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Glycosylation is a fundamental post-translational modification of proteins that occurs in all eukaryotes, but the biological significance of specific glycosylation structures is still largely unknown. We have chosen to study this process by cloning and characterizing the genes involved in the N-linked glycosylation pathway in Drosophila melanogaster and analysing mutants of these genes. Since this biochemical pathway for the production of N-linked glycans is considerably conserved, studying it in Drosophila, a useful organism for genetic research, should yield results applicable to other eukaryotes. This thesis describes the attempt to identify a Drosophila homologue of rat ER mannosidase and how this investigation resulted in the identification of a Drosophila cDNA with considerable homology to calnexin, an enzyme involved in the folding of N-glycosylated proteins. It also describes the cloning and characterization of the genomic sequence for the putative Golgi mannosidase II (GmII)* in Drosophila. The expression and control of expression of GmII have been investigated further, and have suggested expression of this gene throughout Drosophila development. Analysis of the GmII promoter region has shown expression from a TATA-less promoter contained within 288bp of sequence 5' to the start of transcription.

During development DNA-binding transcription factors are deployed downstream of patterning events to enable specific gene regulatory programs that define diverse cell identities. Within a given eukaryotic cell only a subset of potential binding targets in the genome, called cis-regulatory modules, are available due to the distribution of nucleosomes which restrict access to the underlying DNA. The accessible landscape of cells is highly dynamic over time and across different cell types, although how this process is regulated and influences the function of transcription factors in patterning of complex tissues is not well understood. In this thesis I focused on dissecting the cell type-specific chromatin accessibility landscapes that distinguishes different cell populations within the Drosophila dorsal appendages. The patterning of this system is extremely well characterized allowing for a detailed understanding of how transcription factors at the top of cell fate hierarchies influence, or respond to, the chromatin landscape during development. In Chapter 2 I describe the differences in chromatin accessibility along the proximal-distal axis of the wing imaginal disc which gives rise to distinct populations of the thoracic body wall and appendage in the second thoracic segment (T2). I found that a major driver of chromatin differences in these populations is the repressive input of the conserved insect wing marker Nubbin, whose function in the appendage is associated with decreasing accessibility of select chromatin regions relative to their conformation in body wall cells. In Chapter 3 I characterized the serially homologous body wall and appendage cells in the adjacent third thoracic body segment (T3), which diverge extensively in morphology from the T2 state due to influence of a single gene, Ultrabithorax (Ubx). Ubx is a member of the Hox gene family which functions to provide cells with spatial identity along the anterior-posterior axis. I show this function for Ubx in specifying T3 cells coincides with widespread changes to chromatin accessibility which contribute to a segment and cell type-specific regulatory program.

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.

Interest in the mechanisms of homeo domain specificity led to a screen that identified Drosophila proteins able to bind a consensus homeo domain site. One clone isolated in this screen produced no homeo domain and was selected for further characterisation as a protein with an unknown DNA binding domain and the potential to interact with homeo domain proteins on the DNA. This thesis describes the characterisation of the Drosophila gene dead ringer ( dri ) corresponding to this clone. Isolation of overlapping cDNA clones and sequence analysis allowed the identification of a complete open reading frame in the dri message that gave a predicted protein of 901 amino acids. Database searches and multiple sequence alignment revealed a widely conserved motif in the Dri sequence that is found in proteins from organisms as diverse as yeast, nematodes, flies and humans. Biochemical analysis of the properties of this conserved motif revealed that it could function as a DNA binding domain when expressed in a fusion protein. The in vitro specificity of the Dri DNA binding domain was determined by selection and sequencing of target sites. The Dri consensus site obtained was strikingly similar to that of the Qfo class of homeo domains, although the sequence and predicted secondary structure of the Dri DNA binding domain do not resemble a homeo domain. Analysis of the developmental expression pattern of dri showed a ubiquitous maternal deposit gradually refined to localisation in the mesoderm at germ band extension, then further restriction to a diverse set of tissues including the salivary gland ducts, parts of the gut and a subset of the central nervous system. The phenotype of P - element insertion and deletion mutations of dri were identified as causing embryonic lethality preceded by a disruption of the hindgut and loss of Dri expression in the ring gland. The identification of the novel, conserved DNA - binding domain in Dead ringer offers an explanation for the regulatory activity of several important related proteins and presents an opportunity to use the advantages of the Drosophila model system to clarify the role of these proteins in transcriptional control. / Thesis (Ph.D.)--Departments of Biochemistry and Genetics, 1996.

Daily (circadian) rhythms exist at molecular, physiological, and behavioral levels and coordinate many life functions. This coordination is believed to contribute to an organism's fitness, however, such contributions have not been convincingly demonstrated in any animal. The most significant measure of fitness is the reproductive output of the individual and species. In this thesis I examine the consequences of loss of clock function on reproductive fitness in Drosophila melanogaster. I demonstrated that single mating among couples with mutated period (per���), timeless (tim���), cycle (cyc���), and Clock (Clk[superscript Jrk]) genes resulted in approximately 40% fewer progeny compared to wild-type flies. Male and female contribution to this phenotype was demonstrated by a decrease in reproductive capacity among per��� and tim��� flies mated with wild-type flies of the opposite sex. The important role of clock genes for reproductive fitness was confirmed by reversal of the low fertility phenotype in flies with rescued per or tim function. These results prompted an investigation to determine the relative contribution of each sex to the fertility phenotype. Males lacking a functional clock showed a significant decline in the quantity of sperm released from the testes to seminal vesicles (SV), suggesting that this peripheral oscillator is involved in sperm maturation. We found that clock genes are rhythmically expressed in these tissues and the cycling of per and tim expression continued in vitro, hence the testes and SV complex contained an autonomous circadian clock. In contrast to males, PER and TIM were constantly present in the cytoplasm of follicular cells in fly ovaries. Ovarian expression of per and tim is not disrupted by constant light and females lacking per and tim produced nearly 50% fewer mature oocytes then wild-type flies. These results suggest that per and tim are acting in a non-circadian pathway in the ovaries. Taken together, this data demonstrates that circadian clock genes significantly contribute to the fitness of Drosophila melanogaster by affecting the fecundity of both sexes. / Graduation date: 2003

The remodeling of the central nervous system (CNS) during metamorphosis in Drosophila melanogaster is a prime model system in which to study the genetic control of the sexual dimorphisms in the abdominal ganglion of the CNS. I have been using a P[tau-lacZ] enhancer trap line, 4.078, to label a segmentally repeated subset of abdominal motorneurons in order to assess the function of the sex determination hierarchy in controlling sex-specific development of the adult nervous system. In both the male and female larva there are 8 sets of these labeled abdominal motorneurons but only six sets in males and five sets in females survive in the adult. When this P[tau-lacZ] reporter construct is placed into a doublesex (dsx) mutant background, all 8 sets of these labeled abdominal motorneurons survive in both male and female adults. These results strongly suggest that dsx plays a role in the sex-specific survival of larval neurons that have functions in the adult. During the construction of mutant strains containing the sex determining genes transformer (tra) and transformer-2 (tra2), a genetic interactor was discovered in the P[tau-lacZ] 4.078 line. Female flies heterozygous for either tra or tra-2 alleles and the P[tau-lacZ] 4.078 developed with masculinized external and internal sex-specific structures. The external sex-specific structures, such as the genitalia, and ventral muscles are dependent on dsx gene function and a dorsal sex-specific muscle is dependent on fruitless (fru) gene function. From standard genetic crosses, I have characterized and demonstrated that the genetic interaction is linked to the P-element insertion site, which maps to the 85-87 region on the right arm of the third chromosome. By genetic analysis, this new genetic interactor appears to interfere with the tra and tra2 regulated female specific functions of both dsx and fru, potentially by reducing the female-specific splicing of the primary transcripts of the genes dsx and fru. To test the possibility that this newly described genetic interactor was allelic to a known gene, B52, that maps to the same region of the chromosome and alters dsx splicing, complementation tests were conducted which showed that the P[tau-lacZ] is not allelic B52. Additional phenotypes were observed in the crosses that first detected the interaction, suggesting that this newly described locus may affect other gene functions as well. Among the phenotypes observed were XX intersexes, male-female gynandromorphs (XX//XO mosaics), and non-disjunction events evident as XO males and XXY females. This new locus may represent a new member of the family of genes that influence regulated splicing events. / Graduation date: 1999

Graduation date: 1995

Errata is pasted onto back end paper. / Includes bibliographical references (26 leaves). / 115, [68] leaves, [8] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study entailed work towards the isolation of the pbl gene and preliminary characterisation of a candidate pbl transcript. Plasmid rescue of the genomic DNA flanking the inserted P element led to the isolation of a third candidate p61 cDNA, the 1A cDNA. This data suggests that the IA cDNA is encoded by the p61 gene. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1998

Errata is pasted onto back end paper. / Includes bibliographical references (26 leaves). / 115, [68] leaves, [8] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study entailed work towards the isolation of the pbl gene and preliminary characterisation of a candidate pbl transcript. Plasmid rescue of the genomic DNA flanking the inserted P element led to the isolation of a third candidate p61 cDNA, the 1A cDNA. This data suggests that the IA cDNA is encoded by the p61 gene. / Thesis (Ph.D.)--University of Adelaide, Dept. of Genetics, 1998