Ken-ichi Tsutsumi

Triploid form of Fasciola in Japan : genetic relationships between Fasciola hepatica and Fasciola gigantica determined by ITS-2 sequence of nuclear rDNA

The ITS-2 sequences from seven worms of the Japanese triploid form of Fasciola, two of Fasciola hepatica from Uruguay and four of Fasciola gigantica from Zambia and Indonesia were determined. The ITS-2 sequences of the Japanese triploid worms were divided into two distinct types, one of which was almost identical to that of F. hepatica and the other to F. gigantica from Indonesia.

Taxonomic status of the Japanese triploid forms of Fasciola : comparison of mitochondrial ND1 and COI sequences with F. hepatica and F. gigantica

Because it is difficult to identify morphologically the Japanese forms of Fasciola, additional taxonomic criteria are required. In order to clarify the genetic relationships between Fasciola hepatica, Fasciola gigantica, and the Japanese triploid forms of Fasciola, we compared nucleotide sequences of mitochondrial NADH dehydrogenase subunit 1 (ND1) and cytochrome c oxidase subunit I (COI) genes. Intraspecific variation in the ND1 and COI sequences was low, except for 1 specimen of F. gigantica in the ND1 sequence. The ND1 and COI sequences of Japanese triploid forms of Fasciola were nearly identical to those found in F. gigantica but were different from those of F. hepatica. Thus, the Japanese triploid forms of Fasciola are thought to be categorized as F. gigantica.

The syncytium-specific expression of the Orysa;KRP3 CDK inhibitor: implication of its involvement in the cell cycle control in the rice (Oryza sativa L.) syncytial endosperm

During rice (Oryza sativa L.) seed development, the primary endosperm nucleus undergoes a series of divisions without cytokinesis, producing a multinucleate cell, known as a syncytium. After several rounds of rapid nuclear proliferation, the syncytium ceases to undergo mitosis; thereafter, the syncytium is partitioned into individual cells by a specific type of cytokinesis called cellularization. The transition between syncytium and cellularization is important in determining the final seed size and is a model for studying the cell cycle and cytokinesis. The involvement of cyclin-dependent kinase (CDK) inhibitors (CKIs) in cell cycle control was investigated here during the transition between syncytium and cellularization. It was found that one of the rice CKIs, Orysa;KRP3, is strongly expressed in the caryopsis at 2 d after flowering (DAF), and its expression is significantly reduced at 3 DAF. The other CKI transcripts did not show such a shift at 2 DAF. In situ hybridization analysis revealed that Orysa;KRP3 is expressed in multinucleate syncytial endosperm at 2 DAF, but not in cellularized endosperm at 3 DAF. Two-hybrid assays showed that Orysa;KRP3 binds Orysa;CDKA;1, Orysa;CDKA;2, Orysa;CycA1;1, and Orysa;CycD2;2. By contrast, Orysa;CDKB2;1 and Orysa;CycB2;2 do not show binding to Orysa;KRP3. Orysa;KRP3 was able to rescue yeast premature cell division due to the dominant positive expression of mutant rice CDKA;1 indicating that Orysa;KRP3 inhibited rice CDK. These data suggest that Orysa;KRP3 is involved in cell cycle control of syncytial endosperm.

Binding of AlF-C, an Orc1-Binding Transcriptional Regulator, Enhances Replicator Activity of the Rat Aldolase B Origin

ABSTRACT A region encompassing the rat aldolase B gene (aldB) promoter acts as a chromosomal origin of DNA replication (origin) in rat aldolase B-nonexpressing hepatoma cells. To examine replicator function of the aldB origin, we constructed recombinant mouse cell lines in which the rat aldB origin and the mutant derivatives were inserted into the same position at the mouse chromosome 8 by cre-mediated recombination. Nascent strand abundance assays revealed that the rat origin acts as a replicator at the ectopic mouse locus. Mutation of site C in the rat origin, which binds an Orc1-binding protein AlF-C in vitro, resulted in a significant reduction of the replicator activity in the mouse cells. Chromatin immunoprecipitation (ChIP) assays indicated that the reduction of replicator activity was paralleled with the reduced binding of AlF-C and Orc1, suggesting that sequence-specific binding of AlF-C to the ectopic rat origin leads to enhanced replicator activity in cooperation with Orc1. Involvement of AlF-C in replication in vivo was further examined for the aldB origin at its original rat locus and for a different rat origin identified in the present study, which contained an AlF-C-binding site. ChIP assays revealed that both replication origins bind AlF-C and Orc1. We think that the results presented here may represent one mode of origin recognition in mammalian cells.

Antiproliferative Activity of Root Extract from Gentian Plant (Gentiana triflora) on Cultured and Implanted Tumor Cells

We describe a novel pharmacological activity of the gentian root, an ingredient of Chinese medicines. Root extract from Gentiana triflora triggered cell death of human Daudi cells in culture. In addition, daily administration of the extract to mice inhibited growth of implanted solid tumors. Extract treatment of cultured cells resulted in the appearance of shranken, fragmented, or condensed cell and nuclear morphologies, and in chromosomal DNA degradation. But, the extract-treated cells did not show DNA fragmentation, which exhibits a nucleosome ladder, suggesting that extract-triggered cell death is not mediated through a typical apoptotic pathway.

The promoter from the rice nuclear gene encoding chloroplast aldolase confers mesophyll-specific and light-regulated expression in transgenic tobacco

The rice genome contains at least four separate loci that encode aldolase isozymes. Among these, the aldolase P (AldP) gene, a nuclear gene coding for chloroplast aldolase, is expressed predominantly in the leaf blade mesophyll cells in rice. To dissect promoter elements that regulate such tissue- or cell type-specific expression, we constructed variousAldP promoter-β-glucuronidase (GUS) fusion genes and transferred them intoNicotiana tabacum (tobacco) plants. Analysis of GUS activities in the transgenic tobacco revealed the presence of at least two elements within 2.0 kbAldP promoter region. One is located within the segment from position − 2.0 kb to − 1.2 kb and acts as a negative element. The other is a positive element located between − 1.2 kb and − 0.31 kb that confers developmentally regulated, mesophyll cellspecific expression. In addition, the 1.2 kb rice promoter segment flanking the transcription start site contains an element(s) that serves as target for light induction in tobacco. The results suggest that theAIdP gene promoter of rice, a monocot promoter, can function in an essentially physiological manner in the dicot tobacco plant.

Structure and allele-specific expression variation of novel α/β hydrolase fold proteins in gentian plants

Previously, we identified two closely related proteins termed W14 and W15 that were enriched in the overwinter buds of the gentian plant Gentiana triflora. Expression of the latter protein W15 has been implicated in its association with cold hardiness, because of its absence in a cold-sensitive mutant. Here, we characterized these two proteins and the genes encoding them. Amino acid sequences of the W14 and W15 proteins showed difference at only three amino acid positions, and both of them showed homologies to α/β hydrolase fold superfamily. Consistently, GST-fused W14 and W15 proteins expressed in bacteria showed hydrolase activity toward 1-naphtyl acetate. Structural analysis of these two genes in seven different gentian strains/cultivars including an anther culture-derived homozygous diploid revealed that W14 and W15 genes are allelic. Three genotypes were found; two strains carried both alleles (W14/W15), one carried the W15 genes in both alleles (W15/W15), and others were homozygous of W14 (W14/W14). Interestingly, expression of the two proteins exhibited allele-specificity. In one W14/W15 strain, expression of the W15 allele was almost repressed. In addition, organ specific expression of the alleles was observed in different cultivars. These observations were discussed in relation to winter hardiness of the gentian plants.

Genomic structure of the rice aldolase isozyme C-1 gene and its regulation through a Ca2+-mediated protein kinase-phosphatase pathway

Complementary and genomic DNA clones coding for aldolase C-1, the fourth-type isozyme of aldolase in rice Oryza sativa L., have been characterized. The organization of the gene is quite similar to those encoding rice aldolase C-a and a maize cytoplasmic-type aldolase, in that introns are located in the same position. Amino acid sequences are highly conserved among cytoplasmic aldolases in plants. Expression of the gene in rice callus is activated by a protein phosphatase inhibitor okadaic acid, and is inhibited in the presence of thapsigargin, a reagent which increases calcium influx into the cytoplasm. The inhibition is rescued by the simultaneous addition of protein kinase inhibitor H-7. Thus, it is suggested that expression of the aldolase C-1 gene is regulated through a signal transduction pathway involving a Ca2+-mediated protein kinase-protein phosphatase system.

W14/15 esterase gene haplotype can be a genetic landmark of cultivars and species of the genus Gentiana L

We have identified multiple alleles for a single gene termed W14/15. This gene encodes closely related but not identical proteins W14 and W15 that accumulate in overwinter buds of Gentiana triflora (Takahashi et al. in Breed Sci 56:39–46, 2006; Hikage et al. in Mol Genet Genomics 278:95–104, 2007). In this study, structural analysis of the W14/15 gene was carried out for 21 different gentian lines/cultivars consisting of 5 different species, to survey species- or line/cultivar-specific haplotypes. Within the samples examined, multiple variant forms were found. Those were categorized into seven major types (type I–VII) and ten subtypes based on the presence of three short insertion/deletion sites, three RFLP sites, and several SNP sites. Each line/cultivar had a distinct set of W14/15 gene variants for an allelic pair. Phylogenetic analysis showed that the W14/15 alleles cluster into groups that are characteristic of gentian species, i.e., G. triflora, G. scabra, G. pneumonanthe, G. septemfida and an unknown species other than the former four. In addition, within the same gentian species, different sets of haplotypes were found. Thus, the W14/15 alleles provide useful landmarks to resolve phylogenies of the genus or section Gentiana, as well as to analyze pedigree and breeding history of the cultivars derived from those Gentiana sp.

Comparison of plastid DNA replication in different cells and tissues of the rice plant.

In a previous study, we mapped replication origin regions of the plastid DNA around the 3′ end of the 23S rRNA gene in rice suspension-cultured cells. Here, we examined initiation of the plastid DNA replication in different rice cells by two-dimensional agarose gel electrophoresis. We show for the first time, to our knowledge, that the replication origin region of the plastid DNA differs among cultured cells, coleoptiles and mature leaves. In addition, digestion of the replication intermediates from the rice cultured cells with mung bean nuclease, a single-strand-specific nuclease, revealed that both two single strands of the double-stranded parental DNA were simultaneously replicated in the origin region. This was further confirmed by two-dimensional agarose gel analysis with single-stranded RNA probes. Thus, the mode of plastid DNA replication presented here differs from the unidirectional replication started by forming displacement loops (D-loops), in which the two D-loops on the opposite strands expand toward each other and only one parental strand serves as a template.