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Hormonal regulation of xenopus nuclear receptors and their target genes

Esslemont, Graeme Murray; (1995) Hormonal regulation of xenopus nuclear receptors and their target genes. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Metamorphosis in amphibia is obligatorily dependent upon the thyroid hormones triiodothyronine and thyroxine (T3 and T4, respectively), which can precociously induce metamorphosis. This has been exploited advantageously to study both the mode of action of these hormones and the biochemical and molecular basis of gene switching and cellular modifications which occur during metamorphosis. Recent work from our laboratory on thyroid hormone receptors (TRs) has shown that the two isoforms of TR known in the frog Xenopus laevis, TRα and TRβ, are upregulated differentially at the mRNA level, both during natural metamorphosis and precociously in tadpoles exposed to T3. However, very little is known about the properties and dynamics of the Xenopus TR proteins. The major goal of my project is to study the role of thyroid hormone receptor proteins, which are members of a superfamily of ligand dependent transcription factors, in Xenopus during metamorphosis. To study the ligand and DNA binding properties of Xenopus TR proteins, I have prepared in vitro translated Xenopus TRα and -β, as well as nuclear extracts from the T3-responsive Xenopus cell line XTC-2. For high affinity, sequence specific DNA binding to occur I have shown that Xenopus TRs require heterodimerisation with retinoid X receptors (RXR), which were also synthesised in a cell-free transcription- translation system. Polyclonal antibodies against bacterially expressed Xenopus TRs and synthetic peptides were generated for use in Western blotting and "supershifting" in electrophoretic mobility shift assays (EMSA) with various thyroid hormone response elements (TREs). To explain the phenomenon of autoinduction of Xenopus TR genes, the binding of TRα and β proteins to the Xenopus TRβ promoter was examined in parallel with transfection of various promoter constructs in XTC-2 cells. A TRE with the sequence 5'-AGGTCATTTCAGGACA-3' spanning the promoter at -5/+11 was discovered, to which TR/RXR heterodimers bound with high affinity. To further analyse autoregulation of the Xenopus TRβ gene, these studies were extended to examining DNA binding properties of dominant negative TR mutants (human and Xenopus), and their ability to heterodimerise with Xenopus RXR. Taken together, my studies emphasise the importance of heterodimerization of two nuclear hormone receptor proteins in furthering our understanding of how thyroid hormones initiate and regulate amphibian metamorphosis.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Hormonal regulation of xenopus nuclear receptors and their target genes
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
URI: https://discovery.ucl.ac.uk/id/eprint/10103065
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