RNA-seq of dmGluRA[112b] mutant Drosophila against controls
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ABSTRACT: The transcriptional response in 10-day old adult female flies homozygous for the dmGluRA[112b] mutation was compared to age-matched controls.
Project description:Many protein complexes are involved in gene regulation during Drosophila spermatogenesis. tplus3a and tplus3b code for Plus3 domain proteins and are enriched in spermatocytes. Male flies ∆tplus3a/b carrying deletions of both genes in trans to deficiency Df(2L)BSC151 show severely reduced fertility. tbrd-1 is known to regulate hundreds of genes during spermatogenesis in cooperation with tTAFs. To gain more insight into the regulatory mechanisms during spermatogenesis we used RNA from testes of ∆tplus3a/b/Df(2L)BSC151 and tbrd-11 mutant flies for RNAseq in comparison to w1118 wild-typic control. RNA was isolated with Trizol from 200 Testes dissected from w1118, homozygous tbrd-11 mutants or ∆tplus3a/b/Df(2L)BSC151. After DNAse I digestion RNA was purified and RNAseq was performed in three replicates. Genotype Description: w1118: wild-typic control tbrd-11: homozygous tbrd-11 mutants (knock-out through P{EPgy2}tbrd-11, as described in Leser et al., 2012) ∆tplus3a/b/Df(2L)BSC151: CRISPR/Cas9 deletion mutants, representing knock-out of both tplus3 and tplus3b. Male flies y1cho2v1;∆tplus3a/b/CyO were crossed with virgin females w1118;Df(2L)BSC151/CyO. Male offspring with w1118;∆tplus3a/b/Df(2L)BSC151 were used for RNAseq.
Project description:Tsetse flies (Glossina spp.) are major vectors of African trypanosomes, causing either Human or Animal African Trypanosomiasis (HAT or AAT). Several approaches are developed to control the disease among which the anti-vector Sterile Insect Technique. Another approach in the frame of anti-vector strategies could consist in controlling the fly’s vector competence which needs identifying factors (genes, proteins, biological pathways, …) involved in this process. The present work aims to verify whether protein candidates identified under experimental controlled conditions on insectary-reared tsetse flies have their counterpart in field-collected flies. Glossina palpalis palpalis flies naturally infected with Trypanosoma congolense were sampled in two HAT/AAT foci in Southern Cameroon. After dissection, the proteome from guts of parasite-infected flies were compared to that from uninfected flies in order to identify quantitative and/or qualitative changes associated to infection. A total of 3291 proteins were identified of which 1818 could be quantified. The comparative analysis allowed identifying 175 proteins with significant decreased abundance in infected as compared to uninfected flies, while 61 proteins displayed increased abundance. Among the former are RNA binding proteins, kinases, actin, ribosomal proteins, endocytosis proteins, oxido-reductases, as well as proteins that are unusually found such as tsetse salivary proteins (Tsal) or Yolk proteins. Among the proteins with increased abundance are fructose-1,6-biphosphatase, serine proteases, membrane trafficking proteins, death proteins (or apoptosis proteins), and SERPINs (inhibitor of serine proteases, enzymes considered as trypanosome virulence factors) that displayed highest increased abundance. Sodalis, Wiggleswothia and Wolbachia proteins are strongly under-represented, particularly when compared to data from similar experimentation conducted under controlled conditions on T. brucei gambiense infected (or uninfected) G. palpalis gambiensis insectary reared flies. Comparing the overall recorded data, 364 proteins identified in gut extracts from field flies were shown to have a homologue in insectary flies. Discrepancies between the two studies may arise from differences in the species of studied flies and trypanosomes as well as in differences in environmental conditions in which the two experiments were carried out. Finally, the present study together with former proteomic and transcriptomic studies on the secretome of trypanosomes, on the gut extracts from insectary reared and on field collected tsetse flies, provide a pool of data and information on which to draw in order to perform further investigations on, for example, mammal host immunization or on fly vector competence modification via para-transgenic approaches.
Project description:Profiled the transcriptional response to over-expression Manganese-SOD (MnSOD) in adult Drosophila. A doxycycline-regulated system was employed to induce MnSOD over-expression, resulting in mean and maximal lifespan increases of ~ 20%. Examination of the genome-wide response to this lifespan intervention provided key insights into the molecular basis of aging. Experiment Overall Design: 20 Affymetrix DrosGenome1 arrays were employed with four replicates for each of five conditions. Cohorts of treated and untreated MnSOD transgenic flies were sampled at the same chronological age (~50% survival of -DOX flies, day 73) as well as, at the same physiological age (~50% survival for +DOX and -DOX flies, day 83 and day 73, respectively). The effect of DOX was controlled for by sampling control flies treated with or without DOX at the same chronological age (~50% survival of -DOX flies, day 78).
Project description:We report the application of next generation RNA sequencing to analyze the transcriptional response of Drosophila adult flies to infection by the insect pathogenic nematodes Heterorhabditis bacteriophora and their mutualistic bacteria Photorhabdus luminescens, either separately or together. We find that Heterorhabditis and Photorhabdus differentially modulate a large number of genes, many of which participate in metabolic functions, stress responses, repression of gene transcription and neuronal activities. We have also identified Drosophila genes with potential role in nematode recognition and others with putative anti-nematode properties. These findings generate novel insights into how the host immune function is shaped to respond against nematode parasites and their associated bacteria. Transcriptional profiles of Drosophila wild-type adult flies infected with Heterorhabditis bacteriophora carrying or lacking Photorhabdus or the bacteria alone were generated at 12 and 30 hours post infection using Illumina deep sequencing technology.
Project description:Expression of dCoREST or dLSD1 was reduced by RNAi in Drosophila testes and changes to gene transcription were determined by RNA-seq.
Project description:Comparing the transcriptome of wildtype and kdm5 mutant flies in normal conditions revealed a total of 4787 genes that were significantly downregulated and thus require KDM5 for their activation, and 3269 upregulated genes that are normally repressed by KDM5 (p<0.05, FDR <0.05). Because kdm5 mutants are sensitive to the oxidizer paraquat, we also carried out RNA-seq from wildtype and kdm5 mutant adults in oxidative stress conditions. Paraquat treatment of wildtype flies lead to the upregulation of 2481, and downregulation of 3103 genes adult mRNA profiles of 1-3-days old wild type (WT) and kdm5 mutant under normal condition and oxitative stress were generated by deep sequencing, using Illumina HisSeq 2000.
Project description:We observed several hundreds of thousand reads matching the IIV6 genome in IIV6 infected S2 and wild-type flies. The large majority of these reads have a size of 21 nt. This peak was absent from the library prepared from S2 non-infected and infected Dcr-2-/- mutant flies. The virus-derived siRNAs were not uniformally distributed along the viral genome, but rather several hotspots were observed, revealing that specific regions of the viral genome generate the siRNAs. Small RNA profiles of IIV6 infected S2 cells and adult flies were generated by deep sequencing using Illumina 2G Analyzer.
Project description:Because pink1-mutant flies exhibit a global shutdown of protein synthesis, we decided to measure the levels of individual proteins in adult flies through quantitative proteomics.
Project description:Manganese is considered essential for animal growth. Manganese ions serve as cofactors to three mitochondrial enzymes: superoxide dismutase (Sod2), arginase and glutamine synthase. In Drosophila melanogaster, manganese has also been implicated in the formation of ceramide phosphoethanolamine, the insect’s sphingomyelin analogue, a structural component of membranes. Manganese overload leads to neurodegeneration and toxicity in both humans and Drosophila. Here, we describe a Drosophila model of manganese deficiency. Due to the lack of manganese-specific chelators, we used chemically defined media to grow the flies and deplete them of the metal. Dietary manganese depletion reduces Sod2 activity. We then examined gene and protein expression changes in the intestines of manganese depleted flies. We found adaptive responses to the lack of the known manganese-dependent enzymatic activities and alterations in genes/enzymes of carbohydrate metabolism and glycosylations, similar to earlier reports of manganese deficiency in vertebrate animals.
Project description:Protein expression profiles in the whole tissue lysate extracted from the thorax of O1 and O3 long-lived flies with the control B3 strain flies and analyzed by tandem mass tag (TMT)–based mass spectrometry.