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.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.38071 |
| Date | January 2001 |
| Creators | Lachance, Pascal E. D. |
| Contributors | Lasko, Paul (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biology.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001846109, proquestno: NQ75650, Theses scanned by UMI/ProQuest. |
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