"June 2003". / Bibliography: p. 189-196. / xv, 196 p. : ill. (some col.), plates (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis aimed to characterise the G1-S phase role of Drosophila Eb1, dEb1, which was identified in a cyclin E genetic screen. A dominant modifier screen was undertaken to identify novel regulators of cyclin E, and cell cycle progression using a hypomorphic allele of cyclin E. One suppressor from this screen, Su(DmcycEjp)2.5, was identified as a potential Drosophila homologue of the microtubule binding protein Eb1, dEb1. Southern and northern analysis confirmed that Su(DmcycEjp)2.5 is an allele of dEb1, and other dEb1 alleles were shown to also be able to suppress cyclin Ejp. / Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Sciences, 2003

by Pui-Ki Kwan. / Year shown on spine: 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 134-143). / Acknowledgments --- p.i / Abstract --- p.ii / Abbreviations --- p.iii / Table of content --- p.v / Chapter CHAPTER 1 --- General Introduction / Chapter 1.1 --- The formation of body axes --- p.1 / Chapter 1.2 --- Maternal genes are essential for development --- p.8 / Chapter 1.3 --- Maternal gene bicoid is required for formation of anterior structures in the embryo --- p.9 / Chapter 1.4 --- Establishment of an anterior to posterior bcd protein gradient --- p.12 / Chapter 1.5 --- The bcd protein gradient regulates the downstream zygotic target genes in a concentration-dependent manner --- p.12 / Chapter 1.6 --- bcd protein acts as transcriptional regulators --- p.14 / Chapter 1.7 --- The anterior localization of bcd mRNA --- p.17 / Chapter 1.8 --- Components required for the localization of bcd mRNA --- p.17 / Chapter 1.8.1 --- Cis-acting elements --- p.17 / Chapter 1.8.1.1 --- BLE1 at 3' UTR directs localization of bcd mRNA --- p.19 / Chapter 1.8.2 --- Trans-acting elements --- p.21 / Chapter 1.8.2.1 --- "exuperantia, swallow and staufen are necessary for localization of bcd mRNA" --- p.21 / Chapter 1.8.2.2 --- exu protein is an absolute requirement for the localization --- p.24 / Chapter 1.8.2.3 --- Potential functions of exu based on the coding sequence --- p.25 / Chapter 1.8.2.4 --- Microtubules dependence of the localization --- p.26 / Chapter 1.8.2.5 --- Microtubules polarity directs localization of bcd RNAs --- p.27 / Chapter 1.9 --- Functions of exu in localization of bcd mRNA --- p.27 / Chapter CHAPTER 2 --- Characterization of deletion mutants of exu / Chapter 2.1 --- Introduction --- p.30 / Chapter 2.2 --- Construction of deletion mutants of exu --- p.31 / Chapter 2.2.1 --- Materials and Methods --- p.31 / Chapter 2.2.2 --- Results --- p.33 / Chapter 2.3 --- Analysis of exu protein in deletion mutants --- p.35 / Chapter 2.3.1 --- Materials and Methods --- p.35 / Chapter 2.3.1.1 --- Preparation of total ovary protein from the transgenic flies --- p.35 / Chapter 2.3.1.2 --- Analysis of protein content by SDS Polyacrylamide Gel Electrophoresis and immunoblotting --- p.35 / Chapter 2.3.2 --- Results --- p.36 / Chapter 2.4 --- Localization of bcd mRNA and exu protein in oogenesis --- p.39 / Chapter 2.4.1 --- Introduction --- p.39 / Chapter 2.4.2 --- Spatial and temporal distribution of exu protein in the deletion mutants --- p.41 / Chapter 2.4.2.1 --- Materials and Methods --- p.41 / Chapter 2.4.2.2 --- Results --- p.43 / Chapter 2.4.3 --- Spatial and temporal distribution of bcd mRNA in the deletion mutants --- p.56 / Chapter 2.4.3.1 --- Materials and Methods --- p.56 / Chapter 2.4.3.1.1 --- Principles of DIG-labeling and in situ hybridization --- p.56 / Chapter 2.4.3.1.2 --- Synthesis of DIG-labeled bcd DNA probe --- p.59 / Chapter 2.4.3.1.3 --- in situ hybridization of bcd mRNA in egg chambers using DIG-labeled DNA probe --- p.59 / Chapter 2.4.3.2 --- Results --- p.62 / Chapter 2.5 --- Discussion --- p.70 / Chapter CHAPTER 3 --- Determination of the interactions between exu and microtubules / Chapter 3.1 --- Introduction --- p.79 / Chapter 3.2 --- Localization of bcd mRNA and exu protein in the presence of drugs which destabilize cytoskeleton --- p.81 / Chapter 3.2.1 --- Materials and Methods --- p.81 / Chapter 3.2.2 --- Results --- p.82 / Chapter 3.3 --- Analysis of interactions between exu and microtubules by immunoprecipitation --- p.88 / Chapter 3.3.1 --- Materials and Methods --- p.88 / Chapter 3.3.1.1 --- Immunoprecipitation of exu protein and binding of microtubules --- p.88 / Chapter 3.3.1.2 --- Purification of tubulin from bovine or rat brains --- p.89 / Chapter 3.3.1.3 --- Determination of protein concentration of the tubulin stock by Folin-Lowry method --- p.90 / Chapter 3.3.1.4 --- Taxol-stabilized microtubules --- p.90 / Chapter 3.3.2 --- Results --- p.91 / Chapter 3.4 --- Analysis of interactions between exu and microtubules by cosedimentation --- p.94 / Chapter 3.4.1 --- Materials and Methods --- p.94 / Chapter 3.4.2 --- Results --- p.97 / Chapter 3.5 --- Analysis of interactions between exu and microtubules using detergent extracted ovary extract for co sedimentation --- p.100 / Chapter 3.5.1 --- Materials and Methods --- p.100 / Chapter 3.5.2 --- Results --- p.101 / Chapter 3.6 --- Analysis of intracellular distribution of exu protein and Release of exu protein by sodium carbonate treatment for cosedimentation with microtubules --- p.104 / Chapter 3.6.1 --- Materials and Methods --- p.104 / Chapter 3.6.1.1 --- Subcellular fractionation of ovary extracts --- p.104 / Chapter 3.6.1.2 --- Release of contents from fractions by sodium carbonate treatment --- p.105 / Chapter 3.6.1.3 --- Co sedimentation of exu protein with microtubules --- p.105 / Chapter 3.6.2 --- Results --- p.108 / Chapter 3.6.2.1 --- Intracellular distribution of exu protein --- p.108 / Chapter 3.6.2.2 --- Cosedimentation of exu protein with microtubules using Na2CO3 released extracts --- p.108 / Chapter 3.7 --- Cosedimentation of exu protein and microtubules in high ATP concentration --- p.113 / Chapter 3.7.1 --- Materials and Methods --- p.113 / Chapter 3.7.1.1 --- Preparation of ovary extracts and microtubules sedimentation --- p.113 / Chapter 3.7.1.2 --- Western blot using a chemiluminescent detection system --- p.114 / Chapter 3.7.2 --- Results --- p.115 / Chapter 3.8 --- Discussion --- p.122 / Chapter CHAPTER 4 --- Future Prospects --- p.125 / Appendix A Supplementary protocols --- p.126 / Appendix B Reagents --- p.131 / References --- p.134

Cellularisation is a specialised form of cytokinesis in Drosophila melanogaster. Cellularisation occurs after the first 13 syncytial cell cycles of the embryo and involves targeted insertion of membrane to form the blastoderm, which represents a polarised epithelium made out of about 6000 cells. The molecular machinery driving cellularisation is complex and not well understood. In this work a novel gene regulating this process is identified and characterised. The mutation drop out causes defects in intracellular transport, cell polarity and nuclear positioning. Previous work provided evidence that dop1 is an allele of the RNA silencing gene argonaute2 (ago2). However, results presented in this thesis showed that ago2 functions are unimpaired in dop mutant embryos using genetic and biochemical tools. Moreover genetic and molecular mapping revealed that dop mutants carry a mutation in a gene within close proximity to ago2.This work demonstrates that dop encodes the sole Drosophila homolog of the mammalian MAST (microtubule associated serine/threonine) kinase family. The molecular lesion in the dop1 allele of dop leads to an amino acid exchange in the kinase domain and results in a significant reduction of Dop protein levels. A detailed investigation of the mutant phenotype indicated that dop1 affects microtubule rigidity and Dynein-dependent microtubule associated transport. Search for possible Dop targets revealed reduced phosphorylation of the Dynein intermediate chain (DIC). DIC is a subunit of Dynein and has been shown to be involved in the binding of cargo to the Dynein complex. Therefore, a possible function for Dop might be the phosphorylation of DIC to regulate microtubule dependent transport by controlling Dynein-cargo interaction.

Drosophila models for human diseases have helped in advancing our knowledge on human diseases and the discovery of potential treatments. Angelman syndrome is a rare neurological disorder that results in severe mental retardation and loss of motor coordination. The disease is caused by loss-of-function mutations in the UBE3A gene encoding a HECT domain ubiquitin protein ligase. Drosophila dube3a is the fly homolog of human UBE3A and their protein products share ~55% similarity in amino acid sequence along the entire length of the proteins. My goal was to develop a Drosophila AS model that will allow us to identify the AS-associated substrate(s) of the Drosophila UBE3A homolog and ultimately, to determine why the lack of UBE3A protein causes Angelman syndrome in humans. Dube3a is present in the embryonic, larval and adult central nervous system, including the adult mushroom bodies, which is the center for learning and memory. I have generated dube3a knock-out flies and they appear normal externally, but display abnormal locomotor behaviors. Flies that overexpress wild-type dube3a in the nervous system also display locomotion defects, and these overexpression phenotypes are dependent on the presence of a conserved cysteine residue essential for HECT domain E3 enzymatic activity. Targeted overexpression of dube3a in the eye, the wing, or ubiquitously causes rough eyes, curly wings and lethality, respectively. These morphological abnormalities in the eye or wing depend on the critical catalytic cysteine of Dube3a. Overexpression of mutant dube3a carrying AS-associated point mutations does not elicit such defects, suggesting they act as loss-of-function mutants. Taken together, dube3a mutants are a candidate fly model for Angelman syndrome, and the flies that overexpress dube3a in the eye or wing are useful for genetic screens to identify the elusive UBE3A substrates relevant to Angelman syndrome.

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