Biomedical subjects

T Eki

Publications and source records attributed to T Eki.

59 records · Page 4Linked to original sources

PubMed1987

Characterization of chromosome aberrations induced by incubation at a restrictive temperature in the mouse temperature-sensitive mutant tsFT20 strain containing heat-labile DNA polymerase alpha.

tsFT20 cells derived from a mouse mammary carcinoma cell line, FM3A, which has temperature-sensitive DNA polymerase alpha activity (Y. Murakami, H. Yasuda, H. Miyazawa, F. Hanaoka, and M. Yamada, Proc. Natl. Acad. Sci. USA, 82:1761-1765, 1985) were rapidly committed to death after temperature upshift to 39 degrees C. tsFT20 cells synchronized in S phase were more sensitive to the restrictive temperature than exponentially growing cells. In order to gain insight into the processes from the interruption of DNA synthesis to cell death, we analyzed chromosome aberrations induced in tsFT20 cells which had been incubated for 2 or 4 h at the restrictive temperature and then cultured at the permissive temperature. The majority of metaphase cells showed extensive chromosome aberrations such as chromatid gaps, breaks, and exchanges; chromosome pulverizations; their mixed types; and ring chromosomes. Analyses with the use of cell synchronization and autoradiography revealed that chromosome aberrations were induced only in the cells which synthesized DNA during incubation at 39 degrees C. We classified the chromosome aberrations into five types: gap or break type; exchange type; pulverization type; complex type; and ring type. The temporal order of the appearance of these types of chromosome aberrations was found to be the above described order. It was further found that cycloheximide dramatically repressed the induction of chromosome aberrations, and metaphases with many chromosome aberrations exhibited a large number of sister chromatid exchanges. These results indicate that abnormal cessation of DNA replication in tsFT20 cells at the restrictive temperature due to the inactivation of DNA polymerase alpha results in cell death via induction of double-strand breaks which lead to chromosome aberrations as well as sister chromatid exchanges.

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PubMed1987

Characterization of revertants derived from a mouse DNA temperature-sensitive mutant strain, tsFT20, which contains heat-labile DNA polymerase alpha activity.

One spontaneous and four N-methyl-N'-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase alpha activity and cannot grow at 39 degrees C, were isolated and characterized with respect to the thermolability of their DNA polymerase alpha activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase alpha activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 degrees C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 degrees C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 degrees C. At 39 degrees C, the rate for the wild-type increased considerably compared to that at 33 degrees C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 degrees C.

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PubMed1986

Characterization of DNA replication at a restrictive temperature in a mouse DNA temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha activity.

tsFT20 cells derived from a mouse mammary carcinoma cell line FM3A have temperature-sensitive DNA polymerase alpha activity (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1761-1765). DNA replication in tsFT20 cells at the restrictive temperature (39 degrees C) has been characterized in detail. DNA-synthesizing ability of these cells was measured by [3H] thymidine incorporation and autoradiography. The incorporation of [3H]thymidine decreased rapidly after temperature shift-up, and the incorporation was less than 20% of the initial level after 4 h at 39 degrees C. The rapid decrease correlated well with the decrease in the grain number in the individual nucleus but not with the number of cells with labeled nuclei. Alkaline sucrose gradient sedimentation analysis and DNA fiber autoradiography revealed that DNA chain elongation proceeded normally within a replicon in the temperature-sensitive cells incubated at the restrictive temperature and the DNA elongation rate did not change during the incubation at the restrictive temperature up to at least 6 h. On the other hand, the maturation of replicon-sized DNA to higher molecular weight DNA was retarded or inhibited in the temperature-sensitive cells at the restrictive temperature. The analysis of the center to center distance between replicons by DNA fiber autoradiography revealed that the frequency of replicon initiation decreased in tsFT20 cells at 39 degrees C.

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PubMed1986

Further characterization of a murine temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha-activity.

tsFT20 cells, which have temperature-sensitive DNA polymerase alpha-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase alpha in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [3H]thymidine incorporation. At 39 degrees C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 degrees C, but could grow at 37 degrees C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 degrees C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 degrees C----39 degrees C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase alpha plays a key role in DNA replication in mammalian cells.

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PubMed1986

Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.

In vitro replication of DNA containing the polyoma (Py) virus origin of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase alpha in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. The addition of purified DNA polymerase alpha-primase complex isolated from FM3A cells enabled HeLa cell extracts to replicate Py DNA with the same efficiency as FM3A cell extracts. Complementary experiments have shown that FM3A cell extracts do not support SV40 DNA replication unless supplemented with DNA polymerase alpha-primase complex from HeLa cells [Murakami, Y., Wobbe, C.R., Weissbach, L., Dean, F.B. & Hurwitz, J. (1986) Proc. Natl. Acad. Sci. USA 83, 2869-2873]. These results indicate that the host-cell source of the DNA polymerase alpha-primase complex plays an important role in discriminating between SV40 T antigen- and Py T antigen-dependent replication of their homologous DNA in vitro. This may explain the host-range specificity of these viruses in vivo.

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