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In eukaryotes, the process of nuclear migration is critical in fusion of haploid pronuclei after fertilization, in separation of daughter nuclei during mitosis, and in nuclear positioning in interphase cells. Experiments in several organisms have identified the basic protein requirements for nuclear migration and positioning: molecular motors that provide motive force; the cytoskeleton along which motors move nuclei, or to which the nuclei are anchored; and proteins of the outer and inner nuclear envelopes. These nuclear membrane proteins interact with the motors, the nuclear lamina and each other to effect nuclear migration and positioning. Proteins containing a SUN domain, which were first characterized in S. pombe Sad1 and C. elegans UNC-84, are inner nuclear envelope linkers of the nucleus to the cytoskeleton. In fungi, C. elegans, D. discoideum and vertebrates, these proteins are required not only for nuclear positioning, but also for maintaining the connection of the nucleus to the MTOC, for centrosomal duplication, for homologous pairing of chromosomes in meiosis, for distribution of nuclear pore complexes and for connecting the centrosome to chromatin to ensure genomic stability. The D. melanogaster genome has two genes, CG18584 and CG6589, which encode SUN domain proteins. The specific aims of my dissertation research were to generate null mutants in these genes, to characterize their null phenotypes, and to analyze where the genes are expressed. CG18584 = klaroid mutants are grossly normal, but adult eyes are mildly rough due to a defect in nuclear positioning that occurs during larval eye development. Klaroid protein is perinuclear in every cell of the eye, and functions by localizing the MTOC connector Klarsicht to the outer nuclear envelope. CG6589 = dspag4 null mutants are male sterile. In mature sperm, Dspag4 protein localizes rostrally to the sperm centriole. In the absence of Dspag4, most steps of gametogenesis occur normally, however, prior to the final steps of sperm maturation, the sperm nucleus dissociates from its centriole. Klaroid and Dspag4 thus have cellular roles typical for SUN domain proteins, and Dspag4 is unique in that its function is to attach nuclei to centrioles exclusively in maturing spermatids in the male germline. / text

Early Drosophila development is a coordinated series of temporal and spatial events leading to specific localized gene expression. The maternally expressed gene Bicaudal-C (Bic-C) encodes a KH-domain RNA binding protein required in the developing oocyte for anterior-posterior patterning and follicle cell migration. The dominant heterozygous phenotype results in the development of embryos with bicaudal and head defects. A two-hybrid screen using BIC-C as "bait" identified the novel protein ANTIMEROS (ATMS) and the SH3-domain containing protein MILE END (MILE). / ATMS is highly conserved between humans and mice, its expression is almost entirely female-specific, and is limited to certain developmental stages. Mutant alleles for atms are able to dominantly enhance the phenotype of Bic-C heterozygotes confirming the Bic-C-atms interaction. Here I show that NOS mislocalization causes the trans-heterozygous phenotype, as introduction of a nos mutation strongly suppresses the bicaudal phenotype. nos transcripts show a hyper-polyandenylation in atms mutant ovaries, an indicator of translational activation, suggesting that ATMS and BIC-C function as translational repressors of nos through changes in its poly(A) tail length. / MILE, contains two highly conserved SH3 domains at the C-terminus. Experiments involving the analysis of mutant alleles and overexpression mile transgenic lines show that MILE is a negative regulator of both Torso and Egfr RTK signaling. Its not clear what functional role BIC-C may have with RTK signaling, but recent evidence suggests that posterior group gene expression influence terminal pole RTK signaling.

Drosophila are used extensively for genetic, developmental and now molecular biology research. At present, germline transformation of these organisms can only be achieved by microinjection of P-element vectors into the pole cells of young embryos. The technique of microinjection however, requires a delicate touch and is quite laborious. Therefore, the development of a rapid and simple technique was investigated. Electroporation, like microinjection, is a physical means of introducing DNA into a cell and is therefore potentially applicable to all cell types. Electroporation involves the use of an electrical current to create pores in the membrane of a cell. Macromolecules, such as DNA may enter a cell via these pores. Electroporation is a quick, reproducible, and efficient method for transforming cells. Through studies of the survival and permeability of Drosophila melanogaster embryos exposed to electrical currents, it was discovered that although the survival of the embryos decreased steadily as field strength increased, the embryos did not become permeable to a water soluble dye unless a pulse of 10 kV/cm was applied. Few embryos survived this extreme voltage required for dye uptake. Attempts to introduce DNA into dechorionated Drosophila embryos utilizing this technique however, produced no transformants. These results suggested that the remaining protective coatings of the dechorionated embryo were obstructing efficient pore formation, thus preventing DNA penetration. In view of these results, methods to eliminate the wax layer, present between the chorion and vitelline membrane of laid eggs, were examined. Wax removal by detergent solubilization, solvent extraction and melting by heating were investigated, yet did not produce a satisfactory procedure. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

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