Project description:piRNAs are 26-30nt germ-line specific small non-coding RNAs that have evolutionarily conserved function in mobile genetic element silencing and maintenance of genome integrity. It has been shown that Drosophila Hsp70/90 Organizing Protein Homolog (Hop) – a co-chaperone interacts with piRNA binding protein Piwi and mediates silencing of phenotypic variations. However, it is not known if Hop has a direct role in piRNA biogenesis and transposon silencing. Here, we show that knock down of Hop in the germ-line nurse cells (GLKD) of Drosophila ovaries leads to activation of transposable elements. Females without germ-line Hop can lay eggs but the eggs do not hatch into larvae. GLKD of Hop leads to accumulation of γ-H2Av foci in the germline indicating increased DNA damage in the ovary. We also show that Hop is required for efficient piRNA biogenesis. Based on these results, we conclude that Hop is a critical component of piRNA pathway and it maintains genome integrity by silencing transposable elements.
Project description:The hop plant, Humulus lupulus L., contains an exceptionally high content of secondary metabolites, the hop iso-α-acids, which possess a range of beneficial properties including antiseptic action. Studies performed on the mode of action of hop iso-α-acids have hitherto been restricted to lactic acid bacteria. The present study investigates molecular mechanisms of hop iso-α-acid resistance in the model eukaryote Saccharomyces cerevisiae. Growth inhibition occurred at concentrations of hop iso-α-acids that were an order of magnitude higher than those found with hop-tolerant prokaryotes. Chemostat-based transcriptome analysis and phenotype screening of the S. cerevisiae haploid gene deletion collection were used as complementary methods to screen for genes involved in hop iso-α-acids detoxification and tolerance. Further analysis of deletion mutants confirmed that yeast tolerance to hop iso-α-acids involves two major processes: active export of iso-α-acids across the plasma membrane and active proton pumping into the vacuole by the V-ATPase to enable vacuolar sequestration of iso-α-acids. Furthermore, iso-α-acids were shown to affect cellular metal homeostasis by acting as strong zinc and iron chelator.
