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Title: The molecular characterisation of the gene trap GT411
Author: Axton, R. A.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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In a screen designed to identify novel cardiovascular development genes, the gene trap GT411 was isolated. This thesis describes the characterisation of the gene trap integration GT411. Initially, 5’ RACE (rapid amplification of cDNA ends) generated a 206 bp sequence that aligned with EST (expressed sequence tag) data to build a contig with a partial open reading frame (ORF) of 177 amino acids. Additional rounds of 5’RACE generated a full length open reading frame of 823 amino acids. Bioinformatic analysis of the nucleotide and protein sequence showed that it was highly conserved between mouse, rat and human. The gene encodes a protease domain and belongs to the M1 family of aminopeptidases. Recently the human homolog was cloned and named Aminopeptidase O (APO). The C-terminus of the gene contains an armadillo repeat (ARM) and a putative nuclear localisation signal (NLS). The latter was experimentally confirmed by transfection of eGFP-ApO constructs demonstrating that the signal localised the fusion protein to the nucleoli. RT-PCR and Northern blot expression have confirmed this gene has alternative isoforms which can remove the functional domains. Germline transmission of the gene trap resulted in normal, healthy, viable mice with no apparent phenotype. Expression analysis of the reporter gene (LacZ) indicated that the gene trap was restricted to the cardiovasculature in both embryonic and adult tissues. The possibility that the ApO may be involved in the process of angiogenesis was investigated using the aortic ring assay. An antibody was raised against the full length murine APO protein. It was immunopurified and tested by Western blot, immunoprecipitation and immunohistochemistry.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available