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Biomedical subjects

T Seno

Publications and source records attributed to T Seno.

At least 109 records · Page 6Linked to original sources
PubMed1984

Chromosome breakage induced by thymidylate stress in thymidylate synthase-negative mutants of mouse FM3A cells.

In thymidylate synthase-negative mutants of mouse FM3A cells, thymidine starvation rapidly decreased mitotic activity and resulted in cell death (thymineless death). When the thymidine starvation was reversed by an addition of thymidine, mitotic activity was recovered, but the majority of mitotic cells exhibited extensive chromosome aberrations, including chromatid breaks, chromatid exchanges, and pulverizations. Autoradiographic examination revealed that chromosome instability was induced only in cells arrested in the S phase during thymidine starvation. Furthermore, the most sensitive sites to the chromosome-damaging effect appeared to be sites which had replicated just prior to thymidine starvation. During thymidine starvation, cells at other stages in the cell cycle were accumulated at the G1-S boundary, and they were insensitive to the chromosome-damaging effect. Thymidine starvation was also found to be recombinagenic. Complete removal from the medium of a thymidine analogue, 5-bromo-2'-deoxyuridine, resulted in a dramatic increase in the frequency of sister chromatid exchanges. These results support the view that thymidine starvation in mammalian cells results in thymineless death via induction of DNA double-strand breaks, leading to chromosome fragmentation as well as rearrangements in the cells synthesizing DNA.

Animals↗
PubMed1983

Novel form of DNA polymerase alpha associated with DNA primase activity of vertebrates. Detection with mouse stimulating factor.

With a specific stimulating factor of mouse DNA replicase for its detection, a novel form of DNA polymerase alpha (DNA replicase) associated with DNA primase activity was partially purified from several vertebrates, i.e. the cherry salmon Oncorhyncus masou, the frog Xenopus laevis, the chick, and human (HeLa cells). Activity similar to DNA replicase was also partially purified from embryos of the sea urchin Anthocidaris crassispina. In all vertebrates examined, two forms of DNA polymerase alpha were separated by chromatography on ion-exchange columns; one form (DNA replicase) was associated with DNA primase activity and could utilize unprimed single-stranded DNAs as template, and the other could not utilize unprimed single-stranded DNAs. The sedimentation coefficient of the former, the novel form, obtained from each vertebrate in a glycerol gradient at high ionic strength was slightly larger than that of the other form which had no primase activity, except in the case of chick embryos where the sedimentation coefficients of the two forms were almost the same. The initiator RNA synthesized with the DNA primase activity associated with DNA replicase obtained from salmon, chick, HeLa cells, and sea urchin was 8 to 10 nucleotides long. The stimulating factor obtained from Ehrlich ascites cells has been found to stimulate both the activities of DNA primase and DNA polymerase in DNA replicase obtained from all the vertebrates examined, when unprimed single-stranded DNA was used as template, while the factor failed to stimulate both the activities of the enzyme of sea urchin embryos. This factor thus should be an effective tool in studies on the mechanism of vertebrate DNA replication.

Animals↗
PubMed1983

Accumulation of DNA strand breaks during thymineless death in thymidylate synthase-negative mutants of mouse FM3A cells.

Thymidylate synthase-negative mutants of cultured mouse cells were immediately committed to cell death upon thymidine deprivation, especially when the cells were synchronized in the S phase. Thymidylate deprivation induced single strand breaks in chromosome-size DNA strands, as measured by alkaline sucrose gradient sedimentation, giving rise to two peaks, one with large and the other with small fragments, the latter about the size of T4 DNA. An increase in the small DNA fragments paralleled that of thymineless death. Thymidine deprivation also produced double strand DNA fragments as determined by a method of neutral filter elution, and their extent paralleled that of cell death. Double-stranded DNA eluted through the filter sedimented as a single peak both in a neutral and in an alkaline sucrose gradient that coincided with that of the above small DNA fragments. Therefore, the strand breaks seemed to occur in some defined portions of the genome and in a specific manner compared to breaks induced by x-rays, which occurred rather randomly. Cycloheximide blocked both thymineless death and the production of the small DNA fragments. The strand breaks induced by thymidine starvation were not repaired but instead advanced on subsequent incubation of the cells in growth medium containing thymidine.

Animals↗
PubMed1983

Mechanism of stimulation by a specific protein factor of de novo DNA synthesis by mouse DNA replicase with fd phage single stranded circular DNA.

Mouse DNA replicase is a functional multienzyme complex consisting of DNA polymerase and DNA primase. The DNA and initiator RNA syntheses by DNA replicase with single stranded DNA as template are stimulated by a stimulating factor (T. Yagura, T. Kozu and T. Seno, 1982, J. Biochem. (Tokyo).91, 607-618). The action mechanism of the stimulating factor on this novel DNA synthesis with fd phage single stranded circular DNA as template was studied. The stimulating factor directly stimulated initiator RNA synthesis but did not change the length of either initiator RNA (8 to 10 nucleotides long) or the product DNA (300 to 1,000 nucleotides long). Kinetic studies and analysis of the products by neutral agarose gel electrophoresis show that the stimulating factor increased the affinity of DNA replicase for template DNA without changing the apparent Km values for deoxy- and ribonucleotide substrates. Thus, in combination with a sufficient amount of the stimulating factor, DNA replicase quantitatively converted the template DNA to the position of double-stranded circular replicative form II DNA, as shown by agarose gel electrophoresis.

Animals↗
PubMed1983

Nucleotide sequence of Dictyostelium small nuclear RNA Dd8 not homologous to any other sequenced small nuclear RNA.

The cellular slime mold Dictyostelium discoideum, a lower eukaryote, was shown to contain several species of small nuclear RNA (Takeishi, K., and Kaneda, S. (1981) J. Biochem. (Tokyo) 90, 299-308; Wise, J. A., and Weiner, A. M. (1981) J. Biol. Chem. 256, 956-963). One of these RNAs, Dd9 or D2, was sequenced and found to be homologous to mammalian nucleolar U3 RNA. In the present study, the nucleotide sequence of another Dictyostelium small nuclear RNA Dd8 was determined by direct analysis. The sequence is: (formula; see text) Dd8 RNA contains high proportions of A (34%) and U (30%). No modified nucleotide could be detected in the internal region. Computer-assisted analysis of sequence homology indicated that Dd8 RNA is not homologous to any other small nuclear RNA species sequenced so far.

Base Sequence↗
PubMed1983

Tight association of DNA primase with a subspecies of mouse DNA polymerase alpha.

Evidence was obtained for tight association of DNA primase activity with a subspecies of mouse DNA polymerase alpha by study with immunoadsorption assay using two monoclonal antibodies specific for human DNA polymerase alpha that have been shown to react with mouse murine myeloma DNA polymerase alpha (Tanaka, S., Hu, S.-Z., Wang, T.-S.-F. & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390). This result was supported by the finding that ethidium bromide at concentrations of less than 20 microM somewhat stimulated the syntheses of DNA and initiator RNA on unprimed poly(dT) by the novel subspecies of DNA polymerase alpha, but strongly inhibited DNA synthesis with poly(dT) X oligo(rA), suggesting that the conversion of synthesis from initiator RNA to DNA is continuous. Furthermore, the results of neutralization assay with the antibodies and experiment using aphidicolin suggested that the primase site is functionally distinguishable from the catalytic site of DNA polymerase activity.

Animals↗
PubMed1983

Unusual aspects of human thymidylate synthase in mouse cells introduced by DNA-mediated gene transfer.

Thymidylate synthase-negative mutants of mouse FM3A cells were transformed to thymidine prototrophs by human DNA. The stable transformants had only human thymidylate synthase and segments of human DNA. They grew normally but had unusually high levels of the human enzyme. In two transformants examined, however, neither was the dTTP pool elevated nor the dCTP pool decreased. DNA synthesis in permeabilized cells of a transformant was more efficient than that in the wild type with dATP, dGTP, dCTP, and dUMP as substrates, but this was not so when dUMP was replaced by dTTP. Unlike the mouse enzyme, the human enzyme in the transformants did not co-sediment with DNA polymerase alpha and thymidine kinase in a sucrose gradient, suggesting that the human enzyme is not incorporated into a multienzyme complex for DNA replication. The high levels of the human enzyme in the transformants were suppressed to various degrees by fusion with a wild type mouse line. No active hybrid dimer enzyme was found between the human and mouse enzymes, which each consist of two identical subunits. Thus, the human enzyme in the transformants seems to behave differently from the mouse enzyme and its overproduction seems to be necessary for supporting the normal growth of the transformants.

Animals↗
PubMed1983

Size maturation process of nascent DNA intermediates into chromosomal-sized DNA in Tetrahymena pyriformis macronuclear DNA replication.

An analysis was made of the size maturation process of nascent DNA intermediates in macronuclear DNA replication of Tetrahymena pyriformis. The first discrete size class of nascent intermediates larger than Okazaki fragments were replicon-sized DNA (about 2 X 10(7) D single-stranded (ss) DNA) and accumulated in cells treated with cycloheximide. On removal of cycloheximide, the replicon-sized intermediates were converted to middle-sized intermediates (about 10 X 10(7) D ssDNA) and then merged into chromosomal-sized DNA. As indicated by either aphidicolin inhibition or the technique of the photolysis of bromodeoxyuridine (BrdU)-substituted DNA with long-wave ultraviolet light, four to eight replicon-sized intermediates were joined together to form a middle-sized intermediate after rapid sealing by DNA synthesis of the late-replicating regions located between adjacent replicon-sized intermediates. The late-replicating regions may represent the short gaps or terminal regions where DNA synthesis was retarded by cycloheximide, since the size of late-replicating regions was suggested to be shorter than the replicon size by DNA fiber autoradiography. Therefore, it is probable that four to eight completed replicons are joined as a group such as a replicon cluster, as has been reported in DNA replication of other eukaryotic cells.

Animals↗
PubMed1983

OSa, a 'new' low-frequency red cell antigen.

A new low-frequency red cell antigen, OSa, in a Japanese blood donor is inherited as a Mendelian dominant. A family study showed that OSa is not controlled by ABO, P1, Rh, Kidd, or Diego loci, nor is the antigen X- or Y-borne.

Antigen-Antibody Reactions↗
PubMed1983

Unusual sensitivity to bleomycin and joint resistance to 9-beta-D-arabinofuranosyladenine and 1-beta-D-arabinofuranosylcytosine of mouse FM3A cell mutants with altered ribonucleotide reductase and thymidylate synthase.

This paper describes unusual sensitivity to bleomycin; resistance to aphidicolin, 9-beta-D-arabinofuranosyladenine, 1-beta-D-arabinofuranosylcytosine, and hydroxyurea; and a mutator phenotype as common phenotypes of two classes of previously reported mouse FM3A cell mutants with distinct biochemical defects. One class of mutants, selected for aphidicolin resistance, contains altered ribonucleotide reductase desensitized to deoxyadenosine triphosphate, a negative allosteric effector, and the other class contains temperature-sensitive thymidylate synthase. Abnormal levels of deoxyribonucleoside triphosphate pools, especially those of deoxyadenosine triphosphate, were the common biochemical trait for both classes of mutants. The aphidicolin-resistant thymidylate synthase mutants, isolated independently of aphidicolin resistance, showed reduced levels of thymidylate synthase and increased levels of ribonucleotide reductase. Bleomycin was more lethal to both classes of mutants than to wild-type cells, as determined by colony-forming ability. It also caused more marked scissions of DNA in the ribonucleotide reductase mutant cells than in the wildtype cells as analyzed by sedimentation on an alkaline sucrose gradient. Thus, the activity or level of ribonucleotide reductase seems to be one factor determining the bleomycin sensitivity of cells by potentiating the capacity of bleomycin to cleave DNA strands.

Animals↗
PubMed1982

Mouse DNA replicase. DNA polymerase associated with a novel RNA polymerase activity to synthesize initiator RNA of strict size.

De novo DNA synthesis on poly(dT) by a novel mouse DNA polymerase, here named "DNA replicase," was examined for the synthesis of RNA which functions as a primer in the subsequent synthesis of DNA. As has been reported previously (Yagura, T., Kozu, T., and Seno, T. (1982) J. Biochem. (Tokyo) 91, 607-618), a novel RNA polymerase activity, which is distinguished from those of classical RNA polymerases, is associated with DNA replicase. The synthesis of RNA and DNA by DNA replicase (Mr = 16 X 10(4), by glycerol gradient sedimentation analysis) was greatly stimulated by a specific stimulating factor (Mr = 13 X 10(4), by glycerol gradient sedimentation analysis) which was found to consist of two subunits (Mr = 63 X 10(3), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Nearest neighbor analysis in which transfer of 32P from alpha-labeled nucleoside triphosphates to ribo- and deoxyribonucleotides was examined, showed th at RNA of 8-10 nucleotides long was covalently linked to the 5'-end of the DNA product molecule. This RNA, named initiator RNA, had a triphosphate group at its 5' terminus and its size and synthesis were little affected by the addition of high concentrations of deoxynucleoside triphosphate, while in these conditions deoxyribonucleotides were incorporated into initiator RNA to a limited extent. The characteristics of the DNA replicase and stimulating factor that cooperate to synthesize initiator RNA for subsequent DNA synthesis on single-stranded DNA are important because these components seem to be involved in a reaction required to initiate the synthesis of discontinuous earliest DNA intermediates (Okazaki fragments) in chromosomal DNA replication of eukaryotic cells.

Animals↗
PubMed1982

Mouse DNA polymerase accompanied by a novel RNA polymerase activity: purification and partial characterization.

A mouse DNA polymerase accompanied by a novel RNA polymerase activity and its specific protein factor (stimulating factor) were purified from Ehrlich ascites tumor cells and partially characterized. The DNA polymerase was thought to be a subspecies of DNA polymerase alpha, and to be accompanied by or copurified with RNA polymerase activity capable of synthesizing RNA, which was probably utilized as a primer for subsequent DNA polymerization on a template of poly(dT) or poly(dC). This coupled reaction by RNA and DNA polymerase activities required the stimulating factor in addition to ribo- and deoxyribonucleotide substrates, although the degree of requirement depended on the kind of template and ribonucleotide substrate: the activity to incorporate dATP with poly(dT) plus ATP depended greatly on the stimulating factor, while the activity to incorporate dGTP with poly(dC) did not when GTP was added at high concentrations. GDP could be substituted for GTP, but the activity with poly(dC) plus GDP depended largely on the stimulating factor. Involvement of known RNA polymerases in the activity with poly(dT) was excluded, because addition of purified mouse RNA polymerases I and II had no effect on the incorporation of dATP, and alpha-amanitin (100 micrograms/ml) did not inhibit the incorporations of dATP and ATP. Analysis of the inhibition by the nucleotide analog 2',3'-dideoxynucleoside 5'-triphosphate (ddNTP) further supported the involvement of new RNA polymerase; ddNTPs inhibited the activities with poly(dT) and poly(dC) significantly more than RNA polymerases I and II or DNA polymerase alpha activity with poly(dT) . oligo(rA) and poly(dC) . oligo(dG) as template. Lineweaver-Burk analysis of the inhibitions showed that ddATP inhibited competitively with respect to ATP, and ddGTP inhibited competitively with respect to GDP but noncompetitively with respect to GTP.

Animals↗
PubMed1982

Arrest of chain growth of replicon-sized intermediates by aphidicolin during rat fibroblast cell chromosome replication.

The effect of aphidicolin, a specific inhibitor of DNA polymerase alpha, on size maturation of nascent DNA intermediates was studied in cultured rat fibroblast cells. Results provided the first evidence of DNA synthesis associated with merging of intermediates of larger than replicon size. Aphidicolin at a concentration (1.4 micrograms/ml) causing 90-95% inhibition of [3H]thymidine incorporation, resulted in accumulation of intermediates of nearly the same size as the replicon (2-5 x 10(-7) Da); although the synthesis of short nascent fragments (referred to as Okazaki fragments) continued in the presence of aphidicolin, the rate of their elongation to the replicon size was greatly decreased. On removal of aphidicolin, these accumulated intermediates merged into high-molecular-weight DNA. This merging of the intermediates was associated with DNA synthesis in gaps between adjacent intermediates, as revealed by photolysis of bromodeoxyuridine-DNA leader with long-wave ultraviolet light; when the cells had been pulse-labeled for 5 min with bromodeoxyuridine immediately after removal of the drug, the large DNA arising from aphidicolin-arrested intermediates was cut into fragments of the original size by long-wave ultraviolet light irradiation. The arrest of chain elongation at the replicon-size by aphidicolin might be due to inhibition of this DNA synthesis in gaps, because aphidicolin did not cause degradation of nascent DNAs.

Animals↗