A proteomics approach: Identification of proteins mediating different steps in the specification and differentiation of Drosophila melanogaster oocytes.

摘要

In this thesis I present the genetic and biochemical characterization of the Drosophila melanogaster (Drosophila) mago nashi (mago) and tsunagi (tsu) genes during the development of the oocyte. The expression of their proteins and genetic analysis of null alleles was study to determine what early functions Mago and Tsu have in oogenesis, and do they always function together?; Previously Mago and Tsu have been shown to form a complex and regulate axis formation and the localization of proteins/mRNAs in the oocyte. The experiments show that Mago and Tsu are expressed in all germline and somatic cells of the ovary. The magonull blocks the formation of the germline cystoblast. Furthermore, Mago is necessary only in Germline Stem Cell (GSC) not the Somatic Stem Cell. The tsunull does not affect cystoblast formation, in a tsunull mutant cystoblasts form. Therefore, Mago and Tsu do not always function together. Since Mago and Tsu do not always function together a biochemical study to identify protein interactors of Mago was preformed. The additional interactors were identified by immunoprecipitation (IP) and mass spectroscopy. The IPed proteins showed enrichment for proteins involved in cell signaling, intracellular transport, and nucleic acid metabolism.; Since Mago is necessary for cell signaling and protein/mRNA localization, these new interactors should shed light on the molecular pathways that Mago regulates. Several of the interactors functioned with Mago during oogenesis based on their ability to genetically interact with mago alleles. Characterization of three of the interactors shows that the molecular motors dynein heavy chain at 64C (dhc64C) and gamma-dynein affect axis formation, and dhc64C affects oocyte formation, while the transcription factor and actin binding protein sallimus (sls) is necessary for the regeneration of the GSC and the development of egg chambers beyond stage 4. Therefore, Mago regulates oocyte development in two ways; by affect microtubule based transport and affecting gene expression and/or actin binding.