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Biomedical subjects

M Obinata

Publications and source records attributed to M Obinata.

At least 163 records · Page 9Linked to original sources
PubMed1983

Identification of the DNA binding subunit of RNA polymerase II from Ehrlich ascites tumor cells.

The DNA binding subunits of RNA polymerase II from Ehrlich ascites tumor cells were investigated in the following three ways. (1) RNA polymerase II was dissociated in urea and the binding of the dissociated subunits to DNA was investigated. (2) The RNA polymerase II: DNA complex was dissociated progressively with various concentrations of urea, and the subunits firmly attached to DNA were investigated. (3) RNA polymerase II was dissociated into subunits in a SDS-polyacrylamide gel containing urea and blotted onto a nitrocellulose filter. The filter was then incubated with 32P-nick-translated DNA to identify the DNA binding subunits. These procedures all showed that the largest subunit a of RNA polymerase II had strong affinity to DNA. It was found that a portion of subunits b and c could be recovered in DNA fraction when analyzed by procedures (1) and (2), but no significant DNA binding activity was detected when analyzed by procedure (3), suggesting that these subunits have either a much weaker affinity toward DNA compared to a or have affinity to a itself.

Animals↗
PubMed1982

Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina.

Storage-protein mRNA was found to be abundant in poly(A)-containing RNA extracted from the fat-body of third-instar larvae of Sarcophaga peregrina (fleshfly). This RNA sedimented at the position of 19S on sucrose-density-gradient centrifugation and the product of its translation in vitro was 75K protein (protein of mol.wt. 75 000), which was precipitated specifically with antibody against storage protein. This product was suggested to contain a signal sequence that is missing in mature storage protein. The poly(A)-containing RNA was also found to contain much of another mRNA coding for 25K protein (protein of mol.wt. 25 000), but the function of this protein is unknown.

Animals↗
PubMed1982

The 'pricking' method. A new efficient technique for mechanically introducing foreign DNA into the nuclei of culture cells.

Using the thymidine kinase gene-containing plasmid pTK-1 and the mouse fibroblast thymidine kinase-deficient LTK- cell line, we obtained results indicating that the plasmid molecules suspended in the external medium were introduced into the nuclei by pricking cells on the nuclei with a microneedle. Approx. 25% of the cells pricked in the presence of the plasmid pTK-1 showed TK gene expression, and 2% of the cells became stable TK+ transformants. This 'pricking' method is applicable for introducing DNA into cell nuclei and is more efficient than the conventional calcium phosphate transformation method.

Animals↗
PubMed1982

Tissue-specific distribution of DNA-binding nuclear proteins.

The DNA-binding capacity of nuclear proteins of mouse cells was examined by the protein-blotting method. Under conditions in which the lac repressor specifically binds to the lac operator, the DNA-binding nuclear proteins from different tissues showed a tissue-specific distribution, suggesting that the species and amounts of nuclear proteins with DNA binding activity differ in different tissues. When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor.

Animals↗
PubMed1981

Stimulation of messenger ribonucleic acid synthesis in isolated nuclei by a protein that stimulates RNA polymerase II.

A factor stimulating RNA polymerase II purified from Ehrlich ascites tumor cells was found to stimulate alpha-amanitin-sensitive RNA synthesis in nuclei isolated from spleen cells of anemic mice, though less than it stimulated purified RNA polymerase II. The fidelity of the resulting RNA synthesis was monitored by measuring the stimulation of globin mRNA synthesis. Globin mRNA was measured quantitatively by DNA-RNA hybridization by using plasmid DNA containing globin DNA sequences. Results showed that the synthesis of globin mRNA was enhanced in isolated nuclei in the presence of this factor coinciding with an increase of overall alpha-amanitin-sensitive RNA synthesis. Thus, it was concluded that an externally added factor did not stimulate random transcription but meaningful RNA synthesis in isolated nuclei.

Anemia↗
PubMed1980

Change in message sequences during erythrodifferentiation.

The change in the poly A(+) mRNA population during erythrodifferentiation was analyzed by cDNA-RNA hybridization. Poly A(+) RNA was isolated from spleen erythroblasts. When mice became anemic, the amount of globin mRNA increased to 50% of the total poly A(+) mRNA. cDNA from anemic spleen erythroblasts that did not contain globin mRNA sequences was cross-hybridized with mRNAs from mouse reticulocytes and cultured Friend leukemia (FL) cells. Only half the spleen cDNA hybridized with reticulocyte mRNA, whereas most of it hybridized with mRNA from FL cells. The results suggest that decrease in the complexity of the message population and increase in the concentration of globin mRNA are important in erythrodifferentiation.

Anemia↗
PubMed1980

Sequence differences between mouse embryonic y- and adult beta-globin messenger ribonucleic acids.

Poly(A)-containing RNA was isolated from yolk sac cells of BDF1 hybrid mice on the 12th day of gestation. In vitro translation of the poly(A)-containing RNA by a messenger RNA (mRNA) dependent reticulocyte lysate system revealed that this RNA contained a high concentration of mRNAs from two embryonic globin chain subunits, y-1 and y-2. Cross hybridization of complementary DNA (cDNA) to the embryonic globin mRNAs with adult reticulocyte globin mRNAs and with cloned cDNAs for adult alpha- and beta-globin chain mRNAs showed that the sequence of embryonic globin y-chain mRNA differed from that of adult beta-chain mRNA.

Animals↗
PubMed1980

Measurement of a stimulatory protein of RNA polymerase II in various mouse organs by the complement fixation test.

The amount of S-II, a protein specifically stimulating RNA polymerase II, was measured in various mouse organs by a micro complement fixation assay. The amount was almost the same in brain, liver, kidney, spleen, and Ehrlich ascites tumor cells on the basis of DNA, being about 3.8 micrograms/mg DNA, which corresponds to 3.6 X 10(5) molecules/cell. However, the amount of S-II decreased greatly during erythro-differentiation of Friend leukemia cells and no S-II was detected matured erythrocytes.

Animals↗
PubMed1977

Induction of globin gene expression in cultured erythroleukemia cells by butyric acid.

Butyric acid induces erythroid differentiation of cultured Friend leukemia cells when added to the culture medium. A high level of globin messenger RNA (mRNA) was detected in Friend leukemia cells treated with butyric acid by a liquid hybridization method using radioactive DNA complementary to reticulocyte globin mRNA. The content of globin mRNA molecules induced in the cytoplasm of the butyric acid-treated Friend leukemia cells paralleled the hemoglobin content determined by the benzidine staining method. Therefore, this hemoglobin synthesis may be most reasonably explained in terms of transcriptional activation of globin genes, as previously proposed in the case of dimethylsulfoxide, another inducer of erythroid differentiation. Induced accumulation of globin mRNA in the nuclei also supports this interpretation.

Animals↗
PubMed1975

Nuclear receptors for thyroid hormone: evidence for nonrandom distribution within chromatin.

Chromatin receptor proteins appear to mediate some actions of thyroid hormone. In this study, sheared mammalian chromatin containing [125I]triiodothyronine (T3) bound by these receptors was separated using sucrose gradient velocity sedimentation. T3-receptor complexes were distributed throughout the chromatin fractions, but were enriched in the slowly sedimenting fractions. The latter contain most of the template capacity for RNA synthesis and most of the endogenous RNA polymerase activity but a minor portion of the total DNA. Formaldehyde treatment of chromatin containing receptor-bound [125 I ]T3 resulted in fixation of radioactivity, as evidenced by its migration with chromatin after equilibrium density gradient sedimentation in both cesium chloride and Conray. This fixation implies that the T3 receptor protein is closely associated with chromatin. These results suggest that proteins involved in the regulation of gene function may be nonrandomly distributed within chromatin subfractions, and are consistent with a direct role for thyroid hormone in regulating genetic expression.

Animals↗