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

Lookup NU author(s): Professor Zofia Chrzanowska-Lightowlers, Dr Richard Temperley, Professor Robert Lightowlers

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Abstract

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 intraorganellar transcription and translation.


Publication metadata

Author(s): Chrzanowska-Lightowlers ZM, Temperley RJ, McGregor A, Bindoff LA, Lightowlers RN

Publication type: Article

Publication status: Published

Journal: Gene

Year: 1999

Volume: 230

Issue: 2

Pages: 241-247

Print publication date: 16/04/1999

ISSN (print): 0378-1119

ISSN (electronic): 1879-0038

Publisher: Elsevier

URL: http://dx.doi.org/10.1016/S0378-1119(99)00082-7

DOI: 10.1016/S0378-1119(99)00082-7

PubMed id: 10216263


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