| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1991: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1990: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
In order to clarify the relationship between the progress of cell cycle events and tubulin isoforms, changes in tubulin isoforms and their mRNA were investigated on a synchronized culture of higher plant cells.
Carrot cells were initially planned to investigate for this purpose but they were found to be relatively resistant to aphidicolin which was best of the known synchronizing agents for the present purpose. Therefore, we began the experiment by using tobacco BY2 cells as the experimental material because the synchronous culture of this strain induced by aphidicolin treatment had been well established. IEF-immunoblotting of the extract indicated that similar to carrot tubulins, tobacco tubulins reacted with monoclonal antibodies against chick brain alpha- and beta-tubulins. Using the anti-beta-tubulin antibody, we resolved by IEF-immunoblotting, at least three beta-tubulin isoforms ranging from pI = 5.25-5.50. The relative ratio changed according to the progress in cell cycle stage. At S, G2 and M phase, the isoforms with pI = 5.30 and 5.32 were major components, while at G1 phase, that of pI = 5.50 increased. Dot blot hybridization analysis of RNA from the cells at different cell cycle stages revealed that beta-tubulin mRNA existed throughout a cell cycle and showed no prominent changes in their content. However, the mRNA tended to increase at M phase and decrease at G1 phase. To investigate the possibility that differential expression of some specific tubulin isotype to occur, Northern blotting analysis of beta-tubulin RNA was performed. Only a single positive band was observed throughout a cell cycle. Therefore, although different kind of beta-tubulin mRNA with similar size is still possible to exist, the data so far as we have obtained in the present experiments suggest that the change in the relative amount of beta-tubulin isoforms during cell cycle progression are the event of post-translational level rather than at the transcriptional level.
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