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:Dietary amino acids restriction extends lifespan in diverse species ranging from flies to mammals. The evolutionarily conserved serine/threonine kinase General Control Nonderepressible 2 (GCN2) is a key sensor of amino acid deficiency and has been implicated in lifespan regulation upon dietary restriction. However, the role of individual essential amino acids (EAA) in modulating organismal lifespan and the underlying molecular mechanisms through which EAA mediate these effects are only partially understood. We generated a novel Drosophila GCN2 null mutant and systematically analyzed its response to individual amino acid deficiency.
Project description:Temperature reduction has been one of the most robust environmental manipulations used to increase lifespan in invertebrates. However, the underlying molecular mechanisms leading to temperature-mediated lifespan extension remains largely unknown. Here, we evaluate how genes and metabolites are altered in response to changes in temperature in the fruit fly, Drosophila melanogaster. Our data show that environmental temperature affects transcriptome much more than the metabolome. Intriguingly, the most affected candidate genes, the Turandot (tot) family genes, have direct effects on longevity in flies. Specifically, knockdown of tot expression at all temperatures leads to lifespan extension in a sex-specific fashion. Overall, our results imply that individual genes, specifically, the Turandot family of genes that may play a novel role in lifespan regulation in the fruit fly.
Project description:Limitation of essential amino acids, such as tyrosine or methionine/cysteine, causes upregulation of exogenous integrated transgene expression in mammalian cells. This phenomenon is mediated by histone acetylation and chromatin remodelling, since histone deacetylase (HDAC) inhibitors reproduce starvation-induced transgene upregulation and chromatin immunoprecipitation analysis of amino acid-deprived cells reveals significant changes in total core histones detectable at the CMV promoter. Expression profiling of HeLa cells starved for 5 days in medium without tyrosine or methionine/cysteine provides important information on the cellular response to amino acid deprivation and suggests the involvement of HDAC4 (class II HDAC) in transgene derepression during amino acid starvation. Total RNA obtained from HeLa and HeLaOA1myc cells (HeLa cells with a human ocular albinism type 1 (OA1)+myc tag transgene) subjected to 5 days of tyrosine or methionine/cysteine starvation compared to control cells. Twelve samples are analyzed: HeLa and HeLaOA1myc grown for 5 days in RPMI deprived of tyrosine (Y) vs. complete RPMI; technical duplicates of HeLa and HeLaOA1myc grown for 5 days in DMEM deprived of methionine/cysteine (MC) vs. complete DMEM.
Project description:we performed proteome sequencing in Drosophila at day 7 (young) and day 42 (old) under dietary restriction (DR)and ad libitum (AL) conditions.