title: 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.
authors: Fox Chase Cancer Center, Philadelphia, Pennsylvania, United States of America.
authors: Mikael
authors: M
authors: Garabedian
authors: Michael
authors: M
authors: Jarnik
authors: Elena
authors: E
authors: Kotova
authors: Alexei V
authors: AV
authors: Tulin
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics; D029721:Drosophila Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D004331:Drosophila melanogaster / Q000166:cytology* / Q000235:genetics*; D055785:Gene Knockdown Techniques / Q000379:methods*; D006570:Heterochromatin / Q000235:genetics*; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics; D008297:Male; D026901:Membrane Transport Proteins / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D021122:Protein Subunits / Q000172:deficiency / Q000235:genetics* / Q000378:metabolism; D021381:Protein Transport / Q000235:genetics; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*
chemical_list: D029721:Drosophila Proteins; D006570:Heterochromatin; D008164:Luminescent Proteins; D026901:Membrane Transport Proteins; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; C569919:Tim17b protein, Drosophila; C413663:fluorescent protein 583