The main aim of this work was to identify the important cis-acting regulatory sequences, and the trans-acting factors with which they interact, which are required for the tissue-specific expression of the Xenopus borealis skeletal actin gene. All sequences necessary and sufficient for the correct spatial and temporal expression of the Xenopus borealis skeletal actin gene are located in a 156 bp fragment of the gene that spans from nucleotide residues -197 to -42 in its promoter. This region of the skeletal actin promoter contains three imperfect repeats of a CArG sequence motif that has been demonstrated to be important in the expression of other sarcomeric actin genes. Deletion analysis of the promoter of the Xenopus borealis skeletal actin gene, using Xenopus micro-injection techniques as a transient assay system for promoter activity, have identified that CArG box3 is essential for skeletal actin gene expression. By using band shift assays I have demonstrated that, under my assay conditions, CArG box2 is unable to bind any proteins in vitro . Conversely, the CArG boxl sequence exhibits two binding activities on band shift analysis. One of these is antigenically related to the transcription factor SRF, whilst the second appears to be distinct from this protein. CArG box3 also interacts with a protein in vitro. Although this sequence exhibits a similar shift to that of the CArG boxl/SRF complex on band shift analysis, my experiments suggest that this protein is distinct from SRF. A combination of the CArG boxl and CArG box3 motifs is unable to confer muscle-specific gene expression on a heterologous promoter. Furthermore, I have identified an upstream regulatory element (URE) in the Xenopus borealis skeletal actin gene promoter that spans from nucleotides -197 to -167 that is required for the expression of the gene, at least when sequences between nucleotide -42 and +28 are absent. The URE of the Xenopus borealis skeletal actin gene is capable of interacting with a trans-acting factor(s) in vitro. In addition to this a further region of the gene which spans from nucleotide residues -83 to -42 is also capable of interacting with a factor(s) in vitro. The mechanisms by which these multiple regulatory elements control the tissue-specific expression of the Xenopus borealis skeletal actin gene will be discussed.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:293923 |
| Date | January 1991 |
| Creators | Lakin, Nicholas David |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/108299/ |
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