Project description:Abstract: During Drosophila oogenesis, germline stem cell (GSC) identity is maintained largely by preventing the expression of factors that promote differentiation. This is accomplished via the activity of several genes acting either in the GSC or its niche. The translational repressors, Nanos and Pumilio, act in GSCs to prevent differentiation, likely by inhibiting translation of early differentiation factors, while niche signals prevent differentiation by silencing transcription of the differentiation factor Bam. We have found that the DNA-associated protein Stonewall (Stwl) is also required for GSC maintenance. stwl is required cell-autonomously; clones of stwl- germ cells were lost by differentiation, and ectopic Stwl caused an expansion of GSCs. stwl mutants acted as Suppressors of Variegation, indicating stwl normally acts in chromatin-dependent gene repression. In contrast to several previously described GSC maintenance factors, Stwl likely functions epigenetically to prevent GSC differentiation. Stwl-dependent transcriptional repression does not target bam, but rather Stwl represses the expression of many genes, including those that may be targeted by Nanos/Pumilio translational inhibition. Keywords: Genetic modification; gene chip hybridization (Affymetrix Drosophila Genome 2.0)
Project description:Abstract: During Drosophila oogenesis, germline stem cell (GSC) identity is maintained largely by preventing the expression of factors that promote differentiation. This is accomplished via the activity of several genes acting either in the GSC or its niche. The translational repressors, Nanos and Pumilio, act in GSCs to prevent differentiation, likely by inhibiting translation of early differentiation factors, while niche signals prevent differentiation by silencing transcription of the differentiation factor Bam. We have found that the DNA-associated protein Stonewall (Stwl) is also required for GSC maintenance. stwl is required cell-autonomously; clones of stwl- germ cells were lost by differentiation, and ectopic Stwl caused an expansion of GSCs. stwl mutants acted as Suppressors of Variegation, indicating stwl normally acts in chromatin-dependent gene repression. In contrast to several previously described GSC maintenance factors, Stwl likely functions epigenetically to prevent GSC differentiation. Stwl-dependent transcriptional repression does not target bam, but rather Stwl represses the expression of many genes, including those that may be targeted by Nanos/Pumilio translational inhibition. Experiment Overall Design: Genetic variation comparison between germ cells of Drosophila ovaries from bam mutant and stwl bam double mutant females. Six total samples were analyzed, with each genotype performed in triplicates.
Project description:Germline stem cells (GSCs) self-renew and differentiate to sustain a continuous production of gametes. In the female Drosophila germ line, two differentiation factors, bag of marbles (bam) and benign gonial cell neoplasm (bgcn), work in concert in the stem cell daughter to promote the generation of eggs. In GSCs, bam transcription is repressed by signaling from the niche and is activated in stem cell daughters. In contrast, bgcn is transcribed in both the GSCs and stem cell daughters, but little is known about how bgcn is transcriptionally modulated. Here, we find that the conserved protein Nipped_A acts through the Tat interactive protein 60kDa (Tip60) Histone acetyl transferase (HAT) complex in the germ line to promote GSC differentiation. We find that Nipped_A is required for efficient exit from the gap phase 2 (G2) of cell cycle of the GSC daughter and for expression of differentiation factor bgcn. Loss of Nipped_A results in accumulation of GSC daughters. Forced expression of bgcn, in Nipped_A germline-depleted ovaries rescues this differentiation defect. Together, our results indicate that Tip60 complex coordinates cell cycle progression and expression of bgcn to help drive GSC daughters toward a differentiation program.
Project description:Transfer RNAs (tRNAs) are vital in determining the specificity of translation. Mutations in tRNAs can result in the mis-incorporation of amino acids into nascent polypeptides in a process known as mistranslation. Here, our goal was to test the impacts of different types of mistranslation in the model organism Drosophila melanogaster, as impact of mistranslation depends on the type of amino acid substitution. We created two fly lines - one expressing a serine tRNA variant with valine anticodon and the other with a serine tRNA variant with a threonine anticodon. Using mass spectrometry, we measure the amount of mistranslation at various points in fly development.