics. In the nineteenth century it was thought that symmetric overturning circulation cells were located on either side of the equator in the Atlantic. As new hydrographic measurements were obtained in the late nineteenth century and early twentieth century, circulation schematics in the early twentieth century began to show the inter-hemispheric overturning circulation in the Atlantic. In the second half of the twentieth century schematics showed the global ocean overturning circulation including connections between the Atlantic and the Pacific and Indian Oceans. Some recent schematics of the overturning circulation show its complexities, but as more information is included these schematics have also become complex and not as easy to understand as the simple Broecker 1987 version