Researcher Database


KUMAZAWA Yoshinori

FacultyGraduate School of Natural Sciences Division of Biological Science
Last Updated :2020/01/09

Researcher Profile and Settings

Profile & Settings

    Research funding number:60221941


  •   1982 04  - 1984 03 , University of Tokyo, Faculty of Engineering, Department of Industrial Chemistry
  •   1984 04  - 1986 03 , University of Tokyo, Graduate School of Engineering, Master of Engineering
  •   1986 04  - 1989 12 , University of Tokyo, Graduate School of Engineering, Doctor of Engineering (Ph. D)


  • Ph. D, University of Tokyo

Association Memberships

  • Society of Evolutionary Studies, Japan
  • Palaeontological Society of Japan
  • The Molecular Biology Society of Japan

Research Activities

Research Areas

  • Basic biology, Biodiversity/Systematics
  • Basic biology, Evolutionary biology
  • Genome science, Genome biology
  • Conservation of biological resources, Conservation of biological resources

Research Interests

    Mitochondrion, Molecular Phylogeny, Biological Diversity, Historical Biogeography, tRNA, High-throughput sequencing, DNA Barcoding

Published Papers

  • Karyotype analysis of four blind snake species (Reptilia: Squamata: Scolecophidia) and karyotypic changes in Serpentes., Kazumi Matsubara, Yoshinori Kumazawa, Hidetoshi Ota, Chizuko Nishida and Yoichi Matsuda, Cytogenetic and Genome Research, 157, (1-2) 98 - 106, 04 , Refereed, Invited
  • DNA barcoding of freshwater molluscs in Nagoya, Yoshinori Kumazawa, Mieko Suzuki-Matsubara, Yuri Yokoyama, Tadahiro Teramoto, Sachio Murase, Kenichiro Nasu, Yao Sun, Akihiko Moriyama and Motohiro Kawase, Bulletin of Nagoya Biodiversity Center, 6, 1 - 14, 03 , Refereed
  • Intraspecific variations of mitochondrial DNA sequences in Stereophaedusa japonica from central and western Japan and phylogenetic position of newly discovered specimens from Nagoya, central Japan., Motohiro Kawase, Kazuhisa Nishio, Mieko Matsubara-Suzuki, Takashi Ichihara, Akihiko Moriyama, Yoshinori Kumazawa, Bulletin of Nagoya Biodiversity Center, 5, 11 - 22, 03 , Refereed
  • The complete mitochondrial DNA sequence of Pectenocypris sp. (Actinopterygii: Cyprinidae) from Serkap River, Sumatra, Indonesia, Dwi Atminarso, Arif Wibowo, Wahyu Endra Kusuma, Eko Prianto, Harald Ahnelt, Anti Vasemägi, Yoshinori Kumazawa, Mitochondrial DNA Part B: Resources, 3, (1) 122 - 124, 01 , Refereed, The whole mitochondrial genome of a small cyprinid freshwater fish Pectenocypris sp. collected from Serkap River, Central Sumatra, Indonesia was sequenced. This mitochondrial genome consisted of 16,589 bp and included 37 genes in the same order as in many other vertebrates including the human. Phylogenetic analysis suggested that this taxon clusters with Boraras maculatus among several Rasbora species.
  • Variation and evolution of polyadenylation profiles in sauropsid mitochondrial mRNAs as deduced from the high-throughput RNA sequencing, Yao Sun, Masaki Kurisaki, Yasuyuki Hashiguchi, Yoshinori Kumazawa, BMC Genomics, 18, (1) , 08 , Refereed, Background: Genes encoded in vertebrate mitochondrial DNAs are transcribed as a polycistronic transcript for both strands, which is later processed into individual mRNAs, rRNAs and tRNAs, followed by modifications, such as polyadenylation at the 3' end of mRNAs. Although mechanisms of the mitochondrial transcription and RNA processing have been extensively studied using some model organisms, structural variability of mitochondrial mRNAs across different groups of vertebrates is poorly understood. We conducted the high-throughput RNA sequencing to identify major polyadenylation sites for mitochondrial mRNAs in the Japanese grass lizard, Takydromus tachydromoides and compared the polyadenylation profiles with those identified similarly for 23 tetrapod species, featuring sauropsid taxa (reptiles and birds). Results: As compared to the human, a major polyadenylation site for the NADH dehydrogenase subunit 5 mRNA of the grass lizard was located much closer to its stop codon, resulting in considerable truncation of the 3' untranslated region for the mRNA. Among the other sauropsid taxa, several distinct polyadenylation profiles from the human counterpart were found for different mRNAs. They included various truncations of the 3' untranslated region for NADH dehydrogenase subunit 5 mRNA in four taxa, bird-specific polyadenylation of the light-strand-transcribed NADH dehydrogenase subunit 6 mRNA, and the combination of the ATP synthase subunit 8/6 mRNA with a neighboring mRNA into a tricistronic mRNA in the side-necked turtle Pelusios castaneus. In the last case of P. castaneus, as well as another example for NADH dehydrogenase subunit 1 mRNAs of some birds, the association between the polyadenylation site change and the gene overlap was highlighted. The variations in the polyadenylation profile were suggested to have arisen repeatedly in diverse sauropsid lineages. Some of them likely occurred in response to gene rearrangements in the mitochondrial DNA but the others not. Conclusions: These results demonstrate structural variability of mitochondrial mRNAs in sauropsids. The efficient and comprehensive characterization of the mitochondrial mRNAs will contribute to broaden our understanding of their structural and functional evolution.
  • Complete mitogenome sequence of rasbora argyrotaenia (Actinopterygii: Cyprinidae), Wahyu Endra Kusuma, Pratama Deffi Samuel, Dewa Gede Raka Wiadnya, Anik Martinah Hariati, Yoshinori Kumazawa, Mitochondrial DNA Part B: Resources, 2, (2) 373 - 374, 07 , Refereed, The mitochondrial genome of a small freshwater fish Rasbora argyrotaenia from Java Island, Indonesia, was completely sequenced. This mitochondrial genome had 16,740 bp in length and consisted of 37 genes in the typical vertebrate mitochondrial gene arrangement. Phylogenetic analysis showed that R. argyrotaenia is more closely related to R. borapetensis than to other Javanese rasboras, R. aprotaenia and R. lateristriata.
  • DNA barcoding of weevils from Aichi and neighboring prefectures centering on Nagoya City in Japan., Syoji Inoue, Yoshinori Kumazawa, Bulletin of Nagoya Biodiversity Center, 4, 23 - 29, 03 , Refereed
  • Molecular phylogeny and historical biogeography of the Indonesian freshwater fish Rasbora lateristriata species complex (Actinopterygii: Cyprinidae): Cryptic species and west-to-east divergences, Wahyu Endra Kusuma, Sahat Ratmuangkhwang, Yoshinori Kumazawa, Molecular Phylogenetics and Evolution, 105, 212 - 223, 12 , Refereed, Rasbora lateristriata is a primary freshwater fish described from Java Island of Indonesia but its taxonomy, phylogeny, and distributional boundary have not been fully studied. Rasbora baliensis was described as a species endemic to Balinese lakes but its taxonomic status has been controversial in relation to R. lateristriata. Here, we collected Rasbora fishes from various freshwater localities of Java Island, as well as five neighboring islands to conduct molecular and morphological analyses on their phylogenetic relationships. Both molecular analyses using two mitochondrial and two nuclear gene sequences and morphological analyses featuring the body color pattern consistently support that the currently recognized R. lateristriata forms a species complex including at least four major lineages that possibly represent different species. In one of the major lineages, Balinese individuals cluster with those from East Javanese, Lombok and Sumbawa localities, calling for taxonomic revision on R. baliensis. The other three major lineages occur in distinct regions of central, west-central, and western Java and they can be clearly distinguished by the combination of pigmentation patterns in the basicaudal blotch and the supra anal pigment. Our molecular phylogeny suggests west-to-east divergences of the R. lateristriata species complex in Java Island from the late Miocene to Plio-Pleistocene before it finally crossed Wallace's Line, colonizing Lombok and Sumbawa Islands very recently.
  • Complete mitochondrial genome sequences of two Indonesian rasboras (Rasbora aprotaenia and Rasbora lateristriata), Wahyu Endra Kusuma, Yoshinori Kumazawa, Mitochondrial DNA Part A: DNA Mapping, Sequencing, and Analysis, 27, (6) 4222 - 4223, 11 , Refereed, Complete mitochondrial genome sequences were determined for two Indonesian freshwater fishes, Rasbora aprotaenia and Rasbora lateristriata. These genomes are 16,541 bp and 16,539 bp in length, respectively and encode 37 genes in the typical vertebrate gene arrangement. Phylogenetic analyses supported a view that these species are very closely related to each other.
  • Sex chromosome evolution in snakes inferred from divergence patterns of two gametologous genes and chromosome distribution of sex chromosome-linked repetitive sequences., Kazumi Matsubara, Chizuko Nishida, Yoichi Matsuda and Yoshinori Kumazawa, Zoological Letters, 2, 08 , Refereed
  • Genetic diversity of Hynobius nebulosus in Nagoya City and its neighboring regions revealed using mitochondrial DNA sequences., Takeshi Fujitani, Morihiro Notohara, Yoshinori Kumazawa, Bulletin of the Herpetological Society of Japan, 2016, (1) 1 - 12, 05 , Refereed
  • DNA barcoding of japanese click beetles (Coleoptera, Elateridae), Yuichi Oba, Hitoo Ôhira, Yukio Murase, Akihiko Moriyama, Yoshinori Kumazawa, PLoS ONE, 10, (1) , 01 , Refereed, Click beetles (Coleoptera: Elateridae) represent one of the largest groups of beetle insects. Some click beetles in larval form, known as wireworms, are destructive agricultural pests. Morphological identification of click beetles is generally difficult and requires taxonomic expertise. This study reports on the DNA barcoding of Japanese click beetles to enable their rapid and accurate identification. We collected and assembled 762 cytochrome oxidase subunit I barcode sequences from 275 species, which cover approximately 75% of the common species found on the Japanese main island, Honshu. This barcode library also contains 20 out of the 21 potential pest species recorded in Japan. Our analysis shows that most morphologically identified species form distinct phylogenetic clusters separated from each other by large molecular distances. This supports the general usefulness of the DNA barcoding approach for quick and reliable identification of Japanese elaterid species for environmental impact assessment, agricultural pest control, and biodiversity analysis. On the other hand, the taxonomic boundary in dozens of species did not agree with the boundary of barcode index numbers (a criterion for sequence-based species delimitation). These findings urge taxonomic reinvestigation of these mismatched taxa.
  • Molecular phylogeny and biogeography of air sac catfishes of the Heteropneustes fossilis species complex (Siluriformes: Heteropneustidae), Sahat Ratmuangkhwang, Prachya Musikasinthorn, Yoshinori Kumazawa, Molecular Phylogenetics and Evolution, 79, 82 - 91, 10 , Refereed, The air sac catfish, Heteropneustes fossilis (Siluriformes: Heteropneustidae), is widely distributed in freshwaters of the Indian subcontinent and mainland southeast Asia. No comprehensive molecular studies that cover the broad distributional areas have been carried out to date. Here, we conducted molecular phylogenetic analyses using both mitochondrial and nuclear gene sequences to suggest that the Heteropneustes fossilis species complex consists of three clades that may potentially be separate species with distinct geographical distribution (southeast Asia, northeastern India, and southwestern India). The first and second clades are more closely related to each other than they are to the third clade. Within the first clade there is a basal divergence of a subclade consisting of individuals from the Upper Irrawaddy River basin of Myanmar, which share some morphological traits with members of the Indian clades. Our molecular and morphological data are congruent with hypotheses that the Early-Middle Miocene disconnection between the paleo-Tsangpo River and the Irrawaddy River caused the vicariant divergence between southeast Asian and northeastern Indian clades, and that the southeast Asian Heteropneustes originated from the Upper Irrawaddy.
  • Gene rearrangements in gekkonid mitochondrial genomes with shuffling, loss, and reassignment of tRNA genes, Yoshinori Kumazawa, Saaya Miura, Chiemi Yamada, Yasuyuki Hashiguchi, BMC Genomics, 15, (1) , 01 , Refereed, Background: Vertebrate mitochondrial genomes (mitogenomes) are 16-18 kbp double-stranded circular DNAs that encode a set of 37 genes. The arrangement of these genes and the major noncoding region is relatively conserved through evolution although gene rearrangements have been described for diverse lineages. The tandem duplication-random loss model has been invoked to explain the mechanisms of most mitochondrial gene rearrangements. Previously reported mitogenomic sequences for geckos rarely included gene rearrangements, which we explore in the present study. Results: We determined seven new mitogenomic sequences from Gekkonidae using a high-throughput sequencing method. The Tropiocolotes tripolitanus mitogenome involves a tandem duplication of the gene block: tRNAArg, NADH dehydrogenase subunit 4L, and NADH dehydrogenase subunit 4. One of the duplicate copies for each protein-coding gene may be pseudogenized. A duplicate copy of the tRNAArggene appears to have been converted to a tRNAGln gene by a C to T base substitution at the second anticodon position, although this gene may not be fully functional in protein synthesis. The Stenodactylus petrii mitogenome includes several tandem duplications of tRNALeugenes, as well as a translocation of the tRNAAla gene and a putative origin of light-strand replication within a tRNA gene cluster. Finally, the Uroplatus fimbriatus and U. ebenaui mitogenomes feature the apparent loss of the tRNAGlugene from its original position. Uroplatus fimbriatus appears to retain a translocated tRNAGlugene adjacent to the 5' end of the major noncoding region. Conclusions: The present study describes several new mitochondrial gene rearrangements from Gekkonidae. The loss and reassignment of tRNA genes is not very common in vertebrate mitogenomes and our findings raise new questions as to how missing tRNAs are supplied and if the reassigned tRNA gene is fully functional. These new examples of mitochondrial gene rearrangements in geckos should broaden our understanding of the evolution of mitochondrial gene arrangements.
  • Intra-genomic GC heterogeneity in sauropsids: Evolutionary insights from cDNA mapping and GC3 profiling in snake, Kazumi Matsubara, Shigehiro Kuraku, Hiroshi Tarui, Osamu Nishimura, Chizuko Nishida, Kiyokazu Agata, Yoshinori Kumazawa, Yoichi Matsuda, BMC Genomics, 13, (1) , 11 , Refereed, Background: Extant sauropsids (reptiles and birds) are divided into two major lineages, the lineage of Testudines (turtles) and Archosauria (crocodilians and birds) and the lineage of Lepidosauria (tuatara, lizards, worm lizards and snakes). Karyotypes of these sauropsidan groups generally consist of macrochromosomes and microchromosomes. In chicken, microchromosomes exhibit a higher GC-content than macrochromosomes. To examine the pattern of intra-genomic GC heterogeneity in lepidosaurian genomes, we constructed a cytogenetic map of the Japanese four-striped rat snake (Elaphe quadrivirgata) with 183 cDNA clones by fluorescence in situ hybridization, and examined the correlation between the GC-content of exonic third codon positions (GC3) of the genes and the size of chromosomes on which the genes were localized.Results: Although GC3 distribution of snake genes was relatively homogeneous compared with those of the other amniotes, microchromosomal genes showed significantly higher GC3 than macrochromosomal genes as in chicken. Our snake cytogenetic map also identified several conserved segments between the snake macrochromosomes and the chicken microchromosomes. Cross-species comparisons revealed that GC3 of most snake orthologs in such macrochromosomal segments were GC-poor (GC3 < 50%) whereas those of chicken orthologs in microchromosomes were relatively GC-rich (GC3 ≥ 50%).Conclusion: Our results suggest that the chromosome size-dependent GC heterogeneity had already occurred before the lepidosaur-archosaur split, 275 million years ago. This character was probably present in the common ancestor of lepidosaurs and but lost in the lineage leading to Anolis during the diversification of lepidosaurs. We also identified several genes whose GC-content might have been influenced by the size of the chromosomes on which they were harbored over the course of sauropsid evolution. © 2012 Matsubara et al.; licensee BioMed Central Ltd.
  • Characterization of squamate olfactory receptor genes and their transcripts by the high-throughput sequencing approach, Yuki Dehara, Yasuyuki Hashiguchi, Kazumi Matsubara, Tokuma Yanai, Masahito Kubo, Yoshinori Kumazawa, Genome Biology and Evolution, 4, (4) 602 - 616, 09 , Refereed, The olfactory receptor (OR) genes represent the largest multigene family in the genome of terrestrial vertebrates. Here, the high-throughput next-generation sequencing (NGS) approach was applied to characterization of OR gene repertoires in the green anole lizard Anolis carolinensis and the Japanese four-lined ratsnake Elaphe quadrivirgata. Tagged polymerase chain reaction (PCR) products amplified from either genomic DNA or cDNA of the two species were used for parallel pyrosequencing, assembling, and screening for errors in PCR and pyrosequencing. Starting from the lizard genomic DNA, we accurately identified 56 of 136 OR genes that were identified from its draft genome sequence. These recovered genes were broadly distributed in the phylogenetic tree of vertebrate OR genes without severe biases toward particular OR families. Ninety-six OR genes were identified from the ratsnake genomic DNA, implying that the snake has more OR gene loci than the anole lizard in response to an increased need for the acuity of olfaction. This view is supported by the estimated number of OR genes in the Burmese python's draft genome (∼280), although squamates may generally have fewer OR genes than terrestrial mammals and amphibians. The OR gene repertoire of the python seems unique in that many class I OR genes are retained. The NGS approach also allowed us to identify candidates of highly expressed and silent OR gene copies in the lizard's olfactory epithelium. The approach will facilitate efficient and parallel characterization of considerable unbiased proportions of multigene family members and their transcripts from nonmodel organisms. © The Author(s) 2012.
  • Mitochondrial genomes of two African geckos of genus Hemitheconyx (Squamata: Eublepharidae), Pierre Jonniaux, Yasuyuki Hashiguchi, Yoshinori Kumazawa, Mitochondrial DNA, 23, (4) 278 - 279, 08 , Refereed, Mitochondrial genomes of two eyelid geckos (Hemitheconyx caudicinctus and Hemitheconyx taylori) were sequenced. Although these genomes conserve a typical vertebrate gene organization, tRNAGlngene of the former appears to have been pseudogenized. A very extensive RNA editing may restore its function in the RNA level or a functional tRNAGlnencoded in the nuclear chromosome may be imported into mitochondria. © 2012 Informa UK, Ltd.
  • Mitochondrial DNA variability within Uromastyx ornata philbyi (Agamidae: Squamata) from southwestern Saudi Arabia, Sayed A.M. Amer, Mohamed M. Ahmed, Thomas M. Wilms, Mohammed Shobrak, Yoshinori Kumazawa, Comparative and Functional Genomics, 2012, 02 , Refereed, Approximately 2.4kbp of mitochondrial DNA was sequenced from 9 individuals of Uromastyx ornata philbyi originating from Taif, Namas, Al-Baha, and Jazan in southwestern Saudi Arabia. The sequenced regions cover eight tRNA genes (tRNAGln, tRNAIle, tRNAMet, tRNATrp, tRNAAla, tRNAAsn, tRNACys, and tRNATyr) and two protein-coding genes (NADH dehydrogenase subunit 2 and cytochrome b). U. ornata philbyi had an insertion of 170bp length between tRNAGlnand tRNAIlegenes. The first 128bp of this insertion was similar to the one identified earlier in U. ornata ornata and can be folded into a stem-and-loop structure, which was less stable in U. ornata philbyi than in U. ornata ornata, or the second tRNA Gln gene. The next 42bp of the insertion was unique in U. ornata philbyi and additionally retained a stable stem-and-loop structure. Most base substitutions found in the sequenced genes were synonymous transitions rather than transversions. Tree analyses supported the sister group relationship between the two U. ornata subspecies and divided U. ornata philbyi into two groups: Taif+Namas group in the east of Sarawat and Al-Baha+Jazan group in the west of Sarawat. These molecular data are in agreement with current classification of U. ornata. © 2012 Sayed A. M. Amer et al.
  • Preliminary molecular variability within uromastyx aegyptia microlepis (reptilia: Agamidae) inhabiting Saudi Arabia, Sayed A M Amer, Metwally M. Montaser, Yoshinori Kumazawa, World Applied Sciences Journal, 12, (11) 1955 - 1961, 12 , Refereed, Approximately 2229 nucleotides representing partial 12S ribosomal RNA, complete NADH dehydrogenase subunit 2 and partial cytochrome b genes from mitochondrial DNA and partial 18S nuclear gene were sequenced in this study. These data were used to investigate the genetic variation within Uromastyx aegyptia microlepis from Saudi Arabia. Nine individuals inhabiting four localities were studied and these localities were two from the west (Tabok and Taif) and two from the east (Riyadh and Dammam). The mtDNA sequences showed six substitutions (2 transversions and 4 transitions) among the different haplotypes. Two transitions among them were non-synonymous showing different amino acids. The mutations that have been found in 18S gene were six deletions in the samples of Taif possibly due to habitat difference. The observed genetic structure of Uromastyx a. microlepis has management implications for the conservation of this subspecies. Until more information is available, we recommend that Uromastyx in Saudi Arabia should not be hunted severely and should receive more attention from conservational point of view. © IDOSI Publications, 2011.
  • Mitochondrial genomes of acrodont lizards: Timing of gene rearrangements and phylogenetic and biogeographic implications, Yasuhisa Okajima, Yoshinori Kumazawa, BMC Evolutionary Biology, 10, (1) , 05 , Refereed, Background. Acrodonta consists of Agamidae and Chamaeleonidae that have the characteristic acrodont dentition. These two families and Iguanidae sensu lato are members of infraorder Iguania. Phylogenetic relationships and historical biogeography of iguanian lizards still remain to be elucidated in spite of a number of morphological and molecular studies. This issue was addressed by sequencing complete mitochondrial genomes from 10 species that represent major lineages of acrodont lizards. This study also provided a good opportunity to compare molecular evolutionary modes of mitogenomes among different iguanian lineages. Results. Acrodontan mitogenomes were found to be less conservative than iguanid counterparts with respect to gene arrangement features and rates of sequence evolution. Phylogenetic relationships were constructed with the mitogenomic sequence data and timing of gene rearrangements was inferred on it. The result suggested highly lineage-specific occurrence of several gene rearrangements, except for the translocation of the tRNAPro gene from the 5' to 3' side of the control region, which likely occurred independently in both agamine and chamaeleonid lineages. Phylogenetic analyses strongly suggested the monophyly of Agamidae in relation to Chamaeleonidae and the non-monophyly of traditional genus Chamaeleo within Chamaeleonidae. Uromastyx and Brookesia were suggested to be the earliest shoot-off of Agamidae and Chamaeleonidae, respectively. Together with the results of relaxed-clock dating analyses, our molecular phylogeny was used to infer the origin of Acrodonta and historical biogeography of its descendant lineages. Our molecular data favored Gondwanan origin of Acrodonta, vicariant divergence of Agamidae and Chamaeleonidae in the drifting India-Madagascar landmass, and migration of the Agamidae to Eurasia with the Indian subcontinent, although Laurasian origin of Acrodonta was not strictly ruled out. Conclusions. We detected distinct modes of mitogenomic evolution among iguanian families. Agamidae was highlighted in including a number of lineage-specific mitochondrial gene rearrangements. The mitogenomic data provided a certain level of resolution in reconstructing acrodontan phylogeny, although there still remain ambiguous relationships. Our biogeographic implications shed a light on the previous hypothesis of Gondwanan origin of Acrodonta by adding some new evidence and concreteness. © 2010 Okajima and Kumazawa; licensee BioMed Central Ltd.
  • Mitogenomic perspectives into iguanid phylogeny and biogeography: Gondwanan vicariance for the origin of Madagascan oplurines, Yasuhisa Okajima, Yoshinori Kumazawa, Gene, 441, (1-2) 28 - 35, 07 , Refereed, Complete or nearly complete nucleotide sequences of mitochondrial genomes (mtDNAs) were determined from eight species which, together with previous mtDNA data for two other taxa, cover most subfamilies of Iguanidae sensu lato. These iguanid mtDNAs were found to be rather conservative with respect to gene arrangements and molecular evolutionary rates, which contrasts with mtDNAs of Acrodonta (Agamidae and Chamaeleonidae) in which several gene rearrangements and highly accelerated molecular evolutionary rates have been known. Phylogenetic analyses consistently suggested the earliest shoot-off of a Malagasy subfamily Oplurinae and an affinity of Polychrotinae and Tropidurinae sensu stricto. However, even with the ample molecular characters derived from complete mtDNA sequences, phylogenetic relationships between iguanid subfamilies were poorly resolved in general, presumably due to the rapid ancient cladogenesis. Divergence time estimation without assuming the molecular clock suggested the Late Triassic/Early Jurassic divergence of Iguanidae from acrodonts and the Middle/Late Jurassic divergence of Oplurinae from the other iguanids. Together with geological and paleontological evidence, these results led us to propose Gondwanan vicariance for the origin of Malagasy oplurines without invoking a land bridge connection between South America/Antarctica and drifting Madagascar/India. © 2008 Elsevier B.V. All rights reserved.
  • The historical biogeography of the freshwater knifefishes using mitogenomic approaches: A Mesozoic origin of the Asian notopterids (Actinopterygii: Osteoglossomorpha), Jun G. Inoue, Yoshinori Kumazawa, Masaki Miya, Mutsumi Nishida, Molecular Phylogenetics and Evolution, 51, (3) 486 - 499, 06 , Refereed, The continental distributions of freshwater fishes in the family Notopteridae (Osteoglossomorpha) across Africa, India, and Southeast Asia constitute a long standing and enigmatic problem of freshwater biogeography. The migrational pathway of the Asian notopterids has been discussed in light of two competing schemes: the first posits recent transcontinental dispersal while the second relies on distributions being shaped by ancient vicariance associated with plate-tectonic events. In this study, we determined complete mitochondrial DNA sequences from 10 osteoglossomorph fishes to estimate phylogenetic relationships using partitioned Bayesian and maximum likelihood methods and divergence dates of the family Notopteridae with a partitioned Bayesian approach. We used six species representing the major lineages of the Notopteridae and seven species from the remaining osteoglossomorph families. Fourteen more-derived teleosts, nine basal actinopterygians, two coelacanths, and one shark were used as outgroups. Phylogenetic analyses indicated that the African and Asian notopterids formed a sister group to each other and that these notopterids were a sister to a clade comprising two African families (Mormyridae and Gymnarchidae). Estimated divergence time between the African and Asian notopterids dated back to the early Cretaceous when India-Madagascar separated from the African part of Gondwanaland. Thus, estimated time of divergence based on the molecular evidence is at odds with the recent dispersal model. It can be reconciled with the geological and paleontological evidence to support the vicariance model in which the Asian notopterids diverged from the African notopterids in Gondwanaland and migrated into Eurasia on the Indian subcontinent from the Cretaceous to the Tertiary. However, we could not exclude an alternative explanation that the African and Asian notopterids diverged in Pangea before its complete separation into Laurasia and Gondwanaland, to which these two lineages were later confined, respectively. © 2009 Elsevier Inc. All rights reserved.
  • Molecular affinity of Somali and Egyptian mastigures among the Afro-Arabian Uromastyx., Sayed A. M. Amer, Yoshinori Kumazawa, Egyptian Journal of Experimental Biology (Zoology), 5, 1 - 7, Refereed
  • Complete sequence of the mitochondrial genome of the endangered Nile soft-shelled turtle Trionyx triunguis., Sayed A M Amer, Yoshinori Kumazawa, Egyptian Journal of Experimental Biology (Zoology), 5, 43 - 50, Refereed
  • Molecular structures of centromeric heterochromatin and karyotypic evolution in the Siamese crocodile (Crocodylus siamensis) (Crocodylidae, Crocodylia), Taiki Kawagoshi, Chizuko Nishida, Hidetoshi Ota, Yoshinori Kumazawa, Hideki Endo, Yoichi Matsuda, Chromosome Research, 16, (8) 1119 - 1132, 12 , Refereed, Crocodilians have several unique karyotypic features, such as small diploid chromosome numbers (30-42) and the absence of dot-shaped microchromosomes. Of the extant crocodilian species, the Siamese crocodile (Crocodylus siamensis) has no more than 2n = 30, comprising mostly bi-armed chromosomes with large centromeric heterochromatin blocks. To investigate the molecular structures of C-heterochromatin and genomic compartmentalization in the karyotype, characterized by the disappearance of tiny microchromosomes and reduced chromosome number, we performed molecular cloning of centromeric repetitive sequences and chromosome mapping of the 18S-28S rDNA and telomeric (TTAGGG)nsequences. The centromeric heterochromatin was composed mainly of two repetitive sequence families whose characteristics were quite different. Two types of GC-rich CSI-HindIII family sequences, the 305 bp CSI-HindIII-S (G+C content, 61.3%) and 424 bp CSI-HindIII-M (63.1%), were localized to the intensely PI-stained centric regions of all chromosomes, except for chromosome 2 with PI-negative heterochromatin. The 94 bp CSI-DraI (G+C content, 48.9%) was tandem-arrayed satellite DNA and localized to chromosome 2 and four pairs of small-sized chromosomes. The chromosomal size-dependent genomic compartmentalization that is supposedly unique to the Archosauromorpha was probably lost in the crocodilian lineage with the disappearance of microchromosomes followed by the homogenization of centromeric repetitive sequences between chromosomes, except for chromosome 2. © 2008 Springer Science+Business Media B.V.
  • Mitogenomic evaluation of the historical biogeography of cichlids toward reliable dating of teleostean divergences, Yoichiro Azuma, Yoshinori Kumazawa, Masaki Miya, Kohji Mabuchi, Mutsumi Nishida, BMC Evolutionary Biology, 8, (1) , 08 , Refereed, Background. Recent advances in DNA sequencing and computation offer the opportunity for reliable estimates of divergence times between organisms based on molecular data. Bayesian estimations of divergence times that do not assume the molecular clock use time constraints at multiple nodes, usually based on the fossil records, as major boundary conditions. However, the fossil records of bony fishes may not adequately provide effective time constraints at multiple nodes. We explored an alternative source of time constraints in teleostean phylogeny by evaluating a biogeographic hypothesis concerning freshwater fishes from the family Cichlidae (Perciformes: Labroidei). Results. We added new mitogenomic sequence data from six cichlid species and conducted phylogenetic analyses using a large mitogenomic data set. We found a reciprocal monophyly of African and Neotropical cichlids and their sister group relationship to some Malagasy taxa (Ptychochrominae sensu Sparks and Smith). All of these taxa clustered with a Malagasy + Indo/Sri Lankan clade (Etroplinae sensu Sparks and Smith). The results of the phylogenetic analyses and divergence time estimations between continental cichlid clades were much more congruent with Gondwanaland origin and Cretaceous vicariant divergences than with Cenozoic transmarine dispersal between major continents. Conclusion. We propose to add the biogeographic assumption of cichlid divergences by continental fragmentation as effective time constraints in dating teleostean divergence times. We conducted divergence time estimations among teleosts by incorporating these additional time constraints and achieved a considerable reduction in credibility intervals in the estimated divergence times. © 2008 Azuma et al; licensee BioMed Central Ltd.
  • Timing of a mtDNA gene rearrangement and intercontinental dispersal of varanid lizards, Sayed A.M. Amer, Yoshinori Kumazawa, Genes and Genetic Systems, 83, (3) 275 - 280, 08 , Refereed, The mitochondrial genomes of the Komodo monitor (Varanus komodoensis) and the Nile monitor (V. niloticus) were previously shown to have an extensive gene rearrangement. Here, we show that this gene arrangement widely occurs in varanid taxa originated from Africa, Asia and Australasia. Based on phylogenetic relationships of the varanids constructed using mitochondrial DNA sequences encoding the NADH dehydrogenase subunit 2 gene and seven flanking tRNA genes, we estimated their divergence times by the Bayesian method without assuming the molecular clock. The results suggested that the mitochondrial DNA gene rearrangement took place once in an ancestral varanid lineage in the Paleocene or earlier. Our results are more consistent with Cenozoic over-water dispersal of Southeast Asian varanids across the Indonesian Archipelago rather than the Cretaceous Gondwanan vicariance for the origin of Australasian varanids.
  • Molecular phylogenetic and dating analyses using mitochondrial DNA sequences of eyelid geckos (Squamata: Eublepharidae), Pierre Jonniaux, Yoshinori Kumazawa, Gene, 407, (1-2) 105 - 115, 01 , Refereed, Mitochondrial DNA sequences of approximately 2.3 kbp including the complete NADH dehydrogenase subunit 2 gene and its flanking genes, as well as parts of 12S and 16S rRNA genes were determined from major species of the eyelid gecko family Eublepharidae sensu [Kluge, A.G. 1987. Cladistic relationships in the Gekkonoidea (Squamata, Sauria). Misc. Publ. Mus. Zool. Univ. Michigan 173, 1-54.]. In contrast to previous morphological studies, phylogenetic analyses based on these sequences supported that Eublepharidae and Gekkonidae form a sister group with Pygopodidae, raising the possibility of homoplasious character change in some key features of geckos, such as reduction of movable eyelids and innovation of climbing toe pads. The phylogenetic analyses also provided a well-resolved tree for relationships between the eublepharid species. The Bayesian estimation of divergence times without assuming the molecular clock suggested the Jurassic divergence of Eublepharidae from Gekkonidae and radiations of most eublepharid genera around the Cretaceous. These dating results appeared to be robust against some conditional changes for time estimation, such as gene regions used, taxon representation, and data partitioning. Taken together with geological evidence, these results support the vicariant divergence of Eublepharidae and Gekkonidae by the breakup of Pangea into Laurasia and Gondwanaland, and recent dispersal of two African eublepharid genera from Eurasia to Africa after these landmasses were connected in the Early Miocene. © 2007 Elsevier B.V. All rights reserved.
  • The mitochondrial genome of the lizard Calotes versicolor and a novel gene inversion in South Asian draconine agamids, Sayed A M Amer, Yoshinori Kumazawa, Molecular Biology and Evolution, 24, (6) 1330 - 1339, 06 , Refereed, A complete mitochondrial DNA (mtDNA) sequence was determined for the lizard Calotes versicolor (Reptilia; Agamidae). The 16,670-bp genome with notable shorter genes for some protein-coding and tRNA genes had the same gene content as that found in other vertebrates. However, a novel gene arrangement was found in which the proline tRNA (trnP) gene is located in the light strand instead of its typical heavy-strand position, providing the first known example of gene inversion in vertebrate mtDNAs. A segment of mtDNA encompassing the trnP gene and its flanking genes and the control region was amplified and sequenced for various agamid taxa to investigate timing and mechanism of the gene inversion. The inverted trnP gene organization was shared by all South Asian draconine agamids examined but by none of the other Asian and African agamids. Phylogenetic analyses including clock-free Bayesian analyses for divergence time estimation suggested a single occurrence of the gene inversion on a lineage leading to the draconine agamids during the Paleogene period. This gene inversion could not be explained by the tandem duplication/random loss model for mitochondrial gene rearrangements. Our available sequence data did not provide evidence for remolding of the trnP gene by an anticodon switch in a duplicated tRNA gene. Based on results of sequence comparisons and other circumstantial evidence, we hypothesize that inversion of the trnP gene was originally mediated by a homologous DNA recombination and that the de novo gene organization that does not disrupt expression of mitochondrial genes has been maintained in draconine mtDNAs for such a long period of time. © The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.
  • Mitochondrial genomes from major lizard families suggest their phylogenetic relationships and ancient radiations, Yoshinori Kumazawa, Gene, 388, (1-2) 19 - 26, 02 , Refereed, In placental mammals and birds, molecular data generally support a view that they diverged into their ordinal groups in good response to mid-Cretaceous continental fragmentations. However, such divergence patterns have rarely been studied for reptiles for which phylogenetic relationships among their major groups have not yet been established molecularly. Here, I determined complete or nearly complete mitochondrial DNA sequences from seven lizard families and reconstructed phylogenetic relationships between major lizard families. When snakes were included, maximum likelihood analysis did not support a morphological view of the snakes-varanoids affinity, although several other competing hypotheses on the position of snakes still cannot be discriminated presumably due to extremely long branches of the snake lineages. I also conducted clock-free Bayesian analyses to show that divergence times between major lizard families were centered in Triassic-Jurassic times. Thus, lizards include much deeper divergences than the mammals and birds and they appear to have already radiated into various families prior to the mid-Cretaceous major continental fragmentation. © 2006 Elsevier B.V. All rights reserved.
  • Molecular phylogenetic analyses of snakeheads (Perciformes: Channidae) using mitochondrial DNA sequences, Xia Li, Prachya Musikasinthorn, Yoshinori Kumazawa, Yoshinori Kumazaw, Ichthyological Research, 53, (2) 148 - 159, 05 , Refereed, Mitochondrial DNA sequences of approximately 1.5∈kbp including the NADH dehydrogenase subunit 2 (ND2) gene and its flanking gene regions were determined for 20 species from the freshwater fish family Channidae and 3 species from Nandidae, Badidae, and Osphronemidae. Channa orientalis and C. gachua had an approximately 170-bp insertion between the tRNAMet and ND2 genes, where a 5′-half of the insertion was similar to the 5′-end portion of the ND2 gene and a 3′-half was homologous to the tRNAMet gene. This insertion may thus have originated from a tandem gene duplication that occurred in a common ancestor of these two sister species. Molecular phylogenetic analyses from different tree-building methods consistently suggested the mutual monophyly of the African and Asian taxa and the existence of several clades within the Asian taxa, some of which correspond to distinct morphological features. Our molecular phylogeny clearly supported multiple independent losses of pelvic fins on Asian lineages in parallel. Divergence time estimation based on some reasonable assumptions without assuming the molecular clock suggested the early Cretaceous divergence of the African and Asian channids. The results thus support an ancient vicariant divergence of the African and Asian channids, rather than the more recent dispersal between African and Eurasian continents. ©The Ichthyological Society of Japan 2006.
  • Complete mitochondrial DNA sequences of six snakes: Phylogenetic relationships and molecular evolution of genomic features, Songyu Dong, Yoshinori Kumazawa, Journal of Molecular Evolution, 61, (1) 12 - 22, 07 , Refereed, Complete mitochondrial DNA (mtDNA) sequences were determined for representative species from six snake families: the acrochordid little file snake, the bold boa constrictor, the cylindrophiid red pipe snake, the viperid himehabu, the pythonid ball python, and the xenopeltid sunbeam snake. Thirteen protein-coding genes, 22 tRNA genes, 2 rRNA genes, and 2 control regions were identified in these mtDNAs. Duplication of the control region and translocation of the tRNALeu gene were two notable features of the snake mtDNAs. The duplicate control regions had nearly identical nucleotide sequences within species but they were divergent among species, suggesting concerted sequence evolution of the two control regions. In addition, the duplicate control regions appear to have facilitated an interchange of some flanking tRNA genes in the viperid lineage. Phylogenetic analyses were conducted using a large number of sites (9570 sites in total) derived from the complete mtDNA sequences. Our data strongly suggested a new phylogenetic relationship among the major families of snakes: ((((Viperidae, Colubridae), Acrochordidae), (((Pythonidae, Xenopeltidae), Cylindrophiidae), Boidae)), Leptotyphlopidae). This conclusion was distinct from a widely accepted view based on morphological characters in denying the sister-group relationship of boids and pythonids, as well as the basal divergence of nonmacrostomatan cylindrophiids. These results imply the significance to reconstruct the snake phylogeny with ample molecular data, such as those from complete mtDNA sequences. © Springer Science+Business Media, Inc. 2005.
  • Mitochondrial DNA sequences of the Afro-Arabian spiny-tailed lizards (genus Uromastyx; family Agamidae): Phylogenetic analyses and evolution of gene arrangements, Sayed A M Amer, Yoshinori Kumazawa, Biological Journal of the Linnean Society, 85, (2) 247 - 260, 06 , Refereed, Approximately 1.7 kbp of mitochondrial DNA were sequenced from 29 individuals assignable to 11 Uromastyx species or subspecies and two other agamids. U. ocellata and U. ornata had an insertion between the glutamine and isoleucine tRNA genes, which could be folded into a stable stem-and-loop structure, and the insertion for U. ornata additionally retained a sequence similar to the glutamine tRNA gene. This corroborates the role of tandem duplication in reshaping mitochondrial gene arrangements, and supports the idea that the origin of light-strand replication could be relocated within mitochondrial genomes. Molecular phylogeny from different tree-building methods consistently placed African and Arabian taxa in mutually monophyletic groups, excluding U. hardwickii inhabiting India and Pakistan. Unlike previous studies based on morphology, U. macfadyeni did not cluster with morphologically similar Arabian taxa, suggesting convergent evolution to be responsible for the morphological similarities. Divergence times estimated among the Uromastyx taxa, together with geological and palaeontological evidence, suggest that the Uromastyx agamids originated from Central Asia during the Eocene and colonized Africa after its connection with Eurasia in the early Miocene. Their radiation may have been facilitated by repeated aridification of North Africa since the middle Miocene, and geological events such as the expansion of the Red Sea and the East African Rift Valley. © 2005 The Linnean Society of London.
  • Sister group relationship of turtles to the bird-crocodilian clade revealed by nuclear DNA-coded proteins, Naoyuki Iwabe, Yuichiro Hara, Yoshinori Kumazawa, Kaori Shibamoto, Yumi Saito, Takashi Miyata, Kazutaka Katoh, Molecular Biology and Evolution, 22, (4) 810 - 813, 04 , Refereed, The phylogenetic position of turtles is a currently controversial issue. Recent molecular studies rejected a traditional view that turtles are basal living reptiles (Hedges, S. B., and L. L. Poling. 1999. A molecular phylogeny. Science 83:998-1001; Kumazawa, Y., and M. Nishida. 1999. Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink, statistical evidence for archosaurian affinity of turtles. Mol. Biol. Evol. 16:784-792). Instead, these studies grouped turtles with birds and crocodiles. The relationship among turtles, birds, and crocodiles remained unclear to date. To resolve this issue, we have cloned and sequenced two nuclear genes encoding the catalytic subunit of DNA polymerase α and glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase from amniotes and an amphibian. The amino acid sequences of these proteins were subjected to a phylogenetic analysis based on the maximum likelihood method. The resulting tree showed that turtles are the sister group to a monophyletic cluster of archosaurs (birds and crocodiles). All other possible tree topologies were significantly rejected. © Society for Molecular Biology and Evolution 2004; all rights reserved.
  • Mitochondrial genome of Pogona vitticepes (Reptilia; Agamidae): Control region duplication and the origin of Australasian agamids, Sayed A.M. Amer, Yoshinori Kumazawa, Gene, 346, 249 - 256, 02 , Refereed, The complete mitochondrial DNA sequence for an Australian agamid Pogona vitticepes was determined. Twenty-two tRNA genes, two rRNA genes, thirteen protein-coding genes, and two control regions were identified in this mitochondrial genome. The second control region was inserted between NADH dehydrogenase subunits 5 and 6 genes. The duplication of the control region was found in all Australasian agamids examined and was not found in other Asian or African taxa. The two control regions had nearly identical sequences within species but they were divergent among species, suggesting their concerted sequence evolution. Phylogenetic analyses including divergence time estimation without assuming the molecular clock suggested that the duplication of the control region occurred on a lineage leading to the Australasian agamids 25-45 million years ago after their divergence from a Southeast Asian Physignathus cocincinus. Our finding thus supports the recent dispersal origin of Australasian agamids in connection with plate tectonic movement of Australia to the proximity of Southeast Asia. © 2004 Elsevier B.V. All rights reserved.
  • Mitochondrial genome of the Komodo dragon: Efficient sequencing method with reptile-oriented primers and novel gene rearrangements, Yoshinori Kumazawa, Hideki Endo, DNA Research, 11, (2) 115 - 125, 12 , Refereed, The mitochondrial genome of the Komodo dragon (Varanus komodoensis) was nearly completely sequenced, except for two highly repetitive noncoding regions. An efficient sequencing method for squamate mitochondrial genomes was established by combining the long polymerase chain reaction (PCR) technology and a set of reptile-oriented primers designed for nested PCR amplifications. It was found that the mitochondrial genome had novel gene arrangements in which genes from NADH dehydrogenase subunit 6 to proline tRNA were extensively shuffled with duplicate control regions. These control regions had 99% sequence similarity over 700 bp. Although snake mitochondrial genomes are also known to possess duplicate control regions with nearly identical sequences, the location of the second control region suggested independent occurrence of the duplication on lineages leading to snakes and the Komodo dragon. Another feature of the mitochondrial genome of the Komodo dragon was the considerable number of tandem repeats, including sequences with a strong secondary structure, as a possible site for the slipped-strand mispairing in replication. These observations are consistent with hypotheses that tandem duplications via the slipped-strand mispairing may induce mitochondrial gene rearrangements and may serve to maintain similar copies of the control region.
  • Mitochondrial DNA sequences of five squamates: Phylogenetic affiliation of snakes, Yoshinori Kumazawa, DNA Research, 11, (2) 137 - 144, 12 , Refereed, Complete or nearly complete mitochondrial DNA sequences were determined from four lizards (Western fence lizard, Warren's spinytail lizard, Terrestrial arboreal alligator lizard, and Chinese crocodile lizard) and a snake (Texas blind snake). These genomes had a typical gene organization found in those of most mammals and fishes, except for a translocation of the glutamine tRNA gene in the blind snake and a tandem duplication of the threonine and proline tRNA genes in the spinytail lizard. Although previous work showed the existence of duplicate control regions in mitochondrial DNAs of several snakes, the blind snake did not have this characteristic. Phylogenetic analyses based on different tree-building methods consistently supported that the blind snake and a colubrid snake (akamata) make a sister clade relative to all the lizard taxa from six different families. An alternative hypothesis that snakes evolved from a lineage of varanoids was not favored and nearly statistically rejected by the Kishino-Hasegawa test. It is therefore likely that the apparent similarity of the tongue structure between snakes and varanoids independently evolved and that the duplication of the control region occurred on a snake lineage after divergence of the blind snake.
  • Tempo of mitochondrial gene evolution: can mitochondrial DNA be used to date old divergences?, Yoshinori Kumazawa, Yoichiro Azuma, Mutsumi Nishida, Endocytobiosis and Cell Research, 15, 136 - 142, Refereed
  • Non-AUG translation initiation of mRNA encoding plastid-targeted phage-type RNA polymerase in Nicotiana sylvestris, Yuki Kobayashi, Yuko Dokiya, Yoshinori Kumazawa, Mamoru Sugita, Biochemical and Biophysical Research Communications, 299, (1) 57 - 61, 12 , Refereed, A third nuclear gene encoding a bacteriophage T7-type RNA polymerase, NsRpoT-C, was isolated and characterized from Nicotiana sylvestris. The gene, NsRpoT-C, consists of 21 exons and 20 introns and encodes apolypeptide of 977 amino acid residues. The predicted NsRpoT-C protein shows the highest identity (72% amino acid identity) with Arabidopsis thaliana RpoT;3 which is a plastid-targeted protein. Surprisingly, comparison of the deduced amino acid sequence of NsRpoT-C with that of A. thaliana RpoT;3 predicted that the NsRpoT-C starts at a CUG triplet, a rare translation initiation codon. Transient expression assays in protoplasts from tobacco leaves demonstrated that the putative N-terminal transit peptide of NsRpoT-C encodes a targeting signal directing the protein into chloroplasts. This strongly suggests that NsRpoT-C functions as an RNA polymerase transcribing plastid- encoded genes. We have designated this protein NsRpoTp. © 2002 Elsevier Science (USA). All rights reserved.
  • Phylogenetic position of turtles among amniotes: Evidence from mitochondrial and nuclear genes, Ying Cao, Michael D. Sorenson, Yoshinori Kumazawa, David P. Mindell, Masami Hasegawa, Gene, 259, (1-2) 139 - 148, 12 , Refereed, Maximum likelihood analysis, accounting for site-heterogeneity in evolutionary rate with the Γ-distribution model, was carried out with amino acid sequences of 12 mitochondrial proteins and nucleotide sequences of mitochondrial 12S and 16S rRNAs from three turtles, one squamate, one crocodile, and eight birds. The analysis strongly suggests that turtles are closely related to archosaurs (birds + crocodilians), and it supports both Tree-2: (((birds, crocodilians), turtles), squamates) and Tree-3: ((birds, (crocodilians, turtles)), squamates). A more traditional Tree-1: (((birds, crocodilians), squamates), turtles) and a tree in which turtles are basal to other amniotes were rejected with high statistical significance. Tree-3 has recently been proposed by Hedges and Poling [Science 283 (1999) 998-1001] based mainly on nuclear genes. Therefore, we re-analyzed their data using the maximum likelihood method, and evaluated the total evidence of the analyses of mitochondrial and nuclear data sets. Tree-1 was again rejected strongly. The most likely hypothesis was Tree-3, though Tree-2 remained a plausible candidate. © 2000 Elsevier Science B.V.
  • Molecular phylogeny of osteoglossoids: A new model for Gondwanian origin and plate tectonic transportation of the Asian arowana, Yoshinori Kumazawa, Mutsumi Nishida, Molecular Biology and Evolution, 17, (12) 1869 - 1878, 01 , Refereed, One of the traditional enigmas in freshwater zoogeography has been the evolutionary origin of Scleropages formosus inhabiting Southeast Asia (the Asian arowana), which is a species threatened with extinction among the highly freshwater-adapted fishes from the order Osteoglossiformes. Dispersalists have hypothesized that it originated from the recent (the Miocene or later) transmarine dispersal of morphologically quite similar Australasian arowanas across Wallace's Line, but this hypothesis has been questioned due to their remarkable adaptation to freshwater. We determined the complete nucleotide sequences of two mitochondrial protein genes from 12 osteoglossiform species, including all members of the suborder Osteoglossoidei, with which robust molecular phylogeny was constructed and divergence times were estimated. In agreement with previous morphology-based phylogenetic studies, our molecular phylogeny suggested that the osteoglossiforms diverged from a basal position of the teleostean lineage, that heterotidines (the Nile arowana and the pirarucu) form a sister group of osteoglossines (arowanas in South America, Australasia, and Southeast Asia), and that the Asian arowana is more closely related to Australasian arowanas than to South American ones. However, molecular distances between the Asian and Australasian arowanas were much larger than expected from the fact that they are classified within the same genus. By using the molecular clock of bony fishes, tested for its good performance for rather deep divergences and calibrated using some reasonable assumptions, the divergence between the Asian and Australasian arowanas was estimated to date back to the early Cretaceous. Based on the molecular and geological evidence, we propose a new model whereby the Asian arowana vicariantly diverged from the Australasian arowanas in the eastern margin of Gondwanaland and migrated into Eurasia on the Indian subcontinent or smaller continental blocks. This study also implicates the relatively long absence of osteoglossiform fossil records from the Mesozoic.
  • Complete mitochondrial DNA sequences of the green turtle and blue- tailed mole skink: Statistical evidence for archosaurian affinity of turtles, Yoshinori Kumazawa, Mutsumi Nishida, Molecular Biology and Evolution, 16, (6) 784 - 792, 01 , Refereed, Turtles have highly specialized morphological characteristics, and their phylogenetic position has been under intensive debate. Previous molecular studies have not established a consistent and statistically well supported conclusion on this issue. In order to address this, complete mitochondrial DNA sequences were determined for the green turtle and the blue-tailed mole skink. These genomes possess an organization of genes which is typical of most other vertebrates, such as placental mammals, a frog, and bony fishes, but distinct from organizations of alligators and snakes. Molecular evolutionary rates of mitochondrial protein sequences appear to vary considerably among major reptilian lineages, with relatively rapid rates for snake and crocodilian lineages but slow rates for turtle and lizard lineages. In spite of this rate heterogeneity, phylogenetic analyses using amino acid sequences of 12 mitochondrial proteins reliably established the Archosauria (birds and crocodilians) and Lepidosauria (lizards and snakes) clades postulated from previous morphological studies. The phylogenetic analyses further suggested that turtles are a sister group of the archosaurs, and this untraditional relationship was provided with strong statistical evidence by both the bootstrap and the Kishino-Hasegawa tests. This is the first statistically significant molecular phylogeny on the placement of turtles relative to the archosaurs and lepidosaurs. It is therefore likely that turtles originated from a Permian-Triassic archosauromorph ancestor with two pairs of temporal fenestrae behind the skull orbit that were subsequently lost. The traditional classification of turtles in the Anapsida may thus need to be reconsidered.
  • The complete nucleotide sequence of a snake (Dinodon semicarinatus) mitochondrial genome with two identical control regions, Yoshinori Kumazawa, Hidetoshi Ota, Mutsumi Nishida, Tomowo Ozawa, Genetics, 150, (1) 313 - 329, 09 , Refereed, The 17,191-bp mitochondrial DNA (mtDNA) of a Japanese colubrid snake, akamata (Dinodon semicarinatus), was cloned and sequenced. The snake mtDNA has some peculiar features that were found in our previous study using polymerase chain reaction: duplicate control regions that have completely identical sequences over 1 kbp, translocation of tRNA(Leu)(UUR) gene, shortened TψC arm for most tRNA genes, and a pseudogene for tRNA(Pro). Phylogenetic analysis of amino acid sequences of protein genes suggested an unusually high rate of molecular evolution in the snake compared to other vertebrates. Southern hybridization experiments using mtDNAs purified from multiple akamata individuals showed that the duplicate state of the control region is not a transient or unstable feature found in a particular individual, but that it stably occurs in mitochondrial genomes of the species. This may, therefore, be regarded as an unprecedented example of stable functional redundancy in animal mtDNA. However, some of the examined individuals contain a rather scanty proportion of heteroplasmic mtDNAs with an organization of genes distinct from that of the major mtDNA. The gene organization of the minor mtDNA is in agreement with one of models that we present to account for the concerted evolution of duplicate control regions.
  • Molecular phylogenetic characterization of Streptomyces protease inhibitor family, Seiichi Taguchi, Shuichi Kojima, Mahito Terabe, Yoshinori Kumazawa, Hiroshi Kohriyama, Masayuki Suzuki, Kin-Ichiro Miura, Haruo Momose, Journal of Molecular Evolution, 44, (5) 542 - 551, 06 , Refereed, We previously found that proteinaceous protease inhibitors homologous to Streptomyces subtilisin inhibitor (SSI) are widely produced by various Streptomyces species, and we designated them 'SSI-like proteins' (Taguchi S, Kikuchi H, Suzuki M, Kojima S, Terabe M, Miura K, Nakase T, Momose H [1993] Appl Environ Microbiol 59:4338-4341). In this study, SSI-like proteins from five strains of the genus Streptoverticillium were purified and sequenced, and molecular phylogenetic trees were constructed on the basis of the determined amino acid sequences together with those determined previously for Streptomyces species. The phylogenetic trees showed that SSI-like proteins from Streptoverticillium species are phylogenetically included in Streptomyces SSI-like proteins but form a monophyletic group as a distinct lineage within the Streptomyces proteins. This provides an alternative phylogenetic framework to the previous one based on partial small ribosomal RNA sequences, and it may indicate that the phylogenetic affiliation of the genus Streptoverticillium should be revised. The phylogenetic trees also suggested that SSI-like proteins possessing arginine or methionine at the PI site, the major reactive center site toward target proteases, arose multiple times on independent lineages from ancestral proteins possessing lysine at the PI site. Most of the codon changes at the PI site inferred to have occurred during the evolution of SSI-like proteins are consistent with those inferred from the extremely high G + C content of Streptomyces genomes. The inferred mini mum number of amino acid replacements at the PI site was nearly equal to the average number for all the variable sites. It thus appears that positive Darwinian selection, which has been postulated to account for accelerated rates of amino acid replacement at the major reaction center site of mammalian protease inhibitors, may not have dictated the evolution of the bacterial SSI-like proteins.
  • Gene rearrangements in snake mitochondrial genomes: Highly concerted evolution of control-region-like sequences duplicated and inserted into a tRNA gene cluster, Yoshinori Kumazawa, Hidetoshi Ota, Mutsumi Nishida, Tomowo Ozawa, Molecular Biology and Evolution, 13, (9) 1242 - 1254, 01 , Refereed, Mitochondrial DNA (mtDNA) regions corresponding to two major tRNA gene clusters were amplified and sequenced for the Japanese pit viper, himehabu. In one of these clusters, which in most vertebrates characterized to date contains three tightly connected genes for tRNA(Ile), tRNA(Gln), and tRNA(Met), a sequence of approximately 1.3 kb was found to be inserted between the genes for tRNA(Ile) and tRNA(Gln). The insert consists of a control-region-like sequence possessing some conserved sequence blocks, and short flanking sequences which may be folded into tRNA(Pro), tRNA(Phe), and tRNA(Leu) genes. Several other snakes belonging to different families were also found to possess a control-region-like sequence and tRNA(Leu) gene between the tRNA(Ile) and tRNA(Gln) genes. We also sequenced a region surrounded by genes for cytochrome b and 12S rRNA, where the control region and genes for tRNA(Pro) and tRNA(Phe) are normally located in the mtDNAs of most vertebrates. In this region of three examined snakes, a control-region- like sequence exists that is almost completely identical to the one found between the tRNA(Ile) and tRNA(Gln) genes. The mtDNAs of these snakes thus possess two nearly identical control-region-like sequences which are otherwise divergent to a large extent between the species. These results suggest that the duplicate state of the control-region-like sequence and tRNA(Leu) gene into the tRNA gene cluster, which occurred in the early stage of the divergence of snakes. It is also suggested that the duplicated control-region-like sequences at two distant locations of mtDNA have evolved concertedly by a mechanism such as frequent gene conversion. The secondary structures of the determined tRNA genes point to the operation of simplification pressure on the T ψ C arm of snake mitochondrial tRNAs.
  • Variations in mitochondrial tRNA gene organization of reptiles as phylogenetic markers, Yoshinori Kumazawa, Mutsumi Nishida, Molecular Biology and Evolution, 12, (5) 759 - 772, 01 , Refereed, Amplification and sequencing of mitochondrial DNA regions corresponding to three major clusters of transfer RNA genes from a variety of species representing major groups of birds and reptiles revealed some new variations in tRNA gene organization. First, a gene rearrangement from tRNA(His)- tRNA(Ser)(AGY)-tRNA(Leu)(CUN) to tRNA(Ser)(AGY)-tRNA(His)-tRNA(Leu)(CUN) occurs in all three crocodilians examined (alligator, caiman, and crocodile). In addition an exceptionally long spacer region between the genes for NADH dehydrogenase subunit 4 and tRNA(Ser)(AGY) is found in caiman. Second, in congruence with a recent finding by Seutin et al., a characteristic stem- and-loop structure for the putative light-strand replication origin located between tRNA(Asn) and tRNA(Cys) genes is absent for all the birds and crocodilians. This stem-and-loop structure is absent in an additional species, the Texas blind snake, whereas the stem-and-loop structure is present in other snakes, lizards, turtles, mammals, and a frog. The disappearance of the stem-and-loop structure in the blind snake most likely occurred independently of that on the lineage leading to birds and crocodilians. Finally, the blind snake has a novel type of tRNA gene arrangement in which the tRNA(Gln) gene moved from one tRNA cluster to another. Sequence substitution rates for the tRNA genes appeared to be somewhat higher in crocodilians than in birds and mammals. As regards the controversial phylogenetic relationship among the Aves, Crocodilia, and Mammalia, a sister group relationship of birds and crocodilians relative to mammals, as suggested from the common loss of the stem-and-loop structure, was supported with statistical significance by molecular phylogenetic analyses using the tRNA gene sequence data.
  • Higher-order structure of bovine mitochondrial tRNASerUGA: Chemical modification and computer modeling, Yoh-ichi Watanabe, Gota Kawai, Takashi Yokogawa, Nobuhiro Hayashi, Yoshinori Kumazawa, Takuya Ueda, Kazuya Nishikawa, Ichiro Hirao, Kin-ichiro Miura, Kimitsuna Watanabe, Nucleic Acids Research, 22, (24) 5378 - 5384, 12 , Refereed, On the basis of enzymatic probing and phylogenetlc comparison, we have previously proposed that mammalian mitochondrial tRNAsSer(anticodon UGA) possess a slightly altered cloverleaf structure In which only one nucleotide exists between the acceptor stem and D stem (usually two nucleotides) and the anticodon stem consists of six base pairs (usually five base pairs) [Yokogawa et al. (1991) Nucleic Acids Res. 19, 6101-6105]. To ascertain whether such tRNAsSercan be folded Into a normal L-shaped tertiary structure, the higher-order structure of bovine mitochondrial tRNASerUGA was examined by chemical probing using dimethylsulfate and dlethylpyrocarbonate, and on the basis of the results a tertiary structure model was obtained by computer modeling. It was found that a one-base-pair elongation In the anticodon stem was compensated for by multiple-base deletions In the D and extra loop regions of the tRNASerUGA, which resulted In preservation of an L-shaped tertiary structure similar to that of conventional tRNAs. By summarizing the findings, the general structural requirements of mitochondrial tRNAs necessary for their functioning in the mitochondrial translation system are considered. © 1994 Oxford University Press.
  • Batchwise purification of specific tRNAs by a solid-phase DNA probe, Hiromichi Tsurui, Yoshinori Kumazawa, Reiko Sanokawa, Yoichi Watanabe, Tohru Kuroda, Akiyoshi Wada, Kimitsuna Watanabe, Toshikazu Shirai, Analytical Biochemistry, 221, (1) 166 - 172, 08 , Refereed, A simple and efficient method for purifying a specific tRNA in a single microcentrifuge tube was developed. Oligodeoxyribonucleotides (about 30 mer) with sequences complementary to the 3′ side of target tRNAs were synthesized with an aminohexyl linker at the 5′ end, immobilized on a silica gel at a high concentration, and used as a solid-phase probe. A mixture of tRNAs was added to a suspension of the solid-phase probe in 2.4 M tetraethylammonium chloride and incubated for 10-30 min. Only a target tRNA hybridized with the immobilized probe at appropriate temperatures and was eluted out by heating. The solid-phase probe showed a large hybridization capacity (up to 17 A260units/g dry gel) and specific and quantitative recovery of the target tRNA. The intactness of recovered tRNAs was ascertained by both Donis-Keller sequencing and aminoacylation experiments. These features show the usefulness of the solid-phase probe method as a reliable tool for purifying tRNAs whose gene sequences are known. © 1994 Academic Press, Inc.
  • Higher-order structure of bovine mitochondrial tRNAPhelacking the 'conserved' GG and TψCG sequences as inferred by enzymatic and chemical probing, Kawori Wakita, Yoh-ichi Watanabe, Takashi Yokogawa, Yoshinori Kumazawa, Shingo Nakamura, Takuya Ueda, Kimitsuna Watanabe, Kazuya Nishikawa, Nucleic Acids Research, 22, (3) 347 - 353, 02 , Refereed, Bovine mitochondrial (mt) phenylalanine tRNA (tRNAPhe), which lacks the 'conserved' GG and TΨYCG sequences, was efficiently purified by the selective hybridization method using a solid phase DNA probe. The entire nucleotide sequence of the tRNA, including modified nucleotides, was determined and its higher-order structure was investigated using RNaseT2and chemical reagents as structural probes. The D and T loop regions as well as the anticodon loop region were accessible to RNaseT2, and the N-3 positions of cytidines present in the D and T loops were easily modified under the native conditions in the presence of 10mM Mg2+. On the other hand, the nucleotides present in the extra loop were protected from the chemical modification under the native conditions. From the results of these probing analyses and a comparison of the sequences of mitochondrial tRNAPhegenes from various organisms, it was inferred that bovine mt tRNAPhelacks the D loop/T loop tertiary interactions, but does have the canonical extra loop/D stem interactions, which seem to be the main factor for bovine mt tRNAPheto preserve its L-shaped higher-order structure. © 1994 Oxford University Press.
  • Sequence evolution of mitochondrial tRNA genes and deep-branch animal phylogenetics, Yoshinori Kumazawa, Mutsumi Nishida, Journal of Molecular Evolution, 37, (4) 380 - 398, 01 , Refereed, Mitochondrial DNA sequences are often used to construct molecular phylogenetic trees among closely related animals. In order to examine the usefulness of mtDNA sequences for deep-branch phylogenetics, genes in previously reported mtDNA sequences were analyzed among several animals that diverged 20–600 million years ago. Unambiguous alignment was achieved for stem-forming regions of mitochondrial tRNA genes by virtue of their conservative secondary structures. Sequences derived from stem parts of the mitochondrial tRNA genes appeared to accumulate much variation linearly for a long period of time: nearly 100 Myr for transition differences and more than 350 Myr for transversion differences. This characteristic could be attributed, in part, to the structural variability of mitochondrial tRNAs, which have fewer restrictions on their tertiary structure than do nonmitochondrial tRNAs. The tRNA sequence data served to reconstruct a well-established phylogeny of the animals with 100% bootstrap probabilities by both maximum parsimony and neighbor joining methods. By contrast, mitochondrial protein genes coding for cytochrome b and cytochrome oxidase subunit I did not reconstruct the established phylogeny or did so only weakly, although a variety of fractions of the protein gene sequences were subjected to tree-building. This discouraging phylogenetic performance of mitochondrial protein genes, especially with respect to branches originating over 300 Myr ago, was not simply due to high randomness in the data. It may have been due to the relative susceptibility of the protein genes to natural selection as compared with the stem parts of mitochondrial tRNA genes. On the basis of these results, it is proposed that mitochondrial tRNA genes may be useful in resolving deep branches in animal phylogenies with divergences that occurred some hundreds of Myr ago. For this purpose, we designed a set of primers with which mtDNA fragments encompassing clustered tRNA genes were successfully amplified from various vertebrates by the polymerase chain reaction. © 1993, Springer-Verlag New York Inc. All rights reserved.
  • Effect of the higher-order structure of tRNAs on the stability of hybrids with oligodeoxyribonucleotides: Separation of tRNA by an efficient solution hybridization, Yoshinori Kumazawa, Takashi Yokogawa, Hiromichi Tsurui, Kin-ichiro Miura, Kimitsuna Watanabe, Nucleic Acids Research, 20, (9) 2223 - 2232, 05 , Refereed, In the course of developing a method to purify a single tRNA species efficiently, we have examined hybridization effciencies between some tRNAs and short oligodeoxyribonucleotide probes both by the filter and solution hybridization methods without denaturants. The hybridization efficiencies varied considerably among probes which are complementary to different regions of the tRNAs, although there was little efficiency variation in the probes toward DNA substrates including the same nucleotide sequence. This efficiency variation was shown to be due to tRNA specific higher-order structures as well as a hypermodified nucleotide in the anticodon loop. Characterization of the tRNA-probe hybrids by both nondenaturing gel electrophoresis and chemical modification showed the existence of two stable hybridizing states as a function of ionic strength. Our results indicate that RNA molecules with a number of intramolecular base pairings are able to form stable hybrids with complementary sequences under nondenaturing conditions. On the basis of these data, an appropriate probe was designed to successfully purify yeast tRNAPheby making a tRNAPhe-probe hybrid, which has a longer retention time in hydroxyapatite high performance liquid chromato graphy than the tRNAPheitself. © 1992 Oxford University Press.
  • Rearrangements of mitochondrial transfer RNA genes in marsupials, Svante Pääbo, W. Kelley Thomas, Kristina M. Whitfield, Yoshinori Kumazawa, Allan C. Wilson, Journal of Molecular Evolution, 33, (5) 426 - 430, 11 , Refereed, The nucleotide sequences of the mitochondrial origin of light-strand replication and the five tRNA genes surrounding it were determined for three marsupials. The region was found to be rearranged, leaving only the tRNATyrgene at the same position as in placental mammals and Xenopus. Distribution of the same rearranged genotype among two marsupial families indicates that the events causing the rearrangements took place in an early marsupial ancestor. The putative mitochondrial light-strand origin of replication in marsupials contains a hairpin structure similar to other vertebrate origins and, in addition, extensive flanking sequences that are not found in other vertebrates. Sequence comparisons among the marsupials as well as placentals indicate that the tRNATyrgene has been evolving under more constraints than the other tRNA genes. © 1991 Springer-Verlag New York Inc.
  • A novel cloverleaf structure found in mammalian mitochondrial tRNASer(UCN), Takashi Yokogawa, Yoh-ichi Watanabe, Yoshinori Kumazawa, Takuya Ueda, Ichiro Hirao, Kin-ichiro Miura, Kimitsuna Watanabe, Nucleic Acids Research, 19, (22) 6101 - 6105, 11 , Refereed, Bovine mitochondrial tRNASer(UCN) has been thought to have two U-U mismatches at the top of the acceptor stem, as inferred from its gene sequence. However, this unusual structure has not been confirmed at the RNA level. In the course of investigating the structure and function of mitochondrial tRNAs, we have isolated thebovine liver mitochondrial tRNASer(UCN) and determined its complete sequence including the modified nucleotides. Analysis of the 5′-terminal nucleotide and enzymatic determination of the whole sequence of tRNASer(UCN) revealed that the tRNA started from the third nucleotide of the putative tRNASer(UCN) gene, which had formerly been supposed. Enzymatic probing of tRNASer(UCN) suggests that the tRNA possesses an unusual cloverleaf structure with the following characteristics. (1) There exists only one nucleotide between the acceptor stem with 7 base pairs and the D stem with 4 base pairs. (2) The anticodon stem seems to consist of 6 base pairs. Since the same type of cloverleaf structure as above could be constructed only for mitochondrial tRNASer(UCN) genes of mammals such as human, rat and mouse, but not for those of nonmammals such as chicken and frog, this unusual secondary structure seems to be conserved only in mammalian mitochondria. © 1991 Oxford University Press.
  • Interactions of bovine mitochondrial phenylalanyl-tRNA with ribosomes and elongation factors from mitochondria and bacteria, Yoshinori Kumazawa, Caryl J. Schwartzbach, Hua Xin Liao, Kiyohisa Mizumoto, Yoshito Kaziro, Kin-ichiro Miura, Kimitsuna Watanabe, Linda L. Spremulli, BBA - Gene Structure and Expression, 1090, (2) 167 - 172, 10 , Refereed, A homologous in vitro poly(U)-directed translation system has been established using animal mitochondrial ribosomes, elongation factors (EF) and phenylalanyl-tRNAPhe. The rate of incorporation of phenylalanine into polyphenylalanine in the mitochondrial system is slower than that observed for the homologous Escherichia coli system. E. coli ribosomes can be used in place of mitochondrial ribosomes in this system with only a slight decrease in the efficiency of phenylalanine incorporation from mitochondrial Phe-tRNA. However, E. coli elongation factor Tu (EF-Tu) cannot replace the mitochondrial EF-Tu in promoting the use of mitochondrial Phe-tRNA. The interaction between EF-Tu and mitochondrial Phe-tRNA was investigated by using the ability of EF-Tu to protect the aminoacyl-tRNA bond from hydrolysis. These results showed that both mitochondrial and E. coli EF-Tus are capable of interacting with mitochondrial Phe-tRNA. However, ribosomal A-site binding assays demonstrated that efficient binding of the mitochondrial Phe-tRNA to the ribosomal A-site was only obtained with the homologous mitochondrial EF-Tu. © 1991.
  • Strand-specific nucleotide composition bias in echinoderm and vertebrate mitochondrial genomes, Shuichi Asakawa, Yoshinori Kumazawa, Takeyoshi Araki, Hyouta Himeno, Kin-ichiro Miura, Kimitsuna Watanabe, Journal of Molecular Evolution, 32, (6) 511 - 520, 06 , Refereed, The gene organization of starfish mitochondrial DNA is identical with that of the sea urchin counterpart except for a reported inversion of an approximately 4.6-kb segment containing two structural genes for NADH dehydrogenase subunits 1 and 2 (ND 1 and ND 2). When the codon usage of each structural gene in starfish, sea urchin, and vertebrate mitochondrial DNAs is examined, it is striking that codons ending in T and G are preferentially used more for heavy strand-encoded genes, including starfish ND 1 and ND 2, than for light strand-encoded genes, including sea urchin ND 1 and ND 2. On the contrary, codons ending in A and Care preferentially used for the light strand-encoded genes rather than for the heavy strand-encoded ones. Moreover, G-U base pairs are more frequently found in the possible secondary structures of heavy strandencoded tRNAs than in those of light strand-encoded tRNAs. These observations suggest the existence of a certain constraint operating on mitochondrial genomes from various animal phyla, which results in the accumulation of G and T on one strand, and A and C on the other. © 1991 Springer-Verlag New York Inc.
  • Unilateral aminoacylation specificity between bovine mitochondria and eubacteria, Yoshinori Kumazawa, Hyouta Himeno, Kin-ichiro Miura, Kimitsuna Watanabe, Journal of Biochemistry, 109, (3) 421 - 427, 01 , Refereed, The present study shows unilateral aminoacylation specificity between bovine mitochondria and eubacteria (Escherichia coli and Thermus thermophilus) in five amino acid-specific aminoacylation systems. Mitochondrial synthetases were capable of charging eubacterial tRNA as well as mitochondrial tRNA, whereas eubacterial synthetases did not efficiently charge mitochondrial tRNA. Mitochondrial phenylalanyl-, threonyl-, arginyl-, and lysyl-tRNA synthetases were shown to charge and discriminate cognate E. coli tRNA species from noncognate ones strictly, as did the corresponding E. coli synthetases. By contrast, mitochondrial seryl-tRNA synthetase not only charged cognate E. coli serine tRNA species but also extensively misacylated noncognate E. coli tRNA species. These results suggest a certain conservation of tRNA recognition mechanisms between the mitochondrial and E. coli amionoacyl-tRNA synthetases in that anticodon sequences are most likely to be recognized by the former four synthetases, but not sufficiently by the seryl-tRNA synthetase. The unilaterality in aminoacylation may imply that tRNA recognition mechanisms of the mitochondrial synthetases have evolved to be, to some extent, simpler than their eubacterial counterparts in response to simplifications in the species-number and the structural elements of animal mitochondrial tRNAs. © 1991 by The Journal of Biochemistry.
  • Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method, Takashi Yokogawa, Yoshinori Kumazawa, Kin-ichiro Miura, Kimitsuna Watanabe, Nucleic Acids Research, 17, (7) 2623 - 2638, 04 , Refereed, For large scale preparation of mitochondrial tRNAs, a new hybridization assay method using synthetic oligodeoxy-ribonucleotide probes (16-17mer) complementary to individual tRNA sequences was developed and applied for the purification of two serine isoacceptor tRNAs (tRNAAGYSer and tRNAUCNSer) from bovine mitochondria. It is about 100 times more sensitive than the conventional aminoacylation assay method. 2-4 A260units each of both tRNASerisoacceptors were purified from 17.5 kg of bovine liver, and they were characterized by means of nuclease digestion, melting profiles and aminoacylation activity. It is suggested that tRNAUCNSer possesses the D loop/T loop interaction like usual L-shaped tRNAs, and that tRNAUCNSer lacking almost an entire D arm is aminoacylated with an efficiency not very much lower than that of tRNAUCNSer. © 1989 IRL Press.
  • The aminoacylation of structurally variant phenylalanine tRNAs from mitochondria and various nonmitochondrial sources by bovine mitochondrial phenylalanyl-tRNA synthetase, Yoshinori Kumazawa, Takashi Yokogawa, Emi Hasegawa, Kin-Ichiro Miura, Kimitsuna Watanabe, Journal of Biological Chemistry, 264, (22) 13005 - 13011, 01 , Refereed, Bovine mitochondrial (mt) phenylalanine tRNA (tRNAPhe) was purified on a large scale using a new hybridization assay method developed by the authors. Although its melting profile suggested a loose higher order structure, presumably influenced by the apparent loss of D loop-T loop interaction necessary for forming a rigid L-shaped tertiary structure, its aminoacylation capacity catalyzed by mt phenylalanyl-tRNA synthetase (PheRS) was nearly equal to that of Escherichia coli tRNAPhe. Misaminoacylation was not observed for the mt tRNAPhe-mt PheRS system. Comparing the aminoacylation efficiencies of several combinations of tRNAPhes and PheRSs from various sources, including bovine mitochondria, bovine and yeast cy-tosols, E. coli, Thermits thermophilics, and Sulfolobus acidocaldarius, it was clarified that mt PheRS was able to aminoacylate all the above mentioned tRNAPhespecies, albeit with varying degrees of efficiency. This broad charging spectrum suggests that mt PheRS possesses a relatively simple recognition mechanism toward its substrate, tRNAPhe.


  • First record of alien species Phyllomimus klapperichi from Japan, Tadahiro Teramoto, Kensuke Miyata, Yuri Yokoyama, Yoshinori Kumazawa, Yûta Naito and Futoshi Ukai, Gekkan Mushi, 580,   2019 06
  • Application of the next-generation DNA sequencing for evolutionary studies of vertebrate olfactory receptor gene family., Yasuyuki Hashiguchi, Yoshinori Kumazawa, Seibutsu Kagaku, 64, (3) 131 - 140,   2013 03 , Refereed, Invited
  • 進化学のすすめ., Yoshinori Kumazawa, Annual Review 2009. Graduate School of Natural Sciences, Nagoya City University, 14,   2010 , Invited
  • Molecular clock estimation in fishes and its application to biogeographical studies, Yoshinori Kumazawa, Fisheries Science, 68, (sup1) 357 - 360,   2002 01 , Using gamma-corrected distances based on amino acid sequences of two mitochondrial protein-coding genes, we have roughly calibrated a molecular dock to estimate deep divergence times for bony fishes, which turned out to be about three times slower than in mammals. Calibrations were based on the reasonable assumption of the vicariant divergence of cichlids, as well as some reliable fossil records for the external calibration. We have applied this clock to studies of the osteoglossiform freshwater fishes. The Asian arowana has been hypothesized to have originated from morphologically quite similar Australasian arowanas via recent transmarine dispersal. However, the molecular data urged us to propose a new model that the former vicariantly diverged from the latter in the eastern margin of Gondwanaland and migrated into Eurasia on the Indian subcontinent or smaller continental blocks. The arowanas are thus likely to have kept a large extent of morphological conservation like the coelacanth. We hope that people will pay more attention to the conservation of this evolutionary spedal spedes. © 2002, The Japanese Society of Fisheries Science. All rights reserved.
  • Need for change in Japan's universities, Yoshinori Kumazawa, Nature, 388,   1997 01 , Invited
  • ミトコンドリアtRNA遺伝子の配置変動と分子系統学., Yoshinori Kumazawa, SHINKA, 7, (2) 49 - 59,   1997 , Invited
  • Structure of mitochondrial tRNA., T. Yokogawa, Y. Watanabe, Y. Yotsumoto, Y. Kumazawa, T. Ueda, I. Hirao, K. Miura, K. Watanabe, Nucleic Acids Symposium Series, 1991, (25) 175 - 176,   1991 12 , Some mitochondrial (mt) tRNAs are presumed to have unusual secondary structures on the basis of DNA sequence analyses. However there are few tRNAs whose structures have been investigated at RNA level. Thus we isolated some mt tRNAs to investigated their structures and also synthesized mt tRNA by run-off transcription. In these experiments, following results have been obtained. 1) Bovine mt tRNA(Ser)(AGY) needs the interaction between the T-loop and D-arm for its aminoacylation activity. 2) The secondary structure of bovine mt tRNA(Ser)(UCN) is difficult from that predicted previously from its gene. 3) It has been confirmed by the hybridization using oligonucleotide probes that some Ascaris suum mt tRNAs possessing the unusual T-arms actually exist in its mitochondria.
  • Sequence specific purification of a particular tRNA by solid phase DNA probe., H. Tsurui, Y. Kumazawa, R. Sanokawa, K. Watanabe, A. Wada, T. Shirai, Nucleic Acids Symposium Series, 1991, (25) 149 - 150,   1991 12 , A novel method for the purification of a specific tRNA using solid phase DNA probe is developed. With this method, the probe DNA immobilized on HPLC gel hybridized with target tRNA within a minute at room temperature. The hybridizing capacity of the solid phase probe was about 20 O.D. per gram dry gel when yeast phenylalanine tRNA was used. The specificity of this method was extremely high and the recovery rate was about 90%.
  • Bovine mitochondrial tRNAPhe, tRNASer (AGY) and tRNASer (UCN): preparation using a new detection method and their properties in aminoacylation., Y. Kumazawa, T. Yokogawa, K. Miura, K. Watanabe, Nucleic Acids Symposium Series, 1988, (19) 97 - 100,   1988 12 , Bovine mitochondrial tRNAPhe, tRNASer (AGY), and tRNASer (UCN) possessing unusual structures were purified using a new hybridization assay system and their properties in aminoacylation were examined. Bovine mitochondrial phenyl-alanyl- and seryl-tRNA synthetases could aminoacylate the same amino acid-specific tRNAs obtained not only from the mitochondria but also from other sources such as E. coli, Thermus thermophilus, bovine and yeast cytosols and archaebacteria, Sulfolobus acidocaldarius. On the contrary, none of both bacterial and cytosolic synthetases could aminoacylate the same amino acid specific tRNAs from the heterologous sources with some exceptions. We consider that the bovine mitochondrial aminoacyl-tRNA synthetases have considerably simple recognition mechanism toward the substrate tRNAs compared with the non-mitochondrial ones. This mechanism may be correlated with the occurrence of structural varieties of the mitochondrial tRNA species with unusual structures.
  • Gene organization of tRNAs and their secondary structures found in starfish mitochondrial genome., Araki T., Asakawa S., Kumazawa Y., Miura K. and Watanabe K., Nucleic Acids Symposium Series, 1988, (20) 93 - 94,   1988

Books etc

  • The Biology of Biodiversity (ed) Kato M., Yoshinori Kumazawa, Motoomi Yamaguchi, Mutsumi Nishida, Contributor, Chapter 3, Mitochondrial molecular clocks and the origin of euteleostean biodiversity: Familial radiation of perciforms may have predated the Cretaceous/Tertiary boundary. pp. 35-52, Springer-Verlag,   1999
  • Current Topics on Molecular Evolution (eds.) Nei M. and Takahata N., Yoshinori Kumazawa, Mutsumi Nishida, Contributor, Phylogenetic utility of mitochondrial transfer RNA genes for deep divergence in animals. pp. 29-35, Institute of Molecular Evolutionary Genetics, The Pennsylvania State University, USA, and Graduate School for Advanced Studies, Hayama, Japan,   1996

Awards & Honors

  •   2004 08 , Young Scientist Initiative Award, Society of Evolutionary Studies, Japan, Historical biogeography of freshwater fishes in Osteoglossidae
  •   2007 10 , The Best Article Award, Ichthyological Society of Japan, Molecular phylogenetic analyses of snakeheads (Perciformes: Channidae) using mitochondrial DNA sequences.

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