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M Shioda

Publications and source records attributed to M Shioda.

At least 55 records · Page 3Linked to original sources
PubMed1989

Changes in activity and subcellular localization of alpha-like DNA polymerase during cell cycle of Physarum polycephalum.

Regulation of DNA replication was given attention by examining the subcellular localization of alpha-like DNA polymerase in Physarum polycephalum. The activity per plasmodium increased by 2-fold in the cytoplasm during the G2-phase, and decreased with a concomitant increase in nuclei prior to DNA replication. This would suggest that the enzyme translocates from the cytoplasm to the nucleus, co-ordinately with DNA replication. In nuclei, the nuclear matrix-bound activity increased during the S-phase, but changes did not always parallel the rate of DNA replication. On the other hand, the activity measured without exogenous templates did change with the rate, although the activity was low. It is, therefore, suggested that part of the enzyme binding with the matrix participates in DNA replication.

Binding Sites↗
PubMed1988

Subcellular localization of DNA polymerase gamma and changes in its activity in sea urchin embryos.

1. Subcellular localization and changes in the activity of DNA polymerase gamma were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The gamma-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase gamma) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.

Animals↗
PubMed1988

Deoxyribonucleic acid polymerase alpha activity in the endometrium of the human uterus during the menstrual cycle.

Deoxyribonucleic acid polymerase alpha (a replicative enzyme of cellular deoxyribonucleic acid) activity in the endometrium of the human uterus changed in parallel (p less than 0.001) with the concentration of estrogen in serum during the proliferative phase of the menstrual cycle. This suggests that estrogen stimulates cell proliferation via regulation of the enzyme activity.

Adult↗
PubMed1988

Activity of deoxyribonucleic acid polymerase alpha stimulated by estrogen in the endometrium of the human uterus during the menstrual cycle.

To provide some insight into how deoxyribonucleic acid (DNA) synthesis occurs in the endometrium of the human uterus during the menstrual cycle, the DNA polymerase activities (alpha and beta) in endometrial samples taken from normal cycling women, and the concentration of estradiol-17 beta (E2) and progesterone in the serum were measured. DNA polymerase alpha activity increased gradually from the beginning of the menstrual cycle, reaching a peak 2-3 days before ovulation, and then showed a decrease. Increase in this activity occurred in parallel with that of the concentration of E2, but not progesterone, in the serum sample in the proliferative phase (correlation coefficient r = 0.924, p less than 0.001). In contrast, DNA polymerase alpha activity stimulated by estrogen in the endometrium of the 2nd grade amenorrheal women decreased abruptly after an injection of 125 mg progesterone. DNA polymerase beta activity showed no significant change during the menstrual cycle or after estrogen and progesterone treatment. These results suggest that estrogen seems to stimulate the induction of DNA polymerase alpha activity during cell proliferation in the endometrium of the human uterus.

Adult↗
PubMed1987

Selective inhibition of DNA polymerase alpha by a polysaccharide purified from slime of Physarum polycephalum.

A polysaccharide was purified from the slime of a myxomycete, Physarum polycephalum, and its inhibitory effect on eukaryotic DNA polymerases was examined. Almost all the calf thymus DNA polymerase alpha activity was inhibited with higher than 0.2 mg/ml of the polysaccharide, when the assay was carried out with activated DNA as a template. The inhibitory effect occurred regardless of the amounts of the enzyme and deoxyribonucleotides, however, kinetic analysis revealed that the inhibition occurs competitively with the template DNA, the Ki value being 4 micrograms/ml. Inhibition was observed for DNA polymerase alpha, but not for DNA polymerases beta and gamma from various eukaryote species.

Animals↗
PubMed1987

Analysis of replicating DNA molecules from embryos of the sea urchin, Hemicentrotus pulcherrimus, by electron microscopy.

DNA was extracted from embryos of the sea urchin, Hemicentrotus pulcherrimus, at the S phase and examined by electron microscopy. We detected replication microbubbles with a mean size of 404 bases, in addition to replication macrobubbles of more than 1.0 kilobase (kb) in length. Seventy-five percent of the center-to-center distances of the microbubbles were 0.6-1.8 kb with a mean of 1.2 kb. Forty-five percent of the microbubbles were arranged as clusters of four or five microbubbles. These results suggest that at least 34% of the initiation sites for DNA replication are present on a DNA molecule in clusters in which the sites are arranged at 1.2-kb intervals.

Animals↗
PubMed1987

DNA synthesis in isolated nuclei from synchronized plasmodia of Physarum polycephalum.

Nuclei were isolated from synchronized plasmodia of a true slime mold, Physarum polycephalum, in S-phase, and DNA synthesis in the nuclei was studied in vitro. The nuclei catalyzed DNA synthesis at the rate of 0.7 ng DNA/1.0 X 10(6) nuclei/30 min at 25 degrees C, which was 5 times higher than that catalyzed in G2-phase nuclei. The DNA synthesis required Mg2+, four kinds of deoxyribonucleoside 5'-triphosphates and ATP, suggesting that the mode of synthesis is a replicative-type, but not a repair-one. Sedimentation analysis of the DNA products revealed that the nuclei produced 2-4S DNA fragments mainly during a 30-sec pulse incubation, and 2-4S, 5-12S and longer fragments during a 15-min incubation. The pulse- and chase-labeling experiments showed that the 2-4S fragments shifted discontinuously to longer fragments. These results indicate that the nuclei catalyze the formation of 2-4S Okazaki fragments first and then their subsequent ligation. Eighty % and 96% of the DNA synthesis was inhibited by 200 micrograms/ml aphidicolin and 40 mM N-ethylmaleimide, respectively, but 80% of the activity was resistant to 100 microM 2',3'-dideoxythymidine 5'-triphosphate. These results suggest that the DNA synthesis is catalyzed by the alpha-type DNA polymerase of Physarum polycephalum.

Aphidicolin↗
PubMed1986

DNA synthesis in vitro with an endoplasmic-reticulum-DNA-polymerase complex from unfertilized sea urchin eggs.

An endoplasmic-reticulum-DNA-polymerase complex was prepared from unfertilized sea urchin eggs and its DNA-synthesizing activity was examined using single-stranded DNA of bacteriophage fd as a template. The complex catalyzed the ribonucleotide-dependent DNA synthesis which required dNTPs, NTPs, Mg2+ and single-stranded DNA. The DNA synthesis was sensitive to aphidicolin and N-ethylmaleimide but was resistant to 2',3'-dideoxyribosylthymine 5'-triphosphate (ddTTP) and alpha-amanitin, suggesting the involvement of DNA polymerase alpha. In parallel with the DNA synthesis, a small amount of RNA was synthesized in the presence of 100 micrograms/ml alpha-amanitin. The Km value of ribonucleotides for the RNA synthesis coincided with that for the DNA synthesis, suggesting a correlation between the DNA and RNA syntheses. Labelling of the products with [gamma-32P]ATP followed by DNA digestion with pancreatic DNase I revealed the attachment of an oligoribonucleotide (7-11 bases in length) at the 5' ends of the DNA products. These observations suggest that in DNA synthesis, primer RNA synthesis occurs first, followed by DNA chain elongation. During 1-90-min incubation, the amount of the DNA synthesized increased but the length was not significantly increased. Over 80% of the number of synthesized DNA molecules comprised a single population of short DNA fragments (60-200 bases, on average 120 bases in length) and the number of fragments increased, depending on the incubation time. However, DNA fragments of various sizes (about 100-6000 bases) were synthesized with DNA polymerase alpha solubilized from the endoplasmic-reticulum-DNA-polymerase complex. All this evidence suggests that in vitro, the complex preferentially synthesizes a particular size of short DNA fragments. The significance of the fragments is discussed.

Animals↗
PubMed1986

Follicle-stimulating hormone and estrogen elevate deoxyribonucleic acid alpha-nucleotidyltransferase activity in relationship to deoxyribonucleic acid synthesis in immature rat ovaries.

The deoxyribonucleic acid polymerase activities and deoxyribonucleic acid contents of ovaries were measured to clarify the relationship between hormone-stimulated cell proliferation and the enzymes in ovaries of immature intact rats (4 to 29 days after birth) and hypophysectomized rats. The specific activity of deoxyribonucleic acid alpha-nucleotidyltransferase (the activity per microgram of deoxyribonucleic acid of the ovaries) drastically increased with an increase in the deoxyribonucleic acid content of the ovaries from 4 to 14 days after birth and then remained constant or slightly decreased after the increase, while the activity decreased gradually after hypophysectomy with no increase in the DNA content. Ovine follicle-stimulating hormone or estradiol-17 beta enhanced the deoxyribonucleic acid synthesis and alpha-nucleotidyltransferase activity in the ovaries of hypophysectomized rats, while deoxyribonucleic acid beta-nucleotidyltransferase activity showed no significant change. Ovine luteinizing hormone, progesterone, and testosterone caused no significant increases. These results suggest that follicle-stimulating hormone or estrogen causes the induction of deoxyribonucleic acid alpha-nucleotidyltransferase accompanied by deoxyribonucleic acid synthesis in cell proliferation in immature rat ovaries.

Animals↗
PubMed1986

Evidence of the existence of a high molecular weight form of DNA polymerase alpha in sea urchin eggs.

DNA polymerase alpha combined with the endoplasmic reticulum (ER) was isolated from unfertilized sea urchin eggs. NaCl treatment of this fraction released DNA polymerase alpha from the ER. The molecular size (the S value) of the ER-free DNA polymerase alpha changed with the concentration of NaCl used; being 23 S, 11-15 S and 6-8 S in the presence of 0.05-0.12 M, 0.12-0.24 M and more than 0.24 M NaCl. DNA polymerase alpha activity decreased concomitantly with the reduction in molecular size. The 6-8 S form of DNA polymerase alpha did not aggregate by itself nor with other cellular components nonspecifically, when the 23 S form was present. These results are evidence of the presence of 6-8 S DNA polymerase alpha as a high molecular weight form (23 S-form) in sea urchin eggs.

Animals↗
PubMed1986

Increase in DNA polymerase alpha activity associated with DNA synthesis due to FSH or oestrogen in ovaries of immature rats.

DNA polymerase activities and DNA content of ovaries from immature intact rats (4-29 days after birth), hypophysectomized rats and hormone-treated hypophysectomized rats were measured. During normal ovarian growth DNA polymerase alpha activity and DNA content of ovaries increased. The polymerase activity decreased gradually after hypophysectomy without any alteration in the DNA content. Administration of ovine FSH (2 micrograms/day) or oestradiol-17 beta (1 mg/day) to hypophysectomized rats enhanced ovarian DNA content and DNA polymerase alpha activity, whereas DNA polymerase beta activity did not change significantly. These results suggest that DNA polymerase alpha participates in DNA synthesis in these ovaries. The specific activity of DNA polymerase alpha (the activity per microgram DNA) in the ovaries increased between 4 and 14 days after birth, and then remained almost constant; the specific activity declined gradually after hypophysectomy. Administration of FSH or oestradiol-17 beta but not of ovine LH, progesterone or testosterone to hypophysectomized rats restored the specific activity. Mixing experiments with different kinds of ovarian extracts suggested that no activators of DNA polymerase alpha were present in the extracts. These results suggest that FSH or oestrogen causes the induction of DNA polymerase alpha accompanied by DNA synthesis during cell proliferation in ovaries of immature rats.

Animals↗
PubMed1985

DNA replication of single-stranded DNA of phage fd microinjected into Xenopus laevis eggs.

Single-stranded circular DNA of phage fd was microinjected into Xenopus laevis eggs and DNA synthesis was analyzed after incubation. An efficient DNA synthesis occurred in the eggs: 2.7 ng of DNA was newly formed in an egg whose volume is approximately 900 nl during a 5-h incubation at 20 degrees C. The DNA synthesis was sensitive to aphidicolin, suggesting that DNA polymerase alpha participates in the synthesis. Agarose gel electrophoresis showed that full size of fd DNA is synthesized and that replicative form I of fd DNA is the major form of the newly produced DNAs. These results suggest that priming of DNA synthesis, elongation of DNA chain and ligation of DNA strands take place co-operatively in the egg with single-stranded DNA as a template.

Animals↗
PubMed1983

Localization of DNA polymerase alpha on the nuclear membrane in sea urchin embryos.

Subnuclear localization of DNA polymerase alpha was studied in sea urchin embryos. Blastula nuclei treated with EDTA and potassium phosphate released subnuclear components bearing most of the nuclear DNA polymerase alpha. These components were suggested to be a part of nuclear membrane based on their buoyant densities (1.177 and 1.136 g/cm3) in isopyknic centrifugation and the nuclear pore-like structure. Contamination with DNA and endoplasmic reticulum membrane to the subnuclear components was shown to be negligible. These results suggested that DNA polymerase alpha associates with nuclear membrane of sea urchin embryos. Nuclear membrane deprived of DNA polymerase alpha was able to associate with nuclear DNA polymerase alpha from blastulae and the cytoplasmic enzyme of unfertilized eggs efficiently, but not with the cytoplasmic enzyme of gastrulae. This result suggests that the nuclear membrane is originates from the endoplasmic reticulum with which DNA polymerase alpha associates in unfertilized eggs.

Animals↗