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.

No description available.

Les éléments transposables (ET) sont des séquences d'ADN trouvées chez tous les organismes vivants, et capables de se déplacer d'un site chromosomique à un autre. Ils sont une source de mutations et doivent donc être finement contrôlés par leurs hôtes. Afin de parer à leur mobilisation, les génomes ont mis en place des mécanismes de régulation (RNAi) impliquant des petits ARNs dont les siRNAs en sont la composante la plus connue. Récemment il a été mis en évidence chez la drosophile deux nouvelles classes de petits ARNs appelés piRNAs et endo-siRNAs. Les piRNAs contrôlent spécifiquement les ET dans les tissus reproducteurs qui comprennent des cellules germinales et des cellules somatiques : les cellules folliculaires. Les endo-siRNAs quant à eux, contrôlent ces ET dans les tissus somatiques. Il a été montré au laboratoire qu'Idefix, un retrotransposon à LTR, était régulé par un mécanisme post-transcriptionnel (PTGS). Celui-ci implique la voie des piRNAs et sa composante majeure, la protéine PIWI, dans les tissus reproducteurs de drosophiles. En revanche dans le reste des tissus de drosophiles cette régulation ne dépend pas de la protéine PIWI. Durant ma thèse je me suis intéressé à savoir si en plus de ce contrôle de type PTGS, il existait une régulation de type transcriptionnelle (TGS) appliquée sur les ET de drosophile dans les différents tissus aussi bien somatiques que germinaux. En étudiant les régulations que subissent divers transgènes composés d'un gène rapporteur et de divers fragments d'ET, j'ai montré que seule une régulation de type post-transcriptionnelle permettait de réguler les éléments transposables dans la lignée germinale femelle de drosophile. Cette régulation ayant une faiblesse précoce dans le développement des ovaires pouvant entrainer une mobilisation des éléments transposables dans certaines conditions sensibilisées. En revanche dans les tissus somatiques j'ai montré qu'une régulation transcriptionnelle s'ajoutait à la répression de type PTGS pour réprimer les ET. Cependant cette régulation transcriptionnelle présente une spécificité tissulaire puisqu'elle est observée dans les tissus somatiques de larves de drosophiles et absente dans les cellules somatiques folliculaires de l'ovaire. En conclusion divers systèmes de régulation mettent sous silence les éléments transposables en fonctions de la balance bénéfice/problèmes qu'ils apportent pour un tissu donné. / Transposable elements (TEs) are DNA sequences found in all living organisms, and able to move from one chromosomal site to another. They are source of mutations and therefore must be finely controlled by their hosts. To counteract their mobilization, genomes have developed regulatory mechanisms (RNAi) involving small RNAs including the best-known siRNAs. Recently two novel classes of small RNAs called piRNAs and endo-siRNAs have been reported in Drosophila. The piRNAs specifically trigger TE repression in reproductive tissues, composed by germ cells and somatic follicular cells. The endo-siRNAs control those in somatic tissues. It has been shown by our group that Idefix, a LTR retrotransposon, is regulated by a posttranscriptional mechanism (PTGS). It implicates the piRNAs pathway and its major component, the PIWI protein, in reproductive tissues of Drosophila. By contrast, in the other Drosophila tissues, the regulation does not depend on the PIWI protein. During my PhD, I was interested to know if in addition to this PTGS, a transcriptional control (TGS) was necessary to control Drosophila TE in both the somatic and germinal tissues. By studying theregulations of sensor-transgenes carrying a reporter gene (GFP) and various fragments of ET acting as a target of the silencing pathways, I have shown that the post-transcriptional silencing is the only regulatory pathway targeting transposable elements in the Drosophila female germline. This regulation has a weakness early in the development of the ovaries that can lead to a mobilization of transposable elements under certain sensitized conditions. In somatic tissues I have shown that a transcriptional regulation is coupled to the PTGS. However, this transcriptional regulation has tissue specificity because it is only observed in somatic tissues of Drosophila outside of the ovaries, a PTGS with no TGS targeting TE in the somatic cells of the ovarian follicle.

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

Graduation date: 1995

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 119-134).

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 132-151).

Thesis (doctoral)--Rijks-Universiteit te Groningen, 1937. / From: "Genetica", deel 19, alf. 6. Includes bibliographical references (p. 535-536).

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 120-140).