| Summary: | The paediatric nephroblastoma Wilms Tumour, affecting 1 in 10,000 children, is a disease where the disruption in the normal events of kidney development leads to tumour formation. The <I>WT1</I> gene has been shown by knockout experiments to be crucial in kidney and gonadal development. The structure of the protein, which contains four zinc fingers of the Kruppel-type, together with <I>in vitro </I>work, suggests WT1 is a transcription factor. As we are uncertain as to what the genuine functions of WT1 are, this project set out to address what WT1 is doing at the molecular level. I aimed to investigate which components of the cellular machinery WT1 is interacting with, as it is only once we comprehend the molecular mechanism behind development that we will begin to understand the link with tumourigenesis. Using a yeast two hybrid screen I identified a protein that interacts will the C-terminus of WT1. This protein is novel, with no homologues found in the yeast or invertebrate databases. Co-immunoprecitation experiments revealed that the interaction is physiologically relevant as it occurs in WT1-expressing cells. Immunofluorescence shows that this novel protein is nuclear with a speckled localisation that resembles the pattern seen for splicing factors. Immunocytochemistry confirms that this protein, like WT1, does indeed co-localise with splicing factors. It will be of interest to see if this protein really is involved in splicing. During development of the kidney and testes, WT1 is confined to the glomerulus and sertoli cells respectively. Using immunohistochemistry I revealed that this novel protein is expressed in all cells of these organs and does not have the specific pattern seen for WT1. RT-PCR and whole mount <I>in situ </I>hybridisation also show that this protein is ubiquitously expressed. I cloned the mouse homologue and, at the amino acid level, is 93% identical to the human clone. This sequence conservation suggests that whatever the role of this novel protein may be, its structure must be important for function. Clearly further work needs to be carried out to determine the true role of this novel protein and the potential significance of its interaction with WT1.
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