Biomedical subjects

Y Ishimi

Publications and source records attributed to Y Ishimi.

64 records · Page 4Linked to original sources

PubMed1987

Simian virus 40 large tumor antigen requires three core replication origin domains for DNA unwinding and replication in vitro.

Simian virus 40 (SV40) large tumor antigen (T antigen) unwinds DNA containing the SV40 origin of replication. The origin requirement for unwinding can be satisfied by the 64-base-pair SV40 core origin that supports T-antigen-dependent DNA replication both in vivo and in vitro. The core origin contains three domains with specific DNA sequence features. These include an inverted repeat, a central T-antigen binding domain, and an adenine- and thymine-rich domain containing a DNA bending focus. The domain and spacer requirements of the core origin for DNA unwinding and replication in vitro are strikingly similar to the origin requirements for DNA replication in vivo. Thus, each of the three functional domains of the core origin contributes directly to the initiation of duplex DNA unwinding by T antigen.

DNA Helicases↗

PubMed1987

The relationship between fusion and proliferation in mouse alveolar macrophages.

We have reported that protein factors separated from conditioned media of Concanavalin A-stimulated spleen cell cultures induce growth and fusion of mouse alveolar macrophages. A macrophages growth factor (MGF) was purified and, at the final step of purification on HPLC, eluted at the same position as a colony-stimulating factor (CSF), suggesting that MGF is identical with CSF. In the present study, we examined the relationship between proliferation and fusion of macrophages using purified CSF (MGF) and 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25-(OH)2D3]. The latter was used in place of a macrophage fusion factor which is supposed to be contained in the same conditioned medium, since a macrophage fusion factor has not yet been isolated. Adding less than 5% unfractionated conditioned medium from Concanavalin A-stimulated spleen cells markedly induced proliferation of alveolar macrophages without inducing fusion. In contrast, adding the same unfractionated conditioned medium at concentrations of 10% or more suppressed proliferation dose dependently, whereas it induced fusion reciprocally. Proliferation of macrophages was similarly enhanced by adding purified CSF or retinoic acid. Fusion of macrophages was induced by 1 alpha,25-(OH)2D3, but not by purified CSF or retinoic acid. Adding 1 alpha,25-(OH)2D3 together with purified CSF or retinoic acid completely suppressed the increase of proliferation induced by either growth factor, whereas that treatment rather potentiated the fusion induced by 1 alpha,25-(OH)2D3 alone. These results indicate that the fusion and proliferation of macrophages occur in a reciprocal fashion.

Animals↗

PubMed1986

Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions.

A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33 degrees C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39 degrees C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39 degrees C for at least 10 h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. This suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.

Animals↗

PubMed1986

Differentiation-inducing factor purified from conditioned medium of mitogen-treated spleen cell cultures stimulates bone resorption.

Spleen cells treated with mitogens produce a potent bone-resorbing factor called osteoclast-activating factor (OAF). To examine the relationship between the bone-resorbing factor and other protein factors produced by spleen cells, the colony-stimulating factor (CSF), the differentiation-inducing factor (DIF), the macrophage fusion factor (MFF), and the macrophage growth factor (MGF) were purified from 2.68 liters of conditioned medium of mouse spleen cell cultures treated with concanavalin A. Purification was performed successively by DEAE-cellulose, Blue Sepharose, and Sephadex G-150 column chromatography and high-pressure liquid chromatography (HPLC). The DIF was successfully separated from CSF and MGF on HPLC. CSF coincided with MGF on HPLC, but MFF disappeared before application to HPLC. Only the DIF exhibited bone-resorbing activity, whereas CSF and MGF did not. The DIFs purified from L929 cells and Ehrlich ascites tumors similarly exhibited bone-resorbing activity. The DIFs purified from spleen cells and Ehrlich ascites tumor cells exhibited neither interleukin 1 (IL-1) activity nor tumor necrosis factor (TNF) activity, though the unfractionated conditioned medium from spleen cells did exhibit them. In the light of recent reports that IL-1 beta and TNF also stimulate bone resorption, the term OAF should refer to a generic activity rather than a single factor.

Animals↗

PubMed1986

Characterization of the 100,000 dalton material produced by chromatin cross-linking reaction with dithio-bis(succinimidyl propionate).

A chemical cross-linking reagent, dithio-bis(succinimidyl propionate), is known to be capable of cross-linking histones in nucleosomes so as to give one major product with the molecular weight of about 100,000. Because the product has been supposed to represent the cross-linked histone octamer, the reaction has been used for studying the movements of core histones in nucleosomes. However, the precise protein composition of the product has not been determined thus far, so that the use of the reaction was limited. We report here that the 100 kilodalton product is composed of the core histones, and does not contain significant amounts of any other proteins. Moreover, quantitative analysis of the content of each core histone confirmed that the four types of core histones participate in the product with an equal molar ratio. As one can specifically observe the behaviors of histone octamers with this reaction, it should be useful for research in various fields related to the dynamics and functions of the nucleosome.

Animals↗

PubMed1985

Rapid purification of nucleosome assembly protein (AP-I) and production of monoclonal antibodies against it.

A nucleosome assembly protein (AP-I) was purified approximately 50% from the cytosol of HeLa S3 cells by three purification steps. Using this protein fraction as an antigen, we established three stable hybridomas that secrete monoclonal antibodies specific for AP-I by the conventional method of cell fusion. Immunoblotting of the HeLa S3 cytosol, proved AP-I exists as a 58-kDa peptide in vivo, not as the 53-kDa peptide previously identified as active in nucleosome assembly (Ishimi, Y., et al., Eur. J. Biochem., 142, 431-439, 1984). An immunocytochemical study using the monoclonal antibody with the highest specificity against AP-I pin pointed the intranuclear localization of AP-I in HeLa S3 cells.

Animals↗

PubMed1984

Purification and initial characterization of a protein which facilitates assembly of nucleosome-like structure from mammalian cells.

A protein, which facilitates assembly of a nucleosome-like structure in vitro, was previously partially purified from mouse FM3A cells [Ishimi, Y. et al. (1983) J. Biochem. (Tokyo) 94, 735-744]. The protein has been purified to approximately 80% from FM3A cells by using histone-Sepharose column chromatography. It sedimented at 4.6 S and had a molecular mass of 53kDa. A preincubation of core histones with the 53-kDa peptide before DNA addition was necessary for the nucleosome assembly. The 53-kDa peptide bound to core histones and formed a 12-S complex. This complex contained stoichiometrical amounts of the 53-kDa peptide and core histones, and the core histones in this complex were composed of equal amounts of H2A, H2B, H3 and H4 histones. The nucleosomes were assembled by adding pBR322 DNA to the 12-S complex. When mononucleosome DNA and core histones were mixed in the presence of the 53-kDa peptide, formation of a 10.5-S complex was observed. The complex contained DNA and core histones in equal amounts, while no 53-kDa peptide was detected in the complex. From above results it is suggested that the 53-kDa peptide facilitates nucleosome assembly by mediating formation of histone octamer and transferring it to DNA. Rat antibody against the 53-kDa peptide did not bind to nucleoplasmin from Xenopus eggs. The relationship between the 53-kDa peptide and nucleoplasmin is discussed.

Animals↗

PubMed1983

A protein which facilitates assembly of nucleosome-like structures in vitro in mammalian cells.

An activity which facilitates assembly of nucleosome-like structures in vitro at physiological ionic strength was detected both in human HeLa S3 cells and mouse FM3A cells. The assembly protein was purified from FM3A cells by fractionation with ammonium sulfate, DEAE-cellulose, phosphocellulose, Sephadex G-150 column chromatography, and sucrose gradient centrifugation. In the sucrose gradient, the activity was detected at 5S and the active fraction contained three peptides of 59,000, 65,000, and 102,000 daltons. When core histones were mixed with these peptides, the 59,000 peptide sedimented at the 6S and 10S positions, where the histones co-sedimented. The 6S fraction contained H2A, H2B, and A24 proteins, and the 10S fraction contained four kinds of core histones in equal amounts. Nucleosomes were formed by mixing DNA with the 10S fraction, but were not formed with the 6S fraction. The nucleosome structure assembled was assessed using the sensitivity to micrococcal nuclease.

Animals↗

PubMed1981

The reassociation with chromatin of H1 fragments bisected with thrombin.

When fragments of H1 histone formed by bisection of H1 histone with thrombin were allowed to reassociate with H1 histone-depleted chromatin, the carboxyl-terminal segment reassociated in such a manner as to protect the micrococcal nuclease-sensitive site(s) of the nucleosome core. On the other hand, the NH2-terminal segment of H1 histone protected 20 base pairs of linker DNA adjacent to the nucleosome core particle. These data provide strong evidence that the NH2-terminal portion and the carboxyl-terminal portion of H1 histone interact with 20 base pairs of linker DNA and the core DNA of the nucleosome, respectively.

Animals↗

PubMed1981

The interaction of H1 histone with nucleosome core.

The structural changes caused by H1 histone depletion of calf thymus chromatin or nucleosome monomer were studied by using the criterion of accessibility to micrococcal nuclease. When H1 histone was depleted from monomer, the DNS structure unfolded and became sensitive to the nuclease at a particular site on the nucleosome core, 105 base pairs from a chain terminus of core DNA. In reconstitution experiments with chromatin, this sensitive site on the core could be protected from the attack of the nuclease only when H1 histone was present. These results indicate that H1 histone interacts not only with the nucleosome linker region, but also with the core particle.

Animals↗