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

Extreme genetic fragility of the HIV-1 capsid

Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

Archaeological signatures of landscape and settlement change on the Isle of Harris

Between 2004 and 2011, a programme of archaeological investigation by the University of Birmingham on the Isle of Harris, a distinctive island forming part of the Western Isles of Scotland, has allowed the archaeological remains of this enigmatic place to be further characterised and understood. Despite intensive archaeological interest in the archipelago for a number of decades, the Isle of Harris has been overlooked and only now are we beginning to identify the archaeological resource and make comparisons to the wealth of published data from islands such as the Uists, Barra and Lewis. This paper highlights some generic overall patterns of archaeological signatures on the Isle which has been identified through a range of archaeological methods including field walking, intrusive excavation, aerial reconnaissance, geophysical and topographical survey, and documentary research. Several key case studies will be introduced including upland shieling complexes and mulitperiod settlement sites on the west coast machair systems. The purpose of the paper is not to present a gazetteer of the results of the work to date, but to highlight some of the key findings with a view to demonstrating that the Isle of Harris is directly comparable with the archaeologically rich landscapes of the other islands

Phage P22 lysis genes: nucleotide sequences and functional relationships with T4 and lambda genes

Wild-type and amber mutant alleles of the lysis genes of P22 were cloned and sequenced. Gene 13 encodes an 11,520-Da basic hydrophobic protein that has 89% amino acid homology to lambda S protein. Gene 19 encodes a protein that has a small degree of amino acid homology with T4 lysozyme, but we could detect no homology to lambda R or RZ proteins. The protein product of gene 19 was purified; its amino terminal amino acid sequence is as predicted by the DNA sequence. It starts with a single amino terminal methionine residue and is a basic protein with a molecular weight of 15,968. Plasmids expressing P22 gene 19, lambda genes R and RZ, and T4 gene e were constructed. All of these plasmids were able to complement both lambda R- and P22 19-

Genetic Analysis of Bacteriophage P22 Lysozyme Structure

The suppression patterns of 11 phage P22 mutants bearing different amber mutations in the gene encoding lysozyme (19) were determined on six different amber suppressor strains. Of the 60 resulting single amino acid substitutions, 18 resulted in defects in lysozyme activity at 30°; an additional seven were defective at 40°. Revertants were isolated on the ``missuppressing'' hosts following UV mutagenesis; they were screened to distinguish primary- from second-site revertants. It was found that second-site revertants were recovered with greater efficiency if the UV-irradiated phage stocks were passaged through an intermediate host in liquid culture rather than plated directly on the nonpermissive host. Eleven second-site revertants (isolated as suppressors of five deleterious substitutions) were sequenced: four were intragenic, five extragenic; three of the extragenic revertants were found to have alterations near and upstream from gene 19, in gene 13. Lysozyme genes from the intragenic revertant phages were introduced into unmutagenized P22, and found to confer the revertant plating phenotype

Functional significance of conserved amino acid residues

A systemic study of single amino acid substitutions in bacteriophage T4 lysozyme permitted a test of the concept that conserved amino acid residues are more functionally important than nonconserved residues. Substitutions of amino acid residues that are conserved among five bacteriophage-encoded lysozymes were found to lead more frequently to loss of function than substitutions of nonconserved residues. Of 163 residues tested, only 74 (45%) are sensitive to at least one substitution; however, all 14 residues that are fully conserved are sensitive to substitutions

Structure of phage P22 gene 19 lysozyme inferred from its homology with phage T4 lysozyme. Implications for lysozyme evolution

The amino acid sequence of the lysozyme from phage P22 is shown to be homologous (26% identity) with the lysozyme from bacteriophage T4. The sequence correspondence suggests that the structure of P22 lysozyme is similar to the known structure of T4 lysozyme within the core of the molecule, including the active site cleft. However, P22 lysozyme appears to lack two surface loops present in T4 lysozyme. It is possible that P22 lysozyme may provide an evolutionary link between the phage-type lysozymes and the goose-type lysozymes

Alteration of T4 lysozyme structure by second-site reversion of deleterious mutations.

Mutations that suppress the defects introduced into T4 lysozyme by single amino acid substitutions were isolated and characterized. Among 53 primary sites surveyed, 8 yielded second-site revertants; a total of 18 different mutants were obtained. Most of the restorative mutations exerted global effects, generally increasing lysozyme function in a number of primary mutant contexts. Six of them were more specific, suppressing only certain specific deleterious primary substitutions, or diminishing the function of lysozymes bearing otherwise nondeleterious primary substitutions. Some variants of proteins bearing primary substitutions at the positions of Asp 20 and Ala 98 are inferred to have significantly altered structures

Systematic mutation of bacteriophage T4 lysozyme

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161)