5] In Organello RNA synthesis system from HeLa cells

Abstract

Publisher Summary Alterations in mitochondrial gene expression occur in response to energetic conditions within the cell and possibly cytoplasmic and nuclear control factors. Hence, to simplify investigations of the potential control provided by the energetic states of the cell and cytoplasmic factors on mitochondrial DNA (mtDNA) transcription and processing of the mtRNA, a set of experimental protocols is developed that physically separates the mitochondria from the remaining cellular constituents. This separation is achieved by mechanically lysing HeLa cells, isolating the intact mitochondria by centrifugation, and removing the nonmitochondrial RNA through enzyme treatment and washings. The isolated mitochondria can then be assayed for transcriptional capacity and processing fidelity by analyzing the incorporation of radioactive nucleotides into their RNA using gel electrophoretic techniques. As described in the chapter, it is possible to isolate mitochondria easily and quickly that are competent in transcription events and the processing of RNA, comparable to that found in the in vivo situation. Therefore, it is possible to study the effects of changes in the energetic or metabolic environments and maturation of the mtRNA. The molecular analysis of these effects is possible because of the preexisting knowledge about HeLa cell mitochondrial DNA and its in vivo transcription products.