In organello gene expression and RNA editing studies by electroporation-mediated transformation of isolated plant mitochondria.
Plant mitochondrial gene expression is a complex process involving multiple steps such as transcription, cis- and trans-splicing, RNA trimming, RNA editing, and translation. One of the main hurdles in understanding more about these processes has been the inability to incorporate engineered genes into mitochondria. We recently reported an in organello approach on the basis of the introduction of foreign DNA into isolated plant mitochondria by electroporation. This procedure allows the investigation of transcriptional and posttranscriptional processes, such as splicing and RNA editing, by use of site-directed mutagenesis. Foreign gene expression in organello is strongly dependent on the functional status of mitochondria, thus providing relevant information in conditions closer to the situation found in vivo. The study of mutants that affect RNA splicing and editing provides a novel and powerful method to explain the role of specific sequences involved in these processes. Here we describe a protocol to "transform" isolated plant mitochondria that has allowed us to investigate successfully some aspects of RNA editing.
Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, California, USA.
Jean-Claude
JC
Farré
David
D
Choury
Alejandro
A
Araya
D001671:Biochemistry / Q000379:methods*; D018274:Electroporation; D018506:Gene Expression Regulation, Plant; D005821:Genetic Techniques; D008928:Mitochondria / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D017393:RNA Editing / Q000235:genetics*; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D011198:Solanum tuberosum / Q000235:genetics; D014908:Triticum / Q000235:genetics
D010940:Plant Proteins; D012313:RNA