The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary knowledge about its protein composition. One powerful tool for Drosophila biologists has been a group of genes that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several of these genes are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers resemble a fraction of that is tightly associated with the chromatin of interphase nuclei. Like the HP1 in these two cytoplasmic oligomers, this tightly bound nuclear fraction of HP1 is underphosphorylated and is associated with subunits of the origin recognition complex (ORC). We also found the localization of HP1 into heterochromatin to be disrupted in mutants for the ORC2 subunit. This phenotype supports a role for ORC in HP1 targeting and heterochromatin assembly. This proposed role for Drosophila ORC suggests striking similarities to the ability of ORC to recruit the Sir1 protein to silencing nucleation sites at the silent mating type loci in S. cerevisiae.

Bicaudal-C is a dominant maternal effect mutation which shows incomplete penetrance. Females are fertile and not all their progeny are affected. Those embryos which do not hatch show defects in their antero-posterior polarity, and give rise to bicaudal embryos which are duplicated for posterior structures. Twelve alleles of the genes have been phenotypically analysed. The penetrance of each allele has been determined and the phenotypes of embryonic defects such as mouth/head defect, bicaudal and uncellularized embryos classified and scored, in order to use this information to analyse these alleles at the cellular and molecular level. The bicaudal phenotype results from the mislocalization of the oskar and nanos RNA at the anterior end of the embryos. The gene also has a recessive phenotype which makes the females sterile. The phenotype is the result of specific defects in follicle cell migration. The alleles have been subdivided into two classes according to their phenotype: weak and strong. The gene encoding Bicaudal-C has been cloned and sequenced. It is expressed in the germline and appears to encode a member of the KH domain family of putative RNA-binding proteins.

DRMT4 (Drosophila Arginine MethylTransferase 4) is an arginine methyltransferase in Drosophila (Boulanger et al. 2004). It shows the highest identities with mammalian PRMT4/CARM1 (Protein Arginine MethylTransferase 4) (59% identity, 75% similarity). HPLC analysis demonstrated that DRMT4 belongs to the type I class of methyltransferases (Boulanger et al. 2004), meaning that DRMT4 catalyzes asymmetrical dimethylarginine formation. A polyclonal antibody against DRMT4 was generated and used to study DRMT4 expression using western blots and immunostainings. In order to study DRMT4 function in Drosophila using genetic methods, we created three kinds of DRMT4 transgenes: a genomic DRMT4 under its own control, a genomic DRMT4-GFP fusion gene and a cDNA DRMT4 under UAS control. We investigated DRMT4 localization in wild type flies using the DRMT4-GFP transgenic line and immunostaining.

Oocyte loss and meiotic prophase progression was studied in XY sex-reversed and XO female mice, two mouse models that lack pairing between their sex chromosomes. An arrest at the pachytene stage of meiosis was not observed, nor was a significant loss of oocytes at this stage compared to normal XX control mice. Thus, it was concluded that a pairing checkpoint either does not exist in oocytes or is not as stringent as the one observed in males. / The effect of mutating the pro-apoptotic Bax molecule was studied at three distinct ages corresponding to the time when female germ cells are premeiotic, in meiotic prophase, and arrested in dictyotene. Although it appeared that more germ cells were retained in the Bax homozygous mutant compared to the wild-type and heterozygous mice at 18.5 dpc, by 24.5 dpc all of the mice possessed similar numbers of germ cells. These results indicate a role for Bax in germ cell death but also support the idea that an alternative pathway can compensate for the elimination of this molecule.

Translational control is an important means of regulating gene expression. Development of the Drosophila germ line relies on translational regulation to differentially express maternal mRNAs, allowing it to develop distinctly from the soma. One of the critical factors required for germ cell development and function is the conserved DEAD-box RNA helicase Vasa (Vas). The research presented in this thesis examines the role of Vas in translational regulation during Drosophila germ line development. A two-hybrid screen conducted with Vas identified a translation initiation factor eIF5B (dIF2), as a direct interactor. Mutations were created in eIF5B and were found to enhance the vas mutant phenotypes of reduced germ cell numbers, and posterior segmentation defects, suggesting a functional interaction between these factors in vivo. In order to further understand the biological significance of the Vas-eIF5B interaction, the region of Vas required for eIF5B-binding was mapped and then specifically disrupted. Reduction of Vas-eIF5B binding using a transgenic approach, virtually eliminated germ cell formation, while having only a moderate effect on the somatic requirement of Vas in posterior segmentation. In addition, Vas-eIF5B interaction was found to be required for the establishment of polarity within the egg during oogenesis, likely through direct regulation of gurken (grk) mRNA. We concluded that through interaction with eIF5B, Vas plays a critical role in translational regulation in the germ line. In addition, another Drosophila DEAD-box protein, highly similar to Vas, called Belle (Bel) was characterized. Mutations in bel were found to also affect the germ line, leading to both female and male sterility. Like Vas, Bel is implicated in translation initiation, however bel is an essential gene, with a requirement for growth, whose function is not restricted to the germ line. Our data suggest that Bel may be a nucleocytoplasmic shuttling protein,

Ubiquinone (UQ) is a lipid found in all cellular membranes. It is involved in multiple cellular processes, either directly or through its effect on the redox status of the cell. clk-1 encodes a highly conserved hydroxylase required for UQ biosynthesis. In C. elegans, mutations in clk-1 result in the accumulation of an UQ precursor, DMQ, and a pleiotropic phenotype in the mutants characterized by the slowing down of development, behaviors and aging. Additionally, in the absence of dietary UQ, clk-1 mutants also show a transient growth arrest and are sterile. Mutants that can suppress both sets of phenotypes in the point mutant clk-1(e2519) have been isolated. Their suppression patterns indicate that various aspects of the clk-1 phenotype can be uncoupled from each other. Furthermore, the analysis of their quinone content suggests that the phenotypes on UQ-producing bacteria are caused by the inability of dietary UQ to completely substitute for endogenous UQ. These suppressors carry mutations in tRNA genes, and thus to our knowledge, they are the first tRNA missense suppressors found in any metazoan.

Bibliography: leaves 142-165. / xii, 165, [13] leaves, [18] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1991

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (Ph.D.)--Murdoch University, 2003. / Thesis submitted to the Division of Science and Engineering. Bibliography: leaves 229-232.

Thesis (Ph. D.)--Carleton University, 2003. / Includes bibliographical references (p. 200-228). Also available in electronic format on the Internet.