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Title: Identification of a novel protein interacting with RPGR
Author: Boylan, James
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2001
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X-linked retinitis pigmentosa is a severe form of human retinal degeneration and is most commonly caused by mutations in the RPGR gene, which maps to chromosomal region Xp21.1. The RPGR gene product is a novel protein containing an amino terminal domain that is homologous to the guanine nucleotide exchange factor for a small GTP-binding protein. No other domains of known function are recognisable. The principal aim of this thesis was to identify proteins that interact with RPGR. This was done in an attempt to obtain structural or functional information about this novel protein that could account for its roles in health and disease. The yeast two-hybrid method was used to screen a variety of expression libraries made from human pancreas and bovine retina. A novel interacting protein, called RPGRIP, was identified using a bovine retina library and the human homologue identified by database screening and primer extension methods. The specificity of the interaction between RPGR and RPGRIP was confirmed by co-immunoprecipitation. Further evidence for a functionally significant interaction was obtained using six RPGR mutants, corresponding to known disease-causing mutations, all of which abolished or severely reduced the interaction. The expression of human and bovine RPGRIP was examined using northern analysis and/or RT-PCR. This revealed strong expression of the human transcript in testis and retina but more widespread expression in bovine tissues. At least three alternatively spliced forms were identified. The human gene structure was identified and shown to consist of 15 exons spanning 34 kilobases and to code for alternatively spliced proteins of 586 amino acids, 837 amino acids, and 902 amino acids. A structure-function analysis of RPGRIP by database searching identified two strongly predicted coiled-coil domains and two calcium-dependent phospholipid-binding C2-domains. The gene was mapped to chromosomal region 14q11 by radiation hybrid mapping and screening a human monochromosomal hybrid panel. Mutation analysis of three autosomal recessive retinitis pigmentosa families previously shown to be linked to the 14q11 region failed to identify disease-causing mutations. The function of RPGRIP remains to be elucidated but it is a strong candidate gene for causing another form of human retinopathy.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available