寒冷バイオシステム研究センター

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Takahashi, M., Hikage, T., Yamashita, T., Saitoh, Y., Endou, M. and Tsutsumi, K. （2006）
Stress-Related Proteins Are Specifically Expressed under Non-Stress Conditions in the Overwinter Buds of the Gentian Plant Gentiana triflora.
Breeding Science, in press.
The overwinter bud of the gentian plant, Gentiana triflora, is an organ that develops from the basal tuber in late spring and becomes dormant and freezing-tolerant for overwintering. Here, proteins specifically enriched in the overwinter buds were identified using two-dimensional polyacrylamide gel electrophoresis followed by amino acid sequencing. Enriched proteins included those so far identified as stress- and cold-inducible in other plants, i.e., two isoforms of ethylene-induced esterase, a dehydration-induced protein, glyoxalase I, thioredoxin peroxidase, two isoforms of ascorbate peroxidase and a membrane pore protein. Interestingly, the stress-related proteins already accumulated in the overwinter buds under non-stress conditions in early summer. Accumulation of these proteins was also examined in a mutant gentian, which normally develops the overwinter buds but lacks cold tolerance and dies under cold conditions below 3 ^oC. In this mutant, several proteins including one of the putative esterase, enolase and ascorbate peroxidase were absent or greatly decreased. Thus, it appears that these stress-related proteins are expressed under non-stress conditions to prepare cold tolerance before overwintering.

Shimotai, Y., Minami, H., Saitoh, Y., Onodera, Y., Mishima, Y., Kelm Jr. R. J. and Tsutsumi, K.（2006）
A binding site for Pura and Purb is structurally unstable and is required for replication in vivo from the rat aldolase B origin.
Biochem, Biophys. Res. Commun., 340, 517-525. (published online December 20, 2005)
The rat aldolase B promoter acts as a replication origin in vivo, as well as an autonomously replicating sequence (ARS). Here, we examined roles of a polypurine stretch (site PPu) in this origin, which is indispensable to the ARS activity. Purification of site PPu-binding protein revealed that site PPu binds Purα and Purβ, i.e., single-stranded DNA binding proteins whose roles in replication have been implicated, but less clear. Biochemical analyses showed that site PPu even in a longer DNA fragment is unstable in terms of double-helix, implying that Purα/β may stabilize single-stranded state. Deletion of site PPu from the origin DNA, which was ectopically positioned in the mouse chromosome, significantly reduced replicator activity. Chromatin immunoprecipitation experiments showed that deletion of site PPu abolishes binding of the Purα/β proteins to the origin. These observations suggest functional roles of site PPu and Purα/β proteins in replication initiation.

Matsukawa, K., Ogata, M., Hikage, T., Minami, H., Shimotai, Y., Saitoh, Y., Yamashita, T., Ouchi, A., Tsutsumi, R., Fujioka, T. and Tsutsumi, K. (2006)
Antiproliferative activity of root extract from gentian plant (Gentiana triflora) on cultured and implanted tumor cells.
Biosci. Biotech. Biochem. 　(in press)
We describe a novel pharmacological activity of the gentian root, an ingredient of Chinese medicines. The 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 the implanted solid tumors. The extract treatment of cultured cells resulted in appearance of shrank, fragmented or condensed cell and nuclear morphologies, and in chromosomal DNA degradation. However, the extract-treated cells did not show DNA fragmentation that exhibits nucleosome ladder, suggesting that the extract-triggered cell death is not mediated through a typical apoptotic pathway.

Yoshino, M., Nagamatsu, A., Tsutsumi, K. and Kanazawa, A.（2006）
The regulatory function of the upstream sequence of the β-conglycinin α-subunit gene in seed-specific transcription is associated with the presence of RY sequence.
Genes & Genetic Systems (in press)
β-conglycinin, a major component of seed-storage proteins in soybean, comprises three subunits: α, α' and β. Expression of these genes is spatially regulated in a stringent manner and occurs during seed development. To understand the mechanisms that control expression of the subunit gene, we analyzed the nucleotide sequence of the 2.9-kb region upstream of the gene. The upstream sequence up to -1357 or a series of its 5'-deleted derivatives was fused to the β-glucuronidase (GUS) gene. These reporter gene constructs were introduced into Arabidopsis thaliana plants via Agrobacterium-mediated gene transfer. Prominent GUS activity was detected in developing seeds of the T3 generation when 245 bp or longer sequences of the upstream region were fused to the GUS gene. We found a clear association of decreased GUS activity with a stepwise deletion of a region containing the RY sequence from the original construct. These results are consistent with the notion that multiple sequence elements including the RY sequences are involved in the seed-specific transcriptional activation of the β-conglycinin α subunit gene in soybean.