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Title: Immunological and molecular approaches to characterisation of a new member of the epidermal growth factor gene family
Author: Wu, Guochin
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1990
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The aim of this project was to analyse the tissue distribution and function of a novel gene cloned by Jenny Dunne within ICRF. Sequence data revealed that the gene structure is homologous to the EGF-like protein family in the arrangement of cysteine and other conserved amino acids. The cloned cDNA was truncated at both the N- and C-termini. To study the function, tissue distribution and the relationship with other proteins of this gene product, an antibody reacting with the native form of the gene product was considered to be essential. Several strategies have been followed in attempting to achieve this aim. Initially synthetic peptides derived from the gene sequence were used as immunogen, the reactivity of the polyclonal and monoclonal antibodies from immunised mice and rats did not correlate with the results of northern blot analysis and the antibodies did not react with native proteins. In a second series of experiments the truncated gene was linked in frame with a pseudo N-terminus and cloned into a vector which upon transfection can confer resistance to G418. The gene was stably integrated and transcribed. However, sera from mice immunised with the transfectants failed to stain living or fixed cells in a specific fashion. Surface iodination and internal labelling followed by one-D-gel analysis of cell associated or supernatant proteins did not show any difference between untransfected and transfected cells. This might suggest that the products were not processed onto the cell surface as expected. A third approach was to make several TrpE fusion proteins as immunogens. Antisera from rats showed no specific reactivity with the fusion proteins. The difficulty of raising antibody to the gene product is discussed. Northern blot analysis of various established lymphoid, myeloid, and epithelial cell lines, normal lymphoid tissues and activated lymphocytes showed that the gene is predominantly expressed in T-ALL cell lines. A striking feature of the northern analysis was the large number of different message sizes seen in different T cell lines. Cell surface phenotype analysis of these cells suggests that the pattern of transcripts is correlated with different stages of maturation. The implications of these findings for the function of this gene product and its possible role in malignant transformation are discussed.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available