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The genetic and biochemical analysis of Drosophila Wwox protein · PDF file The genetic and biochemical analysis of Drosophila Wwox protein function A thesis submitted for the degree

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    The genetic and biochemical analysis of Drosophila Wwox

    protein function

    A thesis submitted for the degree of Doctor of Philosophy, August 2008

    Alexander Colella, B. Sc. (Hons)

    School of Molecular and Biomedical Science, Discipline of Genetics,

    ARC Special Centre for the Molecular Genetics of Development,

    The University of Adelaide

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    Table of contents

    Index of Figures and Tables...........................................................................................................................x

    Declaration ................................................................................................................................................. xiv

    Acknowledgements...................................................................................................................................... xvi

    Abbreviations ..............................................................................................................................................xviii

    Abstract ................................................................................................................................................ xxii

    1 Chapter 1: Introduction ................................................................................................4

    1.1 Chromosomal fragile sites ....................................................................................................................4

    1.1.1 Rare fragile sites.......................................................................................................................4

    1.1.2 Common fragile sites................................................................................................................5

    1.1.3 Fragile site FRA16D .................................................................................................................5

    1.2 WW-domain containing oxidoreductase (WWOX) ..............................................................................6

    1.2.1 WWOX – Genomic location and Structurte .............................................................................6

    1.2.2 WWOX transcripts and protein products .................................................................................7

    1.2.3 Wwox / WWOX localisation......................................................................................................8

    1.2.4 WWOX protein structure / function ..........................................................................................9

    1.2.4.1 Short chain dehydrogenase/reductase enzymes (SDRs) .............................................10

    1.2.5 WWOX / Wwox binding proteins............................................................................................12

    1.3 WWOX and cancer .............................................................................................................................12

    1.3.1 WWOX expression profile and gene status in cancer cells ..................................................12

    1.3.2 WWOX / Wwox proapoptotic activity .....................................................................................15

    1.3.2.1 Wwox overexpresion enhances TNF killing...................................................................15

    1.3.2.2 Wwox physically interacts with p53 and is required for p53 dependent killing.............16

    1.3.2.3 Wwox and p73 induce apoptosis synergistically ...........................................................16

    1.3.2.4 WWOX / Wwox enhances tumor necrosis factor (TNF) cytotoxicity.............................16

    1.3.2.5 WWOX physically interacts with JNK1 which blocks WWOX mediated cell death......17

    1.3.3 WWOX / Wwox as a tumor suppressor .................................................................................17

    1.3.3.1 Ectopic expression of WWOX / Wwox suppresses cancer cell growth........................17

    1.3.3.2 Wwox knockout and hypomorph mice exhibit increase in tumorigenesis ....................18

    1.3.4 Drosophila Wwox....................................................................................................................18

    1.3.4.1 Wwox null flies exhibit increased sensitivity to ionising radiation .................................18

    1.3.5 Summary .................................................................................................................................20

    1.4 Project aims and approaches.............................................................................................................20

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    1.4.1 Aim #1: To determine the functional domains / regions in the Wwox protein .....................20

    1.4.2 Aim #2: To determine whether the various functional domains / regions of Wwox lead to

    quantitative / qualitative changes in other proteins in vivo. ...............................................................21

    2 Chapter 2: Materials & Methods............................................................................... 24

    2.1 Oligonucleotide primers......................................................................................................................24

    2.1.1 Cloning Primers......................................................................................................................24

    2.1.1 Sequencing & Diagnostic Primers.........................................................................................25

    2.2 Enzymes .............................................................................................................................................25

    2.3 Antibiotics............................................................................................................................................25

    2.4 Plasmids..............................................................................................................................................25

    2.5 Bacterial strains .................................................................................................................................. 26

    2.6 Kits 26

    2.7 Molecular weight markers ..................................................................................................................26

    2.8 Antibodies ...........................................................................................................................................26

    2.9 Bacterial Media ................................................................................................................................... 27

    2.10 Drosophila Media..............................................................................................................................27

    2.11 Buffers and Solutions .......................................................................................................................27

    2.12 PCR amplification of DNA................................................................................................................29

    2.13 Generation of recombinant plasmids...............................................................................................29

    2.14 Transformation of bacteria ...............................................................................................................29

    2.15 Isolation of plasmid DNA..................................................................................................................29

    2.16 Genomic DNA preparations .............................................................................................................29

    2.17 Agarose gel electrophoresis ............................................................................................................30

    2.18 Automated DNA sequencing............................................................................................................30

    2.19 Generation of deletion constructs.................................................................................................... 30

    2.20 In vitro site directed mutagenesis .................................................................................................... 31

    2.21 P-element transformation of Drosophila..........................................................................................31

    2.21.1 Fly strains .............................................................................................................................31

    2.22 Irradiation of Drosophila ...................................................................................................................32

    2.23 One-dimensional polyacrylamide gel electrophoresis .................................................................... 32

    2.24 Two-dimensional electrophoresis .................................................................................................... 32

    2.24.1 Drosophila protein preparations ..........................................................................................32

    2.24.2 Cy Dye Labelling of proteins................................................................................................33

    2.24.3 Rehydration loading isoelectric focusing.............................................................................33

    2.24.4 Cup loading isoelectric focusing..............