Conversion of a reporter gene for mitochondrial gene expression using iterative mega-prime PCR

Mammalian mitochondria possess their own multicopy genome, mtDNA. Although much is known about mtDNA replication and transcription, our knowledge of t he mechanisms governing mt-RNA processing, stability and translation remain s rudimentary. We have taken a step towards addressing these issues by alte ring the luciferase reporter gene to accommodate the variation in mitochond rial codon recognition. 19 essential substitutions have been generated by a n iterative mega-primer PCR technique. To mimic mt-mRNA species and to opti mise intramitochondrial translation, further engineering has produced a tem plate which, when transcribed in vitro, generates an RNA species with only two nucleotides upstream from the initiation codon, an absence of a 3' untr anslated region and a polyadenylated tail of 40 residues. It is intended th at mt-luciferase (mt-luc) RNA will be an excellent reporter for revealing c is-acting elements essential for in organello RNA processing, maturation an d expression. Additionally, the mt-luc gene can be readily incorporated int o any novel mitochondrial transducing vectors to assess intraorganellar tra nscription and translation. (C) 1999 Elsevier Science B.V. All rights reser ved.