Expression and Functional Analysis of the Transcription Factor DMAHP

Harris, Sarah Elizabeth (1999) Expression and Functional Analysis of the Transcription Factor DMAHP. PhD thesis, University of Glasgow.

Abstract

Myotonic dystrophy (DM) is the most prevalent form of adult muscular dystrophy and is characterised by myotonia and muscle weakness and atrophy. The genetic defect associated with DM is an unstable (CTG)n repeat in the 3' untranslated region (UTR) of a protein kinase gene myotonic dystrophy protein kinase (DMPK) and the promoter region of a homeobox gene myotonic dystrophy associated homeodomain protein (DMAHP). These genes are both expressed in a wide range of tissues and their levels of expression have been shown to be altered in DM patients. DMAHP is a member of the Six subfamily of genes of which 6 mammalian and 3 Drosophila genes have been identified. All members of this family encode a homeodomain that is highly diverged from other known homeodomains and a second homologous domain, the Six domain, which lies immediately upstream of the homeodomain. Homeodomain proteins are known to be transcription factors that control gene expression by binding to DNA and activating or repressing transcription. The work presented in this thesis investigated the DNA binding properties of the DMAHP protein. Human foetal cDNA libraries were screened for the presence of a full length DMAHP cDNA but none was identified. Therefore, a cloned RT-PCR product and a genomic cosmid were used to subclone the Six domain, the homeodomain and both domains together into the bacterial expression vector pGEX4T3. From these clones, 3 glutathione S-transferase (GST)-DMAHP fusion proteins were produced and affinity purified by chromatography with a glutathione ligand. Affinity purified recombinant proteins and cell lysates from bacteria expressing the recombinant proteins were used in gel retardation assays with DNA oligonucleotides representing putative DNA binding sites. The putative sites included 2 in the promoter region of DMPK and the known DNA binding site of another Six subfamily member, AREC3/Six4 (the ARE). The recombinant fusion proteins showed no affinity for the 2 putative sites in DMPK. However, both the recombinant fusion proteins containing the homeodomain bound to the ARE, with the recombinant protein containing both domains (GST-Six+HD) forming 2 complexes, one of which was presumed to be a dimer complex. A cofactor present in the bacterial cell lysate increased the affinity of GST-Six+HD for the ARE. Finally, a whole genome PCR based screen was used to identify genomic DNA sequences to which DMAHP binds, as an initial stage in the identification of genes regulated by DMAHP.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Keith Johnson
• Keywords: Genetics
• Date of Award: 1999
• Depositing User: Enlighten Team
• Unique ID: glathesis:1999-74789
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 27 Sep 2019 16:18
• Last Modified: 27 Sep 2019 16:18
• URI: https://theses.gla.ac.uk/id/eprint/74789