Reported here is the isolation and molecular characterization of two novel alleles of the DIP 1 gene; GE89 and GE77. As well, a third deletion of the DIP 1 gene, EY*4, isolated by our collaborators in France was characterized. PCR and sequencing analysis confirms all three alleles to be molecular deletions of the DIP 1 gene. However, in none of these cases is the entire gene excised. Also, immunohistochemistry of ovaries from each of these strains does not demonstrate a complete lack of DIPl protein expression in any of the deletion strains. Thus, it appears that some protein product is being formed in each case. However, it is not clear whether this protein is functional. An assay was also conducted to investigate a function for DIPl in mechanisms of epigenetic gene silencing. Although the findings of these experiments are incomplete, it appears that DIPl may play a functional role in heterochromatin formation and/or post-transcriptional gene silencing. Interestingly, appendage formation phenotypes were observed in the original P-element insertion line as well as a female sterility phenotype in the GE77 allele. Overall, DIP 1 is an interesting double stranded RNA binding protein. Newly isolated alleles of the DIP 1 gene will be useful tools for further investigation of the functional role of this gene. / Thesis / Master of Science (MSc)

Most studies of insect Malpighian tubules (MTs) have examined transport of monovalent ions (K^+, Na^+, Cl^-). Isolated Drosophila melanogaster MTs also transport Ca^2+ from bath to lumen and transport is stimulated by cAMP. The lower segment of the MTs transports Ca^2+ at a higher rate per unit length than does the main segment known to produce the primary urine. This study examines both whole animal calcium regulation in larvae, pupae and adults and the mechanisms of Ca^2+ transport by isolated MTs. Drosophila melanogaster appears to regulate its calcium content and haemolymph calcium level. Calcium content of the whole fly only increased 10% with a 6.2-fold increase in dietary calcium. Anterior MTs can contain as much as 50% of the whole animal calcium content. The difference in MTs accumulation is due primarily to the enlarged initial segment of the anterior MTs. This segment, absent from the posterior MT, contains calcium-containing concretions. Whole fly calcium content does not increase continuously with the age implying that calcium is eventually being excreted. Haemolymph calcium concentrations do not change in response to changes in dietary calcium, suggesting that calcium concentration is regulated either by the rate of absorption or by the rate of excretion. The midgut and the enlarged initial segment of the anterior MTs may play important roles in haemolymph calcium regulation. Isolated MTs show sensitivity to both Ca^2+ channel blockers and Ca^2+ -ATPAse inhibitors on the basolateral and apical membranes respectively. Voltage-gated calcium channels appear to mediate calcium movement from bath to cell. A ruthenium red sensitive Ca^2+ -ATPAse may be used to transport calcium against the electrochemical gradient from cell to lumen. Lastly, the dissolution of luminal concretions plays a large role in net calcium secretion. / Thesis / Master of Science (MSc)

This study investigated the position, ultrastructure and life history of glia in the midline of the Drosophila melanogaster Central Nervous System (CNS) by using enhancer traps (AA142, X81, argoswll, pointed1277) and reporter constructs (EEl, slilacZ 1.0, slilacZ 4.5) as glial-specific markers. Previous work has established that glia are necessary for proper formation and morphogenesis of longitudinal and commissural axon tracts, and axon ensheathment (Jacobs, 1993; Klambt et al., 1991; Jacobs and Goodman, 1989). By the end of embryogenesis there are three midline glial (MG) cells remaining in each segment (Sonnenfeld and jacobs, in press) which this study verifies. In the third instar larval MG cells proliferate to 24 cells per segment as followed with the E. coli lacZ expressing pointed1277 strain. These E. coli lacZ expressing pointed1277 MG cells begin dividing 57 hours after hatching as seen with 5 -bromodeoxyuridine and hydroxyurea treatment. Some MG genes cease midline expression before MG proliferation (seen with EEl, X81), others (seen with AA142) continue to be expressed until the beginning of MG proliferation. Only the argoswll, slilacZ 1.0, slilacZ 4.5, and pointed1277 expression strains continue E. coli lacZ expression to the end of the larval stages. In the first larval stage a few perineuropilar glia begin to express the E. coli lacZ gene and increase to 400 cells per CNS in the third ins tar as seen in the pointed1277 marker strain. pointed1277 EM micrographs show that E. coli lacZ labeled cells have a glial-like ultrastructure. There - was no co-localization of the E. coli lacZ expression in pointed12 77 and an anti-RK2 (repo) antibody in third instar larvae. In pointed1277 pupae the MG cell E. coli lacZ expression stops after 48- 72 hours and the prerineuropilar signal stops after 24 hours. In newly hatched pointed1277 adults perineuropilar E. coli lacZ expression is present with a cluster of 12 cells in the center of the neuropil. To summarize, after embryogenesis, in the pointed1277 marker strain, the MG cells begin dividing after 57 hours and the E. coli lacZ gene expression ends after the second day of the pupal stage. In the first instar, perineuropilar glia begin to label for the E. coli lacZ product and this expression ends by one day into the pupal stage, with re-appearance in the adult CNS. / Thesis / Master of Science (MSc)

Cytochrome P450 was examined in both insecticide resistant and insecticide susceptible strains of Drosophila melanogaster. Much higher levels were observed in the resistant strain IIID when compared to the susceptible strain Canton S. This increase appeared to be the result of an overproduction of a few existing forms. Two heme-staining microsomal proteins found in strain IIID were identified as putative cytochrome P450 isozymes. Polyclonal antibodies produced against these two proteins were used in the immunoanalysis of microsomal proteins from both strains. A lambda gtll cDNA expression vector library was created by inserting cDNA fragments from a Drosophila lambda gt10 library into lambda gtll arms. The library was screened with the polyclonal antiserum. Three clones were isolated, of which one, gtll-Al, was most highly reactive with the antiserum. Analysis of the gtll-Al lysogen indicated a 130 kd fusion protein was produced of which 16 kd was coded for by the cDNA insert. A .5 kb cDNA insert was isolated from the clone as part of a 1.5 kb Kpnl/EcoRl fragment and was used in the analysis of Drosophila genomic DNA and total RNA. Southern analysis revealed an EcoRl polymorphism existed between strain IIID and Canton S. RNA analysis suggested strain IIID produced more coding message for the Al insert in the larval and adult stages than did Canton S. / Thesis / Master of Science (MSc)

One function of programmed cell death in the developing CNS is the removal of excess cells that provide transient function. Previous studies in Drosophila observed an overpopulation of midline glia cells in embryos deficient for apoptosis. Where do these extra glia cells come from? Using different enhancer traps and antibodies as cell identity markers, the cell number of different CNS midline lineages was assessed in both wild type and Df(3L)H99 embryos which are deficient for apoptosis. The results show that at stage 16 there are approximately 3 cells labeled by midline glia specific markers (AA142 enhancer trap & P[slit1.0/lacZ] reporter construct) in wild type while there are up to 12 cells in embryos deficient for apoptosis. Comparing the number of midline neurons of mutants with that of wild type embryos, there are no detectable changes labeled by the enhancer trap P223, antibody 22C10, or an antibody to Engrailed. Exceptionally, there is one more neuron labeled by enhancer trap XS 5 in Df(3L)H99 embryos. Therefore, apoptosis is restricted to the midline glia lineage. Using P[slit1.0/lacZ] as MG marker, I observed that the extra midline glia in Df(3L)H99 initially appear at late stage 12 or early stage 13. The expression of reaper mRNA precedes programmed cell death. In wild type embryos, the initial expression of reaper mRNA of midline cells is at late stage 11 as revealed by in situ hybridization. These indicate that the first programmed cell death in the midline occurs approximately at stage 12. The supernumerary cells labeled by midline glia specific markers in Df(3L)H99 embryos share featur,es ofthe midline glia. These extra midline glia may be divided into two groups according to their differentiation. The cells of the first group strongly express the AA142 enhancer trap and ensheath the commissures. These cells are functional midline glia corresponding to the surviving midline glia in wild type embryos. The cells of the second group weakly express the AA142 and associate with but do not ensheath the commissures. These are likely the cells which normally undergo apoptosis in wild type. The results of this study indicate that the supernumerary midline glia come from neither midline glia proliferation nor other lineages. They may come from a midline glia progenitor pool in which midline glia marker expression begins at different stages. In wild type embryos, these potential midline glia die by apoptosis before activating midline glia specific genes. In Df(3L)H99 embryos, these midline glia survive and express midline glia markers. All the midline glia die in embryos mutant for spitz group genes. In embryos double mutant for spitz group genes and Df(3L)H99, supernumerary midline glia cells survive. These cells cannot totally rescue the axon tract phenotype of spitz group gene mutants indicating that spitz group genes are necessary for producing 'mature' midline glia. In Df(3L)H99 embryos, approximately 12 midline cells labeled with the midline glia specific marker P[slit1.0/lacZ]. However, there is not a significant increase in the number of midline glia expressingpnt or argos compared with wild type. Therefore, the survival of supernumerary midline glia in embryos deficient for apoptosis does not require DER signaling. However, the DER pathway seems to specify which and how many midline glia progenitors avoid apoptosis. / Thesis / Master of Science (MSc)

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No description available.

No description available.

The Rho GTPases are molecular switches principally known for their pivotal role in regulating the actin cytoskeleton throughout the phylogeny of eukaryotes. One major function for RhoA is regulation of the actin-myosin contractile apparatus in developing epithelia. Epithelial morphogenesis in Drosophila imaginal discs is regulated by RhoA signaling and the steroid hormone ecdysone. In order to learn more about the connection between ecdysone signaling and actin cytoskeletal dynamics during epithelial morphogenesis I have characterized mutations in 5 genes [designated en(zip)] that interact genetically with mutations in the Drosophila genes encoding myosin, RhoA, and an ecdysone-induced type II transmembrane serine protease required for epithelial morphogenesis. Two of the en(zip) mutations have been previously identified as alleles of RhoA and DRhoGEF2, two members of the RhoA signaling pathway. I have employed genetic complementation assays to characterize the three unidentified en(zip) genes and narrowed the putative location of two of these genes (18-5 and 31-6) to two small genomic regions. To further characterize the en(zip) mutants I have also determined the developmental lethal phase for each homozygous mutant. Homozygous RhoA 12 - 6 and DRhoGEF212 - 3 animals die during embryogenesis. In contrast, the en(zip) mutants 12-5, 31-6, and 18-5 are pupal lethals, suggesting that the primary role of their gene products may be to regulate epithelial morphogenesis during pupal development.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of requirements for the degree of Master of Science. 2015. / SNAMA, the protein of interest in this thesis is found in the common model organism Drosophila melanogaster, also known as the fruit fly it is also found in all eukaryotic organisms but not in prokaryotes. SNAMA is a 1231 amino acid protein that belongs to the RbBP6 superfamily. Members of this family are characterized by a zinc finger motif, a DWNN domain (domain with no name) and a RING finger motif. The human RbBP6 contains the Rb-binding and p53-binding domains in addition. The mammalian RbBP6 hence interacts with p53 and Rb and it is important for the development and tumorigenesis as a negative regulator of p53. Bioinformatics studies show that transcription of the Snama gene is driven by a single TATA-less promoter which give rise to a single 3.9 kb transcript. However, experimental evidence confirming the promoter region has not being published. The main aim of this study was to examine the regulation of Snama by identifying the maximal promoter sequence that shows promoter activity in mammalian cell line. This was achieved by using specifically designed primers to amplify the putative Snama promoters, ligating promoters in reporter vector (pGL3 basic). The recombinant products used to transfect eukaryotic cells (Cos7, African green monkey cells) and determining the maximal promoter sequence that expresses luciferase activity. The promoter sequences were labelled with biotin attached to the primers and Electrophoretic mobility shift assay (EMSA) was conducted to confirm binding of proteins on the putative promoter fragments. The segment designated promoter 6 has maximal positive activity and many proteins in the cell extract bind to it shown by EMSA. Interestingly the longer fragment designated promoter 7 has less promoter activity. This may suggest that this fragment also contains some repressive elements.