Nakao Kubo

Targeting presequence acquisition after mitochondrial gene transfer to the nucleus occurs by duplication of existing targeting signals.

We have cloned a gene for mitochondrial ribosomal protein S11 (RPS11), which is encoded in lower plants by the mitochondrial genome, in higher plants by the nuclear genome, demonstrating genetic information transfer from the mitochondrial genome to the nucleus during flowering plant evolution. The sequence s11–1 encodes an N‐terminal extension as well as an organelle‐derived RPS11 region. Surprisingly, the N‐terminal region has high amino acid sequence similarity with the presequence of the beta‐subunit of ATP synthase from plant mitochondria, suggesting a common lineage of the presequences. The deduced N‐terminal region of s11–2, a second nuclear‐encoded homolog of rps11, shows high sequence similarity with the putative presequence of cytochrome oxidase subunit Vb. The sharing of the N‐terminal region together with its 5′ flanking untranslated nucleotide sequence in different proteins strongly suggests an involvement of duplication/recombination for targeting signal acquisition after gene migration. A remnant of ancestral rps11 sequence, transcribed and subjected to RNA editing, is found in the mitochondrial genome, indicating that inactivation of mitochondrial rps11 gene expression was initiated at the translational level prior to termination of transcription.

An integrated high-density linkage map of soybean with RFLP, SSR, STS, and AFLP markers using A single F2 population.

Abstract Soybean [Glycine max (L.) Merrill] is the most important leguminous crop in the world due to its high contents of high-quality protein and oil for human and animal consumption as well as for industrial uses. An accurate and saturated genetic linkage map of soybean is an essential tool for studies on modern soybean genomics. In order to update the linkage map of a F2 population derived from a cross between Misuzudaizu and Moshidou Gong 503 and to make it more informative and useful to the soybean genome research community, a total of 318 AFLP, 121 SSR, 108 RFLP, and 126 STS markers were newly developed and integrated into the framework of the previously described linkage map. The updated genetic map is composed of 509 RFLP, 318 SSR, 318 AFLP, 97 AFLP-derived STS, 29 BAC-end or EST-derived STS, 1 RAPD, and five morphological markers, covering a map distance of 3080 cM (Kosambi function) in 20 linkage groups (LGs). To our knowledge, this is presently the densest linkage map developed from a single F2 population in soybean. The average intermarker distance was reduced to 2.41 from 5.78 cM in the earlier version of the linkage map. Most SSR and RFLP markers were relatively evenly distributed among different LGs in contrast to the moderately clustered AFLP markers. The number of gaps of more than 25 cM was reduced to 6 from 19 in the earlier version of the linkage map. The coverage of the linkage map was extended since 17 markers were mapped beyond the distal ends of the previous linkage map. In particular, 17 markers were tagged in a 5.7 cM interval between CE47M5a and Satt100 on LG C2, where several important QTLs were clustered. This newly updated soybean linkage map will enable to streamline positional cloning of agronomically important trait locus genes, and promote the development of physical maps, genome sequencing, and other genomic research activities.

A ribosomal protein L2 gene is transcribed, spliced, and edited at one site in rice mitochondria

The mitochondrial ribosomal protein L2 gene (rpl2) is coded by two exons of 840 and 669 bp separated by an intron sequence of 1481 bp in the rice mitochondrial genome. The rpl2 gene is located three nucleotides upstream of the ribosomal protein S19 gene (rps19) and both genes are co-transcribed. cDNA sequence analysis indentified splicing of the intron sequence from the rpl2 mRNA as well as RNA editing events. The deduced secondary structure of the rpl2 intron sequence shows the characteristic features of a group-II intron. A single RNA editing site is identified in rpl2 and six editing sites in rps19 transcripts. In addition, one editing site is observed in the 3 nucleotide intergenic region. Analysis of individual cDNA clones showed a different extent of RNA editing. The rice rpl2 intron is located at a different site and shows no significant nucleotide sequence similarity with the rpl2 intron of liverwort. However, 60% nucleotide sequence identity is observed between the rice rpl2 intron and the Oenothera nad5 intron in a 234 nucleotide region. The mitochondrial rpl2 sequence is absent from the pea mitochondrial genome and we consequently propose that the mitochondrial RPL2 protein is encoded by a nuclear gene in pea.

A promiscuous chloroplast DNA fragment is transcribed in plant mitochondria but the encoded RNA is not edited

The RNA editing processes in chloroplasts and mitochondria of higher plants show several similarities which are suggestive of common components and/or biochemical steps between the two plant organelles. The existence of various promiscuous DNA fragments of chloroplast origin in plant mitochondrial genomes allowed us to test the possibility that chloroplast sequences are also edited in mitochondria. AnrpoB fragment transferred from chloroplasts to mitochondria in rice was chosen as it contains several editing sites, two of which match sequence motifs surrounding even non-homologous editing sites in both chloroplast and mitochondrial transcripts. Rice chloroplast and mitochondrialrpoB DNA and cDNA sequences were selectively amplified and the editing status of the cDNA sequences was determined. Three of the four potentialrpoB editing sites previously detected in maize were found to be edited in the rice chloroplastrpoB transcript, whereas the fourth was found to remain unedited. In mitochondria, however, all four editing sites remain unmodified at the cDNA level. This indicates that the editing processes of higher plant mitochondria and chloroplasts are not identical and that organelle-specific factors are required for eliciting the respective editing events.

Involvement of 5′ flanking sequence for specifying RNA editing sites in plant mitochondria

Unsuccessful insertion of foreign DNA into plant mitochondrial genomes has hindered scientific evaluation of cis‐elements needed for RNA editing. Both a normal atp6 gene and a chimeric atp6 sequence are present in rice mitochondria. The chimeric atp6 contains one‐half of the normal atp6 sequence in its 5′ portion and an unknown sequence in its downstream portion. The C‐nucleotide at position 511, located just upstream of the unknown sequence recombined in the chimeric atp6 sequence, is edited, as are other possible editing sites upstream from position 511. We report here that the 5′ sequence adjacent to the editing site of atp6 contains cis‐information required for RNA editing and that the 3′ sequence flanking the editing site provides little contribution to editing‐site recognition.

Involvement of N-terminal region in mitochondrial targeting of rice RPS10 and RPS14 proteins

Abstract We have investigated targeting signal of rice mitochondrial ribosomal proteins, RPS10 and RPS14. Their predicted protein structures show that RPS14 has a cleavable N-terminal extension whereas RPS10 does not, and that the both proteins contain several α-helix structures. To know which regions are involved in protein targeting into mitochondria, subcellular localization of the two proteins was examined in vivo by using a green fluorescent protein (GFP). A portion of RPS10 or RPS14 was fused to the GFP and introduced into tobacco BY-2 cells. Localization of fusion proteins was visualized by GFP fluorescence. When the N-terminal part of RPS10 (amino acid position 1–56) was fused to GFP, resultant GFP fusion proteins were detected specifically in mitochondria. In contrast, no such localization was found when the C-terminal part of RPS10 was fused to GFP. GFP fusion proteins were clearly localized to mitochondria when the N-terminal region of RPS14 (amino acid position 1–48) was fused to GFP. Introduction of sequence alterations into their N-terminal regions abolished the specificity of mitochondrial targeting. These results strongly suggest that the N-terminal region plays an important role for the targeting of the ribosomal proteins into plant mitochondria irrespective of the N-terminal extension.

Mitochondrial sequence migrated downstream to a nuclear V-ATPase B gene is transcribed but non-functional

A promiscuous nuclear sequence containing a mitochondrial DNA fragment was isolated from rice. Nucleotide sequence analysis reveals that the cDNA clone #21 carries a mitochondrial sequence homologous to the 3 portion of the rps19 gene followed by the 5 portion of the rps3 gene. The mitochondrial sequence is present in an antisense orientation. Sequence comparison of the #21 cDNA with the original mitochondrial sequence shows 99% similarity, suggesting a recent transfer event. Moreover, evidence for a lack of an RNA editing event and retaining of the group II intron sequence strongly suggests that the sequence was transferred from mitochondrion to the nucleus via DNA rather than RNA as an intermediate. The upstream region to the mitochondria-derived sequence shows homology to part of the vacuolar H(+)-ATPase B subunit (V-ATPase B) gene. Isolation of a functional V-ATPase B cDNA and its comparison with the #21 cDNA reveal a number of nucleotide substitutions resulting in many translational stop codons in the #21 cDNA. This indicates that the #21 cDNA sequence is not functional. Analysis of genomic sequences shows the presence of five intron sequences in the #21 cDNA, whereas the functional V-ATPase B gene has 14 introns. Of these, three exons and their internal two introns are homologous to each other, suggesting a duplication event of V-ATPase B genomic DNA. The results of this investigation strongly suggest that the mitochondrial sequence was integrated in an antisense orientation into the pre-existing V-ATPase B pseudogene that can be transcribed and spliced. This represents a case of unsuccessful gene transfer from mitochondrion to the nucleus.

Transfer of rice mitochondrial ribosomal protein L6 gene to the nucleus: acquisition of the 5'-untranslated region via a transposable element

BackgroundThe mitochondria of contemporary organisms contain fewer genes than the ancestral bacteria are predicted to have contained. Because most of the mitochondrial proteins are encoded in the nucleus, the genes would have been transferred from the mitochondrion to the nucleus at some stage of evolution and they must have acquired cis-regulatory elements compatible with eukaryotic gene expression. However, most of such processes remain unknown.ResultsThe ribosomal protein L6 gene (rpl6) has been lost in presently-known angiosperm mitochondrial genomes. We found that each of the two rice rpl6 genes (OsRpl6-1 and OsRpl6-2) has an intron in an identical position within the 5-untranslated region (UTR), which suggests a duplication of the rpl6 gene after its transfer to the nucleus. Each of the predicted RPL6 proteins lacks an N-terminal extension as a mitochondrial targeting signal. Transient assays using green fluorescent protein indicated that their mature N-terminal coding regions contain the mitochondrial targeting information. Reverse transcription-PCR analysis showed that OsRpl6-2 expresses considerably fewer transcripts than OsRpl6-1. This might be the result of differences in promoter regions because the 5-noncoding regions of the two rpl6 genes differ at a point close to the center of the intron. There are several sequences homologous to the region around the 5-UTR of OsRpl6-1 in the rice genome. These sequences have characteristics similar to those of the transposable elements (TE) belonging to the PIF/Harbinger superfamily.ConclusionThe above evidences suggest a novel mechanism in which the 5-UTR of the transferred mitochondrial gene was acquired via a TE. Since the 5-UTRs and introns within the 5-UTRs often contain transcriptional and posttranscriptional cis-elements, the transferred rice mitochondrial rpl6 gene may have acquired its cis-element from a TE.

Detection of quantitative trait loci for heading traits in Brassica rapa using different heading types of Chinese cabbage

Summary Brassica species exhibit a wide diversity of leaf and floral morphologies.There are many cultivars of heading Chinese cabbage in East Asia, but the genetic mechanism of head formation remains unclear. In this study, we generated an F2 population derived from a cross between two Chinese cabbages of different heading type (i.e., the cylindrical-head type, ‘Chihili 70’, and the round-head type, inbred line ‘Y-54’). The F2 population was grown in 2010 and in 2011 to score morphological traits for head formation [i.e., HT, degree of head top leaf overlap; PW, plant fresh weight (FW); HW, head FW; HW:PW, head FW to plant FW ratio; HH, head height; HD, head diameter; HH:HD, head height to head diameter ratio; and HDS, head density].We constructed linkage maps in both years so that quantitative trait locus (QTL) analyses could be performed. Thirteen and ten QTLs were detected in 2010 and in 2011, respectively. QTL clusters for the different heading traits were found on linkage groups A1, A5, A7, and A8 in either one of the years, or in both years. Among these, four QTLs (for HT, HH:HD, HW, and HH) were detected in both years under different growth conditions, suggesting environmentally-stable QTLs for head formation in this F2 population of Chinese cabbage. Given that no overlap was found between the positions of the QTLs in previous reports and in the present study, QTLs for head formation might be cultivar-specific.

CHROMOSOMAL ASSIGNMENT OF RADISH LINKAGE GROUPS USING A COMPLETE SET OF DISOMIC RAPESEED-RADISH CHROMOSOME ADDITION LINES

Radish, Raphanus sativus L., is a vegetable with high economic importance, especially in Asia. A genetic map with 322 markers and a total length of 673 cM was anchored to chromosomes using a complete set of disomic rapeseed-radish chromosome addition lines. Eighteen SSRs and one CAPS marker, at least two for each linkage group, were assigned to the individual radish chromosomes. Three of the radish chromosomes (C, D and H) were characterized cytogenetically by fluorescence in situ hybridization (FISH). The anchored linkage map can be used for QTL analysis of morphological characters and other traits.