寒冷バイオシステム研究センター

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S. Miyagi, Y. Zhao, Y. Saitoh, K. Tamai and K. Tsutsumi (2001)
Replication of the rat aldolase B locus differs between aldolaseB-expressing and non-expressing cells. FEBS Lett. 505: 332-336.
We previously reported a rat chromosomal origin of DNA replication (oriA1) that encompassed the aldolase B (AldB) gene promoter. Here, we examined utilization of oriA1 in AldB-expressing and non-expressing cells. The results suggested the occurrence of mutually exclusive regulation between DNA replication and transcription. Nascent strand abundance as assayed bycompetitive PCR using BrdU-labeled nascent DNA indicated that oriA1 is notutilized in AldB-expressing cells, while it is fired in non-expressing cells. In the latter non-expressing cells, replication fork seemed to slowat 20 to 22 kb downstream of oriA1.

T. Yabuki, S. Miyagi, H.Ueda, Y. Saitoh and K. Tsutsumi (2001)
A novel growth-related nuclear protein binds and inhibits rat aldolase B gene promoter. Gene 264: 123-129.
The promoter of the rat aldolase B (AldB) gene that confers liver-specific transcription has an additional role.　It functions in vivo as an originregion of DNA replication in the cells in which the gene is repressed　(Zhao,Y., Tsutsumi, R., Yamaki, M., Nagatsuka, N., Ejiri, S. and Tsutsumi,K., 1994. Initiation zone of DNA replication at the rat aldolase B locus encompasses transcription promoter region. Nucl. Acids. Res. 22,5385-5390). This promoter/origin region has multiple protein-binding sites and, thus, binding of a particular set of protein factors in AldB-expressing or non-expressing cells seems to correlate with functional switch of this promoter/origin region. In the present study, we characterized two closely related proteins, termed AlF-C1 and AlF-C2,which are assumed to be involved in repression of the AldB gene. These two proteins share an identical amino acid sequence except for a 47-residue-insertion in AlF-C1, and are members of a gene family including heterogeneous nuclear ribonucleoprotein (hnRNP) and CCAAT-binding factor subunit A (CBF-A) genes.　Bacterially expressed AlF-C1 can bind sequence-specifically to the AldB gene promoter, whereas AlF-C2 can only weakly. Transfection experiments using mammalian expression vectors showed that AlF-C1 down-regulates the AldB gene promoter in rat hepatoma cells,while AlF-C2 had no or little effect. Expressions of mRNAs encoding these two proteins are enriched in fetal livers and in regenerating livers.These results implied that AlF-C1 and/or C2 is involved in growth-regulated repression of the AldB gene.

M. Yoshino, A. Kanazawa, K. Tsutsumi, I. Nakamura and Y. Shimamoto (2001)
Structure and characterization of the gene encoding a subunit of soybean β-conglycinin. Genes Genet. Syst. 76: 99-105.
β-conglycinin, a soybean seed storage protein, is comprised of three different subunits α, α', and β. Several candidates for the a subunitgene have been isolated, however, the structure of the α subunit gene has not been completely determined. Here we have determined the nucleotide sequence and transcription start site of the α subunit gene, and compared the structural components with those of other subunit or other seed protein genes. The α subunit gene, which is located on a 7.6-kb EcoRI fragment, was composed of six exons that had the same organization as those for the α' subunit gene. Within a 400 bp upstream region of the transcription start site, four regions (designated as boxes I, II, III, and IV) were found to be conserved among the α and α' and other seed protein genes. Genomic Southern blot analysis of soybean varieties lacking the α subunit gene candidate indicated that the gene characterized in this paper actually encodes the α subunit and is functionally active. In addition, these experiments revealed the presence of an additional gene which is also responsible for the expression of the α subunit.

K.-J. Wang, Y. Takahata, K. Ito, Y. Zhao, K. Tsutsumi and N. Kaizuma (2001)
Genetic characterization of a novel soybean kunitz trypsin inhibitor. Breeding Sci. 51: 185-190.
Genetic and nucleotide sequence studies were performed on a new variant of soybean Kunitz trypsin inhibitor (SKTI) detected in wild soybean (Glycine soja) and showing a slightly slower electrophoretic mobility than the Tia type. The segregation analysis of SKTI bands in F ₂ seeds from crosses of the new variant type with Tia or Tib type showed that the variant type is controlled by an allele codominant to Tia and Tib at an SKTI locus. Nucleotide sequence analysis showed that this variant has 217 amino acids composed of 181 amino acid residues of mature SKTI and extra 25 and 11amino acids at N- and C- terminal regions, respectively. This sequence was identical to that of Tia (= KTi3), except that a G → A transitional mutation occurred at position 500 of Tia, which results in the translational change from Arg to Lys. The result of isoelectric focusing-PAGE coincided with this change. In addition, three nucleotides GCT were inserted at the N-terminus, which leads to an Ala addition in the precursor of this SKTI protein. From these results, we propose the genetic symbol Tie for the new variant of SKTI.