published_or_final_version / abstract / Zoology / Master / Master of Philosophy

DNA intrinsic curvature is, at present, one of the major topics of research concerning DNA conformation and dynamics because of its implications in phenomena of great biological relevance including transcription activation and chromatin packaging. DNA sequences containing runs of adenines residues (A-tracts) are of central importance to this phenomenon since the length of the A-tracts and their phasing with the helix screw significantly alter the curvature profile of DNA. X-ray crystallography of DNA single crystals has provided a wealth of information about the local, short range conformational features of general-sequence and also A-tractcontaining DNA oligomers but it lacks the same strength in the analysis of long range conformational features of DNA. On the other hand, gel-electrophoresis analysis of DNA has not only uncovered the macroscopic curvature of DNA but it also provides most of the available data on DNA intrinsic curvature. However, gel electrophoresis can not identify features of DNA structure at the nucleotide or atomic level. This work is an attempt of bridging the gap between such techniques by using the method of molecular dynamics (MD) simulations. MD simulations were performed on 5 A-tract-containing dodecamers, 3 of which have available crystal structures, and on a 51 base-pair DNA fragment from the kinetoplast DNA of Leishmania tarentolae. The results shows that A-tracts can be strongly curved, contrary to what is expected from X-ray analysis of DNA single crystals. The detailed analysis of the MD trajectories also shows that DNA curves towards the major groove. An explanation which demonstrate the consistence between major groove curvature and uncurving of DNA upon binding of the antibiotic distamycin is provided. Furthermore, regions of hyperflexibility or junctions between A-tracts and general-sequence DNA are identified and shown to modulate DNA intrinsic curvature. The flexibility pattern of the sugar rings can provide an alternative explanation for the cleavage pattern of A-tract-containing DNA by the hydroxyl radical. An attempt to predict the curvature of other A-tract-containing DNA sequences is also presented. It is shown that, in order to realistically represent the curvature of A-tract-containing DNA, the intrinsic structure of individual A-tracts must be preserved and not averaged as in the Wedge model. Finally, some computer experiments are suggested in order to refine our knowledge of DNA conformation and dynamics using MD simulations before such technique can be routinely used in the investigation of phenomena of biological and medicinal relevance.

engrailed-related genes are homeobox containing transcription factors and have been cloned in numerous taxa. Their roles, in segmentation of the early embryo and in neural development, have been extensively described. Within the insects, some species have a single engrailed-related gene whilst others have two copies, raising the question of when and how often this duplication occurred. Here I report the cloning of two engrailed-related genes cren 1 and cren 2 in the cockroach Periplaneta americana. By comparing conserved domains and by carrying out a phylogenetic analysis, I conclude that these two genes are likely to be the product of a recent duplication in the cockroach lineage. Differentially spliced cren 2 isoforms that lack some regulatory domains have also been isolated. The roles these isoforms could play in regulating developmental processes are discussed. cren 1 and cren 2 are co-expressed during early embryogenesis; their segmental pattern of expression appears in an anteriorposterior progression. Double-stranded RNA interference was used to abolish the expression of Engrailed which is a putative positional determinant in an array of sensory neurones in the cockroach cereal system. This treatment radically alters the axonal arborization and choice of postsynaptic interneural partners of an identified Engrailed-positive sensory afferent. The neurone now resembles a neighbouring, Engrailed-negative cell, which is itself unaffected by the double-stranded RNA.

The way in which scientific research is conceived, organized and practiced in the context of shifting policies and institutional structures is the main focus of this thesis. The empirical component examines the development of the UK Human Genome Mapping Project (HGMP), the British contribution to the international plan to locate all of the genes in the human species. A comparative case-study examines the unsuccessful attem pts by Australian scientists to establish a national genome mapping initiative. Two main sets of research questions are posed. Why was the HGMP set up in the UK at the end of the 1980s when, prima facie, a number of factors suggested that an organized project would not be established? Allied to this question is the comparative one of how and why a project was successfully established in the UK but not in Australia? The second major question asks how policy subsequently developed for the UK programme: what factors shaped policy-making, what aspects of science did the programme shape? Drawing on recent developments in sociology of science and science policy studies, it is argued that in both Australia and the UK debates over whether to have a genome project, and the subsequent developments in UK policy towards the project, were not straightforward administrative choices. In both countries, the question of what was the best science to support was translated into debates over the best way to do science, what scientific knowledge was for and even what was to count as worthwhile knowledge. The implicit and explicit answers to those questions by various groups were embodied in policies and policy recommendations. The thesis concludes that differing expectations concerning the role of science had to be orchestrated together in order to mobilize, and subsequently maintain, support for gene mapping and sequencing.

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