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A number of cytosines are altered to be recognized as uridines in transcripts of the nad3 locus in mitochondria of the higher plant Oenothera. Such nucleotide modifications can be found at 16 different sites within the nad3 coding region. Most of these alterations in the mRNA sequence change codon identities to specify amino acids better conserved in evolution. Individual cDNA clones differ in their degree of editing at five nucleotide positions, three of which are silent, while two lead to codon alterations specifying different amino acids. None of the cDNA clones analysed is maximally edited at all possible sites, suggesting slow processing or lowered stringency of editing at these nucleotides. Differentially edited transcripts could be editing intermediates or could code for differing polypeptides. Two edited nucleotides in an open reading frame located upstream of nad3 change two amino acids in the deduced polypeptide. Part of the well-conserved ribosomal protein gene rps12 also encoded downstream of nad3 in other plants, is lost in Oenothera mitochondria by recombination events. The functional rps12 protein must be imported from the cytoplasm since the deleted sequences of this gene are not found in the Oenothera mitochondrial genome. The pseudogene sequence is not edited at any nucleotide position.

The gene encoding ribosomal protein S14 (rps14) in Oenothera mitochondria is located upstream of the cytochrome b gene (cob). Sequence analysis of independently derived cDNA clones covering the entire rps14 coding region shows two nucleotides edited from the genomic DNA to the mRNA derived sequences by C to U modifications. A third editing event occurs four nucleotides upstream of the AUG initiation codon and improves a potential ribosome binding site. A CGG codon specifying arginine in a position conserved in evolution between chloroplasts and E. coli as a UGG tryptophan codon is not edited in any of the cDNAs analysed. An inverted repeat 3' of an unidentified open reading frame is located upstream of the rps14 gene. The inverted repeat sequence is highly conserved at analogous regions in other Oenothera mitochondrial loci.

In the mitochondrial genome of Arabidopsis thaliana eight mosaic open reading frames arose by recombination of fragments duplicated from one or more mitochondrial genes. These duplications represent unedited sequences, suggesting their derivation from genomic DNA rather than RNA. Five of the chimeric reading frames contain the information for the N-terminus of the original polypeptide and 5' upstream regions. These observations suggest that the generation of novel open reading frames in plant mitochondria can occur rather easily by chance extensions of duplicated gene fragments. The presence of so many mosaic open reading frames in the normal Arabidopsis thaliana mitochondrial genome suggests that such recombined sequences interfere only occasionally and fortuitously with the peak mitochondrial performance presumably required during pollen maturation, and usually do not cause a cytoplasmic male sterile phenotype.

We have determined the complete sequence of the mitochondrial DNA in the model plant species Arabidopsis thaliana, affording access to the first of its three genomes. The 366,924 nucleotides code for 57 identified genes, which cover only 10% of the genome. Introns in these genes add about 8%, open reading frames larger than 100 amino acids represent 10% of the genome, duplications account for 7%, remnants of retrotransposons of nuclear origin contribute 4% and integrated plastid sequences amount to 1%-leaving 60% of the genome unaccounted for. With the significant contribution of duplications, imported foreign DNA and the extensive background of apparently functionless sequences, the mosaic structure of the Arabidopsis thaliana mitochondrial genome features many aspects of size-relaxed nuclear genomes.

In the mitochondrial genome of the flowering plant Arabidopsis thaliana the atp6 open reading frame is located on the border of one of the repeats resulting in two copies with different presequence extensions. The two presequences of 135 and 97 amino acids respectively show no similarity to each other, while the mature protein sequences are identical. Both preproteins are most likely synthesized in Arabidopsis mitochondria from promoter elements upstream of each copy. The presence of two arrangements in the mitochondrial genome of fertile Arabidopsis plants suggests this recombination to be unrelated to a cytoplasmic male sterile phenotype. This recombination precisely at the mature protein terminus is reminiscent of the domain shuffling model in protein evolution.

In Arabidopsis mitochondria the nad2-gene consists of five exons (a-e) which are separated by three cis-splicing introns and one trans-splicing intron. Sequence analysis of the region upstream of exons a and b reveals an open reading frame encoding ribosomal protein S4 (rps4). In the second nad2 coding region (exons c-e) a pseudo tRNA(Tyr) sequence and a fragment of the plastid psbA gene are located upstream of the trans-spliced exon c. Primer extension analysis identifies RNA 5'-termini within the pseudo-tRNA(Tyr) confirming this sequence to be non-functional. Northern blot analysis suggests the rps4-gene to be cotranscribed with at least the first part of the nad2-gene. The rps4 and nad2 coding sequences as well as the first cis-intron and the trans-intron sequences of the nad2 gene are altered by RNA editing. RNA editing in the open reading frames improves in most instances conservation of the specified amino acids.

As part of the worldwide efforts at molecular analysis of Arabidopsis thaliana as a model plant the complete structure of the mitochondrial genome has been determined. The mitochondrial DNA molecules were mapped by restriction fragment analysis of more than 300 cosmid clones and purified mitochondrial DNA. The entire genome of 372 kb is contained in three different configurations of circular molecules and is split into two additional subgenomic molecules of 234 kb and 138 kb, respectively. These arrangements result from recombinations of the two sets of repeats present in combinations of inverted and/or direct orientation. Alignment of YAC clones confirms the in vivo presence of continuous DNA molecules of more than 300 kb in A. thaliana mitochondria. The presence of this comparatively large mitochondrial genome in a plant with one of the smallest nuclear genomes shows that different size constraints act upon the different genomes in plant cells.