Diagnostic Assays for Defects in mtDNA Replication and Transcription in Yeast and Humans

Abstract

Publisher Summary This chapter describes several simple assays for the determination of relative mitochondrial DNA (mtDNA) copy number (as a measure of mtDNA replication and stability) and steady-state levels of mitochondrial transcripts (as a measure of an intact mitochondrial transcription apparatus) in human and yeast cells. These diagnostic assays allow potential defects in mtDNA replication and transcription to be identified in a time- and labor-efficient manner. Once a defect of this type is postulated based on the preliminary assays described, the chapter encourages the use of more intensive in vitro and in organello methods to address the molecular nature of the defect more precisely. In general, the methodologies for assaying mitochondrial transcript levels are similar, if not identical, to those utilized to analyze nucleus-derived transcripts. For example, northern hybridization, nuclease protection (e.g., RNase protection), and reverse-transcriptase polymerase chain reaction assays have been successfully used to monitor steady-state levels of specific mitochondrial transcripts in cultured human and yeast cells. The assays described in this chapter provide an initial assessment of whether mtDNA replication or mitochondrial transcription is occurring normally in human or yeast cells. While these assays are helpful for initial diagnostic purposes, it is noted that a reduction in the steady-state level of mtDNA or mitochondrial RNA in a cell, which is the end point of all the assays described in the chapter, may be due to perturbations in the cell other than those that affect mtDNA replication.