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The polyhomeotic (ph) locus of Drosophila melanogaster has been characterized genetically. Early studies showed that ph is a member of the Polycomb (Pc) group. These genes have similar phenotypes and are required for normal segment determination. Recent analyses of amorphic ph mutations show that the ph locus is complex, has a strong maternal effect and plays a role in cuticular development. To test the function of ph at the molecular level, the cloning of the ph locus was undertaken. One strain had been shown to contain a P element insertion near ph. A genomic library was prepared from this strain and a recombinant phage containing this P element insertion was isolated by transposon tagging. The DNA flanking the insertion was used as a starting point for a chromosomal walk. A series of overlapping phage spanning 170 kilobases was isolated. Southern blot analysis was used to determine the locations of important deficiency breakpoints within the region covered by the walk. A distance of approximately 35 kb was shown to separate the two deficiency breakpoints which include ph. This interval was found to contain rearrangements in four of the seven ph alleles which were examined by Southern blot analysis. The interval also contains a repeated sequence. The relationship between the genetic and molecular structure of ph is discussed. / Science, Faculty of / Zoology, Department of / Graduate

Mutagen-sensitive (mus) mutations in Drosophila melanogaster render developing flies hypersensitive to the lethal effects of DNA-damaging agents. In general, mus mutations identify DNA repair-related genes. In this study, 5 new second chromosome mus mutations (mus205B¹, mus208B¹, mus209B¹, mus210B¹ and mus211B¹), selected on the basis of sensitivity to methyl methanesulfonate (MMS), were characterized using a variety of genetic tests. One test measured the MMS-sensitivity of double mutant mus strains compared to their component single mutants. Mutant interactions were examined in 8 double mus and in 2 triple mus strains containing combinations of mus201D¹, mus205B¹, mus208B¹, mus210B¹ and mus211B¹ (or mus211B²). These analyses have revealed predominantly synergistic and epistatic responses to MMS. Taken together with the findings of previous genetic and biochemical studies of Drosophila mus strains, these results suggest that 3 major repair pathways may operate in flies to correct damage caused by MMS. Mutagen cross-sensitivity data and the results of the interaction studies suggest that mus mutations might serve as rapid and sensitive bioassays of somatic genotoxicity caused by mutagens and carcinogens. To explore this possibility, a simple mutagen test system was devised employing triple mutant mus strains. One strain (mus208B¹ mus210B¹ mus211B²) was tested for sensitivity to 14 mutagens/carcinogens and 2 non-carcinogens. Eleven of the mutagens/carcinogens were readily detected as genotoxic. Both non-carcinogens were non-genotoxic. These preliminary results demonstrate the feasibility (and some limitations) of the proposed somatic genotoxicity assay and emphasize the need for further test validation using a larger chemical data base. The temperature-sensitive lethal mutation mus209B¹ was subjected to extensive genetic analyses and to temperature shift experiments during development. This locus was found to encode a product(s) that (1) is essential for viability at virtually all pre-imaginal developmental stages (the latter half of pupation appears to be an exception), (2) is necessary for wildtype levels of resistance to the genotoxic effects of MMS and ionizing radiation, and (3) is required for female fertility. Confirmation of the pleiotropic nature of this mutation was obtained by meiotic and cytogenetic mapping studies and by complementation tests with a series of allelic mutations. The mus209B¹ phenotypes are similar to ones conferred by mutations in Drosophila and yeast that disrupt various aspects of chromosome metabolism. In this context, some possible roles for mus209B¹ are discussed. / Science, Faculty of / Zoology, Department of / Graduate

Damage to DNA from a variety of sources can lead to damaged proteins, genomic instability, aneuploidy, and cancer. It is therefore essential to repair DNA damage, and to do so a variety of DNA repair mechanisms have evolved. One of the repair mechanisms, known as homologous recombination (HR) repair, uses an undamaged sister chromatid as a template to make error free repairs to double-strand (ds) DNA breaks. While many proteins are involved in HR, this work focuses on testing the interactions of a subset of these proteins known as the Rad51 paralogs. The goal of this study is to determine if the putative Rad51 paralogs in Drosophila melanogaster are sufficiently conserved as to function in the same manner as their human counterparts. This research is part of a larger project to determine if Drosophila melanogaster is a good model organism for studying HR in humans (Hs). The D. melanogaster Rad51 gene, and its four paralogs Spn D, Spn B, Rad51D, XRCC2 (the last 2 identified by sequence homology), and human hsRad51D and hsXRCC2, were cloned into Invitrogen's TOPO protein expression vector. When induced with IPTG, the resulting fusion proteins contains either aN-terminal Xpress TM epitope or a C-terminal V5 epitope. The fusion proteins were used in immunoprecipitation assays with antibodies against the epitope tags to test for proteinprotein interactions. While many of the assays were inconclusive and are still being optimized, the interaction of the C-terminally tagged dmXRCC2 with theN-terminally tagged hsRad51D gave a positive result. This single interspecies result suggests that homologous recombination is highly conserved between D. melanogaster and humans.

Understanding the biological basis of behavior is crucial for gaining insights into human nature, treating behavioral disorders and improving overall well-being. Efforts to understand the biological basis of behavior have largely emphasized the role of neurons. However, examples across life show that behavior can occur in lieu of or in cell types outside of neurons. This thesis presents work exploring the mechanisms underlying multiple behaviors using the model system Drosophila melanogaster. Chapter 2 provides evidence for the influence of glia on courtship, aggression, and sleep. Chapter 3 characterizes a Drosophila model of autism spectrum disorder, finding that genetic neuronal manipulation of a single gene can cause pathologies beyond the nervous system. Chapter 4 investigates the connection between sleep behavior and oxidative stress response, demonstrating metabolism as a probable mediator of this relationship. Altogether, this work supports a wider definition for the biological basis of behavior.

A thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg for the Degree of Doctor of Philosophy. / This thesis reports on the investigation of two genetic aspects of Southern African populations of Drosophila Melanogaster: the " family of transposable elements and the allelic variation present in several enzyme systems. (Abbreviation abstract) / Andrew Chakane 2018

Aging is a biological process that has many detrimental effects due to the accumulation of oxidative damage to key biomolecules due to the action of free radicals. Methionine sulfoxide reductase (Msr) functions to repair oxidative damage to methionine residues. Msr comes in two forms, MsrA and MsrB, each form has been shown to reduce a specific enantiomer of bound and free oxidized methionine. Effects of Msr have yet to be studied in the major developmental stages of Drosophila melanogaster despite the enzymes elevated expression during these stages. A developmental timeline was determined for MsrA mutant, MsrB mutant, and double null mutants against a wild type control. Results show that the Msr double mutant is delayed approximately 20 hours in the early/mid third instar stage while each of the single mutants showed no significant difference to the wild type. Data suggests that the reasoning of this phenomenon is due to an issue gaining mass. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2013. / FAU Electronic Theses and Dissertations Collection

Finding novel compounds that affect neuronal or muscular function is of great interest, as they can serve as potential pharmacological agents for a variety of neurological disorders. For instance, conopeptides have been developed into powerful drugs like the painkiller PrialtTM. Most conopeptides, however, have yet to be characterized, revealing the need for a rapid and straightforward screening method. We have designed a novel bioassay, which allows for unbiased screening of biological activity of compounds in vivo against numerous molecular targets on a wide variety of neurons and muscles in a rapid and straightforward manner. For this, we paired nanoinjection of compounds with electrophysiological recordings from the Giant Fiber System of Drosophila melanogaster, which mediates the escape response of the fly. / by Monica Mejia. / Thesis (Ph.D.)--Florida Atlantic University, 2013. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.

Biological homeostasis relies on protective mechanisms that respond to cellular oxidation caused primarily by free radical reactions. Methionine sulfoxide reductases (Msr) are a class of enzymes that reverse oxidative damage to methionine in proteins. The focus of this study is on the relationship between Msr and dopamine levels in Drosophila. Dopaminergic neurons in Drosophila have comparable roles to those found in humans. A deficit in dopamine leads to the onset of many neurological disorders including the loss of fine motor control—a neurodegenerative condition characteristic of Parkinson’s disease (PD). We found that dopamine levels in the heads of MsrAΔ/ΔBΔ/Δ mutants are significantly reduced in comparison to MsrA ⁺/⁺ B⁺/⁺ heads. In addition, wefound protein and expression levels are markedly reduced in an Msr-deficient system. Our findings suggest an important role for the Msr system in the CNS. / Includes bibliography. / Thesis (M.S.)--Florida Atlantic University, 2014. / FAU Electronic Theses and Dissertations Collection

Aging and disease processes may be due to deleterious and irreversible changes produced by free radical reactions. The enzyme copper-zinc superoxide dismutase (CuZn SOD; superoxide: superoxide oxidoreductase, EC 1.15.1.1) performs a protective function by scavenging superoxide radicals. In order to determine whether additional SOD activity affects longevity and oxygen metabolism in Drosophila, our approach was to clone the Sod gene and introduce additional copies of the gene back into the genome via P element mediated transformation. The effects of increased SOD activity on Drosophila life span and oxygen free radical metabolism were investigated. The CuZn SOD cDNA and gene were cloned from Drosophila melanogaster. The sequence of the Sod cDNA and gene revealed an additional C-terminal triplet coding for valine not found in the mature SOD protein. The nucleotide sequence of the coding region has 56% and 57% identity when compared to the corresponding human and rat Sod genes, respectively. A probe of the cloned gene hybridizes to position 68A4-9 on Drosophila polytene chromosomes. In wild-type Drosophila the Sod cDNA hybridizes to a 0.7-0.8 kb transcript which is greatly diminished in a SOD 'null' mutant that produces only 3.5% of the SOD protein. A 1.8 kb EcoRI gene fragment containing the Sod gene was cloned into the P vector pUChsneo and microinjected into Drosophila embryos. Five transformed lines, each of which contain an additional copy of the Sod gene at different chromosomal sites were constructed. The chromosomal positions of the transposed Sod sequence were determined by in situ hybridization of the Sod gene to salivary gland polytene chromosomes. Analysis of RNA from the transformed flies revealed that the transposed Sod gene was expressed. The range of SOD activity for the five transformed lines was 131% to 170% of the value of wild-type. There was good correlation between the amount of Sod mRNA and the level of SOD activity in the transformed lines. Increased SOD levels in the transformed lines did not confer greater resistance to paraquat-generated superoxide radicals, nor increase their lifespan. The SOD 'null' mutant with 3.5% of the wild-type SOD activity was hypersensitive to paraquat when compared to wild-type, whereas the heterozygous SOD deficiency Df(3L)1xd⁹/TM3SbSer with 50% of the wild-type SOD activity was not. Mutants lacking SOD are dramatically impaired in oxygen metabolism and a few percent of wild-type activity appears to provide significant protection against superoxide, while 50% of the wild-type levels confers essentially the same resistance as wild-type. Despite the observation that the SOD activities found in a wide range of animals correlates directly with their longevity, Drosophila melanogaster appears to be well protected against the toxic effects of oxygen by its native levels of SOD. / Arts, Faculty of / Philosophy, Department of / Graduate