Botcherby, Marc Robert Michel; (2002) Comparative vertebrate genomic sequence analysis studies based on Fugu rubripes. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

This thesis explores the benefits of the comparative analysis of genomic DNA sequence in three vertebrate species: man, mouse and Fugu rubripes. The first region studied is the interval around Fibroblast Growth Factor 2 (FGF2). It falls within the candidate mapping interval for Insulin Dependent Diabetes 3 (Idd3) in mouse, in which Spermatogenesis Associated Factor (SPAF), Fibroblast Growth Factor 2 (FGF2) along with NUDT4 (formerly known as the antisense of FGF2, GFG) and a Centrin related sequence are known to be present. The Ensembl entry for the human draft sequence only contains FGF2. A Fugu cosmid was sequenced to 99.99% accuracy and is shown to contain orthologues to SPAF, FGF2, NUDT4 and a Centrin gene, as well as a novel gene prediction (vader), evolutionarily conserved in mouse and man. Comparison to the available mouse and human genomic sequences indicates the presence of SPAF, FGF2, NUDT4 and a Centrin gene in both the mouse and human FGF2 genomic regions. The second area of study is the conventional Protein Kinase C family (cPKC), which comprises 4 isoenzymes in man: α, β1, β2, and γ. A number of approaches are taken to identify the Fugu cPKC homologues: genomic library screening by hybridisation, sequence scanning, Southern Blotting and degenerate PCR. A PKCα orthologue is identified and evidence for the existence of two further β homologues found. These genes are compared to the PKCs found in other species by means of phylogenetic trees. Two overlapping cosmids containing the PKCα orthologue are identified, sequenced to 99.99% accuracy and analysed. Potential regulatory elements are identified. A calcium channel subunit gene (CACNG5) is also found close to Fugu PRKCA, which reflects an association between all cPKCs and CACNG genes. This is interesting both from an evolutionary point of view and in the light of the association between cPKCs and Retinitis pigmentosa. Comparative sequence analysis is a useful tool for studying the evolution of gene families; the Fugu model provides a particularly effective means for the identification novel genes as well as regulatory elements.

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