Halina Augustyniak

Plant mitochondrial genes can be expressed from mRNAs lacking stop codons

The mRNAs of the nad6 and ccmC genes of Arabidopsis and cauliflower were found to be processed upstream of the inframe stop codons. This result was confirmed by northern hybridization and by RT‐PCR. There is no evidence that an alternative stop codon is created post‐transcriptionally, either by RNA editing or by polyadenylation. The non‐stop mRNAs are found in the high molecular weight polysomal fractions, suggesting that they are translated. Using antibodies directed against CcmC, the corresponding protein was detected in Arabidopsis mitochondrial extracts. These observations raise the question of how the plant mitochondrial translation system deals with non‐stop mRNAs.

A RAD52‐like single‐stranded DNA binding protein affects mitochondrial DNA repair by recombination

The plant mitochondrial DNA-binding protein ODB1 was identified from a mitochondrial extract after DNA-affinity purification. ODB1 (organellar DNA-binding protein 1) co-purified with WHY2, a mitochondrial member of the WHIRLY family of plant-specific proteins involved in the repair of organellar DNA. The Arabidopsis thaliana ODB1 gene is identical to RAD52-1, which encodes a protein functioning in homologous recombination in the nucleus but additionally localizing to mitochondria. We confirmed the mitochondrial localization of ODB1 by in vitro and in vivo import assays, as well as by immunodetection on Arabidopsis subcellular fractions. In mitochondria, WHY2 and ODB1 were found in large nucleo-protein complexes. Both proteins co-immunoprecipitated in a DNA-dependent manner. In vitro assays confirmed DNA binding by ODB1 and showed that the protein has higher affinity for single-stranded than for double-stranded DNA. ODB1 showed no sequence specificity in vitro. In vivo, DNA co-immunoprecipitation indicated that ODB1 binds sequences throughout the mitochondrial genome. ODB1 promoted annealing of complementary DNA sequences, suggesting a RAD52-like function as a recombination mediator. Arabidopsis odb1 mutants were morphologically indistinguishable from the wild-type, but following DNA damage by genotoxic stress, they showed reduced mitochondrial homologous recombination activity. Under the same conditions, the odb1 mutants showed an increase in illegitimate repair bypasses generated by microhomology-mediated recombination. These observations identify ODB1 as a further component of homologous recombination-dependent DNA repair in plant mitochondria.

Mitochondrial DNA from lupine: restriction analysis and cloning of fragments coding for tRNA.

Mitochondrial DNA (mtDNA) was isolated from lupine. Restriction analysis was used to estimate its size, which is about 180 kb. A BamHI bank of this mtDNA was constructed using plasmids pBR322 and pBR327 as vectors. Eight clones containing plasmids hybridizing to mitochondrial tRNA (mttRNA) were isolated. Restriction maps of these plasmids were determined. Six of these plasmids hybridized to unique fragments and two to two fragments of very similar size, all obtained by BamHI cleavage of mtDNA.

Detection of Three DNA‐Binding Proteins from Lupine Mitochondria1

To investigate the mechanism of transcription in plant mitochondria, proteins displaying binding activity to mitochondrial plasmid promoters were studied. We have identified a conserved nonanucleotide motif at the 5′ end of a Lupinus albus mitochondrial plasmid transcript. Three proteins with molecular masses of ca. 25, 30, and 48 kD interacted with ds oligonucleotides, representing different parts of the plasmid promoter. Proteins with molecular masses 25 and 30 kD seem to represent two components of a heterodimer. The DNA‐binding activities of these proteins have been confirmed by electrophoretical mobility shift assay (EMSA), SDS‐PAGE, Southwestern, and UV cross‐linking assays. The specificity of DNA‐binding reaction was examined by competition experiments.