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O Koiwai

Publications and source records attributed to O Koiwai.

At least 55 records · Page 3Linked to original sources
PubMed1988

Isolation of putative promoter region for human terminal deoxynucleotidyltransferase gene.

We have isolated the genomic clone, which contained 5'-flanking region for human terminal deoxynucleotidyltransferase (TdT) gene, by screening Charon 4A library and determined its nucleotide sequence. Two transcription initiation sites (cap sites) were localized at 81 and 106 bp upstream from the third ATG codon which is translation initiation site, by primer extension analysis. Although neither typical TATA nor CAAT sequences were detected within 100 bp upstream region of the 5' cap site, a palindromic structure comprised of 28 bp was formed just upstream of the 5' cap site and double repeats of 5'-CTGGC sequence were found in its palindrom. The typical octamer of 5'-ATGCAAAT and three octamer-like sequences, which are involved in the transcription of lymphoid-specific genes, were detected at 212, 293, 347 and 770 bp upstream from the first cap site.

Base Sequence↗
PubMed1987

Analysis of human terminal deoxynucleotidyl transferase cDNA expressible in mammalian cells.

Human Molt3 cDNA library was constructed using pcD vector system which permits the expression of cDNA inserts in mammalian cells. Nearly full-length human terminal deoxynucleotidyltransferase (TdT) cDNA was cloned using a fragment of bovine TdT cDNA as a probe. The human TdT cDNA contains an open reading frame of 1,557 bp coding for 519 amino acids, including 31 bp and 341 bp from 5' and 3' untranslated regions, respectively. The TdT cDNA was transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 59,495. The cloned TdT cDNA hybridized with poly A+ RNAs of 2,100 b and 3,300 b from stable T-cell leukemia Molt3 and Molt4 cells.

Animals↗
PubMed1986

Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells.

We have isolated nearly full-length cDNA clones of terminal deoxynucleotidyltransferase (TdT) from calf thymus and mouse lymphoma cDNA libraries. The libraries were constructed using the pcD vector system which permits the expression of cDNA inserts in mammalian cells. The bovine TdT cDNA clone contains an open reading frame coding for 520 amino acids, Mr 59,678. The mouse TdT cDNA clone contains an open reading frame of 1,587 bp, whose translated cDNA encodes a 60,004 dalton protein. The mouse TdT cDNA clone contains 60 bp in the 3' end region of the coding sequence not found in the bovine TdT cDNA sequence, otherwise, the clones share about 80% homology. A possible nuclear-localization-sequence (Pro-Arg-Lys-Lys-Arg-Pro-Arg) was conserved in the N-terminal region in the mouse and bovine cDNA clones. Bovine and mouse cDNAs transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 60,000 which was detected immunologically using polyclonal rabbit antibody against bovine TdT. Bovine TdT expressed in COS7 cells by nearly full-length cDNA clone was localized in the nucleus and the translational product of pOK103 lacking the nuclear-localization-sequence was localized in the cytoplasm.

Amino Acid Sequence↗
PubMed1986

Characterization of DNA primase separated from DNA polymerase alpha-DNA primase complex of calf thymus.

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus (Yoshida, S. et al. (1983) Biochim. Biophys. Acta 741, 348-357). In the present study, the primase activity was separated from DNA polymerase alpha by treating purified 10S DNA polymerase alpha with 3.4 M urea followed by a fast column chromatography (Pharmacia FPLC, Mono Q column equilibrated with 2 M urea). Ten to 20 % of the primase activity was separated from 10S DNA polymerase alpha by this procedure but 80-90% remained in the complex. The separated primase activity sedimented at 5.6S through a gradient of glycerol. The separated primase was strongly inhibited by araATP (Ki = 10 microM) and was also sensitive to salts such as KCl (50% inhibition at 30 mM). The primase used poly(dT) or poly(dC) as templates efficiently, but showed little activity with poly(dA) or poly(dI). These properties agree well with those of the primase activity in the DNA polymerase alpha-primase complex (10S DNA polymerase alpha). These results indicate that the calf thymus primase may be a part of the 10S DNA polymerase alpha and its enzymological characters are preserved after separation from the complex.

Animals↗
PubMed1985

Arabinosylnucleoside 5'-triphosphate inhibits DNA primase of calf thymus.

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus and that the ribonucleotide-dependent DNA synthesis is more sensitive to araCTP than DNA-primed DNA synthesis (Yoshida, S., et al. (1983) Biochim. Biophys. Acta 741, 348-357). Here we measured DNA primase activity using poly(dT) template or M13 bacteriophage single-stranded DNA template and primer RNA synthesis was coupled to the reaction by Escherichia coli DNA polymerase I Klenow fragment. By this method, the primer RNA synthesis can be measured independently of the associating DNA polymerase alpha. Using poly(dT) template, it was found that arabinosyladenine 5'-triphosphate (araATP) strongly inhibited DNA primase in competition with rATP. The apparent Ki for araATP was 21 microM and the ratio of Ki/Km (for rATP) was as low as 0.015. With poly(dI, dT) or M13 DNA, it was shown that araCTP also inhibited DNA primase in the similar manner. Product analysis using [alpha-32P]rATP showed that araATP inhibited the elongation of primer RNA. However, it is not likely that arabinosylnucleotides act as chain-terminators, since incubation of primer RNA with araATP did not abolish its priming activity. From these results, it is suggested that arabinosylnucleotide inhibits the initiation as well as elongation of Okazaki fragments in mammalian cells.

Animals↗
PubMed1984

Arrest of DNA elongation by DNA polymerases at guanine adducts on 4-hydroxyaminoquinoline 1-oxide-modified DNA template.

In vitro modification of M13 phage single-stranded DNA with 4-hydroxyaminoquinoline 1-oxide (4HAQO) resulted in four kinds of adducts: three guanine adducts, QGI, QGII, and QGIII; and one adenine adduct, QA, at ratios of 16.4 47.3, 13.7, and 22.6, respectively. The carcinogen-modified DNA, initiated with a sequence-defined oligodeoxynucleotide primer, was replicated in vitro with Escherichia coli DNA polymerase I (Klenow fragment) and calf thymus DNA polymerases alpha and beta. The reaction products were analyzed on a DNA-sequencing gel. DNA elongation by DNA polymerase I was arrested at putative guanine adducts on the template in three ways: at one base prior to guanine; at positions opposite to guanine; and at one base beyond guanine. Similar patterns of elongation arrest were also obtained with the mammalian DNA polymerases alpha and beta. In contrast to guanine adducts, the adenine adduct, QA, might lack the capacity to arrest DNA chain elongation by DNA polymerases.

4-Hydroxyaminoquinoline-1-oxide↗
PubMed1983

DNA primase associated with 10 S DNA polymerase alpha from calf thymus.

Among multiple subspecies of DNA polymerase alpha of calf thymus, only 10 S DNA polymerase alpha had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase alpha through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase alpha. These results indicate that the primase is tightly bound to 10 S DNA polymerase alpha. The RNA polymerizing activity was resistant to alpha-amanitin, required high concentration of all four ribonucleoside triphosphates (800 microM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase alpha because it was strongly inhibited by araCTP, resistant to d2TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.

Animals↗
PubMed1982

Production and characterization of monoclonal antibody against 10S DNA polymerase alpha from calf thymus.

One hybridoma cell line that produces an antibody directed against 10S DNA polymerase alpha purified from calf thymus was obtained. The monoclonality of the antibody was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis, isoelectrofocusing and antibody subclass determination. The antibody specifically recognized the 10S DNA polymerase alpha and 6.5S DNA polymerase alpha-2 from calf thymus, but not 6.5S DNA polymerase alpha-1. The antibody precipitated both polypeptides of 140-150,000 and 46-50,000 dalton of 10S DNA polymerase alpha. The antibody also recognized the DNA polymerase alpha purified from human cells, but did pig DNA polymerase alpha only partially. The antibody did not crossreact with rat DNA polymerase alpha, calf DNA polymerase beta, virus DNA polymerase and E. coli DNA polymerase I. This antibody will be a useful tool for studying the mechanism of DNA replication in eukaryotic cells.

Animals↗
PubMed1982

10 S DNA polymerase alpha of calf thymus shows a microheterogeneity in its large polypeptide component.

The 10 S DNA polymerase alpha from calf thymus (Masaki, S., and Yoshida, S. (1978) Biochim. Biophys. Acta 521, 74-88) has been purified to near homogeneity. The most purified fraction obtained by repeated sucrose rate-zonal centrifugation contained three large polypeptides of 150,000, 145,000, and 140,000 daltons and three to four smaller polypeptides ranging from 43,000 to 50,000 daltons. A good resolution of these polypeptides was achieved on a sodium dodecyl sulfate-polyacrylamide linear gradient gel (5-20%) which was stained by the silver stain method. The three large polypeptides were also observed in the more crude fractions prepared in the presence of three kinds of protease inhibitors. By a peptide mapping analysis, it was revealed that these three polypeptides have a similar primary structure. Treatments of the enzyme with alkaline phosphatase, phosphodiesterase, and neuraminidase did not affect the gel pattern. These results indicate that the 10 S DNA polymerase alpha of calf thymus has a microheterogeneity in terms of the large polypeptide component. Among these three large polypeptides, the two polypeptides of 150,000 and 145,000 daltons disappeared by keeping the sucrose gradient fraction at 4 degrees C in the absence of glycerol, while the 140,000-dalton polypeptide was well preserved. The poly(rA)oligo(dT)-dependent activity of 10 S DNA polymerase alpha was selectively lost under this condition.

Animals↗
PubMed1982

Purification of terminal deoxynucleotidyl transferase from pig thymus: identification of 42,000 and 57, 000 dalton species.

Terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been purified 4365-fold from pig thymus. It was further separated into two molecular forms of 57,000 and 45,000 dalton by Sephadex G-100 gel-filtration. The former sedimented at 4.2s through a sucrose gradient, while the latter, at 3.6s. By a SDS-polyacrylamide gel-electrophoresis, their molecular weights were estimated as 57,000 and 42,000 dalton, respectively. Thus each of the large and small pig TdT consists of a single polypeptide of 57,000 or 42,000 dalton and has no subunit structure. These two forms were indistinguishable in antigenicity by a neutralization assay of 42,000 dalton-TdT antibody. The enzymological properties of 42,000 dalton-TdT from pig thymus were very similar to those of calf TdT which has a two-subunits structure.

Animals↗
PubMed1982

Analysis of calf thymus DNA polymerases alpha and beta and terminal deoxynucleotidyl transferase on two-dimensional polyacrylamide gels.

Calf thymus DNA polymerases alpha and beta [EC 2.7.7.7] and terminal deoxynucleotidyl transferase [EC 2.7.7.31] were analyzed on two-dimensional gel slabs. DNA polymerase beta appeared as a single spot on two-dimensional gel at the position of 40,000 daltons and pI 8.0 using non-equilibrium pH gradient gel electrophoresis for the first-dimensional run. By overlapping gel slabs, it was possible to identify the distinct spot of DNA polymerase beta among many polypeptide spots of a crude enzyme fraction. 10S DNA polymerase alpha showed two clusters of polypeptide spots on two-dimensional gel slab. One cluster was composed of three large polypeptides of 140,000-150,000 daltons and another was composed of four smaller polypeptides of 46,000-50,000 daltons. All these spots were arranged in a narrow pI range (6.5-6.8) although each spot showed a distinct pI value. Purified terminal deoxynucleotidyl transferase showed three polypeptides of 57,000, 42,000, and 33,000 daltons at similar pI values (7.0-7.2). Each polypeptide consisted of plural spots which differed slightly in pI but were the same in molecular weight. These results suggest a microheterogeneity of polypeptides of terminal deoxynucleotidyl transferase as well as those of 10S DNA polymerase alpha.

Animals↗
PubMed1981

Further characterization of a poly(rA) . oligo(dT)-dependent activity of multiple DNA polymerase alpha from calf thymus.

DNA polymerase alpha (EC 2.7.7.7) from calf thymus has been separated into three molecular species, i.e., 10 S DNA polymerase alpha, 6.5 S DNA polymerase alpha-1 and 6.5 S DNA polymerase alpha-2 (Masaki, S. and Yoshida, S. (1978) Biochim, Biophys. Acta 531, 74-88; Yoshida, S., Yamada, M., Masaki S. and Seneyoshi, M. (1979) Cancer Res. 39, 3955-3958). Among these three, 10 S DNA polymerase alpha and 6.5 S DNA polymerase alpha-2 were found to copy efficiently poly(rA) . oligo(dT), a template-primer, which was thought to be specific for DNA polymerase gamma or beta. 6.5 S DNA polymerase alpha-1, however, could not use the ribopolymer as a template. The poly(rA) . oligo(dT)-dependent activities of DNA polymerase alpha species differed markedly from those with activated calf thymus DNA in sensitivity to various reagents: the former was inhibited more than 80% by 80 mM KCl, while the latter was stimulated somewhat. Furthermore, aphidicolin, a specific inhibitor of DNA polymerase alpha, did not inhibit the poly(rA) . oligo(dT)-dependent activity. 2',3'-DideoxyTTP, a potent inhibitor of DNA polymerase beta or gamma, slightly inhibited the reactions with poly(rA) . oligo(dT), while it did not inhibit the reactions with activated DNA. The apparent Km values for dTTP on poly(rA) . oligo(dT) template were 260 and 70 microM for 10 S alpha and 6.5 S alpha-2, respectively; these values were much higher than those obtained on activated DNA template (8-10 microM).

Animals↗
PubMed1981

Purification of terminal deoxynucleotidyl transferase from pig thymus: identification of 42,000 and 57,000 dalton species.

Terminal deoxynucleotidyl transferase [EC 2.7.7.31] has been purified 4,365-fold from pig thymus. It was further separated into two molecular forms of 57,000 and 45,000 daltons by Sephadex G-100 gel-filtration. The former sedimented at 4.2S through a sucrose gradient, while the latter sedimented at 3.6S. By sodium dodecyl sulfate-polyacrylamide gel-electrophoresis, their molecular weights were estimated at 57,000 and 42,000 daltons, respectively. Thus, the large and small pig terminal deoxynucleotidyl transferases both consist of a single polypeptide of 57,000 and 42,000 daltons and have no subunit structure. These two forms were indistinguishable in antigenicity as examined by a neutralization assay with an anti-calf terminal deoxynucleotidyl transferase antibody. The enzymatic properties of 42,000-dalton terminal deoxynucleotidyl transferase from pig thymus were very similar to those of the calf enzyme, which has a two-subunit structure.

Animals↗
PubMed1980

Studies on bacterial chemotaxis. V. Possible involvement of four species of the methyl-accepting chemotaxis protein in chemotaxis of Escherichia coli.

Chemotactic Escherichia coli contains five major methyl-accepting proteins. Three of them were identified as the product of tsr gene, tar gene and peptide elongation factor Tu. Electrophoretic analysis of sulfur-labeled proteins and methyl-labeled proteins from trg mutants, which lost the ability of chemotaxis only towards ribose, galactose and their analogs, showed that the product of trg gene was another methyl-accepting protein i.e. a methyl-accepting chemotaxis protein for ribose and galactose (trg-MCP). The last methylatable protein, named as MCP-IV, seems to be involved in chemosensory transduction (accompanying paper). Thus, it is possible that chemosensory transduction in E. coli involves four species of MCP, although no genetic evidence for MCP-IV has yet been found. A hypothesis relating a change in the methylation of MCP with a movement of ions is presented.

Bacterial Proteins↗
PubMed1979

In vivo methylation of elongation factor Tu of Escherichia coli.

A protein existing mainly in the supernatant fraction of Escherichia coli was found to be methylated by accepting the methyl moiety originating from methionine. The protein was identified as peptide synthesis elongation factor Tu (EF-Tu) by the following criteria. 1) The methylatable protein separated at the same position as purified EF-Tu on two-dimensional gel electrophoresis. 2) The methylatable protein interacted with antiserum specific for EF-Tu. Amino acid analysis of the methyl-labeled protein suggested that the site of methylation was an epsilon-amino group of lysine.

Amino Acids↗
PubMed1979

Studies on bacterial chemotaxis. II. Effect of cheB and cheZ mutations on the methylation of methyl-accepting chemotaxis protein of Escherichia coli.

Radioactive proteins from chemotactic mutants of Escherichia coli with continuous tumbling phenotype (cheB and cheZ) and their otherwise isogenic parent were compared by two-dimensional gel electrophoresis. The system was capable of separating non-methylated methyl-accepting chemotaxis protein (MCP) from its methylated equivalent. The analysis of proteins from the envelope fraction of the bacteria showed that the cheB mutants contained a larger portion of methylated MCP than did the parent. However, the change of MCP methylation level was small, if any, in cheZ strains. The results suggest that the product of cheB gene and the product of cheZ gene are not functional complementary. The product of cheB gene functions in controlling the level of methylation at the stationary state of the organisms. In addition to known MCP species, a new MCP of about 43,000 daltons was found. This MCP appears to be involved in transducing signals of some sugars.

Bacterial Proteins↗