Traditional screens for female sterile mutants have revealed only two genes which when mutant, give a fully penetrant 16 nurse cell phenotype. One way to gain a better understanding of the function of these genes in oocyte determination, is to identify genes which interact with them. Using P-element mutagenesis, I have isolated one dominant suppressor and five dominant enhancers of Bicaudal-D, and begun the phenotypic and molecular characterization of three of these genes. By deficiency screening, I have identified two different loci which act as dominant enhancers of Bic-D, and eight different loci which act as dominant suppressors of Bic-D. Further work on defining the locus responsible for the strong suppression phenotype associated with one of these deficiencies, revealed betaH-spectrin to be a strong dominant suppressor of loss-of-function Bic-D alleles, and a strong dominant enhancer of Bic-D gain-of-function alleles.

Bicaudal-C, a Drosophila gene, is required for centripetal follicle cell migration in the egg chamber and anterior patterning of the embryo; its exact functions are unknown. BICAUDAL-C carries five KH domains required for in vitroRNA-binding and for in vivo activity. Here, I show that Bicaudal-C functions, through RNA-binding, to regulate oskar expression during mid-oogenesis. Females carrying mutations in Bicaudal-C in the regions encoding its KH domains display premature, ectopic translation of OSKAR at the anterior of their stages 8--10 oocytes; this likely accounts for the disruption of anterior patterning in embryos of Bicaudal-C/+ females. Also here, I propose that oskar and gurken share common regulators, including Bicaudal-C. This is based on the discovery of dorsalized progeny of Bicaudal-C/+ females, and on the dominant enhancement of the Bicaudal-C/+ phenotype by specific gurken and cornichon alleles. Finally, I describe a yeast two-hybrid screen used to investigate BICAUDAL-C-protein interaction.

Bicaudal-D (Bic-D) is essential in Drosophila for the establishment of oocyte fate and polarity within the developing oocyte. To study these processes we have engineered a chimeric Bic-D::GFP fusion protein which behaves like the endogenous Bic-D polypeptide. We have identified three genes which are required for the normal subcellular distribution of Bic-D::GFP two genes predicted to encode RNA binding proteins (egalitarian and orb) and Dynein heavy chain. In particular, they affect Bic-D::GFP localization during the early germarial stages of oogenesis during which oocyte fate is established, or later when anterior-posterior polarity is initiated. Our results support the model that Bic-D acts in conjunction with mRNA binding proteins and a negative-end directed microtubule motor in localizing mRNAs. Throughout stages 1--6 of oocyte development, Bic-D::GFP accumulates in the oocyte in a strong posterior cortical focus, resembling a spool, that is aligned with a crater-like indentation in the oocyte nucleus. The aligned focus and crater reveal an early oocyte polarity and a previously undescribed asymmetric subcellular structure that may be involved in tethering the oocyte nucleus. Shape, positioning and orientation of the oocyte nucleus change around stage 6--7, concomitantly with a change in position of the Bic-D::GFP focus to the presumptive dorsoanterior corner. This re-orientation appears to anticipate the establishment of a new dorsoventral polarity in the oocyte and egg chamber. Dhc and Bic-D are both involved in the process of re-orientation of the oocyte nucleus and in polarity formation.

No description available.

No description available.

No description available.

In cell cycle checkpoint control, the Chk2 family protein kinases play a central role in mediating the cellular responses to DNA damage or DNA replication block. However, at the beginning of this project, there was no evidence for a Drosophila homologue of Chk2. loki was identified in a screen for serine/threonine protein kinases that are expressed in the ovary. Using a phylogenetic analysis, I showed that loki is a Drosophila chk2 orthologue. To characterize the checkpoint function of loki in Drosophila development, we created a loki null mutant and generated anti-Loki antibodies. Under normal laboratory conditions, loki null mutants display no apparent defect during the whole life span. Further functional analysis revealed that loki is not required for the meiotic pachytene checkpoint, the essential DNA replication checkpoint control in syncytial embryos and the DNA damage/replication checkpoint during the larval stage. However, in postblastoderm embryos, loki is required for the DNA damage checkpoint activated by gamma irradiation. Embryos lacking loki are not able to arrest the cell cycle in response to gamma irradiation.

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.

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.

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,