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Koh-ichi Kadowaki

Publications and source records attributed to Koh-ichi Kadowaki.

11 recordsLinked to original sources

The Rc and Rd genes are involved in proanthocyanidin synthesis in rice pericarp.

Different colors, such as purple, brown, red and white, occur in the pericarp of rice. Here, two genes affecting proanthocyanidin synthesis in red- and brown-colored rice were elucidated. Genetic segregation analysis suggested that the Rd and A loci are identical, and both encode dihydroflavonol-4-reductase (DFR). The introduction of the DFR gene into an Rcrd mutant resulted in red-colored rice, which was brown in the original mutant, demonstrating that the Rd locus encodes the DFR protein. Accumulation of proanthocyanidins was observed in the transformants by the introduction of the Rd gene into the rice Rcrd line. Protein blot analysis showed that the DFR gene was translated in seeds with alternative translation initiation. A search for the Rc gene, which encodes a transacting regulatory factor, was conducted using available DNA markers and the Rice Genome Automated Annotation System program. Three candidate genes were identified and cloned from a rice RcRd line and subsequently introduced into a rice rcrd line. Brown-colored seeds were obtained from transgenic plants by the introduction of a gene containing the basic helix-loop-helix (bHLH) motif, demonstrating that the Rc gene encodes a bHLH protein. Comparison of the Rc locus among rice accessions showed that a 14-bp deletion occurred only in the rc locus.

Amino Acid Sequence↗

Evidence for transit peptide acquisition through duplication and subsequent frameshift mutation of a preexisting protein gene in rice.

Many proteins synthesized in the cytosol are delivered to their appropriate compartments in the cell by specific targeting signals. Here, we provide new insight into the generation of the chloroplast-targeting signal (called the transit peptide) in rice. First, we identified the mitochondrial ribosomal protein L13 (mt rpl13) gene on chromosome 5. Downstream of the gene, we identified a DNA fragment of 266 bp: a segment within a duplication of mt rpl13. The duplicated region was transcribed and found to encode an open reading frame (ORF) of 160 amino acids (aa) (orf160). The orf160 gene comprises C-terminal 60 aa derived from the mt rpl13 gene and N-terminal 100 aa derived from another duplicated fragment of a pentatricopeptide repeat (ppr)564 gene that encodes 564 aa with ppr motifs on chromosome 1. Examination of the localization of the ORF160 protein tagged with green fluorescent protein (GFP) showed that it is targeted to the chloroplasts. As such, ORF160 clearly contains a transit peptide. Interestingly, this was translated from the alternative reading frame of the duplicated fragment of ppr564. To confirm this, the reading frame of the ppr564 gene was shifted according to that of the orf160 gene, and the frameshifted ppr564 sequence was fused to the gene for GFP. The expressed GFP-fused protein was also located in the chloroplasts. These results provide clear evidence for the generation of the transit peptide through duplication and subsequent frameshifting of a reading frame of a preexisting protein gene. We also demonstrate the importance of sequence redundancy and frameshift mutation in this evolutionary process.

Amino Acid Sequence↗

Promoter shuffling at a nuclear gene for mitochondrial RPL27. Involvement of interchromosome and subsequent intrachromosome recombinations.

The Reclinomonas americana mitochondrial genome contains a mitochondrial ribosomal protein L27 (rpl27) gene, whereas the rpl27 gene is absent from all plant mitochondrial genomes examined to date. This suggests that plant mitochondrial rpl27 genes have been transferred previously from the mitochondrial genome to the nuclear genome. A nuclear cDNA encoding mitochondrial RPL27 was identified in rice (Oryza sativa). Three similar sequences were identified: rpl27-1 and rpl27-2 on chromosome 8 and rpl27-3 on chromosome 4. Harr plot analysis suggests that they were generated by inter- and intrachromosomal duplications. Interestingly, the transcribed rpl27 gene (rpl27-1) acquired a promoter sequence that was derived from the rice spt16 (Osspt16) gene, the homolog of a global transcription factor in yeast (Saccharomyces cerevisiae) located downstream from the rpl27-3 sequence on chromosome 4, after inter- and intrachromosomal recombination. Reverse transcription-PCR and promoter assay revealed that the rpl27 mRNAs were mainly transcribed from rpl27-1. A repeat of seven nucleotides (AATAGTT) was identified at the junction of rpl27-1 and rpl27-2 on chromosome 8, and the same repeat was also identified at the 5' end of rpl27-2 and the 3' end of rpl27-1. This repeat (AATAGTT) contains the hot-spot sequence AGTT, which is preferentially recognized by topoisomerase I in wheat (Triticum aestivum) germ, suggesting the involvement of topoisomerase I in this recombination. We here report the example of promoter shuffling and show that this promoter shuffling resulted from a recent segmental duplication through inter- and intrachromosomal recombination events.

Amino Acid Sequence↗

Metabolic engineering of coenzyme Q by modification of isoprenoid side chain in plant.

Coenzyme Q (CoQ), an electron transfer molecule in the respiratory chain and a lipid-soluble antioxidant, is present in almost all organisms. Most cereal crops produce CoQ9, which has nine isoprene units. CoQ10, with 10 isoprene units, is a very popular food supplement. Here, we report the genetic engineering of rice to produce CoQ10 using the gene for decaprenyl diphosphate synthase (DdsA). The production of CoQ9 was almost completely replaced with that of CoQ10, despite the presence of endogenous CoQ9 synthesis. DdsA designed to express at the mitochondria increased accumulation of total CoQ amount in seeds.

Alkyl and Aryl Transferases↗

[Detection of fish DNA in ruminant feed by PCR amplification].

The Japanese Government has prohibited the use of seafood protein, as well as mammalian protein, in ruminant feed. There is an official method to detect meat and bone meal, but no method is yet available to detect fishmeal in ruminant feed. We tried to develop a suitable method to detect fishmeal in ruminant feed, similar to the official method "PCR detection of animal-derived DNA in feed". Our previously reported primers (fishcon5 and fishcon3-1) showed low sensitivity, so we designed new primers based on a DNA sequence from yellowfin tuna mitchondrial DNA. Among the primers, FM5 and FM3 specifically detected fish DNA (sardine, yellowfin tuna, skipjack tuna, chub mackerel, Pacific saury, salmon, rainbow trout, Japanese anchovy, codfish and Japanese horse mackerel) from fish meat, and did not amplify DNA from animals and plants. The sensitivity for detection of the presence of fishmeal in ruminant feed was 0.01-0.001%.

Animal Feed↗

Translocation of a 190-kb mitochondrial fragment into rice chromosome 12 followed by the integration of four retrotransposons.

A 190-kb mitochondrial DNA sequence interrupted by seven foreign DNA segments was identified in rice chromosome 12. This fragment is the largest mitochondrial fragment translocated into the rice nuclear genome. The sequence is composed of a 190-kb segment of mitochondrial origin corresponding to 38.79% of the mitochondrial genome, 45 kb comprising four segments of retrotransposon origin, and 13 kb comprising three segments of unknown origin. The 190-kb sequence shows more than 99.68% similarity to the current mitochondrial sequence, suggesting that its integration into the nucleus was quite recent. Several sequences in the 190-kb segment have been rearranged relative to the current mitochondrial sequence, suggesting that the past and present arrangements of the mitochondrial genome differ. The four retrotransposons show no mutual sequence similarity and are integrated into different locations, suggesting that their integration events were independent, frequent, and quite recent. A fragment of the mitochondrial genome present in the nuclear genome, such as the 248-kb sequence characterized in this study, is a good relic with which to investigate the past mitochondrial genome structure and the behavior of independent retrotransposons during evolution.

Biological Transport↗

Recognition and processing of a nuclear-encoded polyprotein precursor by mitochondrial processing peptidase.

The nuclear-encoded protein RPS14 (ribosomal protein S14) of rice mitochondria is synthesized in the cytosol as a polyprotein consisting of a large N-terminal domain comprising preSDHB (succinate dehydrogenase B precursor) and the C-terminal RPS14. After the preSDHB-RPS14 polyprotein is transported into the mitochondrial matrix, the protein is processed into three peptides: the N-terminal prepeptide, the SDHB domain and the C-terminal mature RPS14. Here we report that the general MPP (mitochondrial processing peptidase) plays an essential role in processing of the polyprotein. Purified yeast MPP cleaved both the N-terminal presequence and the connector region between SDHB and RPS14. Moreover, the connector region was processed more rapidly than the presequence. When the site of cleavage between SDHB and RPS14 was determined, it was located in an MPP processing motif that has also been shown to be present in the N-terminal presequence. Mutational analyses around the cleavage site in the connector region suggested that MPP interacts with multiple sites in the region, possibly in a similar manner to the interaction with the N-terminal presequence. In addition, MPP preferentially recognized the unfolded structure of preSDHB-RPS14. In mitochondria, MPP may recognize the stretched polyprotein during passage of the precursor through the translocational apparatus in the inner membrane, and cleave the connecting region between the SDHB and RPS14 domains even before processing of the presequence.

Cell Nucleus↗

Phylogenetic analysis of Oryza species, based on simple sequence repeats and their flanking nucleotide sequences from the mitochondrial and chloroplast genomes.

Simple sequence repeats (SSR) and their flanking regions in the mitochondrial and chloroplast genomes were sequenced in order to reveal DNA sequence variation. This information was used to gain new insights into phylogenetic relationships among species in the genus Oryza. Seven mitochondrial and five chloroplast SSR loci equal to or longer than ten mononucleotide repeats were chosen from known rice mitochondrial and chloroplast genome sequences. A total of 50 accessions of Oryza that represented six different diploid genomes and three different allopolyploid genomes of Oryza species were analyzed. Many base substitutions and deletions/insertions were identified in the SSR loci as well as their flanking regions. Of mononucleotide SSR, G (or C) repeats were more variable than A (or T) repeats. Results obtained by chloroplast and mitochondrial SSR analyses showed similar phylogenetic relationships among species, although chloroplast SSR were more informative because of their higher sequence diversity. The CC genome is suggested to be the maternal parent for the two BBCC genome species (O. punctata and O. minuta) and the CCDD species O. latifolia, based on the high level of sequence conservation between the diploid CC genome species and these allotetraploid species. This is the first report of phylogenetic analysis among plant species, based on mitochondrial and chloroplast SSR and their flanking sequences.

Base Sequence↗

Complete nucleotide sequence of the sugarcane (Saccharum officinarum) chloroplast genome: a comparative analysis of four monocot chloroplast genomes.

The complete nucleotide sequence of the chloroplast genome of sugarcane (Saccharum officinarum) has been determined. It is a circular double-stranded DNA molecule, 141,182 bp in size, and is composed of a large single copy of 83,048 bp, a small single copy of 12,544 bp, and a pair of inverted repeat regions of 22,795 bp each. A comparative analysis among monocots showed that the sugarcane chloroplast genome was very similar to maize but not to rice or wheat. Between sugarcane and maize at the rps16-trnQ (UUG) region, however, a length polymorphism was identified. With regard to insertions/deletions equal to or longer than 5 bp, a total of 53 insertion and 31 deletion events were identified in the sugarcane chloroplast genome. Of the 84 loci identified, a pair of direct repeat sequences was located side by side in a tandem fashion in 47 loci (56.0%). A recombination event during plant evolution is discussed at two sites between the sugarcane and tobacco chloroplast genomes.

Base Sequence↗

Rpp16 and Rpp17, from a common origin, have different protein characteristics but both genes are predominantly expressed in rice phloem tissues.

The genes for two types of rice phloem protein (RPP16 and RPP17) were isolated and characterized. Conservation of five exon sizes as well as splicing positions between the two genes suggest that either RPP16 or RPP17 is a resultant of gene duplication. By protein blot analysis, RPP16 and RPP17 proteins were specifically detected in soluble and insoluble fractions of a crude extract of rice plants, respectively, suggesting that these proteins play different roles in individual cells. The expression of Rpp16 and Rpp17 was monitored by the beta-glucuronidase (gusA) reporter-gene method. Rpp16-gusA and Rpp17-gusA were expressed preferentially in the phloem tissues from different parts of the plant, but almost no GUS staining was observed in the rest of the tissues. In roots of both constructs, interestingly, stronger GUS-accumulation was detected in younger vascular tissues than in aged vascular tissues. In situ hybridization also showed that Rpp17 was more strongly expressed in vascular tissues of tiller buds. These results suggest that transcript of these genes was more abundant in young tissues. The presence of two copies of the gene in higher plants, from a common origin, which have different protein characteristics, indicates that evolutionary diversification might have occurred in the gene function.

Amino Acid Sequence↗

Molecular phylogeny of the genus Hordeum using three chloroplast DNA sequences.

The genus Hordeum consists of three cytotypes (2x, 4x, and 6x). Its reproductive isolation has been incomplete between closely related species and hence the genetic relationship is reticulate and complex. We used 32 taxa of Hordeum and three chloroplast DNA sequences, matK, atpB-rbcL, and trnL-trnF in the current study. Molecular phylogenetic analysis based on sequence data of the three chloroplast DNA regions clearly demonstrated genetic relationships among taxa and origin of polypoids. The formation of H. secalinum likely involved hybridization between Hordeum marinum subsp. marinum and a Eurasian diploid possessing the H genome. The formation of hexaploid Hordeum brachyantherum involved hybridization between tetraploid H. brachyantherum and diploid H. marinum subsp. gussoneanum. The formation of three tetraploids, H. brachyantherum, Hordeum jubatum, and Hordeum guatemalense, probably involved hybridization between H. brachyantherum subsp. californicum and an altered H genome diploid. The formation of Hordeum arizonicum involved the two taxa Hordeum pusillum and H. jubatum.

Base Sequence↗