Generating a Knockdown Transgene against Drosophila Heterochromatic Tim17b Gene Encoding Mitochondrial Translocase Subunit

Abstract

Heterochromatic regions of eukaryotic genomes contain multiple functional elements involved in chromosomal dynamics, as well as multiple housekeeping genes. Cytological and molecular peculiarities of heterochromatic loci complicate genetic studies based on standard approaches developed using euchromatic genes. Here, we report the development of an RNAi-based knockdown transgenic construct and red fluorescent reporter transgene for a small gene, Tim17b, which localizes in constitutive heterochromatin of Drosophila melanogaster third chromosome and encodes a mitochondrial translocase subunit. We demonstrate that Tim17b protein is required strictly for protein delivery to mitochondrial matrix. Knockdown of Tim17b completely disrupts functions of the mitochondrial translocase complex. Using fluorescent recovery after photobleaching assay, we show that Tim17b protein has a very stable localization in the membranes of the mitochondrial network and that its exchange rate is close to zero when compared with soluble proteins of mitochondrial matrix. These results confirm that we have developed comprehensive tools to study functions of heterochromatic Tim17b gene.

Bringing down the host: enteropathogenic and enterohaemorrhagicEscherichia colieffector-mediated subversion of host innate immune pathways

“…(2011a) showed that RNAi‐mediated knockout of Tim17b resulted in decreased survival of EPEC‐infected cells, supporting the proposed EspZ–Tim17b mechanism. However, other reports have shown that Tim17b ablation results in decreased cell survival, suggesting that these results cannot definitively support the EspZ–Tim17b mechanism and that further investigation is required (Garabedian et al ., ; Sinha et al ., ).…”