Identification and characterization of an A-form DNA promoter and its downstream novel Rab-Gdi gene in Xenopus
Student thesis: Doctoral Thesis
Despite the incredible technological advancements of the last 100 years much is still to be learnt about the biological function of DNA. In particular, the importance of non-coding DNA sequences and the mechanisms by which these sequences can regulate gene expression is an area of growing research. Genetic information must be decoded by a single cell embryo to allow development into an adult organism. Each cell type resulting in the process of development has a signature of specifically expressed genes and conversely a set of specifically silenced genes. Genes are preceded by non-coding promoter regions which contain the regulatory cis- elements, read by the trans-acting transcription factors, some of these promoters are known to adopt unusual DNA structures. In general, less is known about DNA regions that may adopt non-canonical DNA structures than other sequences and specifically little is known about the non-conventional ways that transcription factors can interact with unusual DNA structures. There are several different DNA conformations, one of which is A-form. DNA sequences with A-form propensity have been proposed to be involved in gene regulation through an indirect readout promoter recognition mechanism. Here a novel bioinformatic tool was used to screen the Xenopus genome for combined promoter sequences consisting of an A-DNA secondary structure in combination with a known sequence motif. Circular dichroism was used to validate the presence A-form elements within five identified promoters and EMSA confirmed these putative cis-acting elements were targets for specific DNA binding proteins. One of these promoters drove a previously unknown gene which was selected for further functional analysis. This novel gene, designated as gdi3, was shown to belong to the gdi family of proteins known to function as Rab-GTPAse effectors. Phylogenetic and synteny analysis supported gdi3 as a novel gene currently found only in Anura and Teleosti. RT-PCR using gdi3 specific primers showed differential expression of gdi3 between Xenopus laevis and Xenopus tropicalis in both embryos and adult frogs. In situ hybridization determined the neural specific expression of gdi3 in both species and was consistent with the knock down phenotype in Xenopus embryos exhibiting a lack of head structures and a shortened A-P axis. Finally further promoter analysis was conducted on an extended gdi3 promoter DNA sequence of 2.8 kb using an in vivo using I-SceI transgenesis approach.
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