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.
Project description:To understand potential downstream signaling molecules that are responsible for wild type-Malt1 induced resistance of tumor cell to CD8 T cell killing , we first use E0771 cells expressing either -Vector control, wild type-Malt1 (M-WT), or Malt1 PD mutant (M-PD) to coculture with activated CD8 T cell at a effctor: target ratio(E:T ration) of 3 for 12 hours. Then wash the cells with PBS, digest the cells by 0.25% trypsin solution, harvest the cells in 15 ml tubes by centrifugation (1000 rpm 5min) and seed cells into the original plate with DMEM+10%FBS+1%P/S medium. 6 hours after sseding, wash cells with PBS, lyse cells with TRIZOL for RNA extraction, perform reverse transcription and cDNA library for RNA-Seq analysis.
Project description:MicroRNAs detected in Drosophila melanogaster unfertilized eggs Bloomington w[1118] flies were kept at 25ºC on cornmeal based media, with 12 hours light/dark cycles. Virgin females were sorted at the pupae stage to avoid any unwanted fertilization. In a population cage I let 80-100 females to lay eggs in apple juice agar plates for 8 hours, collecting 1 hour after dawn. Eggs were collected with a sieve and washed with saline solution. Small RNA was size selected and sequenced.
Project description:In this study we use a combination of proteomics Label-Free quantification methods to monitor protein expression changes over a time course of more than 20 hours of embryo development in Drosophila melanogaster.
Project description:Experimental Design Type of experiment: In order to evaluate the influence of subinhibitory concentrations of the macrolide antibiotic Azithromycin (AZM) on the global Pseudomonas aeruginosa gene expression, we performed a comparative gene expression analysis of AZM-treated versus untreated P. aeruginosa PAO1 cultures. Experimental factors: P. aeruginosa PAO1 cultures with and without addition of 2 µg/ ml AZM were grown until early stationary phase. Total RNA was extracted when the cultures reached an OD600 of 2.8. The transcriptomes of the untreated cultures were taken as reference values. Both of the AFFYMETRIX Chips of AZM-treated PAO1 (GSM45590, GSM45591) were compared with each of the two chips of the untreated PAO1 (GSM45588, GSM45589). Hybridization procedures and parameters: Hybridization of the probes was carried out according to the manufacturer’s “Expression Analysis Protocol (p. 25: Modified Fluidic Protocol for P. aeruginosa cDNA Assay)”, see http://www.affymetrix.com: Hybridization for 16 h at 50°C and 60 rpm. - Post Hyb Wash 1: 10 cycles of 2 mixes/cycle with Wash Buffer A at 25°C - Post Hyb Wash 2: 4 cycles of 15 mixes/cycle with Wash Buffer B at 50°C - Stain: Stain the probe array for 10 minutes in Streptavidin Solution Mix at 25°C - Post Stain Wash: 10 cycles of 4 mixes/cycle with Wash Buffer A at 30°C - 2nd Stain Wash: Stain the probe array for 10 minutes in antibody solution at 25°C - 3rd Stain Wash: Stain the probe array for 10 minutes in SAPE solution at 25°C - Final Wash: 15 cycles of 4 mixes/cycle with Wash Buffer A at 30°C. The holding temperature is 25°C. Measurement of data and specifications: Data transformation and selection procedures: · The entire signals were scaled to 150 (4% capped median); scaling factors of the arrays were within 3.4 fold of each other, as the % genes called present were in the range of 23.7% and 38.1% for three arrays (GSM455889, GSM45589, GSM45591) and 74,4% for one array (GSM45590). The background levels (Average Background) were similar for all arrays (in a range of 47.20 and 55.64). The Q scores were comparable (in a range of 2.38 and 2.81). The Average Signals of the four arrays were similar (between 187.8 and 228.7). · Scanning hardware: AFFYMETRIX Scanner; software: AFFYMETRIX MAS 5 (Statistical Algorithms Reference Guide, http://www.affymetrix.com/support/technical/technotes/statistical_reference_guide.pdf), MICRO DB 3 and DMT 3 · Image analysis software: AFFYMETRIX MAS 5 Data selection: · Threshold of Signal Log ratio: -1/1; change is I (increased), MI (marginally increased), D (decreased), MD (marginally decreased); change p-value >0.999 or <0.001; only genes that are present (Detection=P) in at least one of the compared chipsets. · Only following genes were considered: Minimal threshold of regulation ±2 fold; minimal change p-values of 0.999 and 0.001, respectively; minimal difference of signal intensities of 200. · The final gene expression data table used by the authors to make their conclusions after data selection and transformation will be published as supplementary data. Keywords: other