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A Matsukage

Publications and source records attributed to A Matsukage.

At least 127 records · Page 7Linked to original sources
PubMed1984

Conservation of molecular structure of DNA polymerase beta in vertebrates probed by tryptic peptide mapping.

DNA polymerase beta's from mouse myeloma, chick embryo, and cherry salmon testis were all composed of a single polypeptide of about 40K daltons as judged by a sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified enzyme preparations. Although the enzyme from bullfrog ovary was not fully purified, its molecular weight was estimated to be the same as that of the chick enzyme by immunological detection after electrophoresis. All the enzymes tested cross-reacted immunologically with the antibody against chick DNA polymerase beta, indicating that they have a common molecular structure, at least in part. Two-dimensional maps of radioiodinated tryptic peptides directly showed the presence of highly conserved amino acid sequences among mouse, chick, and cherry salmon enzymes. This conserved structure is thought to be essential for the enzyme activity, which is very similar among all these vertebrates.

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PubMed1984

Tight association of DNA polymerase alpha with granular structures in the nuclear matrix of chick embryo cell: immunocytochemical detection with monoclonal antibody against DNA polymerase alpha.

Immunofluorescent methods using a monoclonal antibody against chick DNA polymerase alpha and a rabbit antibody against chick DNA polymerase beta demonstrated that both DNA polymerases alpha and beta are present mainly in nuclei of cultured chick embryo cells. Fluorescence produced by anti-DNA polymerase alpha was more intense in the small granules than in other parts of the nucleus but, fluorescence produced by anti-DNA polymerase beta was distributed evenly in the nucleus. Cells first were treated with Nonidet P-40, followed by treatment with 50 micrograms/ml pancreatic DNase and 2 M NaCl in order to prepare the nuclear matrix. Fluorescence produced by anti-DNA polymerase alpha was still detectable in the granules after these treatments, but most of the fluorescence produced by anti-DNA polymerase beta disappeared. Our results indicate that a part of DNA polymerase alpha is tightly bound to a special structure present in the nuclear matrix which presumably is the DNA replication machinery.

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PubMed1984

DNA replication.

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DNA Replication↗
PubMed1984

Polyester wax embedding and sectioning technique for immunohistochemistry.

We have developed a method useful for immunohistochemical studies by combining tissue fixation with buffered neutral formalin and polyester wax embedding. Buffered neutral formalin fixation preserves cell and tissue fine structure, and also the antigenicity of unstable enzymes. Polyester wax embedding makes possible thin serial sections of various tissues and preserves antigenicities for at least 6 months. We have demonstrated using this technique the localization of alpha-amylase in mouse salivary gland, parietal-cell specific antigen in mouse glandular stomach, and DNA polymerase alpha and beta in chick tissue.

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PubMed1983

Characterization of messenger RNA for chick-embryo DNA polymerase beta and its translation product in vitro.

A specific immunoprecipitation method, using rabbit anti-(chick DNA polymerase beta) IgG was applied to detect the polypeptide of DNA polymerase beta among translation products obtained in vitro with mRNA extracted from chick embryos. A polypeptide of Mr = 40 000 was specifically immunoprecipitated from [35S]methionine-labeled translation products and was competitive with the purified DNA polymerase beta for the antibody. Furthermore, the 40 000-Mr translation product obtained in vitro had DNA polymerase activity, which was detected by assay in situ after electrophoresis in a polyacrylamide gel containing DNA. The mRNA for DNA polymerase beta was polyadenylated and its content was estimated as the range of 0.001% of total poly(A)-rich RNA on the basis of [35S]methionine incorporation in the translation in vitro. The size of this mRNA was determined to be about 1800 nucleotides by zone sedimentation and agarose gel electrophoresis under denaturating conditions.

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PubMed1983

Immunocytochemical localization of chick DNA polymerases alpha and beta +.

An immunofluorescent method using specific antibodies was employed to detect DNA polymerases alpha and beta in chick cells. With monoclonal antibodies produced by four independent hybridoma clones, most of the DNA polymerase alpha was shown to be present in nuclei of cultured chick embryonic cells. With a polyclonal, but highly specific, antibody against DNA polymerase beta, this enzyme was also shown to be present in nuclei. DNA polymerase alpha was detected in proliferating cells before cell contact and in lesser amount in resting cells after cell contact, indicating that its content is closely correlated with cell proliferation. On the other hand, similar amounts of DNA polymerase beta were detected in proliferating and resting cells. Furthermore, DNA polymerase beta was detected in nuclei of most cells, while DNA polymerase alpha was detected only in large round nuclei in seminiferous tubules of chick testis. DNA polymerase alpha is presumably present in cells that are capable of DNA replication, and during the cell cycle it seems to remain in the nuclei during the G1, S, and G2 phases, but to leave from condensed chromatin for the cytoplasm during the mitotic phase.

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PubMed1983

Detection of DNA replicase activity in rat thymoma cells.

A DNA polymerase which was active with unprimed poly(dC) as a template was detected in the DNA polymerase alpha fraction from rat thymoma tissue. The activity was dependent on rGTP and associated with ribonucleotide polymerizing activity. The activity was partially sensitive to aphidicolin and dideoxy GTP and resistant to alpha-amanitin. Thus, this is a rat "replicase" which is very similar to that found in mouse cells. The replicase activity with poly(dT) or phage fd single stranded circular DNA was greatly enhanced by the mouse replicase-stimulating factor beyond the species difference.

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PubMed1982

Chick embryo DNA polymerase alpha. Polypeptide components and their microheterogeneity.

DNA polymerase alpha was purified from a chick embryo extract by ammonium sulfate fractionation and successive column chromatographies. The final preparation had a specific enzyme activity of 39,000 units/mg of protein with activated calf thymus DNA as a template.primer. Electrophoresis in nondenaturing polyacrylamide gel showed that the preparation contained two proteins, one of which was associated with DNA polymerase activity. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the electrophoretically pure enzyme gave two clusters of polypeptide bands. One was composed of 3-4 polypeptides with similar electrophoretic mobilities corresponding to Mr = 130,000-155,000, and the other consisted of four distinct polypeptide bands corresponding to Mr = 59,000, 56,000, 54,000, and 51,000. Two-dimensional tryptic peptide mapping analysis of these polypeptides after 125I-iodination indicated that the structures of all four Mr = 51,000-59,000 polypeptides were very similar. In addition, polypeptides in the Mr = 145,000-155,000 region had almost identical structure with those in the Mr = 130,000-140,000 region. No significant structural homology was observed between the high molecular weight polypeptides and low molecular weight polypeptides. These findings indicate that the four polypeptides of Mr = 51,000-59,000 are generated by minor modification(s) of a single polypeptide. Similarly, the heterogeneity of the high molecular weight polypeptides is brought about by minor modification(s). Thus, chick embryo DNA polymerase alpha is basically composed of two kinds of subunit polypeptides.

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PubMed1982

Biosynthesis of DNA polymerase beta in chick embryonic cells.

A monospecific antibody to chick embryo DNA polymerase beta was prepared from a rabbit immunized with the homogeneous enzyme preparation. The antibody has high neutralizing ability to the enzyme activity and, in the presence of formalin fixed Staphylococcus aureus, precipitates a Mr = 40,000 polypeptide from the crude extract and the partially purified DNA polymerase beta which were prepared from [35S]methionine-labeled chick embryonic cells. Pulse-chase experiments were carried out to clarify the process of the biosynthesis of DNA polymerase beta. We have attempted to detect a precursor polypeptide which would be expected to have the following properties: 1) a polypeptide which is specifically precipitated by the antibody and has a molecular weight different from 40,000, 2) the amount of the 35S-labeled polypeptide decreases in the chase period, and 3) 35S-labeled polypeptide which is eliminated from the immunoprecipitate by adding an excess amount of purified unlabeled DNA polymerase beta. However, no such polypeptide was detected in the 30-min pulse-labeled cells. A Mr = 40,000 polypeptide was immunoprecipitated from the extract of 30-min pulse-labeled cells and its amount did not change in a 5-hr chase, then decreased. Results suggest that a Mr = 40,000 polypeptide of chick embryo DNA polymerase beta is the initial translation product of the mRNA of DNA polymerase beta.

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PubMed1982

Establishment of hybridoma clones which produce anti-chick embryo DNA polymerase alpha monoclonal antibodies.

Four hybridoma clones which produce monoclonal antibodies against chick embryo DNA polymerase alpha have been established. Antibodies produced by the four clones all belonged to the gamma 1-type IgG and precipitated enzyme activity in the presence of Staphylococcus aureus cells. An affinity column prepared with one of the monoclonal antibodies (clone 6-1E) and protein A-Sepharose retained DNA polymerase alpha activity as well as the 135,000 approximately 150,000 and 50,000 approximately 60,000 dalton polypeptides of this enzyme. Thus, the antibody is specific to DNA polymerase alpha.

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PubMed1981

Identification of a polypeptide component of mouse myeloma DNA polymerase gamma.

Mouse myeloma DNA polymerase gamma was extensively purified to a final specific activity of 156 000 units (nmol dTMP incorporation per h) per mg protein on (rA)n.(dT)12-18 as a template primer. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein fractions obtained by DNA-cellulose column chromatography revealed that the amount of a polypeptide of Mr = 47 000 changed proportionally with DNA polymerase gamma activity. A minor polypeptide of Mr = 140 000 also seemed to change with the enzyme activity, but other polypeptides did not. Analysis by 125I-labeled peptide mapping indicates that the Mr 47 000 polypeptide in the mouse myeloma DNA polymerase gamma preparation is structurally related to the Mr 47 000 polypeptide of chick embryo DNA polymerase gamma (Yamaguchi, M., Matsukage, A. and Takahashi, T. (1980) J. Biol. Chem. 255, 7002-7009). An antibody against chick embryo DNA polymerase gamma cross-reacted with the mouse enzyme, indicating a structural relationship between avian and murine enzymes. Since the Mr 47 000 polypeptide accounts for 31.4% of total protein in the purified preparation, the specific activity is estimated to be about 490 000 units per mg of the Mr 47 000 polypeptide. The rate of poly(dT) elongation by the purified enzyme was 1 260 per nucleotides per min. This value is in the same range as the turnover number (1 530 nucleotides per min per enzyme molecule) which is calculated from the 'expected' specific activity with respect to the Mr 47 000 polypeptide and the molecular weight (Mr = 188 000 on the assumption of a tetrameric structure of the Mr 47 000 polypeptide). Results indicate that the Mr 47 000 polypeptide is a component of the mouse myeloma DNA polymerase gamma.

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PubMed1981

Structural homology of DNA polymerase beta from various mammalian cells.

DNA polymerase beta was purified from various mammalian cells, i.e. mouse myeloma, rat liver, rat ascites hepatoma cells, rabbit liver, pig liver, and calf thymus cells. The apparent molecular weights of the polypeptides composing these enzymes were all about 40,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, tryptic peptide maps of these enzymes after radioiodination with 125I indicated that the molecular structures of the enzymes were basically identical. No difference was detected in the peptide maps of the mouse and rabbit enzymes, and only a few of the 22 spots in the fingerprint of the mouse enzyme were not detected in that of the rat enzyme. Furthermore, the peptide maps of DNA polymerase beta's from normal and malignant rat cells differed in only one spot.

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PubMed1980

Chick embryo DNA polymerase beta. Purified enzyme consists of a single Mr = 40,000 polypeptide.

DNA polymerase (EC 2.7.7.7) beta was purified to near homogeneity from chick embryos. The final preparation has a specific enzyme activity of 1,100,000 units (nanomoles of dTMP incorporation/h) per mg of protein with (rA)n X (dT)12-18 as a template-primer. The molecular weight of DNA polymerase beta is about 40,000 as judged by gel filtration on Sephadex G-150 column. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the polypeptide of Mr = 40,000 accounted for 95% of total protein in the final preparation. The fingerprint analysis of tryptic peptides of polypeptide shows that 14 out of 24 125I-peptides from the Mr = 40,000 polypeptide are the same as those of the Mr = 40,000 polypeptide of DNA polymerase beta purified from rat ascites hepatoma cells. A Mr = 27,000 polypeptide, which was present in the less pure preparation, did not show any structure similar to Mr = 40,000 polypeptides from rat and chick cells. Thus, it is concluded that chick embryo DNA polymerase beta consists of a single polypeptide of Mr = 40,000. The minimal number of DNA polymerase beta molecules per chick embryo cell was estimated as about 5,000.

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