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The completion of a substantial number of complete genome sequencing initiatives has produced more than a million protein sequences. Analysis of these protein sequences is possible using recent advances in computing and bioinformatics techniques. This thesis describes a novel automated protein classification protocol which groups proteins into families and identifies protein domain architectures via domain assignment. This data is presented in the Gene3D database which is used for subsequent analysis. The analysis of the distribution of protein family and protein domain data shows a power-law like distribution that is typically seen in many biological data distributions and is indicative of the small world networks that underlie biological systems biology. Kingdom distribution of superfamilies and protein families in Gene3D has been used to describe the evolutionary mechanisms that determine genome diversity through protein diversity. Domain occurrence profiles have been used to identify protein domain superfamilies that are correlated with genome size in bacteria. These superfamilies are shown to exhibit a balance between metabolic and regulatory roles along microeconomic principles that may determine bacterial genome size. Domain families identified in Gene3D enable a determination of the total number of protein folds in nature. Sub-clustering of domain families permits domain family sub-cluster occurrence profiles to be determined. These profiles are shown to be capable of detecting correlations and anti-correlations between domain families that are undetectable using superfamily occurrence profiles alone. Clusters of correlated domain subclusters are shown to identify functionally linked clusters of proteins. Finally, the data in Gene3D is used to functionally annotate the CATH database and provide functional predictions for un-annotated proteins, providing more comprehensive functional repertoire and greater accuracy than other functional prediction methods.

1. It is becoming increasingly apparent that cell volume, otherwise termed the cellular hydration state, regulates processes such as protein synthesis, lipogenesis and membrane transport. In this study, the effect of cell volume perturbations on mammary cell metabolism has been investigated. Thus, the effect of cell swelling and shrinking on protein synthesis and lipogenesis in rat mammary tissue has been examined. In addition, the effect of cell volume changes on the free cytosolic calcium concentration ([Ca2+]i) in rat mammary acinar cells has been studied. 2. Mammary tissue (explants and acini) isolated from lactating rats during peak lactation was used in this study. Protein synthesis was studied by measuring the incorporation of radiolabelled amino acids into trichloroacetic acid precipitable material. Cell swelling, induced by a hyposmotic shock markedly increased protein synthesis in mammary tissue explants and acini. Conversely, cell shrinking, induced by a hyperosmotic shock markedly inhibited protein synthesis in mammary explants and acini. The effect of cell swelling and shrinking on protein synthesis was a) dependent upon the extent of the osmotic challenge and b) reversible. Swelling- induced mammary protein synthesis was dependent upon the presence of extracellular calcium. In this connection, thapsigargin and tBHQ markedly inhibited volume- sensitive protein synthesis suggesting that protein synthesis is dependent upon luminal calcium stores. Isosmotic cell swelling, induced by using buffers containing urea significantly increased mammary protein synthesis. 3. The effect of cell volume perturbations on the [Ca2+]i in mammary acinar cells isolated from rats during peak lactation was examined. The [Ca2+]i was measured using the fura-2 dye technique. Cell swelling, induced by a hyposmotic challenge, increased the [Ca2+]i in a fashion that was transient. The effect of a hyposmotic shock on the [Ca2+]i was dependent upon the extent of the osmotic perturbation. The hyposmotically-induced increase in the [Ca2+]i could not be attributed to a) a change in the trans-membrane sodium gradient or b) a change in the ionic strength of the incubation buffer.