The complete DNA sequence of the mitochondrial genome of Physarum polycephalum

The complete sequence of the mitochondrial DNA (mtDNA) of the true slime mold Physarun polycephalum has been determined. The mtDNA is a circular 62,862-bp molecule with an A+T content of 74.1%. A search with the program BLAST X identified the protein-coding regions. The mitochondrial genome of P. polycephalum was predicted to contain genes coding for 12 known proteins [for three cytochrome c oxidase subunits, apocytochrome b, two F1Fo-ATPase subunits, five NADH dehydrogenase (nad) subunits, and one ribosomal protein], two rRNA genes, and five tRNA genes. However, the predicted ORFs are not all in the same frame, because mitochondrial RNA in P. polycephalum undergoes RNA editing to produce functional RNAs. The nucleotide sequence of an nad7 cDNA showed that 51 nucleotides were inserted at 46 sites in the mRNA. No guide RNA-like sequences were observed in the mtDNA of P. polycephalum. Comparison with reported Physarum mtDNA sequences suggested that sites of RNA editing vary among strains. In the Physarum mtDNA, 20 ORFs of over 300 nucleotides were found and ORFs 14 19 are transcribed.     

陆地棉线粒体nad6基因mRNA无常规终止密码子

植物线粒体nad6基因编码NADH（还原型辅酶Ⅰ）脱氢酶第6亚基,前期研究发现,该基因可能与棉花细胞质雄性不育相关,但该基因的转录情况尚不清楚.本研究利用PCR测序、Southern印迹方法发现,陆地棉线粒体基因组（mtDNA）中nad6基因长621 bp,且为单拷贝. RT-PCR及环化RT-PCR分析发现,其mRNA在终止密码子前-14或-15 nt处提前终止|虽然该基因mRNA编码区存在12处RNA编辑（C-U）位点,但并未产生新的替代的终止密码子|基因mRNAs尾端poly（A）处含有0、1、2或4个“A”,也并未与前方相邻碱基凑成新的终止密码子,即：该基因mRNA无常规终止密码子（UGA,UAG,UAA）.本研究结果提示,棉花线粒体nad6基因mRNA很可能有其它的终止密码子.针对这种情况对植物线粒体如何翻译无常规终止密码子的mRNA进行了讨论.     

Genes and Processed Paralogs Co-exist in Plant Mitochondria

RNA-mediated gene duplication has been proposed to create processed paralogs in the plant mitochondrial genome. A processed paralog may retain signatures left by the maturation process of its RNA precursor, such as intron removal and no need of RNA editing. Whereas it is well documented that an RNA intermediary is involved in the transfer of mitochondrial genes to the nucleus, no direct evidence exists for insertion of processed paralogs in the mitochondria (i.e., processed and un-processed genes have never been found simultaneously in the mitochondrial genome). In this study, we sequenced a region of the mitochondrial gene nad1, and identified a number of taxa were two different copies of the region co-occur in the mitochondria. The two nad1 paralogs differed in their (a) presence or absence of a group II intron, and (b) number of edited sites. Thus, this work provides the first evidence of co-existence of processed paralogs and their precursors within the plant mitochondrial genome. In addition, mapping the presence/absence of the paralogs provides indirect evidence of RNA-mediated gene duplication as an essential process shaping the mitochondrial genome in plants.     

Mitochondrial rps14 is a transcribed and edited pseudogene in Arabidopsis thaliana

We have isolated and analysed a 2 kb region of the mitochondrial genome of Arabidopsis thaliana (Columbia) showing a high level of nucleotide identity with the mitochondrial (mt) rps14 small-subunit ribosomal protein gene from Oenothera berteriana and Vicia faba, as well as with an open reading frame (ORF) located upstream of the nad3 locus in O. berteriana. The rps14 locus is present as a single copy in the A. thaliana mt genome and has a translational stop codon located near the initiation codon, as well as a deletion of one nucleotide that disturbs the coding sequence. The cloning and sequencing of nine amplified mt rps14 cDNAs clearly demonstrated that this gene is transcribed and that the mRNA precursors are edited at three positions, all involving C-to-U conversions. No editing events changing the stop codon and restoring the correct coding sequence were witnessed within the 9 individual cDNA clones. Therefore, we conclude that the single rps14 sequence of the mitochondrial genome from A. thealiana is in fact a pseudogene that is transcribed and edited but not translated.     

Trans splicing in Oenothera mitochondria: nad1 mRNAs are edited in exon and trans-splicing group II intron sequences.

The complete NADH dehydrogenase subunit 1 (nad1) ORF in Oenothera mitochondria is encoded by five exons. These exons are located in three distant locations of the mitochondrial genome. One genomic region encodes exon a, the second encodes exons b and c, and the third specifies exons d and e. Cis-splicing group II introns separate exons b and c and d and e, while trans-splicing reactions are required to link exons a and b and c and d. The two parts of the group II intron sequences involved in these trans-splicing events can be aligned in domain IV. Exon sequences and the maturase-related ORF in intron d/e are edited by numerous C to U alterations in the mRNA. Two RNA editing events in the trans-splicing intron a/b improve conservation of the secondary structure in the stem of domain VI. RNA editing in intron sequences may thus be required for the trans-splicing reaction.