Biomedical subjects

Hiroyoshi Takano

Publications and source records attributed to Hiroyoshi Takano.

16 recordsLinked to original sources

PubMed2025

Transcriptomic and enzymological evidence for plastid peptidoglycan synthesis in the gymnosperm Picea abies.

It is understood that a cyanobacterium was the progenitor of plastids and that the biosynthesis of cell wall peptidoglycan was lost during chloroplast evolution. However, accumulated data, especially from the moss Physcomitrium patens, suggest that peptidoglycan remains essential for plastid division in some land plants. A fundamental set of peptidoglycan biosynthesis (Mur) genes has been identified in the genomes of these land plants, while many angiosperms no longer encode some core Mur genes, including a bifunctional penicillin-binding protein (PBP). Ten incomplete Mur genes were previously identified in the genome of the gymnosperm Picea abies but these could be pseudogenes or encode proteins that have been repurposed. For instance, mutant albino maize and Arabidopsis seedlings possess a defective UDP-N-acetylmuramoyl-l-alanyl-d-glutamate--2,6-diaminopimelate ligase (MurE), an intact MurE ligase being essential for peptidoglycan synthesis. In this study, we isolated a full set of cDNAs for peptidoglycan biosynthesis from P. abies. GFP fusion proteins with either P. abies (Pa)MurE or PaPBP were detected in chloroplasts. Cross-species complementation assays with PaMurE in Arabidopsis albino MurE mutants and Physcomitrium MurE chloroplast division mutants showed that the gymnosperm MurE completely rescued both mutant phenotypes. Enzymatic assay of recombinant PaMurE proteins revealed they catalyze the same reaction performed by their bacterial MurE homologs. Moreover, the expression of the PaPbp cDNA partially rescued the giant chloroplast phenotype in the moss Pbp knockout line. These results are consistent with the operation of a functional Mur gene set in the Norway spruce genome.

Peptidoglycan↗

PubMed2006

Genes for the peptidoglycan synthesis pathway are essential for chloroplast division in moss.

The general consensus is that a cyanobacterium phagocytosed by a host cell evolved into the plastids of red and green algae, land plants, and glaucophytes. In contrast to the plastids of glaucophytes, which retain a cyanobacterial-type peptidoglycan layer, no wall-like structures have been detected in plastids from other sources. Although the genome of Arabidopsis thaliana contains five genes that are essential for peptidoglycan synthesis, MurE, MurG, two genes for D-Ala-D-Ala ligase (Ddl), and the gene for translocase I (MraY), their functions have not been determined. We report that the moss Physcomitrella patens has nine homologous genes related to peptidoglycan biosynthesis: MurA, B, C, D, E, and F, Ddl, genes for the penicillin-binding protein Pbp, and dd-carboxypeptidase (Dac). Corroborating a computer prediction, analysis of the GFP fusion proteins with the N terminus of PpMurE or of PpPbp suggests that these proteins are located in the chloroplasts. Gene disruption of the PpMurE gene in P. patens resulted in the appearance of macrochloroplasts both in protonema and in leaf cells. Moreover, gene knockout of the P. patens Pbp gene showed inhibition of chloroplast division in this moss; however, no Pbp gene was found in A. thaliana.

Arabidopsis↗

PubMed2006

Reverse transcriptase-like sequences related to retrotransposon in a red alga, Porphyra yezoensis.

Four DNA fragments encoding a reverse transcriptase (RT)-like gene related to that of long terminal repeat (LTR) retrotransposons were isolated from the red alga Porphyra yezoensis by genomic PCR. Southern blot analysis suggested that one clone exists as a single copy per genome. Its full-length cDNA (PyRE2A) contained RT/RNase H-like sequences, which are most closely related to those of the Volvox LTR retrotransposon, although two stop codons were present within the RT region. We did not find any sequence related to LTR retrotransposons other than RT/RNase H in RyRE2A. These results indicate that PyRE2A is a single RT/RNase H-like gene and a defective progenitor of LTR retrotransposons.

Amino Acid Sequence↗

PubMed2005

Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos.

We report a new protocol for the stable transformation of Larix gmelinii. Thirty mature zygotic embryos precultured for 3 days on solid medium supplemented with benzyladenine were bombarded with plasmids pUC-GHG (GUS, HPT, and GFP genes) or pBI221-HPT (HPT and GUS genes). After a 2-month culture on selection medium, hygromycin-resistant calli appeared on the surfaces of the necrotic embryos. The frequencies of embryos with resistant calli were 18.4% and 17.4% in the transformations with pUC-GHG and pBI221-HPT DNA, respectively. More than 20 adventitious shoots formed from each of the transgenic calli. Of 17 elongated shoots selected for culturing on a rooting medium, five shoots rooted after 2 months. Expression of the GFP and GUS genes was detected in the resistant tissues by microscopic observations and by a histological GUS activity assay, respectively. PCR and Southern analysis confirmed the stable insertion of the introduced DNA into the genome.

Biolistics↗

PubMed2004

Efficient plant regeneration from suspension cells of Allium cepa L.

Plant regeneration from calli of three cultivars of Allium cepa (Senshuki, O.Pki and Shojovaka) was investigated. Callus was induced on four variations of BDS medium containing different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzyladenine (BAP). The regeneration frequency of calli of cvs. Senshuki and O.Pki subcultured on solid MS medium supplemented with BAP ranged from 50% to 80%; this frequency decreased to less than 30% after subculture in the dark in liquid BDS medium. By repeating the dark/light transitions of the culture protocol and by selecting for green cell clusters, we were able to increase the regeneration frequency to more than 80% in all three cultivars. These cell clusters maintained a high regeneration capacity in subsequent subcultures in the absence of light for 2 months. Most (97%) of the regenerated plantlets had a normal diploid karyotype (2 n=16) that was identical to that of the mother plants, although 3% of the regenerated plants of cv. Shojovaka had a tetraploid karyotype.

Cell Culture Techniques↗

PubMed2004

Chalcone synthase-like gene in the liverwort, Marchantia paleacea var. diptera.

A chalcone synthase (CHS)-like gene, MpCHSLK1, was isolated from liverwort, Marchantia paleacea var. diptera. Phylogenetic analysis revealed that MpCHSLK1 is closely related to stilbene synthase of the whisk fern, Psilotum nudum. Southern blot analysis using an MpCHSLK1 probe revealed that the gene belongs to a small gene family. Northern blot analysis indicated that CHS-like genes were expressed in either the mother plants or photoautotrophic cells. In photoautotrophic cells, the CHS-like genes were expressed light-dependently, and this expression was completely inhibited by the photosynthetic electron transport inhibitor, DCMU.

Acyltransferases↗

PubMed2004

Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D.

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

Actins↗

PubMed2004

Copper deficiency induced expression of Fe-superoxide dismutase gene in Matteuccia struthiopteris.

Iron-superoxide dismutase (Fe-SOD) activity was not detected in extracts from the leaves of ferns, Equisetum arvense and Matteuccia struthiopteris. To know why ferns lack Fe-SOD activity, the Fe-SOD like gene (MsFeSOD1) was isolated from M. struthiopteris and its expression was investigated with a focus on the metals Fe and Cu using the prothalli of the fern. The expression of MsFeSOD1 mRNA was induced by a deficiency of Cu, but Fe-SOD activity was not detected. The recombinant protein of MsFeSOD1 produced in E. coli showed Fe-SOD activity. These findings suggest that the fern Fe-SOD like gene was transcriptionally regulated by Cu but an additional mechanism is involved in the formation of an active enzyme.

Amino Acid Sequence↗

PubMed2004

Organelle nuclei in higher plants: structure, composition, function, and evolution.

Plant cells have two distinct types of energy-converting organelles: plastids and mitochondria. These organelles have their own DNAs and are regarded as descendants of endosymbiotic prokaryotes. The organelle DNAs associate with various proteins to form compact DNA-protein complexes, which are referred to as organelle nuclei or nucleoids. Various functions of organelle genomes, such as DNA replication and transcription, are performed within these compact structures. Fluorescence microscopy using the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole has played a pivotal role in establishing the concept of "organelle nuclei." This fluorochrome has also facilitated the isolation of morphologically intact organelle nuclei, which is indispensable for understanding their structure and composition. Moreover, development of an in vitro transcription?DNA synthesis system using isolated organelle nuclei has provided us with a means of measuring and analyzing the function of organelle nuclei. In addition to these morphological and biochemical approaches, genomics has also had a great impact on our ability to investigate the components of organelle nuclei. These analyses have revealed that organelle nuclei are not a vestige of the bacterial counterpart, but rather are a complex system established through extensive interaction between organelle and cell nuclear genomes during evolution. Extensive diversion or exchange during evolution is predicted to have occurred for several important structural proteins, such as major DNA-compacting proteins, and functional proteins, such as RNA and DNA polymerases, resulting in complex mechanisms to control the function of organelle genomes. Thus, organelle nuclei represent the most dynamic front of interaction between the three genomes (cell nuclear, plastid, and mitochondrial) constituting eukaryotic plant cells.

Cell Nucleus↗

PubMed2004

Photosynthetic electron transport differentially regulates the expression of superoxide dismutase genes in liverwort, Marchantia paleacea var. diptera.

Liverwort, Marchantia paleacea var. diptera, contains Mn-superoxide dismutase (Mn-SOD) in mitochondria, Fe-SOD in chloroplast and CuZn-SOD in cytosol. An Mn-SOD gene (MpMnSOD) was isolated from the liverwort. Using this clone together with the liverwort Fe-SOD and CuZn-SOD genes as probes, the expression of three SOD genes was investigated. Under heterotrophic conditions, the transcript of three SOD genes was accumulated light independently. On the other hand, under photoautotrophic conditions, the transcript levels of Fe-SOD and Mn-SOD increased in the light while that of CuZn-SOD decreased. The reverse occurred in the dark. In contrast to the transcript level, the activity of the three SODs was barely affected by light. The transcription inhibitor, cordycepin, inhibited either the light-promoted accumulation of Fe-SOD and Mn-SOD transcript or the light-induced reduction of the CuZn-SOD transcript. Photosynthetic electron transport inhibitors, DCMU and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, inhibited the photo-response in three SOD genes. These results suggest that the transcript abundance of three SOD genes in liverwort is regulated by photosynthetic electron transport but the mechanism regulating the transcript abundance of the CuZn-SOD gene is different from that of the Fe-SOD and Mn-SOD genes.

Amino Acid Sequence↗

PubMed2003

Glom is a novel mitochondrial DNA packaging protein in Physarum polycephalum and causes intense chromatin condensation without suppressing DNA functions.

Mitochondrial DNA (mtDNA) is packed into highly organized structures called mitochondrial nucleoids (mt-nucleoids). To understand the organization of mtDNA and the overall regulation of its genetic activity within the mt-nucleoids, we identified and characterized a novel mtDNA packaging protein, termed Glom (a protein inducing agglomeration of mitochondrial chromosome), from highly condensed mt-nucleoids of the true slime mold, Physarum polycephalum. This protein could bind to the entire mtDNA and package mtDNA into a highly condensed state in vitro. Immunostaining analysis showed that Glom specifically localized throughout the mt-nucleoid. Deduced amino acid sequence revealed that Glom has a lysine-rich region with proline-rich domain in the N-terminal half and two HMG boxes in C-terminal half. Deletion analysis of Glom revealed that the lysine-rich region was sufficient for the intense mtDNA condensation in vitro. When the recombinant Glom proteins containing the lysine-rich region were expressed in Escherichia coli, the condensed nucleoid structures were observed in E. coli. Such in vivo condensation did not interfere with transcription or replication of E. coli chromosome and the proline-rich domain was essential to keep those genetic activities. The expression of Glom also complemented the E. coli mutant lacking the bacterial histone-like protein HU and the HMG-boxes region of Glom was important for the complementation. Our results suggest that Glom is a new mitochondrial histone-like protein having a property to cause intense DNA condensation without suppressing DNA functions.

Amino Acid Sequence↗

PubMed2003

Inhibition of plastid division by ampicillin in the pteridophyte Selaginella nipponica Fr. et Sav.

We investigated the effect of the beta-lactam antibiotic, ampicillin, on plastid division in the pteridophyte Selaginella nipponica. Guard cells of plantlets treated with 1 mM ampicillin only often had one plastid, whereas guard cells of untreated plantlets had two to four plastids. We generated a S. nipponica cell culture system and used it to investigate the effects of ampicillin. Treatment with 1 mM ampicillin had no effect on cell division in culture. We classified cultured cells into four types based on the number of plastids they contained: one (Type I), two (Type II), three or four (Type III) and more than five (Type IV). After 3 d in culture, the percentage of each cell type (I-IV) was 29.5, 46.7, 20.9, and 1.9%, respectively. Subsequently, the percentage of Types III and IV increased gradually, reaching 61.9 and 11.4%, respectively, after 15 d in culture in the absence of ampicillin. When 1 mM ampicillin was added, there was a minimal increase in the number of Type III and IV cells, with high percentages of Type I and II cells (32.4 and 45.7%, respectively) after 15 d. These results suggest that ampicillin inhibits plastid division in S. nipponica.

Ampicillin↗

PubMed2003

Effects of antibiotics that inhibit the bacterial peptidoglycan synthesis pathway on moss chloroplast division.

Moss chloroplasts should prove useful for studying the cyanobacteria-derived system in chloroplasts. To determine the effects of antibiotics that inhibit bacterial peptidoglycan synthesis, the numbers of chloroplasts in treated Physcomitrella patens cells were counted. Ampicillin and D-cycloserine caused a rapid decrease in the number of chloroplasts per cell. Fosfomycin affected half of the cells, while vancomycin affected a few cells. Conversely, bacitracin had no effect. With the decrease in chloroplast number, macrochloroplasts appeared in antibiotic-treated cells. Removal of the antibiotics resulted in the recovery of chloroplast number, suggesting that the decrease in number was directly dependent on the antibiotic treatment. Microscopic observations showed that the decrease in the number of chloroplasts resulted from cell division without chloroplast division. These results suggest that enzymes derived from the bacterial peptidoglycan synthesis pathway are related to moss chloroplast division.

Ampicillin↗

PubMed2002

Characterization of a putative fusogen encoded in a mitochondrial plasmid of Physarum polycephalum.

The mitochondrial plasmid mF induces mitochondrial fusion in zygotes and during sporulation in the true slime mold Physarum polycephalum. There are nine open reading frames (ORFs) in the mF plasmid, and it has been suggested that ORF640 encodes the mitochondrial fusogen. We prepared antisera directed against the ORF640 protein (ORF640p) in rabbits, and used it to localize this protein in mitochondria. Western blot analysis showed that ORF640p was produced only in the mitochondria of mF(+) strains, i.e., in cells that carried the mF plasmid. Proteinase K treatment of mitochondria isolated from the mF(+) strain suggested that the C-terminus coiled-coil (CC) region of ORF640p was exported from the matrix to the cytosol. Digitonin treatment confirmed this localization. West-Western blot analysis suggested that the CC region of ORF640p formed multimers and could interfere with an unknown mitochondrial protein in normal mitochondria. These results suggest that ORF640p is related to fusion of the outer mitochondrial membrane.

Journal Article↗

PubMed2002

Evolution of rbcL group IA introns and intron open reading frames within the colonial Volvocales (Chlorophyceae).

Mobile group I introns sometimes contain an open reading frame (ORF) possibly encoding a site-specific DNA endonuclease. However, previous phylogenetic studies have not clearly deduced the evolutionary roles of the group I intron ORFs. In this paper, we examined the phylogeny of group IA2 introns inserted in the position identical to that of the chloroplast-encoded rbcL coding region (rbcL-462 introns) and their ORFs from 13 strains of five genera (Volvox, Pleodorina, Volvulina, Astrephomene, and Gonium) of the colonial Volvocales (Chlorophyceae) and a related unicellular green alga, Vitreochlamys. The rbcL-462 introns contained an intact or degenerate ORF of various sizes except for the Gonium multicoccum rbcL-462 intron. Partial amino acid sequences of some rbcL-462 intron ORFs exhibited possible homology to the endo/excinuclease amino acid terminal domain. The distribution of the rbcL-462 introns is sporadic in the phylogenetic trees of the colonial Volvocales based on the five chloroplast exon sequences (6021 bp). Phylogenetic analyses of the conserved intron sequences resolved that the G. multicoccum rbcL-462 intron had a phylogenetic position separate from those of other colonial volvocalean rbcL-462 introns, indicating the recent horizontal transmission of the intron in the G. multicoccum lineage. However, the combined data set from conserved intron sequences and ORFs from most of the rbcL-462 introns resolved robust phylogenetic relationships of the introns that were consistent with those of the host organisms. Therefore, most of the extant rbcL-462 introns may have been vertically inherited from the common ancestor of their host organisms, whereas such introns may have been lost in other lineages during evolution of the colonial Volvocales. In addition, apparently higher synonymous substitutions than nonsynonymous substitutions in the rbcL-462 intron ORFs indicated that the ORFs might evolve under functional constraint, which could result in homing of the rbcL-462 intron in cases of spontaneous intron loss. On the other hand, the presence of intact to largely degenerate ORFs of the rbcL-462 introns within the three isolates of Gonium viridistellatum and the rare occurrence of the ORF-lacking rbcL-462 intron suggested that the ORFs might degenerate to result in the spontaneous intron loss during a very short evolutionary time following the loss of the ORF function. Thus, the sporadic distribution of the rbcL-462 introns within the colonial Volvocales can be largely explained by an equilibrium between maintenance of the introns by the intron ORF and spontaneous loss of introns when the introns do not have a functional ORF.

Amino Acid Sequence↗

PubMed2002

Photosynthetic electron transport regulates the stability of the transcript for the protochlorophyllide oxidoreductase gene in the liverwort, Marchantia paleacea var. diptera.

The transfer of Marchantia paleacea var. diptera cells to darkness caused a reversible repression in the accumulation of transcript for a gene, por, encoding the NADPH: protochlorophyllide oxidoreductase (EC 1.3.1.33). The photosynthetic inhibitor DCMU and DBMIB repressed the accumulation in light. In the presence of transcription inhibitor cordycepin, not only incubation in the dark but also addition of DCMU or DBMIB in light stimulated the degradation of the por transcript. These findings suggest that photosynthetic electron transport is involved in regulating the stability of the por transcript.

Darkness↗