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 the mechanisms governing mt-RNA processing, stability and translation remains rudimentary. We have taken a step towards addressing these issues by altering the luciferase reporter gene to accommodate the variation in mitochondrial codon recognition. 19 essential substitutions have been generated by an iterative mega-primer PCR technique. To mimic mt-mRNA species and to optimise intramitochondrial translation, further engineering has produced a template which, when transcribed in vitro, generates an RNA species with only two nucleotides upstream from the initiation codon, an absence of a 3' untranslated region and a polyadenylated tail of 40 residues. It is intended that mt-luciferase (mt-luc) RNA will be an excellent reporter for revealing cis-acting elements essential for in organello RNA processing, maturation and expression. Additionally, the mt-luc gene can be readily incorporated into any novel mitochondrial transducing vectors to assess intra-organellar transcription and translation.
Chrzanowska-Lightowlers
Z M
ZM
Molecular Neurology Unit, Department of Neurology, Medical School, University of Newcastle upon Tyne, Framlington Place, Newcastle upon Tyne NE2 4HH, UK.
Temperley
R J
RJ
McGregor
A
A
Bindoff
L A
LA
Lightowlers
R N
RN
D017931:DNA Primers; D004272:DNA, Mitochondrial / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics*; D008156:Luciferases / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D014176:Protein Biosynthesis; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000077278:RNA, Mitochondrial; D014158:Transcription, Genetic
D017931:DNA Primers; D004272:DNA, Mitochondrial; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D012313:RNA; D008156:Luciferases