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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.

S. Kobayashi, S. Kidou and S. Ejiri (2001)
Detection and characterizationof glutathione S-transferase activity in rice EF-1ββ'γ and EF-1γ expressed in E.coli. Biochem. Biophys. Res. Commun. 288: 509-514.
Plant elongation factor EF-1 consists of four subunits (EF-1αββ'γ). EF-1α・GTP catalyses the binding of aminoacyl-tRNA to the ribosome. EF-1β and　EF-1β' catalyze the GDP/GTP exchange on EF-1α・GDP. However, the function of EF-1γ, a subunit detected in eukaryotes, but not in prokaryotes remained unknown. This report demonstrates that rice EF-1ββ'γ and recombinant EF-1γ possess glutathione S-transferase (GST) activity. The EF-1ββ'γ- or EF-1γ-dependent GST activity is about one-fiftieth of the rice GST activity. The Km values of EF-1ββ'γ, EF-1γ and rice GST for glutathione and 1-chloro-2,4-dinitrobenzene are of about the same order. Although recombinant EF-1γ is heat labile, active EF-1γ was obtained by purifying it in the presence of 20 % glycerol.

T. Shiina, M. Morimatsu, H. Kitamura, T. Ito, S. Kidou, K. Matsubara, Y.Matsuda, M. Saito and B. Syuto (2001)
Genomic organization, chromosomal localization, and promoter analysis of the mouse Mail gene. Immunogenetics 53: 649-655.
The Mail (molecule possessing ankyrin repeats induced by lipopolysaccharide) protein is a member of the IkappaB family. It has six ankyrin repeats that are conserved in other IkappaB proteins, such as IkappaB-alpha and Bcl-3. Mail mRNA expression is induced rapidly following lipopolysaccharide (LPS) injection, most notably in the spleen, lung, and lymph nodes of mice, where immune cells, such as lymphocytes and macrophages, are abundant. In this study, we cloned and characterized the Mail gene. The isolated genomic clones span approximately 30 kb and encompass the entire gene. Comparisons with Mail cDNA revealed that the Mail gene consists of 14 exons. Several splice junctions encoding ankyrin repeats are conserved among Mail and other IkappaB family genes. Southern hybridization showed that Mail is a single-copy gene. Using fluorescence in situ hybridization analysis, mouse and rat Mail genes were mapped to Chromosome (Chr) 16C1.2-C1.3 and Chr 11q21.1, respectively. Primer extension determined the transcription start site of Mail. Sequence analysis of the proximal promoter region revealed the presence of a TATA box and putative transcription factor-binding sites, such as those for NF-kappaB and NF-IL6. This region is sufficient to drive high-level reporter gene expression in LPS-stimulated transfected cells.

K. Otsu, K. Ito, T. Kuzumaki and Y. Iuchi (2001)
Differential regulation of liver-specific and ubiquitously expressed genesin primary rat hepatocytes by the extracellular matrix. Cell. Physiol.Biochem. 11: 33-40.
Primary rat hepatocytes were cultured with an extracellular matrix (ECM) overlay, in order to investigate the effect of an ECM on gene expression in hepatocytes. When hepatocytes, isolated by the collagenase-perfusion method, were cultured on type I collagen-coated dishes, the mRNA levels of liver-specific genes (aldolase B, tyrosine aminotransferase and albumin) decreased continuously, while those of ubiquitously-expressed genes (glyceraldehyde 3-phosphate dehydrogenase gene andβ-actin) increased.When a dilute ECM derived from the Engelbreth-Holm-Swarm mouse sarcoma (anEHS gel) was added to the above hepatocytes 3 days after plating, the mRNA levels of liver-specific genes increased, while those of ubiquitously-expressed genes decreased. The effects of a rat liver biomatrix (a physiological ECM for rat hepatocytes) on gene expression inprimary hepatocytes were similar to those of the EHS gel. A nuclear run-on assay, and 5, 6-dichloro-1-β-d-ribofuranosylbenzimidazole or actinomcin D treatments revealed that the transcriptional rates of liver-specific genes were enhanced by the EHS gel overlay, while the apparent stability of the corresponding mRNAs were unchanged. In contrast, the transcriptional rates of ubiquitously-expressed genes were not greatly affected by an EHS gel overlay, while the apparent stability of their mRNAs were decreased. These data suggest that the ECM plays an important role in the maintenance of the differentiated characteristics of liver-specific genes and, also, by destabilizing the mRNAs of ubiquitously-expressed genes.