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O Koiwai

Publications and source records attributed to O Koiwai.

At least 19 recordsLinked to original sources
PubMed2001

Drosophila mitochondrial transcription factor A: characterization of its cDNA and expression pattern during development.

We cloned a cDNA for Drosophila mitochondrial transcription factor A (D-mtTFA) and characterized the recombinant protein. In Drosophila Kc cells, D-mtTFA was localized in the mitochondria, but not in the nucleus. By repetitive precipitation with His-tag and PCR amplification, the consensus nucleotide sequence for D-mtTFA-binding was determined to be 5'-TTATC/G. The binding sequence was found to be clustered in the A + T region of mitochondrial DNA which is suggested to be a replication origin and promoter region for light strand and heavy strand. We found a DNA replication-related element (DRE)-like sequence located upstream of the transcription initiation site of the D-mtTFA gene and obtained results indicating that DRE-binding factor (DREF) can bind to the DRE-like sequence of the D-mtTFA gene. The data suggest that transcription of the D-mtTFA gene is under control of the DRE/DREF regulatory system. Based on these results, the functions of D-mtTFA were discussed in relation to mitochondrial biogenesis of Drosophila melanogaster.

Animals↗
PubMed2001

Terminal deoxynucleotidyltransferase directly interacts with a novel nuclear protein that is homologous to p65.

BACKGROUND: Terminal deoxynucleotidyltransferase (TdT) is a DNA polymerase that enhances Ig and TcR gene diversity in the N region in B- and T-cells. TdT is found as a member of a large protein complex in the lysate of the thymocytes. To elucidate the molecular mechanism of the synthesis of the N region, we first attempted to isolate the genes with products that are interacting directly with TdT. RESULTS: Using a yeast two-hybrid system, we isolated a cDNA clone encoding a novel nuclear protein that interacts with TdT. This protein was designated as TdT interacting factor 1 (TdIF1). TdIF1 has a high degree of homology to the transcription factor p65, which belongs to the nuclear receptor superfamily. TdIF1 contains HMG-I and HMG-Y DNA binding domains (AT-hooks) and can bind to single- and double-stranded DNA. TdT and TdIF1 were co-eluted at position 232 kDa by gel filtration of MOLT4 lysate. TdIF1 can enhance TdT activity fourfold in vitro assay system using oligo(dT)16 as primers. CONCLUSIONS: TdIF1 binds directly to TdT, both in vitro and in vivo. TdIF1 and TdT exist as the members of a 232 kDa protein complex. TdIF1 can enhance TdT activity maximum fourfold in vitro assay system, suggesting that it positively regulates the synthesis of the N region during V(D)J recombination in the Ig and TcR genes.

Amino Acid Sequence↗
PubMed2001

Terminal deoxynucleotidyltransferase is negatively regulated by direct interaction with proliferating cell nuclear antigen.

BACKGROUND: The repertoires of Ig and TcR are generated by a combinatorial rearrangement of variable (V), diversity (D), and joining (J) segments (V(D)J recombination) in B- and T-cells. Terminal deoxynucleotidyltransferase (TdT) adds extra nucleotides (N nucleotides) at the junctions of the gene segments to enhance the Ig and TcR genes diversity. Using an anti-TdT antibody column, TdT has been purified as a member of a megadalton protein complex from rat thymus. The N region would be synthesized with the large protein complex. RESULTS: The cDNAs for proliferating cell nuclear antigen (PCNA) were isolated by yeast two-hybrid screening as the gene products which directly interacted with TdT. The interaction between PCNA and TdT was confirmed by co-immunoprecipitation, both in vitro and in vivo. TdT binds directly to a PCNA trimer, as shown by gel filtration. TdT interacts with PCNA in its DNA polymerization domain (DPD), but not in its BRCA-1 C-terminal (BRCT) domain. TdT activity was reduced to 17% of the maximum value by TdT/PCNA complex formation. CONCLUSION: TdT interacts directly with PCNA through its DPD. A functional consequence of this interaction is the negative regulation of TdT activity. These findings suggest that TdT catalyses the addition of N nucleotides under the negative control of PCNA during V(D)J recombination.

Binding Sites↗
PubMed2001

Cytological examination of rat amniotic epithelial cells and cell transplantation to the liver.

It is hoped that amniotic epithelial cells can be useful in cell-mediated gene therapy. We report here an experimental cell transplantation model of amniotic cells in rats. There is an anatomical difference between human and rodent embryos. We established a method to isolate amniotic cells that are equivalent to human amniotic epithelial cells. An amniotic membrane distinct from the yolk sac was carefully collected and teased in saline containing deoxyribonuclease and hyaluronidase, followed by collagenase digestion. The cell yield was approximately 10(6) cells per pregnant female (10(5) cells per fetus), roughly in proportion to the age of fetus used, and 60% of the isolated cells were attached to the dish under culture conditions. Telomerase activity was higher in the cells isolated from fetuses in the middle stage (day 13.5 to 15.5) than in the late stage (day 17.5 to 21.5). Adherent cells exhibited two to three times more cell division, resulting in a ninefold increase in the number of cells. Immunohistochemical analysis revealed that approximately half of the adherent cells were albumin positive and formed clusters. The senescent cells survived for 2 months without apparent morphological changes. The adherent cells were able to be stored in liquid nitrogen and had a viability of 70% when thawed. Gene transduction with adenovirus vector was highly effective for rat amniotic cells. Transplantation of lacZ transfected amniotic cells into syngeneic rat liver resulted in the integration of the transplanted cells in the liver structure and the cells survived for at least 30 days.

Albumins↗
PubMed2000

Novel role of phosphatidylinositol 3-kinase in CD28-mediated costimulation.

Ligation of the CD28 surface receptor provides a major costimulatory signal for full scale T cell activation. Despite extensive studies, the intracellular signaling pathways delivered by CD28 ligation are not fully understood. A particularly controversial matter is the role of phosphatidylinositol 3-kinase (PI3K) in CD28-mediated costimulation. It is known that the binding site for PI3K and Grb-2 lies nested within the YMNM motif of the CD28 cytoplasmic domain. To elucidate the role of PI3K during CD28-mediated interleukin-2 (IL-2) production, CD28 YMNM point and deletion mutants were expressed in Jurkat cells. We then measured IL-2 promoter activation after CD28 ligation. Our results showed that the Y189F mutant, which disrupts binding by PI3K, and the YMNM deletion mutant both demonstrated reduced but significant activity for IL-2 promoter activation. In contrast, the N191A mutant, which retains PI3K binding ability, resulted in a complete abrogation of activity, suggesting that PI3K mediates a negative effect upon transcriptional activation of the IL-2 gene. Consistent with this idea, we found that the addition of a PI3K pharmacological inhibitor augmented IL-2 promoter activity, whereas coexpression of a constitutively active form of PI3K reduced this activity. Taken together, these data indicate that PI3K, when associated with the YMNM motif, may act as a negative mediator in CD28-mediated IL-2 gene transcription.

Amino Acid Motifs↗
PubMed2000

Molecular cloning and expression during development of the Drosophila gene for the catalytic subunit of DNA polymerase epsilon.

We have cloned the genomic DNA and cDNA of Drosophila DNA polymerase epsilon (pol-epsilon) catalytic subunit (GenBank No. AB035512). The gene is separated into four exons by three short introns, and the open reading frame consists of 6660 base pairs (bp) capable of encoding a polypeptide of 2220 amino acid residues. The calculated molecular mass is 255018, similar to that of mammalian and yeast homologues. The deduced amino acid sequence of the pol-epsilon catalytic subunit shares approximately 41% identity with human and mouse homologues as well as significant homology those of C. elegans, S. cerevisiae and S. pombe. Similar to the pol-epsilon catalytic subunits from other species, the pol-epsilon catalytic subunit contains domains for DNA polymerization and 3'-5' exonuclease in the N-terminal region, and two potential zinc-finger domains in the C-terminal regions. Interestingly, a 38 amino acid sequence in the C-terminal region from amino acid positions 1823 to 1861 is similar to the site for Mycoplasma ATP binding and/or ATPase domain (GenBank No. P47365). Northern hybridization analysis indicated that the gene is expressed at the highest levels in unfertilized eggs, followed by zero to 4h embryos and adult females, and then embryos at other embryonic stages, instar larva stages and adult males. Low levels of the mRNA were also detected at the pupa stage. This pattern of expression is similar to those of DNA replication-related enzymes such as DNA polymerase alpha and delta except for the high level of expression in adult males.

Amino Acid Sequence↗
PubMed2000

The UL34 gene product of herpes simplex virus type 2 is a tail-anchored type II membrane protein that is significant for virus envelopment.

The UL34 gene of herpes simplex virus type 2 (HSV-2) is highly conserved in the herpesvirus family. The UL34 gene product was identified In lysates of HSV-2-infected cells as protein species with molecular masses of 31 and 32.5 kDa, the latter being a phosphorylated product. Synthesis of these proteins occurred at late times post-infection and was highly dependent on viral DNA synthesis. Immunofluorescence assays revealed that the UL34 protein was localized in the cytoplasm in a continuous net-like structure, closely resembling the staining pattern of the endoplasmic reticulum (ER), in both HSV-2-infected cells and in cells transiently expressing UL34 protein. Deletion mutant analysis showed that this colocalization required the C terminus of the UL34 protein. The UL34 protein associated with virions but not with A, B or C capsids. We treated virions, HSV-2-infected cells and cells expressing the UL34 protein with a protease in order to examine the topology of the UL34 protein. In addition, we constructed UL34 deletion mutant proteins and examined their intracellular localization. Our data strongly support the hypothesis that the UL34 protein is inserted into the viral envelope as a tail-anchored type II membrane protein and is significant for virus envelopment.

Animals↗
PubMed1998

Isolation, purification, and characterization of a collagen-associated serpin, caspin, produced by murine colon adenocarcinoma cells.

A 45-kDa serpin secreted by a murine colon adenocarcinoma cell line, colon26, was isolated, purified, and characterized. It was found to bind specifically to type I collagen with high affinity and to type III collagen with lower affinity. Immunohistochemical studies of murine embryonic tissues showed a specific distribution of this collagen-associated serpin, named caspin, in relation to the formation of bone, cartilage, teeth, and basement membrane. The expression of caspin in high and low lung metastatic subclones of colon26 cell lines was inversely correlated with their metastatic capacity: low lung metastatic cells secreted higher amounts of caspin than their high lung metastatic counterparts. Caspin also demonstrated high homology with human pigment epithelium-derived factor/early population doubling level cDNA-1, which reportedly induces neuronal differentiation of human retinoblastoma cells and is expressed in association with G0 growth arrest. These findings suggest that caspin/pigment epithelium-derived factor/early population doubling level cDNA-1 is a novel factor that might play a crucial role in embryogenesis and tumor metastasis through binding to the extracellular matrix.

Amino Acid Sequence↗
PubMed1996

Structure and organization of the gene encoding human selenoprotein.

We have isolated a genomic clone encoding human selenoprotein P including the putative promoter region. The gene spans 12 kb and consists of five exons with a start codon in the second exon. A typical TATA sequence, the recognition motifs for a GATA-binding factor and the liver-specific factors, HNF-1 and HNF-3, were detected upstream from the transcription start point.

Base Sequence↗
PubMed1996

Crigler-Najjar syndrome type II is inherited both as a dominant and as a recessive trait.

Crigler-Najjar syndrome type II (CN-II) is caused by a severely reduced hepatic activity of bilirubin UDP-glucuronosyltransferase (UGT). Recently, by the analysis of the genetic background of CN-II patients, it has been clarified that the patients carry homozygous missense mutations or nonsense plus missense mutations on the gene for UGT, and CN-II was inherited as an autosomal recessive trait. We encountered a new case which had a nonsense mutation caused by a single nucleotide substitution on one allele. This indicates that CN-II is also inherited as a dominant trait as well as a recessive trait. Expression study in vitro strongly suggests that the disease in this case is caused by a dominant negative mutation by forming a heterologous subunit structure.

Amino Acid Sequence↗
PubMed1996

Coexpression of cell-surface immunoglobulin (sIg), terminal deoxynucleotidyl transferase (TdT) and recombination activating gene 1 (RAG-1): two cases and derived cell lines.

While it is generally agreed that in the lymphoid differentiation of B lineage cells there is no stage in which cell-surface immunoglobulin (sIg) and terminal deoxynucleotidyl transferase (TdT) are expressed simultaneously, a few B cell acute lymphoblastic leukemia (B-ALL) cases with this phenotype have been reported. Two such cases and the derived cell lines are reported here, in which the expression of recombination activating gene-1 (RAG-1) was also detected. One case was a CD19+ CD22+ HLA-DR+ sIg+ (gamma, kappa) B-ALL. The cell line (Bay9I) also expressed CD10. Karyotypic analysis revealed t(14;18)(q32;q21) and additional aberrations. In the other case, the fresh leukemia cells expressed CD19, CD24 and HLA-DR antigen. The derived cell line (Tree92) also expressed CD22 and sIg (mu, lambda). The karyotype of the Tree92 cells was t(8;14)(q24;q32) with additional aberrations. Tree92 is the first established cell line having both t(8;14)(q24;q32) and TdT. TdT was detected by Northern blotting as well as indirect immunofluorescence analysis. In addition, both Bay9I and Tree92 expressed RAG-1, as detected by Northern blot analysis. Cross-linking of sIg on Tree92 cells with anti-mu antibody led to significant down-regulation of RAG-1 expression. It seems that there is a sIg+ TdT+ RAG-1+ B lineage differentiation stage, and that signaling through sIg can modulate RAG-1 expression.

Adult↗
PubMed1995

Analysis of genes for bilirubin UDP-glucuronosyltransferase in Gilbert's syndrome.

Gilbert's and Crigler-Najjar syndromes are characterised by unconjugated hyperbilirubinaemia due to complete and partial absence of bilirubin UDP-glucuronosyltransferase (UGT). Nucleotide sequences of the genes for bilirubin UGT were analysed in six patients with Gilbert's syndrome. All patients had a missense mutation caused by a single nucleotide substitution and the mutations were heterozygous. In addition, relatives of patients with Crigler-Najjar syndrome types I and II, and of those with Gilbert's syndrome were analysed. All ten relatives with mild hyperbilirubinaemia were heterozygotes with respect to each defective allele. These results suggest that Gilbert's syndrome is inherited as a dominant trait.

Adolescent↗
PubMed1995

Isolation of cDNAs for mouse phenol and bilirubin UDP-glucuronosyltransferases and mapping of the mouse gene for phenol UDP-glucuronosyltransferase (Ugtla1) to chromosome 1 by restriction fragment length variations.

The mouse gene for phenol UDP-glucuronosyltransferase (UDPGT; Ugtla1) was mapped at 42 cM on chromosome 1, a position identical to that of the gene for bilirubin UDPGT (Ugtla1), from linkage analysis of a three-point cross test with Idh-1, En-1, and Ugtla1 as marker genes. The cDNAs for mouse phenol and bilirubin UDPGTs, isolated after amplification by PCR, shared an identical 3'-half region. Our results strongly suggest that mouse bilirubin and phenol UDPGTs are expressed from a single gene and involve alternative splicing events. We also detected duplication of the gene for phenol UDPGT in all mouse strains examined with the exception of MOL-MIT and SUB-SHH.

Animals↗
PubMed1995

Gilbert's syndrome is caused by a heterozygous missense mutation in the gene for bilirubin UDP-glucuronosyltransferase.

Gilbert's syndrome, which is characterized by chronic, non-hemolytic unconjugated hyperbilirubinemia, is caused by a reduction in the activity of hepatic bilirubin UDP-glucuronosyltransferase (UGT). Here, we report that all examined patients with this disease carried missense mutations in the gene for UGT and that the mutations were heterozygous. An expression study in COS cells in vitro, using the expression vector pcDL that carried the mutated gene for UGT from a patient, indicated that approximately 14% of the normal UGT activity was expressed. However, the UGT activity of the patient with Gilbert's syndrome was unexpectedly < 50% of the normal, perhaps as the result of the dominant negative nature of the mutation.

Adolescent↗
PubMed1995

Mapping of rat bilirubin UDP-glucuronosyl-transferase gene (Ugt1a1) to chromosome region 9q35-->q36.

Bilirubin and phenol UDP-glucuronosyltransferases (UGTs) are located on the same chromosome and comprise the UGT1 gene complex. A 1,763-bp cDNA probe (UGT1*0) specific for rat liver bilirubin UGT was used to localize the UGT1 complex locus (Ugt1a1) to chromosome region 9q35-->q36 by fluorescence in situ hybridization. This assignment is the first report on the location of a gene of the rat UGT1 complex using high-resolution banded metaphase chromosomes.

Animals↗