1 results for Ahokovi Tukia, Richard Katavake Taka-I-Utukakai

  • Investigation of the Wilms' tumour suppressor protein (WT1)

    Ahokovi Tukia, Richard Katavake Taka-I-Utukakai (2011)

    Masters thesis
    University of Otago

    Wilms’ tumour is a paediatric nephroblastoma which affects 1: 10,000 live births, and is the most common form of solid tumour in children under 5 years of age. Approximately 10-20% of cases have a mutated wt1 gene. Wilms’ tumour suppressor protein (WT1) is a transcription factor which has a proline and glutamine rich N-terminus and four contiguous zinc fingers of the Cys2His2 type in the C-terminus. WT1 is important in kidney, gonad, and heart development and is associated with Wilms’ tumour and other diseases such as WAGR (Wilms’ tumour, Anirida, Genitourinary, and mental Retardation), DDS (Denys-Drash syndrome), AML (acute myeloid leukaemia), and Frasier syndrome. WT1 has four functional domains (suppression, activation, self-association, and zinc finger domain) and as a result of alternative splicing, can form 24 potential isoforms. The WT1 protein has both tumour suppressor and oncogenic properties and its functional role has been further complicated by reports that WT1 function is plasmid, vector and/or cell line specific. Understanding the structure and function of WT1 will help us in elucidating its role in Wilms’ tumour and other related diseases. This project consisted of two parts. First we investigated the N-terminus of WT1 to determine the minimal region required for self association. This included a four step process: 1) Clone truncations of the WT1 N-terminal region using the Invitrogen Gateway cloning system 2) Express the constructs in E.coli cell lines 3) Batch purify the protein with GST sepharose beads 4) Conduct pull down experiments to test for self association. All the N-terminus WT1 constructs contained an N-terminal 3c protease cleavage site linked to a GST tag. The constructs 68-180 a.a., 2-135 a.a., and 2-180 a.a. were successfully expressed in the BL21 (DE3) Star pLysS cell line. The constructs were batch purified and pull down experiments were conducted. The second part of this project was to design a functional assay to determine the DNA binding fractional activity of the WT1 zinc finger domain variants used by Fagerlund (2009). This also included a four step process 1) Purify the zinc finger domain variants 2) Conduct electrophoretic mobility shift assay to determine protein: DNA interaction 3) Design a functional assay using the zinc finger domain variants which created a shift 4) Apply the functional assay to Fagerlund’s samples to determine the DNA binding fractional activity. Full length WT1 and variants of the WT1 zinc finger domain were successfully purified. Only Min-zf (minimal zinc finger motif) created a shift and was selected to design the functional assay. The EMSA results for Fagerlund’s samples did not create a shift suggesting that the protein was inactive. This meant that the fractional activity of Fagerlund’s samples could not be determined. However, the fractional activity of Min-zf was estimated to be approximately 25%. Using this estimation and our experience with this complex protein, we suggest that Fagerlund’s (2009) samples were not 100% active.

    View record details