Project description:Absolute (molar) quantification determines proteins stoichiometry in complexes, networks and metabolic pathways. We employed MS Western workflow to determine molar abundances of proteins critical for morphogenesis and phototransduction (PT) in eyes of Drosophila melanogaster using a single chimeric 264 kDa protein standard that covers, in total, 197 peptides from 43 proteins. Each protein was independently quantified with 2 to 4 proteotypic peptides with the coefficient of variation of less than 15 %, better than 1000-fold dynamic range and sub-femtomole sensitivity. We determined molar abundances and stoichiometric ratios of the components of the PT machinery and the rhabdomere, and how they are changing when rhabdomere morphogenesis is perturbed by genetic manipulation of the evolutionary conserved gene crumbs (crb).
Project description:We demonstrate that the cell cycle regulators, E2f /Dp and Rb, control the transcription of ribosomal proteins (RP) in Drosophila embryos. Mutation of E2f1 or Dp and over expression of Rbf1 increase and reduce RP transcription, respectively. Although E2f/Dp/Rb might exert this effect through a repressor complex, the regulatory regions of RP genes do not show an enrichment of canonical E2f binding sites. In addition, E2f1, Dp and Rbf1 also regulate the expression of RACK1, a ribosomal component and a negative regulator of cell cycle progression. These findings strengthen the coupling of cell cycle regulation to protein biosynthesis. Keywords: genotype response
Project description:We demonstrate that the cell cycle regulators, E2f /Dp and Rb, control the transcription of ribosomal proteins (RP) in Drosophila embryos. Mutation of E2f1 or Dp and over expression of Rbf1 increase and reduce RP transcription, respectively. Although E2f/Dp/Rb might exert this effect through a repressor complex, the regulatory regions of RP genes do not show an enrichment of canonical E2f binding sites. In addition, E2f1, Dp and Rbf1 also regulate the expression of RACK1, a ribosomal component and a negative regulator of cell cycle progression. These findings strengthen the coupling of cell cycle regulation to protein biosynthesis. Experiment Overall Design: Our study examines the genomic response of intact Drosophila embryos to the genetic manipulation of E2F/Rb pathway molecules. For over-expression experiments, Arm-Gal4 stocks were crossed to UAS-gene stocks for the following genes: E2f1/Dp, E2f2/Dp, Rbf1, and E2f1336-805 (a dominant negative form of E2f1 lacking the DNA binding domain) [16]. The E2f17172 and E2f191 null alleles were balanced by TM3[Kr-GFP]. Dpa2 and Dpa4 null alleles were balanced by CyO[Kr-GFP]. 14-16 hr E2f17172/E2f191 null mutant embryos were hand selected based on the absence of fluorescent Bolwigâs organs in Kr-GFP balanced stocks using a Zeiss stereomicroscope equipped with epifluorescence. E2f17172/E2f191 and Dpa2/Dpa4 null mutant embryos (8-10h) were selected using the COPASTM SELECT system for automated sorting of multi-cellular organisms. Eighteen RNA samples, two from each cross, were obtained from approximately 200 to 700 embryos per sample. For each cross, 2 h embryo collections were aged for 8 to 14 h at 25oC, quick frozen in an ethanol/dry ice mixture, and stored at -80 oC. The RNA was prepared for microarray analysis in accordance with Affymetrix instructions.
Project description:To verify unannotated translated open reading frames (utORFs) identified from Drosophila melanogaster, we collected data to target them.
Project description:dFOXO targets in adult Drosophila melanogaster females, and the effect of insulin signalling and stress on binding. The experimets determined the binding locations of dFOXO in the whole adult female fly using ChIP-chip. The protocol was validated using mock conditions: pre-immune serum or IP on chromatin from foxo null flies. The response of this binding to stress induced by treatment of flies with paraquat or by their exposure to starvation, as well as the response to an insulin-signalling-reducing genetic manipulation (over-expression of dominant negative form of the insulin receptor), was determined.
Project description:Drosophila melanogaster RNA sequencing with Illumina Genome Analyzer. High-throughput sequencing of Drosophila melanogaster RNAs. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf