Roberts, Gwyndaf Thomas; (1997) Regulation of the <italic>Xenopus</italic> gene Xsna. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Xsna is a zinc finger transcription factor that is expressed in the developing Xenopus embryo in the mesoderm from stage 9 and the prospective neural crest from stage 11. The promoter of this gene contains no sequences that resemble the motifs of known transcription factors. However, it has previously been shown that two adjacent elements within the promoter (approximately 100 base pairs from the transcriptional start site), are responsible for the differential expression pattern of the gene. Using the electrophoretic mobility shift assay (EMSA) specific DNA binding activities have been associated with the sequences within these regions. Their binding preferences have been further defined using a range of mutated oligonucleotides in the EMSA, and by DNA protection analysis. Binding activity has been detected in extracts prepared from ovary, unfertilised eggs and embryos up to stage 35, indicating that their presence is in itself insufficient to initiate Xsna transcription. A combination of gel filtration and UV crosslinking analyses suggest that both activities are about 370 kDa in size, with ~97 kDa in direct contact with the DNA. Several purification techniques have been considered as possible components of a purification protocol but an inherent instability of the complexes has limited the use of these conventional steps. To overcome these problems, a new approach to DNA affinity purification has been developed using biotinylated double-stranded oligonucleotides and streptavidin coated magnetic beads. The protein-DNA complex is released from the beads by reducing a disulphide bond positioned between the biotin moiety and the specific binding motif. The optimal conditions for this technique have been established, and it will now be possible to isolated sufficient material for N-terminal sequencing, from which the proteins responsible for the regulation will be identified.

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