Masao Hyodo

Isolation of temperature-sensitive CHO-K1 cell mutants exhibiting chromosomal instability and reduced DNA synthesis at nonpermissive temperature

Twenty-five temperature-sensitive (ts) mutants were isolated from Chinese hamster CHO-K1 cells after mutagenization withN-methyl-N′-nitro-N-nitrosoguanidine. Of 13 complementation groups identified, nine exhibited chromosomal instability at a nonpermissive temperature. They were classified into three major classes according to inducibility of sister chromatid exchange (SCE) and/or chromosomal aberration (CA): class 1 resulted in predominant SCEs, class 2 manifested both SCEs and CAs, and class 3 exhibited higher induction of CAs. Flow cytometric analysis of the mutants exhibiting chromosomal instability indicated that many of the mutants were arrested in the S or S to G2 phases of the cell cycle at the nonpermissive temperature, accompanied by a decrease in the rate of DNA synthesis. These results imply that ts defects are related to some points in DNA replication and might be responsible for the induction of SCEs and/or CAs at the nonpermissive temperature.

A temperature-sensitive mutant isolated from mouse FM3A cells defective in DNA replication at a non-permissive temperature☆

Abstract Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.

DNA replicatiion and cell-cycle progresssion of cultured mouse FM3A cells after treatment with 8-methoxypsoralen plus near-UV radiation

To investigate the response of cells to one type of DNA damage--namely DNA crosslinks--cell-cycle progression and macromolecular synthesis were studied with cultured mouse FM3A cells. Treatment of the cells with low doses of 8-methoxypsoralen (8-MOP) plus near-UV radiation (0.1 micrograms/ml plus 5 kJ/m2 or 1.0 micrograms/ml plus 1-2.5 kJ/m2) halted the progression of cells through the cell cycle temporarily for the first several hours. Then the cells resumed progression through the cell cycle, and most of the cells reached, and were finally arrested at, the G2 phase of the cycle. There was a rapid decrease of incorporation of [3H]thymidine into cellular Dna immediately after the treatment. Then, after 8 h of incubation, the incorporation of [3H]thymidine recovered to some extent depending on the dose of 8-MOP plus near-UV radiation. Thus the decrease and recovery of the incorporation of [3H]thymidine were correlated with the halt and resumption in the cell-cycle process. Synthesis of RNA and protein was measured by determination of the amounts in the cells or by the incorporation of radioactive precursors after treatment. RNA and protein synthesis were stimulated by low doses of 8-MOP plus near-UV radiation, but inhibited severely by high doses.

Properties of a strain of free-living nematode, Rhabditidae sp.: life cycle and age-related mortality.

Abstract Some properties, especially the life cycle and the temperature-dependency of age-related mortality, of a newly isolated free-living nematode have been investigated. The nematode was identified as a member of the family Rhabditidae, but the genus has not yet been identified. The nematode is bisexual and the male to female ratio was found to be 1 : 15. In all experiments animals were cultured with Escherichia coli cells as a food source. At 20°C, eggs hatched to larvae after about 24 hours and the eggs of the next generation appeared in a population about 5 days after being laid. The longevity of the nematode was greatly influenced by the temperature. The average life span observed for virgin females at 20°C was about 95 days and that at 35°C was 30 days. The life span of the females which had laid eggs was shorter than that of virgin females. Virgin males were also found to have a shorter average life span than virgin females. One female lays about 100 eggs during its life span. It was shown that one third of 100 days old virgin females could mate with males and produce about 100 eggs per female with almost complete hatchability. These results indicate that this particular nematode is a useful animal for the study of aging, like other free-living nematodes currently being used in aging research.

Stability of DNA replicating activity in permeabilized mouse cells during preincubation.

When permeabilized cells, treated with detergent and made permeable to the nucleoside triphosphates, were preincubated briefly without nucleoside triphosphates, the activity of DNA replication was lost rapidly. This loss of DNA replicating activity was prevented when the mixture of nucleoside triphosphates (5 mM ATP and 0.1 mM each of dATP, dGTP, dCTP and TTP, the same concentrations contained in reaction mixture) was added to the permeabilized cells during the incubation. Each of deoxyribonuclesode triphosphates or ribonucleoside triphosphates, when added at 5 mM, was effective to varying degrees, but ATP was the most effective. These results suggests that there exists a process or factor(s) that requires ATP for DNA replication in mammalian cells, and that its decay during the preincubation could be prevented by ATP.

Isolation and characterization of mutator mutants from cultured mouse FM3A cells

A method to select mutator mutants was developed and 3 mutants were isolated from cultured mouse FM3A cells. Fluctuation analyses revealed that these mutator mutants have increased rates of spontaneous mutation at 3 genetic loci tested (resistance to ouabain, blasticidin S and tunicamycin). None of the 3 mutator mutants showed altered sensitivity to aphidicolin or arabinofuranosylcytosine, and so they differed from the mammalian mutator mutants reported previously. Also, all the mutator mutants had the same sensitivity as wild-type to UV or other DNA-damaging agents. Thus, these mutator mutants do not seem to have any deficiency in the DNA-repair process. To determine whether the mutator activity was due to the intracellular dNTP pool imbalance, 4 dNTPs in these mutator mutants were determined by high-pressure liquid chromatography and compared to that of the wild-type cells. The results show that there is no large dNTP pool imbalance in these mutator mutants. Since the mutator activity is not associated with the dNTP pool imbalance, these mutants may have altered protein(s) directly involved in DNA replication.

Yolk Syncytial Layer Independent Expression of no tail (Brachyury) or goosecoid Genes in Cultured Explants from Embryos of Freshwater Fish Medaka

Abstract Formation of embryonic axis is an essential step for animal development but its mechanism is not well understood. For axial determination in the fish embryos, participation of the yolk syncytial layer (YSL; a unique multinucleated structure formed in the yolk cell) has been shown. To investigate relationship between the YSL and axial specification, we examined whether or not expressions of the mesodermal marker genes no tail (Brachyury) or goosecoid are dependent on the YSL in explants isolated from the medaka embryos. The results of whole-mount in situ hybridization showed that these genes were expressed in the explants irrespective of the YSL, indicating that activation of these genes is a separate process from YSL-dependent axis formation.

DNA crosslinks and DNA replication in mouse FM3A cells after treatment with 8-methoxypsoralen plus near-ultraviolet radiation

The rate of DNA-chain elongation was studied in mouse FM3A cells after treatment with 8-methoxypsoralen plus near-ultraviolet radiation using the minimal doses (1 microgram/ml 8-methoxypsoralen plus 1-2.5 kJ/m2 of near-ultraviolet radiation) which inhibited cell-cycle progression or DNA replication. A rapid decrease in incorporation of [3H]thymidine and recovery to some extent during incubation after treatment have been reported (Hyodo, M., Fujita, H., Suzuki, K., Yoshino, K., Matsuo, I. and Ohkido, M. (1982) Mutat. Res. 94, 199-211). The results of the present study showed that the rate was not changed suggesting that the decrease in [3H]thymidine incorporation was not due to the rate of DNA-chain elongation, but was due to change in the frequency of initiation of replication. Formation of DNA crosslinks was then studied by the sedimentation of pre-labeled DNA in an alkaline sucrose gradient. The results showed that, at these doses of 8-methoxypsoralen plus near-ultraviolet radiation, approx. 2-7 crosslinks were formed per 10(9) Da. It was also suggested that some of the DNA crosslinks might be repaired during the prolonged incubation, but unrepaired crosslinks were still present after 24 h incubation.

Isolation of temperature-sensitive mouse FM3A cell mutants exhibiting conditional chromosomal instability.

Fourteen mutants exhibiting conditional induction of sister chromatid exchanges (SCEs) and/or chromosomal aberrations (CAs) were selected out of 65 temperature-sensitive (for growth) mutants from mouse FM3A cells treated with N-methyl-N′-nitro-N-nitrosoguanidine. The mutants with chromosomal abnormality at the nonpermissive temperature were classified into three groups. The group 1 mutant manifested mainly SCEs at the nonpermissive temperature. The group 2 mutants manifested both SCEs and CAs. The group 3 mutants, including ts-131b, a mutant defective in DNA replication and previously described, manifested only CAs. A substantial number of SCEs accompanied by CAs at the site of SCEs were observed in some of the group 2 mutants at the nonpermissive temperature. The results suggest that there are at least three processes involved in the formation of SCEs and CAs, one is common to both phenomena while the others are independent. Some other mutants belonging to the groups 1 and 2 displayed a high incidence of interchromosomal chromatid exchanges at the nonpermissive temperature, suggesting that the same genetic defects leading to high induction of SCEs correlate with induction of chromosomal rearrangements. From these results, the group 2 mutants were subdivided further into five classes, and the mutants in each class had different cytogenetic properties from one another. This indicates defects in at least several genes that participate in the production of SCEs or CAs. The mutants described should be useful for analyzing the mechanisms of SCE or CA formation, and also the mechanism of chromosomal rearrangements.

Maturation of nascent DNA fragment is disturbed after treatment of cultured mouse FM3A cells with 8-methoxypsoralen plus near-ultraviolet radiation.

By the method of sedimentation in 5-20% alkaline sucrose gradient, the process of maturation of the nascent DNA fragment was studied with cultured mouse FM3A cells treated with 8-methoxypsoralen plus near-ultraviolet radiation. This treatment is known to cause crosslinks of the chromosomal DNA strands. The profile of the newly-replicated DNA, labeled for 10 min with [3H]thymidine immediately after treatment, was the same as that of the untreated cells, where the incorporated radioactivity was present in the intermediate DNA fragment (about 50-80 S). But, when the treated cells were labeled after several hours of incubation, the labeled DNA became much shorter due to inhibition of maturation of the initial DNA fragment (the Okazaki fragment) to the intermediate DNA. With the use of aphidicolin, a specific inhibitor of eukaryotic DNA polymerase alpha, it became apparent that, in addition to formation of the crosslinks, further DNA replication is required to cause this inhibition of DNA maturation. Aphidicolin also suppressed the inhibition of incorporation of [3H]thymidine into cellular DNA after treatment, but inhibition of this incorporation resumed after its removal.