Yoshinobu Uno and Kazumi Matsubara : Unleashing diversity through flexibility: The evolutionary journey of sex chromosomes in amphibians and reptiles, Journal of Experimental Zoology. Part A, Ecological and Integrative Physiology, 341, 3, 230-241, 2024.
(要約)
Sex determination systems have greatly diversified between amphibians and reptiles, with such as the different sex chromosome compositions within a single species and transition between temperature-dependent sex determination (TSD) and genetic sex determination (GSD). In most sex chromosome studies on amphibians and reptiles, the whole-genome sequence of Xenopous tropicalis and chicken have been used as references to compare the chromosome homology of sex chromosomes among each of these taxonomic groups, respectively. In the present study, we reviewed existing reports on sex chromosomes, including karyotypes, in amphibians and reptiles. Furthermore, we compared the identified genetic linkages of sex chromosomes in amphibians and reptiles with the chicken genome as a reference, which is believed to resemble the ancestral tetrapod karyotype. Our findings revealed that sex chromosomes in amphibians are derived from genetic linkages homologous to various chicken chromosomes, even among several frogs within single families, such as Ranidae and Pipidae. In contrast, sex chromosomes in reptiles exhibit conserved genetic linkages with chicken chromosomes, not only across most species within a single family, but also within closely related families. The diversity of sex chromosomes in amphibians and reptiles may be attributed to the flexibility of their sex determination systems, including the ease of sex reversal in these animals.
Shigehiro Kuraku, Mana Sato, Kohta Yoshida and Yoshinobu Uno : Genomic reconsideration of fish non-monophyly: why cannot we simply call them all `fish'?, Ichthyological Research, 71, 1, 1-12, 2024.
Yoshinobu Uno, Kazumi Matsubara, Jun Inoue, Johji Inazawa, Akio Shinohara, Chihiro Koshimoto, Kenji Ichiyanagi and Yoichi Matsuda : Diversity and Evolution of Highly Repetitive DNA Sequences Constituting Chromosome Site-Specific Heterochromatin in Two Gerbillinae Species, Cytogenetic and Genome Research, 163, 1-2, 42-51, 2023.
(要約)
Constitutive heterochromatin, consisting of repetitive sequences, diverges very rapidly; therefore, its nucleotide sequences and chromosomal distributions are often largely different, even between closely related species. The chromosome C-banding patterns of two Gerbillinae species, Meriones unguiculatus and Gerbillus perpallidus, vary greatly, even though they belong to the same subfamily. To understand the evolution of C-positive heterochromatin in these species, we isolated highly repetitive sequences, determined their nucleotide sequences, and characterized them using chromosomal and filter hybridization. We obtained a centromeric repeat (MUN-HaeIII) and a chromosome 13-specific repeat (MUN-EcoRI) from M. unguiculatus. We also isolated a centromeric/pericentromeric repeat (GPE-MBD) and an interspersed-type repeat that was predominantly amplified in the X and Y chromosomes (GPE-EcoRI) from G. perpallidus. GPE-MBD was found to contain a 17-bp motif that is essential for binding to the centromere-associated protein CENP-B. This indicates that it may play a role in the formation of a specified structure and/or function of centromeres. The nucleotide sequences of the three sequence families, except GPE-EcoRI, were conserved only in Gerbillinae. GPE-EcoRI was derived from the long interspersed nuclear elements 1 retrotransposon and showed sequence homology throughout Muridae and Cricetidae species, indicating that the repeat sequence occurred at least in the common ancestor of Muridae and Cricetidae. Due to a lack of assembly data of highly repetitive sequences constituting heterochromatin in whole-genome sequences of vertebrate species published to date, the knowledge obtained in this study provides useful information for a deep understanding of the evolution of repetitive sequences in not only rodents but also in mammals.
(キーワード)
C-positive heterochromatin / CENP-B box / Centromeric/pericentromeric repetitive sequence / Concerted evolution / Long interspersed nuclear element 1 retrotransposon
Genomic studies of vertebrate chromosome evolution have long been hindered by the scarcity of chromosome-scale DNA sequences of some key taxa. One of those limiting taxa has been the elasmobranchs (sharks and rays), which harbor species often with numerous chromosomes and enlarged genomes. Here, we report the chromosome-scale genome assembly for the zebra shark Stegostoma tigrinum, an endangered species that has a relatively small genome among sharks (3.71 Gb), as well as for the whale shark Rhincodon typus Our analysis using a male-female comparison identified an X Chromosome, the first genomically characterized shark sex chromosome. The X Chromosome harbors the Hox C cluster whose intact linkage has not been shown for an elasmobranch fish. The sequenced shark genomes show a gradualism of chromosome length with remarkable length-dependent characteristics-shorter chromosomes tend to have higher GC content, gene density, synonymous substitution rate, and simple tandem repeat content as well as smaller gene length and lower interspersed repeat content. We challenge the traditional binary classification of karyotypes as with and without so-called microchromosomes. Even without microchromosomes, the length-dependent characteristics persist widely in nonmammalian vertebrates. Our investigation of elasmobranch karyotypes underpins their unique characteristics and provides clues for understanding how vertebrate karyotypes accommodate intragenomic heterogeneity to realize a complex readout. It also paves the way to dissecting more genomes with variable sizes to be sequenced at high quality.
(キーワード)
Female / Male / Animals / Base Sequence / Chromosome Mapping / Vertebrates / Sharks / Karyotype
Eikichi Kamimura, Yoshinobu Uno, Kazuhiko Yamada, Chizuko Nishida and Yoichi Matsuda : Molecular Cytogenetic Characterization of C-Band-Positive Heterochromatin of the Greater Long-Tailed Hamster (Tscherskia triton, Cricetinae), Cytogenetic and Genome Research, 162, 6, 323-333, 2023.
(要約)
The greater long-tailed hamster (Tscherskia triton, Cricetinae) has a unique karyotype (2n = 28), containing 11 pairs of acrocentric chromosomes with large C-band-positive centromeric heterochromatin blocks. To understand the origin and evolutionary process of heterochromatin in this species, we isolated novel families of chromosome site-specific highly repetitive DNA sequences from TaqI-digested genomic DNA and then characterized them by chromosome in situ and filter hybridization. The TaqI-families of repetitive sequences were classified into 2 types by their genome organization and chromosomal distribution: the 110-bp repeated sequence organized in large tandem arrays (as satellite DNA), localized to centromeric C-positive heterochromatin of acrocentric autosomes (chromosomes 1-11) and submetacentric X chromosome, and the 405-bp repeated sequence that was composed of 30-32-bp internal repeats, distributed in the pericentromeric region on the short arms of X and Y chromosomes. The repetitive sequences did not cross-hybridize with genomic DNA of any genera of Cricetinae (Mesocricetus, Cricetulus, and Phodopus). These results suggest that the 110-bp and 405-bp repeats rapidly diverged in the lineage of T. triton, evolving in a concerted manner among autosomes and X chromosome and within X and Y chromosomes, respectively. The 110-bp centromeric repeat contained a 17-bp motif in which 9 bases are essential for binding with the centromere-associated protein CENP-B, suggesting the possibility that the 110-bp major satellite DNA carrying the 17-bp motif may have a role in the formation of specified structure and/or function of centromeres in T. triton.
Shigehiro Kuraku, Osamu Nishimura, John Rozewicki, Kazuaki Yamaguchi, Kaori Tatsumi, Yuta Ohishi, Tazro Ohta, Masaru Yagura, Taiki Niwa, Chiharu Tanegashima, Akinori Teramura, Shotaro Hirase, Akane Kawaguchi, Milton Tan, Salvatore D'Aniello, Filipe Castro, André Machado, Mitsumasa Koyanagi, Akihisa Terakita, Ryo Misawa, Masayuki Horie, Junna Kawasaki, Takashi Asahida, Atsuko Yamaguchi, Kiyomi Murakumo, Rui Matsumoto, Iker Irisarri, Norio Miyamoto, Atsushi Toyoda, Sho Tanaka, Tatsuya Sakamoto, Yasuko Semba, Shinya Yamauchi, Kazuyuki Yamada, Kiyonori Nishida, Itsuki Kiyatake, Keiichi Sato, Susumu Hyodo, Mitsutaka Kadota and Yoshinobu Uno : Squalomix: Shark and ray genome analysis consortium and its data sharing platform, F1000Research, 11, 1077, 2022.
(要約)
The taxon Elasmobranchii (sharks and rays) contains one of the long-established evolutionary lineages of vertebrates with a tantalizing collection of species occupying critical aquatic habitats. To overcome the current limitation in molecular resources, we launched the Squalomix Consortium in 2020 to promote a genome-wide array of molecular approaches, specifically targeting shark and ray species. Among the various bottlenecks in working with elasmobranchs are their elusiveness and low fecundity as well as the large and highly repetitive genomes. Their peculiar body fluid composition has also hindered the establishment of methods to perform routine cell culturing required for their karyotyping. In the Squalomix consortium, these obstacles are expected to be solved through a combination of in-house cytological techniques including karyotyping of cultured cells, chromatin preparation for Hi-C data acquisition, and high fidelity long-read sequencing. The resources and products obtained in this consortium, including genome and transcriptome sequences, a genome browser powered by JBrowse2 to visualize sequence alignments, and comprehensive matrices of gene expression profiles for selected species are accessible through https://github.com/Squalomix/info.
Yoshinobu Uno, Ryo Nozu, Itsuki Kiyatake, Nobuyuki Higashiguchi, Shuji Sodeyama, Kiyomi Murakumo, Keiichi Sato and Shigehiro Kuraku : Cell culture-based karyotyping of orectolobiform sharks for chromosome-scale genome analysis, Communications Biology, 3, 1, 652, 2020.
(要約)
Karyotyping, traditionally performed using cytogenetic techniques, is indispensable for validating genome assemblies whose sequence lengths can be scaled up to chromosome sizes using modern methods. Karyotype reports of chondrichthyans are scarce because of the difficulty in cell culture. Here, we focused on carpet shark species and the culture conditions for fibroblasts and lymphocytes. The utility of the cultured cells enabled the high-fidelity characterization of their karyotypes, namely 2n = 102 for the whale shark (Rhincodon typus) and zebra shark (Stegostoma fasciatum), and 2n = 106 for the brownbanded bamboo shark (Chiloscyllium punctatum) and whitespotted bamboo shark (C. plagiosum). We identified heteromorphic XX/XY sex chromosomes for the two latter species and demonstrated the first-ever fluorescence in situ hybridization of shark chromosomes prepared from cultured cells. Our protocols are applicable to diverse chondrichthyan species and will deepen the understanding of early vertebrate evolution at the molecular level.
Yoshinobu Uno, Chizuko Nishida, Ayano Hata, Satoshi Ishishita and Yoichi Matsuda : Molecular cytogenetic characterization of repetitive sequences comprising centromeric heterochromatin in three Anseriformes species, PLoS ONE, 14, 3, e0214028, 2019.
(要約)
The highly repetitive DNA sequence of centromeric heterochromatin is an effective molecular cytogenetic marker for investigating genomic compartmentalization between macrochromosomes and microchromosomes in birds. We isolated four repetitive sequence families of centromeric heterochromatin from three Anseriformes species, viz., domestic duck (Anas platyrhynchos, APL), bean goose (Anser fabalis, AFA), and whooper swan (Cygnus cygnus, CCY), and characterized the sequences by molecular cytogenetic approach. The 190-bp APL-HaeIII and 101-bp AFA-HinfI-S sequences were localized in almost all chromosomes of A. platyrhynchos and A. fabalis, respectively. However, the 192-bp AFA-HinfI-L and 290-bp CCY-ApaI sequences were distributed in almost all microchromosomes of A. fabalis and in approximately 10 microchromosomes of C. cygnus, respectively. APL-HaeIII, AFA-HinfI-L, and CCY-ApaI showed partial sequence homology with the chicken nuclear-membrane-associated (CNM) repeat families, which were localized primarily to the centromeric regions of microchromosomes in Galliformes, suggesting that ancestral sequences of the CNM repeat families are observed in the common ancestors of Anseriformes and Galliformes. These results collectively provide the possibility that homogenization of centromeric heterochromatin occurred between microchromosomes in Anseriformes and Galliformes; however, homogenization between macrochromosomes and microchromosomes also occurred in some centromeric repetitive sequences.
(キーワード)
Animals / Anseriformes / Centromere / Chickens / Chromosome Painting / Cytogenetics / DNA / Ducks / Female / Galliformes / Geese / Heterochromatin / In Situ Hybridization, Fluorescence / Male / Repetitive Sequences, Nucleic Acid / Species Specificity
Soichiro Kumamoto, Nozomi Takahashi, Kayo Nomura, Makoto Fujiwara, Megumi Kijioka, Yoshinobu Uno, Yoichi Matsuda, Yusuke Sotomaru and Tomohiro Kono : Overexpression of microRNAs from the Gtl2-Rian locus contributes to postnatal death in mice, Human Molecular Genetics, 26, 19, 3653-3662, 2017.
(要約)
The Dlk1-Dio3 imprinted domain functions in embryonic development but the roles of noncoding RNAs expressed from this domain remain unclear. We addressed this question by generating transgenic (TG) mice harbouring a BAC carrying IG-DMR (intergenic-differentially methylated region), Gtl2-DMR, Gtl2, Rtl1/Rtl1as, and part of Rian. High postnatal lethality (>85%) of the BAC-TG pups was observed in the maternally transmitted individuals (MAT-TG), but not following paternal transmission (PAT-TG). The DNA methylation status of IG-DMR and Gtl2-DMR in the BAC-allele was paternally imprinted similar to the genomic allele. The mRNA-Seq and miRNA-Seq analysis revealed marked expression changes in the MAT-TG, with 1,500 upregulated and 2,131 downregulated genes. The long noncoding RNAs and 12 miRNAs containing the BAC locus were markedly enhanced in the MAT-TG. We identified the 24 target genes of the overexpressed miRNAs and confirmed the downregulation in the MAT-TG. Notably, overexpression of mir770, mir493, and mir665 from Gtl2 in the MAT-TG embryos led to decreased expression of the 3 target genes, Col5a1, Pcgf2, and Clip2. Our results suggest that decreased expression of the 3 target genes concomitant with overexpression of the miRNAs within Gtl2 may be involved in the postnatal death in the MAT-TG. Because this imprinted domain is well conserved between mice and humans, the results of genetic and molecular analysis in mice hold important implications for related human disorders such as Temple syndrome.
Shuuji Mawaribuchi, Shuji Takahashi, Mikako Wada, Yoshinobu Uno, Yoichi Matsuda, Mariko Kondo, Akimasa Fukui, Nobuhiko Takamatsu, Masanori Taira and Michihiko Ito : Sex chromosome differentiation and the W- and Z-specific loci in Xenopus laevis, Developmental Biology, 426, 2, 393-400, 2017.
(要約)
Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W- and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278kb W-specific and 83kb Z-specific sequences on chromosome 2Lq32-33, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X. laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes.
(キーワード)
Allotetraploidization / Homologous recombination / Sex chromosome / Sex-determining gene / Transposable element / Whole genome duplication
Atsushi Suzuki, Yoshinobu Uno, Shuji Takahashi, Jane Grimwood, Jeremy Schmutz, Shuuji Mawaribuchi, Hitoshi Yoshida, Kimiko Takebayashi-Suzuki, Michihiko Ito, Yoichi Matsuda, Daniel Rokhsar and Masanori Taira : Genome organization of the vg1 and nodal3 gene clusters in the allotetraploid frog Xenopus laevis, Developmental Biology, 426, 2, 236-244, 2017.
(要約)
Extracellular factors belonging to the TGF-β family play pivotal roles in the formation and patterning of germ layers during early Xenopus embryogenesis. Here, we show that the vg1 and nodal3 genes of Xenopus laevis are present in gene clusters on chromosomes XLA1L and XLA3L, respectively, and that both gene clusters have been completely lost from the syntenic S chromosome regions. The presence of gene clusters and chromosome-specific gene loss were confirmed by cDNA FISH analyses. Sequence and expression analyses revealed that paralogous genes in the vg1 and nodal3 clusters on the L chromosomes were also altered compared to their Xenopus tropicalis orthologs. X. laevis vg1 and nodal3 paralogs have potentially become pseudogenes or sub-functionalized genes and are expressed at different levels. As X. tropicalis has a single vg1 gene on chromosome XTR1, the ancestral vg1 gene in X. laevis appears to have been expanded on XLA1L. Of note, two reported vg1 genes, vg1(S20) and vg1(P20), reside in the cluster on XLA1L. The nodal3 gene cluster is also present on X. tropicalis chromosome XTR3, but phylogenetic analysis indicates that nodal3 genes in X. laevis and X. tropicalis were independently expanded and/or evolved in concert within each cluster by gene conversion. These findings provide insights into the function and molecular evolution of TGF-β family genes in response to allotetraploidization.
Junko Ishijima, Yoshinobu Uno, Mitsuo Nunome, Chizuko Nishida, Shigehiro Kuraku and Yoichi Matsuda : Molecular cytogenetic characterization of chromosome site-specific repetitive sequences in the Arctic lamprey (Lethenteron camtschaticum, Petromyzontidae), DNA Research, 24, 1, 93-101, 2017.
(要約)
All extant lamprey karyotypes are characterized by almost all dot-shaped microchromosomes. To understand the molecular basis of chromosome structure in lampreys, we performed chromosome C-banding and silver staining and chromosome mapping of the 18S-28S and 5S ribosomal RNA (rRNA) genes and telomeric TTAGGG repeats in the Arctic lamprey (Lethenteron camtschaticum). In addition, we cloned chromosome site-specific repetitive DNA sequences and characterized them by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. Three types of repetitive sequences were detected; a 200-bp AT-rich repetitive sequence, LCA-EcoRIa that co-localized with the 18S-28S rRNA gene clusters of 3 chromosomal pairs; a 364-bp AT-rich LCA-EcoRIb sequence that showed homology to the EcoRI sequence family from the sea lamprey (Petromyzon marinus), which contains short repeats as centromeric motifs; and a GC-rich 702-bp LCA-ApaI sequence that was distributed on nearly all chromosomes and showed significant homology with the integrase-coding region of a Ty3/Gypsy family long terminal repeat (LTR) retrotransposon. All three repetitive sequences are highly conserved within the Petromyzontidae or within Petromyzontidae and Mordaciidae. Molecular cytogenetic characterization of these site-specific repeats showed that they may be correlated with programed genome rearrangement (LCA-EcoRIa), centromere structure and function (LCA-EcoRIb), and site-specific amplification of LTR retroelements through homogenization between non-homologous chromosomes (LCA-ApaI).
Adam M. Session, Yoshinobu Uno, Taejoon Kwon, Jarrod A. Chapman, Atsushi Toyoda, Shuji Takahashi, Akimasa Fukui, Akira Hikosaka, Atsushi Suzuki, Mariko Kondo, Simon Heeringen J. van, Ian Quigley, Sven Heinz, Hajime Ogino, Haruki Ochi, Uffe Hellsten, Jessica B. Lyons, Oleg Simakov, Nicholas Putnam, Jonathan Stites, Yoko Kuroki, Toshiaki Tanaka, Tatsuo Michiue, Minoru Watanabe, Ozren Bogdanovic, Ryan Lister, Georgios Georgiou, Sarita S. Paranjpe, Ila Kruijsbergen van, Shengquiang Shu, Joseph Carlson, Tsutomu Kinoshita, Yuko Ohta, Shuuji Mawaribuchi, Jerry Jenkins, Jane Grimwood, Jeremy Schmutz, Therese Mitros, Sahar V. Mozaffari, Yutaka Suzuki, Yoshikazu Haramoto, Takamasa S. Yamamoto, Chiyo Takagi, Rebecca Heald, Kelly Miller, Christian Haudenschild, Jacob Kitzman, Takuya Nakayama, Yumi Izutsu, Jacques Robert, Joshua Fortriede, Kevin Burns, Vaneet Lotay, Kamran Karimi, Yuuri Yasuoka, Darwin S. Dichmann, Martin F. Flajnik, Douglas W. Houston, Jay Shendure, Louis DuPasquier, Peter D. Vize, Aaron M. Zorn, Michihiko Ito, Edward M. Marcotte, John B. Wallingford, Yuzuru Ito, Makoto Asashima, Naoto Ueno, Yoichi Matsuda, Gert Jan C. Veenstra, Asao Fujiyama, Richard M. Harland, Masanori Taira and Daniel S. Rokhsar : Genome evolution in the allotetraploid frog Xenopus laevis., Nature, 538, 7625, 336-343, 2016.
(要約)
To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.
Penporn Sujiwattanarat, Parinya Pongsanarakul, Yosapong Temsiripong, Theeranan Temsiripong, Charin Thawornkuno, Yoshinobu Uno, Sasimanas Unajak, Yoichi Matsuda, Kiattawee Choowongkomon and Kornsorn Srikulnath : Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene, Comparative Biochemistry and Physiology. Part A: Molecular & Integrative Physiology, 191, 187-195, 2016.
(要約)
Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD.
Akira Kanamori, Yosuke Sugita, Yasufumi Yuasa, Takamasa Suzuki, Kouichi Kawamura, Yoshinobu Uno, Katsuyasu Kamimura, Yoichi Matsuda, A. Catherine Wilson, Angel Amores, H. John Postlethwait, Koushirou Suga and Yoshitaka Sakakura : A genetic map for the only self-fertilizing vertebrate, G3 : Genes - Genomes - Genetics, 6, 4, 1095-1106, 2016.
(要約)
The mangrove killifish Kryptolebias marmoratus, and its close relative Kryptolebias hermaphroditus, are the only vertebrate species known to reproduce by self-fertilization due to functional ovotestis development. To improve our understanding of their genomes, we constructed a genetic map. First, a single F1 fish was made by artificial fertilization between K. marmoratus and K. hermaphroditus strains. F2 progeny were then obtained by self-fertilization of the F1 fish. We used RAD-seq to query genomic DNAs from the two parental strains, the F1 individual and 49 F2 progeny. Results identified 9904 polymorphic RAD-tags (DNA markers) that mapped to 24 linkage groups, corresponding to the haploid chromosome number of these species. The total length of the map was 1248 cM, indicating that about one recombination occurred for each of the 24 homologous chromosome pairs in each meiosis. Markers were not evenly distributed along the chromosomes: in all chromosomes, many markers (> 8% of the total markers for each chromosome) mapped to chromosome tips. Centromeres suppress recombination, and this uneven distribution is probably due to the species' acrocentric chromosomes. Mapped marker sequences were compared to genomic sequences of medaka and platyfish, the next most closely related species with sequenced genomes that are anchored to genetic maps. Results showed that each mangrove killifish chromosome corresponds to a single chromosome of both platyfish and medaka, suggesting strong conservation of chromosomes over 100 million years of evolution. Our genetic map provides a framework for the K. marmoratus/K. hermaphroditus genome sequence and an important resource for understanding the biology of hermaphroditism.
(キーワード)
Centromeres and recombination / Conserved chromosomes / Genetics of sex / Hermaphrodite / Phylogeny by RAD-seq / Teleost
Kazumi Matsubara, Yoshinobu Uno, Kornsorn Srikulnath, Risako Seki, Chizuko Nishida and Yoichi Matsuda : Molecular cloning and characterization of satellite DNA sequences from constitutive heterochromatin of the habu snake (Protobothrops flavoviridis, Viperidae) and the Burmese python (Python bivittatus, Pythonidae), Chromosoma, 124, 4, 529-539, 2015.
(要約)
Highly repetitive DNA sequences of the centromeric heterochromatin provide valuable molecular cytogenetic markers for the investigation of genomic compartmentalization in the macrochromosomes and microchromosomes of sauropsids. Here, the relationship between centromeric heterochromatin and karyotype evolution was examined using cloned repetitive DNA sequences from two snake species, the habu snake (Protobothrops flavoviridis, Crotalinae, Viperidae) and Burmese python (Python bivittatus, Pythonidae). Three satellite DNA (stDNA) families were isolated from the heterochromatin of these snakes: 168-bp PFL-MspI from P. flavoviridis and 196-bp PBI-DdeI and 174-bp PBI-MspI from P. bivittatus. The PFL-MspI and PBI-DdeI sequences were localized to the centromeric regions of most chromosomes in the respective species, suggesting that the two sequences were the major components of the centromeric heterochromatin in these organisms. The PBI-MspI sequence was localized to the pericentromeric region of four chromosome pairs. The PFL-MspI and the PBI-DdeI sequences were conserved only in the genome of closely related species, Gloydius blomhoffii (Crotalinae) and Python molurus, respectively, although their locations on the chromosomes were slightly different. In contrast, the PBI-MspI sequence was also in the genomes of P. molurus and Boa constrictor (Boidae), and additionally localized to the centromeric regions of eight chromosome pairs in B. constrictor, suggesting that this sequence originated in the genome of a common ancestor of Pythonidae and Boidae, approximately 86 million years ago. The three stDNA sequences showed no genomic compartmentalization between the macrochromosomes and microchromosomes, suggesting that homogenization of the centromeric and/or pericentromeric stDNA sequences occurred in the macrochromosomes and microchromosomes of these snakes.
(キーワード)
Animals / Base Sequence / Boidae / Cloning, Molecular / DNA, Satellite / Evolution, Molecular / Heterochromatin / In Situ Hybridization, Fluorescence / Karyotype / Molecular Sequence Data / Sequence Analysis, DNA / Trimeresurus
Yoriko Nakamura, Takehiro Iwasaki, Yosuke Umei, Kazuhiro Saotome, Yukiko Nakajima, Shoichi Kitahara, Yoshinobu Uno, Yoichi Matsuda, Akira Oike, Maho Kodama and Masahisa Nakamura : Molecular cloning and characterization of oocyte-specific Pat1a in Rana rugosa frogs, Journal of Experimental Zoology. Part A, Ecological Genetics and Physiology, 323, 8, 516-526, 2015.
(要約)
The Pat1 gene is expressed in the immature oocytes of Xenopus, and is reportedly involved in regulating the translation of maternal mRNAs required for oocyte-maturation. However, it is still unknown when Pat1a first appears in the differentiating ovary of amphibians. To address this issue, we isolated the full-length Pat1a cDNA from the frog Rana rugosa and examined its expression in the differentiating ovary of this frog. Among eight different tissues examined, the Pat1a mRNA was detectable in only the ovary. When frozen sections from the ovaries of tadpoles at various stages of development were immunostained for Vasa-a germ cell-specific protein-and Pat1a, Vasa-immunopositive signals were observed in all of the germ cells, whereas Pat1a signals were confined to the growing oocytes (50-200 μm in diameter), and absent from small germ cells (<50 μm in diameter). Forty days after testosterone injection into tadpoles to induce female-to-male sex-reversal, Pat1a-immunoreactive oocytes had disappeared completely from the sex-reversed gonad, but Vasa-positive small germ cells persisted. Thus, Pat1a would be a good marker for identifying the sexual status of the sex-reversing gonad in amphibians. In addition, fluorescence in situ hybridization analysis showed Pat1a to have an autosomal locus, suggesting that Pat1a transcription is probably regulated by a tissue-specific transcription factor in R. rugosa.
Akira Hikosaka, Yoshinobu Uno and Yoichi Matsuda : Distribution of the T2-MITE Family Transposons in the Xenopus (Silurana) tropicalis Genome, Cytogenetic and Genome Research, 145, 3-4, 230-242, 2015.
(要約)
The T2 family of miniature inverted-repeat transposable elements (T2-MITE) is a prevalent MITE family found in both Xenopus(Silurana) tropicalis and X. laevis. Some subfamilies, particularly T2-A1 and T2-C, may have originated prior to the diversification of the 2 Xenopus lineages and currently include active members in X. tropicalis, whereas another subfamily, T2-E, may have lost its transposition activity even earlier. The distribution of each T2-MITE subfamily in X. tropicalis was investigated and compared to evaluate the evolutionary dynamics of the T2-MITE subfamilies. The subfamilies showed differences in chromosomal distribution, uniformity of insertion density on scaffolds, ratios of upstream to downstream insertions with respect to genes, and their distance from genes. Among these, the T2-C subfamily was interesting because it was frequently inserted upstream and close to genes and because genes with close insertions of this subfamily showed high correlations in spatial expression patterns. This unique distribution and long-lived transposition activity may reflect a mutual relationship evolved between this subfamily and the host.
(キーワード)
FISH / Miniature inverted-repeat transposable elements / Transposon / Western clawed frog / Xenopus tropicalis
Yoshinobu Uno, Chizuko Nishida, Chiyo Takagi, Takeshi Igawa, Naoto Ueno, Masayuki Sumida and Yoichi Matsuda : Extraordinary Diversity in the Origins of Sex Chromosomes in Anurans Inferred from Comparative Gene Mapping, Cytogenetic and Genome Research, 145, 3-4, 218-229, 2015.
(要約)
Sex determination in frogs (anurans) is genetic and includes both male and female heterogamety. However, the origins of the sex chromosomes and their differentiation processes are poorly known. To investigate diversity in the origins of anuran sex chromosomes, we compared the chromosomal locations of sex-linked genes in 4 species: the African clawed frog (Xenopus laevis), the Western clawed frog (Silurana/X. tropicalis), the Japanese bell-ring frog (Buergeria buergeri), and the Japanese wrinkled frog (Rana rugosa). Comparative mapping data revealed that the sex chromosomes of X. laevis, X. tropicalis and R. rugosa are different chromosome pairs; however, the sex chromosomes of X. tropicalis and B. buergeri are homologous, although this may represent distinct evolutionary origins. We also examined the status of sex chromosomal differentiation in B. buergeri, which possesses heteromorphic ZW sex chromosomes, using comparative genomic hybridization and chromosome painting with DNA probes from the microdissected W chromosome. At least 3 rearrangement events have occurred in the proto-W chromosome: deletion of the nucleolus organizer region and a paracentric inversion followed by amplification of non-W-specific repetitive sequences.
(キーワード)
Anuran / Buergeria / Chromosome homology / Comparative map / FISH / Sex chromosome / Xenopus
Yoichi Matsuda, Yoshinobu Uno, Mariko Kondo, J. Michael Gilchrist, M. Aaron Zorn, S. Daniel Rokhsar, Michael Schmid and Masanori Taira : A New Nomenclature of Xenopus laevis Chromosomes Based on the Phylogenetic Relationship to Silurana/Xenopus tropicalis, Cytogenetic and Genome Research, 145, 3-4, 187-191, 2015.
(要約)
Xenopus laevis (XLA) is an allotetraploid species which appears to have undergone whole-genome duplication after the interspecific hybridization of 2 diploid species closely related to Silurana/Xenopus tropicalis (XTR). Previous cDNA fluorescence in situ hybridization (FISH) experiments have identified 9 sets of homoeologous chromosomes in X. laevis, in which 8 sets correspond to chromosomes 1-8 of X. tropicalis (XTR1-XTR8), and the last set corresponds to a fusion of XTR9 and XTR10. In addition, recent X. laevis genome sequencing and BAC-FISH experiments support this physiological relationship and show no gross chromosome translocation in the X. laevis karyotype. Therefore, for the benefit of both comparative cytogenetics and genome research, we here propose a new chromosome nomenclature for X. laevis based on the phylogenetic relationship and chromosome length, i.e. XLA1L, XLA1S, XLA2L, XLA2S, and so on, in which the numbering of XLA chromosomes corresponds to that in X. tropicalis and the postfixes 'L' and 'S' stand for 'long' and 'short' chromosomes in the homoeologous pairs, which can be distinguished cytologically by their relative size. The last chromosome set is named XLA9L and XLA9S, in which XLA9 corresponds to both XTR9 and XTR10, and hence, to emphasize the phylogenetic relationship to X. tropicalis, XLA9_10L and XLA9_10S are also used as synonyms.
Kornsorn Srikulnath, Yoshinobu Uno, Chizuko Nishida, Hidetoshi Ota and Yoichi Matsuda : Karyotype reorganization in the Hokou Gecko (Gekko hokouensis, Gekkonidae): The process of microchromosome disappearance in Gekkota, PLoS ONE, 10, 8, e0134829, 2015.
(要約)
The Hokou gecko (Gekko hokouensis: Gekkonidae, Gekkota, Squamata) has the chromosome number 2n = 38, with no microchromosomes. For molecular cytogenetic characterization of the gekkotan karyotype, we constructed a cytogenetic map for G. hokouensis, which retains the ancestral karyotype of Gekkota, with 86 functional genes, and compared it with cytogenetic maps for four Toxicofera species that have many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) and that for a lacertid species (Lacerta agilis) with only one pair of autosomal microchromosomes. Ten pairs of G. hokouensis chromosomes [GHO1, 2, 3, Z(4), 6, 7, 8, 13, 14, and 15] showed highly conserved linkage homology with macrochromosomes and/or macrochromosome arms of the four Toxicofera species and corresponded to eight L. agilis macrochromosomes (LAG). However, GHO5, GHO9, GHO10, GHO11, and LAG6 were composed of chromosome segments that have a homology with Toxicofera microchromosomes, and no homology was found in the chromosomes between G. hokouensis and L. agilis. These results suggest that repeated fusions of microchromosomes may have occurred independently in each lineage of Gekkota and Lacertidae, leading to the disappearance of microchromosomes and appearance of small-sized macrochromosomes.
Maho Kodama, Mari Suda, Daiki Sakamoto, Takehiro Iwasaki, Yasuki Matsuo, Yoshinobu Uno, Yoichi Matsuda, Yoriko Nakamura, Shun Maekawa, Yoshinao Katsu and Masahisa Nakamura : Molecular cloning and characterization of anti-Müllerian hormone (AMH) from the Japanese wrinkled frog, Rana rugosa, Endocrinology, 156, 5, 1914-1923, 2015.
(要約)
The role of anti-Müllerian hormone (AMH) during gonad development has been studied extensively in many species of mammal, bird, reptile, and fish but remains unresolved in amphibians. In male mammalian embryos, Sox9 activates AMH expression, which initiates regression of the Müllerian ducts. However, Sox9 (Sry-related HMG box 9) is unlikely to initiate AMH in chicken, because AMH precedes Sox9 expression in this species. To clarify whether AMH is involved in testicular differentiation in amphibians, we cloned the full-length AMH cDNA from the Japanese wrinkled frog, Rana rugosa. The AMH gene, which appears to be autosomal, is exclusively expressed in the testis of adult frog among 8 different tissues examined; Sertoli cells are probably responsible for its expression. AMH expression was found in the undifferentiated gonad of both male and female tadpoles, increasing in the differentiating testis. Moreover, we observed consensus binding sites for Sox9 in the 5'-flanking region of the AMH gene. Sox9 stimulated statistically significant AMH expression in luciferase reporter assays when coexpressed in Xenopus kidney-derived A6 cells. However, Sox9 expression showed no sexual dimorphism when AMH expression was up-regulated in the developing testis. These results, taken together, suggest that AMH is probably involved in testicular differentiation in R. rugosa, although an additional, perhaps tissue-specific, transcription factor may be required for the regulation of AMH transcription.
Kazumi Matsubara, Yoshinobu Uno, Kornsorn Srikulnath, Yoichi Matsuda, Emily Miller and Mats Olsson : No interstitial telomeres on autosomes but remarkable amplification of telomeric repeats on the W sex chromosome in the sand lizard (Lacerta agilis), The Journal of Heredity, 106, 6, 753-757, 2015.
(要約)
Telomeres are repeat (TTAGGG) n sequences that form terminal ends of chromosomes and have several functions, such as protecting the coding DNA from erosion at mitosis. Due to chromosomal rearrangements through evolutionary history (e.g., inversions and fusions), telomeric sequences are also found between the centromere and the terminal ends (i.e., at interstitial telomeric sites, ITSs). ITS telomere sequences have been implicated in heritable disease caused by genomic instability of ITS polymorphic variants, both with respect to copy number and sequence. In the sand lizard (Lacerta agilis), we have shown that telomere length is predictive of lifetime fitness in females but not males. To assess whether this sex specific fitness effect could be traced to ITSs differences, we mapped (TTAGGG) n sequences using fluorescence in situ hybridization in fibroblast cells cultured from 4 specimens of known sex. No ITSs could be found on autosomes in either sex. However, females have heterogametic sex chromosomes in sand lizards (ZW, 2n = 38) and the female W chromosome showed degeneration and remarkable (TTAGGG) n amplification, which was absent in the Z chromosomes. This work warrants further research on sex chromosome content, in particular of the degenerate W chromosome, and links to female fitness in sand lizards.
Kornsorn Srikulnath, Kazumi Matsubara, Yoshinobu Uno, Chizuko Nishida, Mats Olsson and Yoichi Matsuda : Identification of the linkage group of the Z sex chromosomes of the sand lizard (Lacerta agilis, Lacertidae) and elucidation of karyotype evolution in lacertid lizards, Chromosoma, 123, 6, 563-575, 2014.
(要約)
The sand lizard (Lacerta agilis, Lacertidae) has a chromosome number of 2n = 38, with 17 pairs of acrocentric chromosomes, one pair of microchromosomes, a large acrocentric Z chromosome, and a micro-W chromosome. To investigate the process of karyotype evolution in L. agilis, we performed chromosome banding and fluorescent in situ hybridization for gene mapping and constructed a cytogenetic map with 86 functional genes. Chromosome banding revealed that the Z chromosome is the fifth largest chromosome. The cytogenetic map revealed homology of the L. agilis Z chromosome with chicken chromosomes 6 and 9. Comparison of the L. agilis cytogenetic map with those of four Toxicofera species with many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) showed highly conserved linkage homology of L. agilis chromosomes (LAG) 1, 2, 3, 4, 5(Z), 7, 8, 9, and 10 with macrochromosomes and/or macrochromosome segments of the four Toxicofera species. Most of the genes located on the microchromosomes of Toxicofera were localized to LAG6, small acrocentric chromosomes (LAG11-18), and a microchromosome (LAG19) in L. agilis. These results suggest that the L. agilis karyotype resulted from frequent fusions of microchromosomes, which occurred in the ancestral karyotype of Toxicofera and led to the disappearance of microchromosomes and the appearance of many small macrochromosomes.
Taiki Kawagoshi, Yoshinobu Uno, Chizuko Nishida and Yoichi Matsuda : The Staurotypus turtles and aves share the same origin of sex chromosomes but evolved different types of heterogametic sex determination, PLoS ONE, 9, 8, e105315, 2014.
(要約)
Reptiles have a wide diversity of sex-determining mechanisms and types of sex chromosomes. Turtles exhibit temperature-dependent sex determination and genotypic sex determination, with male heterogametic (XX/XY) and female heterogametic (ZZ/ZW) sex chromosomes. Identification of sex chromosomes in many turtle species and their comparative genomic analysis are of great significance to understand the evolutionary processes of sex determination and sex chromosome differentiation in Testudines. The Mexican giant musk turtle (Staurotypus triporcatus, Kinosternidae, Testudines) and the giant musk turtle (Staurotypus salvinii) have heteromorphic XY sex chromosomes with a low degree of morphological differentiation; however, their origin and linkage group are still unknown. Cross-species chromosome painting with chromosome-specific DNA from Chinese soft-shelled turtle (Pelodiscus sinensis) revealed that the X and Y chromosomes of S. triporcatus have homology with P. sinensis chromosome 6, which corresponds to the chicken Z chromosome. We cloned cDNA fragments of S. triporcatus homologs of 16 chicken Z-linked genes and mapped them to S. triporcatus and S. salvinii chromosomes using fluorescence in situ hybridization. Sixteen genes were localized to the X and Y long arms in the same order in both species. The orders were also almost the same as those of the ostrich (Struthio camelus) Z chromosome, which retains the primitive state of the avian ancestral Z chromosome. These results strongly suggest that the X and Y chromosomes of Staurotypus turtles are at a very early stage of sex chromosome differentiation, and that these chromosomes and the avian ZW chromosomes share the same origin. Nonetheless, the turtles and birds acquired different systems of heterogametic sex determination during their evolution.
B. Fhamida Islam, Yoshinobu Uno, Mitsuo Nunome, Osamu Nishimura, Hiroshi Tarui, Kiyokazu Agata and Yoichi Matsuda : Comparison of the chromosome structures between the chickn and three anserid species, the domestic duck (anas platyrhynchos), muscovy duck (cairina moschata), and chinese goose (anser cygnoides), and the delineation of their karyotype evolution by comparative chromosome mapping, The Journal of Poultry Science, 51, 1, 1-13, 2014.
Satoshi Ishishita, Yuri Tsuruta, Yoshinobu Uno, Atsushi Nakamura, Chizuko Nishida, K. Darren Griffin, Masaoki Tsudzuki, Tamao Ono and Yoichi Matsuda : Chromosome size-correlated and chromosome size-uncorrelated homogenization of centromeric repetitive sequences in New World quails, Chromosome Research, 22, 1, 15-34, 2014.
(要約)
Many families of centromeric repetitive DNA sequences isolated from Struthioniformes, Galliformes, Falconiformes, and Passeriformes are localized primarily to microchromosomes. However, it is unclear whether chromosome size-correlated homogenization is a common characteristic of centromeric repetitive sequences in Aves. New World and Old World quails have the typical avian karyotype comprising chromosomes of two distinct sizes, and C-positive heterochromatin is distributed in centromeric regions of most autosomes and the whole W chromosome. We isolated six types of centromeric repetitive sequences from three New World quail species (Colinus virginianus, CVI; Callipepla californica, CCA; and Callipepla squamata, CSQ; Odontophoridae) and one Old World quail species (Alectoris chukar, ACH; Phasianidae), and characterized the sequences by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. The 385-bp CVI-MspI, 591-bp CCA-BamHI, 582-bp CSQ-BamHI, and 366-bp ACH-Sau3AI fragments exhibited tandem arrays of the monomer unit, and the 224-bp CVI-HaeIII and 135-bp CCA-HaeIII fragments were composed of minisatellite-like and microsatellite-like repeats, respectively. ACH-Sau3AI was a homolog of the chicken nuclear membrane repeat sequence, whose homologs are common in Phasianidae. CVI-MspI, CCA-BamHI, and CSQ-BamHI showed high homology and were specific to the Odontophoridae. CVI-MspI was localized to microchromosomes, whereas CVI-HaeIII, CCA-BamHI, and CSQ-BamHI were mapped to almost all chromosomes. CCA-HaeIII was localized to five pairs of macrochromosomes and most microchromosomes. ACH-Sau3AI was distributed in three pairs of macrochromosomes and all microchromosomes. Centromeric repetitive sequences may be homogenized in chromosome size-correlated and -uncorrelated manners in New World quails, although there may be a mechanism that causes homogenization of centromeric repetitive sequences primarily between microchromosomes, which is commonly observed in phasianid birds.
(キーワード)
Centromeric heterochromatin / Genomic compartmentalization / Homogenization / Microchromosomes / New World quails / Old World quails
Junko Ishijima, Yoshinobu Uno, Chizuko Nishida and Yoichi Matsuda : Genomic structures of the kW1 loci on the Z and W Chromosomes in ratite birds: Structural changes at an early stage of W chromosome differentiation, Cytogenetic and Genome Research, 142, 4, 255-267, 2014.
(要約)
The W chromosome of ratite birds shows minimal morphological differentiation and retains homology of genetic linkage and gene order with a substantial stretch of the Z chromosome; however, the molecular structure in the differentiated region is still not well known. The kW1 sequence was isolated from the kiwi as a W-specific DNA marker for PCR-based molecular sexing of ratite birds. In ratite W chromosomes, this sequence commonly contains a ∼200-bp deletion. To characterize the very early event of avian sex chromosome differentiation, we performed molecular cytogenetic analyses of kW1 and its flanking sequences in paleognathous and neognathous birds and reptiles. Female-specific repeats were found in the kW1-flanking sequence of the cassowary (Casuarius casuarius), and the repeats have been amplified in the pericentromeric region of the W chromosomes of ratites, which may have resulted from the cessation of meiotic recombination between the Z and W chromosomes at an early stage of sex chromosome differentiation. The presence of the kW1 sequence in neognathous birds and a crocodilian species suggests that the kW1 sequence was present in the ancestral genome of Archosauria; however, it disappeared in other reptilian taxa and several lineages of neognathous birds.
Kornsorn Srikulnath, Yoshinobu Uno, Chizuko Nishida and Yoichi Matsuda : Karyotype evolution in monitor lizards: Cross-species chromosome mapping of cDNA reveals highly conserved synteny and gene order in the Toxicofera clade, Chromosome Research, 21, 8, 805-819, 2013.
(要約)
The water monitor lizard (Varanus salvator macromaculatus (VSA), Platynota) has a chromosome number of 2n = 40: its karyotype consists of 16 macrochromosomes and 24 microchromosomes. To delineate the process of karyotype evolution in V. salvator macromaculatus, we constructed a cytogenetic map with 86 functional genes and compared it with those of the butterfly lizard (Leiolepis reevesii rubritaeniata (LRE); 2n = 36) and Japanese four-striped rat snake (Elaphe quadrivirgata (EQU); 2n = 36), members of the Toxicofera clade. The syntenies and gene orders of macrochromosomes were highly conserved between these species except for several chromosomal rearrangements: eight pairs of VSA macrochromosomes and/or chromosome arms exhibited homology with six pairs of LRE macrochromosomes and eight pairs of EQU macrochromosomes. Furthermore, the genes mapped to microchromosomes of three species were all located on chicken microchromosomes or chromosome 4p. No reciprocal translocations were found in the species, and their karyotypic differences were caused by: low frequencies of interchromosomal rearrangements, such as tandem fusions, or centric fissions/fusions between macrochromosomes and between macro- and microchromosomes; and intrachromosomal rearrangements, such as paracentric inversions or centromere repositioning. The chromosomal rearrangements that occurred in macrochromosomes of the Varanus lineage were also identified through comparative cytogenetic mapping of V. salvator macromaculatus and V. exanthematicus. Morphologic differences in chromosomes 6-8 between the two species could have resulted from pericentric inversion or centromere repositioning.
(キーワード)
Cytogenetic map / Karyotype evolution / Monitor lizard / Squamata / Synteny and gene order / Toxicofera
Yoshinobu Uno, C. Nishida, C. Takagi, N. Ueno and Y. Matsuda : Homoeologous chromosomes of Xenopus laevis are highly conserved after whole-genome duplication, Heredity, 111, 5, 430-436, 2013.
(要約)
It has been suggested that whole-genome duplication (WGD) occurred twice during the evolutionary process of vertebrates around 450 and 500 million years ago, which contributed to an increase in the genomic and phenotypic complexities of vertebrates. However, little is still known about the evolutionary process of homoeologous chromosomes after WGD because many duplicate genes have been lost. Therefore, Xenopus laevis (2n=36) and Xenopus (Silurana) tropicalis (2n=20) are good animal models for studying the process of genomic and chromosomal reorganization after WGD because X. laevis is an allotetraploid species that resulted from WGD after the interspecific hybridization of diploid species closely related to X. tropicalis. We constructed a comparative cytogenetic map of X. laevis using 60 complimentary DNA clones that covered the entire chromosomal regions of 10 pairs of X. tropicalis chromosomes. We consequently identified all nine homoeologous chromosome groups of X. laevis. Hybridization signals on two pairs of X. laevis homoeologous chromosomes were detected for 50 of 60 (83%) genes, and the genetic linkage is highly conserved between X. tropicalis and X. laevis chromosomes except for one fusion and one inversion and also between X. laevis homoeologous chromosomes except for two inversions. These results indicate that the loss of duplicated genes and inter- and/or intrachromosomal rearrangements occurred much less frequently in this lineage, suggesting that these events were not essential for diploidization of the allotetraploid genome in X. laevis after WGD.
Nampech Chaiprasertsri, Yoshinobu Uno, Surin Peyachoknagul, Ornjira Prakhongcheep, Sudarath Baicharoen, Saranon Charernsuk, Chizuko Nishida, Yoichi Matsuda, Akihiko Koga and Kornsorn Srikulnath : Highly species-specific centromeric repetitive DNA sequences in lizards: Molecular cytogenetic characterization of a novel family of satellite DNA sequences isolated from the water monitor lizard (Varanus salvator macromaculatus, Platynota), The Journal of Heredity, 104, 6, 798-806, 2013.
(要約)
Two novel repetitive DNA sequences, VSAREP1 and VSAREP2, were isolated from the water monitor lizard (Varanus salvator macromaculatus, Platynota) and characterized using molecular cytogenetics. The respective lengths and guanine-cytosine (GC) contents of the sequences were 190 bp and 57.5% for VSAREP1 and 185 bp and 59.7% for VSAREP2, and both elements were tandemly arrayed as satellite DNA in the genome. VSAREP1 and VSAREP2 were each located at the C-positive heterochromatin in the pericentromeric region of chromosome 2q, the centromeric region of chromosome 5, and 3 pairs of microchromosomes. This suggests that genomic compartmentalization between macro- and microchromosomes might not have occurred in the centromeric repetitive sequences of V. salvator macromaculatus. These 2 sequences did only hybridize to genomic DNA of V. salvator macromaculatus, but no signal was observed even for other squamate reptiles, including Varanus exanthematicus, which is a closely related species of V. salvator macromaculatus. These results suggest that these sequences were differentiated rapidly or were specifically amplified in the V. salvator macromaculatus genome.
B. Fhamida Islam, Satoshi Ishishita, Yoshinobu Uno, R. Md Bazlur Mollah, Kornsorn Srikulnath and Yoichi Matsuda : Male hybrid sterility in the mule duck is associated with meiotic arrest in primary spermatocytes, The Journal of Poultry Science, 50, 4, 311-320, 2013.
Yoshinobu Uno, Y. Asada, C. Nishida, Y. Takehana, M. Sakaizumi and Y. Matsuda : Divergence of repetitive DNA sequences in the heterochromatin of medaka fishes: Molecular cytogenetic characterization of constitutive heterochromatin in two medaka species: Oryzias hubbsi and O. celebensis (Adrianichthyidae, Beloniformes), Cytogenetic and Genome Research, 141, 2-3, 212-226, 2013.
Yoshinobu Uno, Chizuko Nishida, Hiroshi Tarui, Satoshi Ishishita, Chiyo Takagi, Osamu Nishimura, Junko Ishijima, Hidetoshi Ota, Ayumi Kosaka, Kazumi Matsubara, Yasunori Murakami, Shigeru Kuratani, Naoto Ueno, Kiyokazu Agata and Yoichi Matsuda : Inference of the Protokaryotypes of Amniotes and Tetrapods and the Evolutionary Processes of Microchromosomes from Comparative Gene Mapping, PLoS ONE, 7, 12, e53027, 2012.
(要約)
Comparative genome analysis of non-avian reptiles and amphibians provides important clues about the process of genome evolution in tetrapods. However, there is still only limited information available on the genome structures of these organisms. Consequently, the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes in tetrapods remain poorly understood. We constructed chromosome maps of functional genes for the Chinese soft-shelled turtle (Pelodiscus sinensis), the Siamese crocodile (Crocodylus siamensis), and the Western clawed frog (Xenopus tropicalis) and compared them with genome and/or chromosome maps of other tetrapod species (salamander, lizard, snake, chicken, and human). This is the first report on the protokaryotypes of amniotes and tetrapods and the evolutionary processes of microchromosomes inferred from comparative genomic analysis of vertebrates, which cover all major non-avian reptilian taxa (Squamata, Crocodilia, Testudines). The eight largest macrochromosomes of the turtle and chicken were equivalent, and 11 linkage groups had also remained intact in the crocodile. Linkage groups of the chicken macrochromosomes were also highly conserved in X. tropicalis, two squamates, and the salamander, but not in human. Chicken microchromosomal linkages were conserved in the squamates, which have fewer microchromosomes than chicken, and also in Xenopus and the salamander, which both lack microchromosomes; in the latter, the chicken microchromosomal segments have been integrated into macrochromosomes. Our present findings open up the possibility that the ancestral amniotes and tetrapods had at least 10 large genetic linkage groups and many microchromosomes, which corresponded to the chicken macro- and microchromosomes, respectively. The turtle and chicken might retain the microchromosomes of the amniote protokaryotype almost intact. The decrease in number and/or disappearance of microchromosomes by repeated chromosomal fusions probably occurred independently in the amphibian, squamate, crocodilian, and mammalian lineages.
Mari Suda, Yoshinobu Uno, Yumiko Mori, Yoichi Matsuda and Masahisa Nakamura : Molecular cytogenetic characterization of telomere-specific repetitive DNA sequences in Rana rugosa, Journal of Experimental Zoology. Part A, Ecological Genetics and Physiology, 315 A, 4, 222-231, 2011.
(要約)
We performed a molecular cloning of the glutamic oxaloacetic transaminase (GOT1) gene from R. rugosa, and determined its chromosomal location. This gene was reportedly localized near the sex-determining region of the ZW sex chromosomes in the frog Buergeria buergeri; however, the GOT1 gene was mapped to the distal end of chromosome 9 in R. rugosa using a GOT1 cDNA FISH probe. This was also the case when a 46.3 kb genomic clone containing exon 8 and 9 and the 3'-neighboring region of the GOT1 gene, designated clone B, was used as probe. However, weak signals were also detected at the telomeric ends of other autosomes and the Z sex chromosome, and near the centromeric region of the W sex chromosome. To intensify the signals, we used eight internal fragments in clone B and applied them to chromosome mapping. Consequently, only two fragments containing repeated sequence blocks produced hybridization signals; those signals were observed on autosomes and ZW sex chromosomes. The 3'-neighboring region contained two types of repeated sequence elements: a 41 bp element, designated 41-REL, localized to telomeric ends of autosomes and a 31 bp element, designated 31-REL, localized to telomeric ends of all autosomes and the ZW sex chromosomes, and also near the centromere on the W long arm. The results collectively suggest that the two repeated sequence elements were independently amplified around the chromosomal telomeres in R. rugosa, indicating that they will be useful cytogenetic markers for studying karyotypic evolution-especially the W chromosome differentiation-in this species.
Mari Suda, Yoshinobu Uno, Jun Fujii, Yoichi Matsuda and Masahisa Nakamura : Isolation and characterization of the CYP17A1 gene and its processed pseudogene in Rana rugosa, Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 160, 1, 54-61, 2011.
(要約)
CYP17A1 expression is up-regulated in the gonad in Rana (Glandirana) rugosa tadpoles treated with androgens to induce female-to-male sex-reversal. In this study, we isolated the CYP17A1 gene and its processed pseudogene from R. rugosa. The former was found to consist of 8 exons, and the latter a single-exon gene, designated CYP17A1P. The sequence of the promoter region of CYP17A1 differed from that of CYP17A1P. We found several consensus binding-sites for candidate transcription factors including androgen receptor (AR), Sox and FoxL2 in the CYP17A1 promoter region, but an AR-binding site was absent from CYP17A1P. When AR was over-expressed in Xenopus A6 cells, it did not increase CYP17A1 transcription in luciferase assays. CYP17A1 was strongly expressed in indifferent male gonads during sex determination and exclusively in testis, among eight adult tissues of R. rugosa. By contrast, CYP17A1P was expressed at very low, and similar levels in the adult tissues of both sexes. Fluorescent In-Situ Hybridization (FISH) analysis showed that CYP17A1P is localized to chromosome 4, while CYP17A1 is on chromosome 9. These results collectively suggest that CYP17A1, but not CYP17A1P is involved in male sex-determination in R. rugosa, and that androgens may not have a direct effect on the CYP17A1 transcription.
Kornsorn Srikulnath, Chizuko Nishida, Kazumi Matsubara, Yoshinobu Uno, Amara Thongpan, Saowanee Suputtitada, Somsak Apisitwanich and Yoichi Matsuda : Karyotypic evolution in squamate reptiles: Comparative gene mapping revealed highly conserved linkage homology between the butterfly lizard (Leiolepis reevesii rubritaeniata, Agamidae, Lacertilia) and the Japanese four-striped rat snake (Elaphe quadrivirgata, Colubridae, Serpentes), Chromosome Research, 17, 8, 975-986, 2009.
(要約)
The butterfly lizard (Leiolepis reevesii rubritaeniata) has the diploid chromosome number of 2n = 36, comprising two distinctive components, macrochromosomes and microchromosomes. To clarify the conserved linkage homology between lizard and snake chromosomes and to delineate the process of karyotypic evolution in Squamata, we constructed a cytogenetic map of L. reevesii rubritaeniata with 54 functional genes and compared it with that of the Japanese four-striped rat snake (E. quadrivirgata, 2n = 36). Six pairs of the lizard macrochromosomes were homologous to eight pairs of the snake macrochromosomes. The lizard chromosomes 1, 2, 4, and 6 corresponded to the snake chromosomes 1, 2, 3, and Z, respectively. LRE3p and LRE3q showed the homology with EQU5 and EQU4, respectively, and LRE5p and LRE5q corresponded to EQU7 and EQU6, respectively. These results suggest that the genetic linkages have been highly conserved between the two species and that their karyotypic difference might be caused by the telomere-to-telomere fusion events followed by inactivation of one of two centromeres on the derived dicentric chromosomes in the lineage of L. reevesii rubritaeniata or the centric fission events of the bi-armed macrochromosomes and subsequent centromere repositioning in the lineage of E. quadrivirgata. The homology with L. reevesii rubritaeniata microchromosomes were also identified in the distal regions of EQU1p and 1q, indicating the occurrence of telomere-to-telomere fusions of microchromosomes to the p and q arms of EQU1.
K. Srikulnath, K. Matsubara, Yoshinobu Uno, A. Thongpan, S. Suputtitada, S. Apisitwanich, Y. Matsuda and C. Nishida : Karyological characterization of the butterfly lizard (leiolepis reevesii rubritaeniata, agamidae, squamata) by molecular cytogenetic approach, Cytogenetic and Genome Research, 125, 3, 213-223, 2009.
(要約)
Karyological characterization of the butterfly lizard (Leiolepis reevesii rubritaeniata) was performed by conventional Giemsa staining, Ag-NOR banding, FISH with the 18S-28S and 5S rRNA genes and telomeric (TTAGGG)n sequences, and CGH. The karyotype was composed of 2 distinct components, macrochromosomes and microchromosomes, and the chromosomal constitution was 2n = 2x = 36 (L(4)(m) + L(2)(sm) + M(2)(m) + S(4)(m) + 24 microchromosomes). NORs and the 18S-28S rRNA genes were located at the secondary constriction of the long arm of chromosome 1, and the 5S rRNA genes were localized to the pericentromeric region of chromosome 6. Hybridization signals of (TTAGGG)n sequences were observed at the telomeric ends of all chromosomes and interstitially at the same position as the 18S-28S rRNA genes, suggesting that in the Leiolepinae tandem fusion probably occurred between chromosome 1 and a microchromosome where the 18S-28S rRNA genes were located. CGH analysis, however, failed to identify sex chromosomes, suggesting that this species may have a TSD system or exhibit GSD with morphologically undetectable cryptic sex chromosomes. Homologues of 6 chicken Z-linked genes (ACO1/IREBP, ATP5A1, CHD1, DMRT1, GHR, RPS6) were all mapped to chromosome 2p in the same order as on the snake chromosome 2p.
(キーワード)
Butterfly lizard / CGH / FISH / Gene mapping / Microchromosome / RNA genes / Sex chromosome / Tandem fusion / Telomere
T. Kawagoshi, Yoshinobu Uno, K. Matsubara, Y. Matsuda and C. Nishida : The ZW micro-sex chromosomes of the chinese soft-shelled turtle (pelodiscus sinensis, trionychidae, testudines) have the same origin as chicken chromosome 15, Cytogenetic and Genome Research, 125, 2, 125-131, 2009.
(要約)
The Chinese soft-shelled turtle (Pelodiscus sinensis, Trionychidae, Testudines) has ZZ/ZW-type micro-sex chromosomes where the 18S-28S ribosomal RNA genes (18S-28S rDNA) are located. The W chromosome is morphologically differentiated from the Z chromosome by partial deletion and amplification of 18S-28S rDNA and W-specific repetitive sequences. We recently found a functional gene (TOP3B) mapped on the P. sinensis Z chromosome, which is located on chicken (Gallus gallus, GGA) chromosome 15. Then we cloned turtle homologues of 4 other GGA15-linked genes (GIT2, NF2, SBNO1, SF3A1) and localized them to P. sinensis chromosomes. The 4 genes all mapped on the Z chromosome, and 2 of them (SBNO1, SF3A1) were also localized to the W chromosome. Our mapping data suggest that at least one large inversion occurred between GGA15 and the P. sinensis Z chromosome, and that there are homologous regions in the distal portions of both the short and long arms between the Z and W chromosomes. W chromosomal differentiation in P. sinensis probably proceeded by the deletion of the proximal chromosomal region followed by 18S-28S rDNA amplification, after a paracentric inversion occurred at the breakpoints between the distal region of 18S-28S rDNA and the proximal region of SBNO1 on the Z chromosome.
(キーワード)
Chicken / Chromosome homology / Chromosome rearrangement / Comparative mapping / FISH / Reptile / Sex chromosome / Sex determination / Turtle / ZW