Mutations in the Pit1 gene severely affect the development and function of the anterior pituitary gland in both mice and humans, resulting in deficiencies of growth hormone, prolactin and thyroid stimulating hormone. These hormone deficiencies are thought to result from the failure to specify the identity of cells, presumably through the inability to activate genes that encode hormones. Furthermore, hypoplasia of the pituitary gland may result from the inability of pituitary endocrine cells to proliferate. The aim of this project is to study the effect of dominant Pit1 gene mutations to determine the mechanism of action of the mutant proteins. The study of mutant PIT1 proteins should allow greater understanding of how wild type PIT1 protein functions. Luciferase reporter assays were used to test the transcriptional activity of dominant negative PIT1 mutant proteins on different target promoters. These assays showed that the mutations had cell and reporter specific effects on luciferase activity that may be due to the combined effects of both direct transcriptional repression and indirect off-target effects, perhaps negative effects on cell viability. Several of the mutations resulted in increased activity in a reporter and cell type specific manner. Mutation of the N-terminal transactivation domain affected protein stability and electrophoretic mobility, strongly suggesting an effect on post-translational modification. However, subsequent analysis was unable to confirm this. Additionally, two novel recessively inhertited mutations were identified in a patient with combined pituitary hormone deficiency. The IVS1+3nt (a>g) mutation severely affected splice donor site definition whereas the R265W missense mutation resulted in reduced protein expression.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:625332 |
| Date | January 2010 |
| Creators | Turton, J. P. G. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/20005/ |
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