Protein synthesis is one of the multiple levels at which gene expression is regulated. The rate-limiting steps of protein synthesis occur during initiation. The binding of the ribosome to the mRNA in translation initiation is catalyzed by the proteins of the eukaryotic initiation factor 4 (eIF4) group. In mammals, the mRNA cap-binding protein eIF4E is present in limiting levels and is regulated by several mechanisms. This thesis examines the regulation of eIF4E during the development of the genetically tractable organism, Drosophila melanogaster. A Drosophila eIF4E gene was cloned, its position was mapped cytologically, and this gene was shown to encode two cap-binding protein isoforms via alternative splicing. Antisera specific to the eIF4E isoforms were raised and purified to characterize the expression of eIF4E during development. Several mutant alleles of eIF4E were identified and demonstrate that this gene is essential for the viability of Drosophila. Furthermore, eIF4E mutants arrest in growth during early larval stages. The lethality and growth defects of eIF4E mutant alleles were rescued by a transgene containing a wild-type copy of eIF4E expressed under the control of its endogenous promoter. Ser251 of Drosophila eIF4E is in a sequence context identical to site on which eIF4E is phosphorylated in response to extracellular stimuli in other organisms. To examine the biological significance of the phosphorylation of eIF4E, transgenic flies were generated in which Ser251 was mutated. We show that eIF4E from Ser251 mutant lines cannot incorporate labeled orthophosphate. Interestingly, flies in which the only source of eIF4E is non-phosphorylatable are semi-lethal and escapers are small in size. These results are evidence that Ser251 of eIF4E is required for the normal growth of a multicellular organism.

A preliminary analysis of translation initiation has been carried out using the model system Drosophila melanogaster. These efforts have focussed on identifying the homolog of mammalian of mammalian eIF-4F, a complex of three subunits; eIF-4E which binds the mRNA cap, eIF-4A which has ATP-dependent RNA helicase activity, and eIF-4$ gamma$, of unknown function. Attempts to clone the genes encoding subunits of eIF-4F have led to the isolation of two novel genes. A molecular screen for members of the DEAD family of RNA helicases that includes eIF-4A led to the isolation of a gene which encodes the putative homolog of a yeast protein involved in ribosome assembly. From a complementation screen for suppressors of mutants of the Saccharomyces cerevisiae cap binding subunit, was isolated a Drosophila cDNA encoding a putative ribosomal protein, RpS15a, of the 40S subunit. Further characterization of the mechanism of suppression has shown that overexpression of RpS15a stabilizes the yeast eIF-4E protein suggesting a direct interaction between eIF-4E and the ribosome. Our work has shown that unlike the mammalian system, two cap binding proteins exist in Drosophila. The molecular analysis of cDNA clones encoding Drosophila eIF-4E suggests that the proteins result from alternatively spliced mRNAs expressed from a single gene. The biochemical characterization of Drosophila eIF-4A has shown that eIF-4A is part of a complex similar in size to mammalian eIF-4F but that unlike mammals, one eIF-4A protein is produced from a single gene and is regulated by phosphorylation. Phosphorylated eIF-4A is present in oocytes and early embryos but not in later embryos. Phosphorylated eIF-4A accumulates on the 48S initiation complex suggesting that a mechanism involving the post-translational regulation of eIF-4A affects the translation of maternal mRNAs in the oocyte and early embryo.

Although many of the genes and pathways involved in Drosophila embryogenesis have been thoroughly investigated, a complete understanding of the mechanisms behind these processes is still lacking. In order to gain a better perspective, the main objective of current research is to identify additional components of the signaling pathways that are crucial for normal Drosophila development. / One such developmental process is germ band retraction, which occurs in mid-embryogenesis and consists of the movement of the tail end of the germ band, or embryo proper, to its final posterior position. One of our primary objectives is to identify the signaling pathways behind this process. To this end, we investigated the 7T2 mutant, which fails to retract. This zygotic lethal mutant was originally uncovered in a screen for maternal-effect U-shaped embryonic phenotypes. Using a combination of meiotic recombination with molecularly mapped P-element insertions and complementation tests with deficiencies, we mapped the 7T2 mutant to the chromosomal region containing the gene pellino. Here, we show that both pellino mRNA and Pellino protein are missing in the 7T2 mutant tissue, indicating that 7T2 is a loss of function allele of pellino. / Further characterization of the 7T2 mutant revealed three distinct phenotypes: germ band retraction defects, twisted germ bands and head defects. Based on these observations, we propose that pellino is involved in several biological processes during early Drosophila development. Here we show that pellino is involved in the JNK pathway through genetic interaction with hemipterous, an upstream member of the JNK pathway. In addition, we provide preliminary evidence suggesting that the expression of Twist, a protein induced by the Toll pathway, is affected in the absence of pellino, suggesting a role for pellino in dorsal-ventral pattern formation.

In Drosophila, neuropeptides have regulatory roles in development, growth, metabolism and reproduction. This study focused on GABA and the neuropeptides Drosophila tachykinin (DTK), short neuropeptide F (sNPF), adipokinetic hormone (AKH), corazonin (CRZ) and Drosophila insulin-like peptides (DILPs) as possible regulators of metabolic stress responses and homeostasis. We showed that metabotropic GABAB receptors (GBRs) are expressed on brain insulin producing cells (IPCs), suggesting an inhibitory regulation of these cells by GABA. Knockdown of GBR on IPCs shortened lifespan and stress resistance, altered carbohydrate and lipid metabolism at stress (paper I). We showed that three different neuropeptides; DTK, sNPF and ITP, are co-expressed in five pairs of adult neurosecretory cells (paper II). ITP-knock down was not studied yet, but sNPF- and DTK-knock down flies showed decreased stress resistance at desiccation and starvation and decreased water levels at desiccation, suggesting that these peptides are involved in water homeostasis during stress conditions. sNPF was previously shown to affect feeding, growth and DILP expression via the IPCs, but it was not known which sNPF-expressing neurons are responsible for these actions. We could identify a specific set of bilateral neurons (DLPs) that co-express sNPF and corazonin that target the IPCs. We showed that these peptides co-released from DLPs regulate DILP transcription and probably release in the adult Drosophila brain and thus have roles in regulation of stress resistance and metabolism (paper III). AKH signaling was previously shown to affect hemolymph carbohydrate levels and lipid stores in Drosophila. Insulin (DILP) signaling and AKH signaling are suggested to have opposing effects on lipid and sugar metabolism in Drosophila. We studied the possible functional relationship between these two systems; do they mutually regulate each other?  Our results suggest action of DILPs via the Insulin Receptor on the IPCs and the AKH producing cells, but we could not provide evidence for AKH action on IPCs or AKH cells (paper IV). / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 3: Epub ahead of print. Paper 4: Manuscript.</p>

Sexual selection is pervasive in nature and plays an important role in the evolution of biological diversity both within and among sexual species. However, while we have a good understanding of how competition for reproductive opportunities and mate choice can drive the evolution of exaggerated secondary sexual traits, much less is known about how sexual selection interacts with other forms of natural selection and the consequences such interactions may have for adaptation to novel environments, the purging of deleterious mutations, and population divergence/speciation. In my thesis, I carried out a series of experiments with the fruit fly Drosophila melanogaster to test hypotheses regarding the operation of sexual selection and to broaden our understanding of how sexual selection may influence adaptation and diversification. Theory suggests that natural and sexual selection may align to promote adaptation and the purging of deleterious mutations, although the harm imposed by sexual conflict may counter this. In two separate experiments, I find no evidence that sexual selection promotes adaptation to a novel environment and, rather than aligning with natural selection, I find that the effects of sexual conflict may cause sexual selection to hamper the purging of deleterious mutations. With respect to diversification, sexual conflict has been suggested to be an important, non-ecological driver of population divergence. However, the traits involved in sexual conflict may also affect nonsexual fitness and natural selection may therefore act to constrain diversification. Using an evolution experiment, I demonstrate ecologically-dependent parallel evolution of traits involved in sexual conflict, providing evidence for ecology’s importance in divergence via sexual conflict. Overall, my work has shed light on the interaction of natural and sexual selection and the consequences this may have beyond the evolution of exaggerated sexual displays and armaments.

The blastoderm embryo of Drosophila melanogaster consists of a unicellular syncytium with a large number of peripheral nuclei. The cytoplasm surrounding each peripheral nucleus is compartmentalised into apical periplasm above each nucleus and basal periplasm below it. The expression of different genes in the syncytial blastoderm is crucial for the genetic control of development. The pair-rule genes are involved in controlling the pattern of metamerisation of the embryo. Pair-rule mRNAs are expressed in alternate metameres, in a pattern of stripes. Within each stripe, mRNA is found in the apical periplasm of the syncytial blastoderm. By analysing the distribution of mRNA of a number of hybrid constructs, I show that the 3' untranslated part of three pair-rule genes are required for the apical localisation of their transcripts. A 1.2kb region in the 3' end of fushi tarazu (ftz), a 700bp region in the 3' end of hairy (h) and a 160bp fragment of the 3' untranslated part of the even-skipped (eve) pair-rule gene are shown to contain apical localisation signals. I show that the mechanism of apical localisation is unlikely to involve a cytoplasmic process and that the 3' untranslated part of the bicoid (bed) gene contains sequences necessary for apical localisation. I propose that apical localisation involves a nuclear mechanism which exports mRNA from the apical side of the nuclear membrane. I demonstrate that apical localisation is achieved by an RNA-mediated process and not by a DNA-mediated mechanism. Finally, I demonstrate that the intracellular localisation of transcripts encoding cytoplasmic proteins influences the distribution of the protein in the periplasm. I propose that the function of apical localisation is to limit the diffusion of pair-rule proteins so that the pattern of protein expression resembles precisely the transcriptional domain.

Variation in condition can result from environmental heterogeneity or genetic variation affecting resource acquisition and processing ability. Although condition should be positively correlated with the fitness of both males and females, the strength of selection on condition may differ between the sexes due to differences in reproductive variance. Sexual selection on male condition has been proposed to reduce mutation load, but only if selection is greater on males than females. To investigate the strength of selection on condition, the quality of the larval environment was used to manipulate the condition of Drosophila melanogaster. Additionally, selection was measured when the availability of key resources for females (live yeast) and males (access to females) were altered. Overall, selection was found to be stronger on males than females. However, selection on males weakened under a female-biased sex ratio, whereas selection on females was not significantly affected by the abundance of live yeast.

Variation in condition can result from environmental heterogeneity or genetic variation affecting resource acquisition and processing ability. Although condition should be positively correlated with the fitness of both males and females, the strength of selection on condition may differ between the sexes due to differences in reproductive variance. Sexual selection on male condition has been proposed to reduce mutation load, but only if selection is greater on males than females. To investigate the strength of selection on condition, the quality of the larval environment was used to manipulate the condition of Drosophila melanogaster. Additionally, selection was measured when the availability of key resources for females (live yeast) and males (access to females) were altered. Overall, selection was found to be stronger on males than females. However, selection on males weakened under a female-biased sex ratio, whereas selection on females was not significantly affected by the abundance of live yeast.

Evaluating the evolutionary role of mutations depends on an understanding of their major properties, including their rate of origin, U, and the distribution of their fitness effects, f(s). While substantial effort has been put into measuring these properties, most studies have only examined their distributions in a single context. In nature, spontaneous mutations are likely to experience heterogeneity in genetic and environmental context, and this could lead to variation in both U and f(s). My thesis investigates the changes in U and f(s) with different genetic and environmental factors in Drosophila melanogaster, in order to elucidate the nature of context-associated variation in mutational properties. Examination of condition-dependent variation in DNA repair showed that high and low conditioned individuals differ in the use of alternative repair pathways. This could ultimately lead to variance in their heritable mutation rates. However, the assumption that condition dependence in repair arises solely due to a presumed trade-off between accuracy and the energetic costs associated with different repair pathways is too simplistic. Instead, physiological considerations appear to mediate condition-dependent changes in DNA repair. Measurements of selection on individual mutations across different genetic and environment contexts showed that context-associated changes in mutational fitness effects are common. I found that heterogeneity in fitness effects across different environments result in changes to the overall mean and variance of f(s). This does not, however, seem attributable to the degree of ‘adaptedness’ of a population to a particular environment (a prediction generated by previous theoretical analysis). On the other hand, f(s) appears to be relatively robust to differences among genotypes, with epistasis averaging close to zero. This finding suggests that genetic and environmental perturbations may affect mutations differently. Overall, my thesis represents the most rigorous empirical investigation to date of the conceptual and theoretical predictions regarding the nature of context-dependent heterogeneity in U and f(s) for multicellular eukaryotes.

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