| Project/Area Number |
14572146
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Human genetics
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| Research Institution | KANAZAWA MEDICAL UNIVERSITY |
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Principal Investigator |
DATE Takayasu KANAZAWA MEDICAL UNIVERSITY, Department of Medicine, Professor, 医学部, 教授 (50019676)
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| Co-Investigator(Kenkyū-buntansha) |
MATSUI Tadashi KANAZAWA MEDICAL UNIVERSITY, Department of Medicine, Assistant Researcher, 医学部, 助手 (60288272)
IWABUCHI Kuniyoshi KANAZAWA MEDICAL UNIVERSITY, Department of Medicine, Associate Professor, 医学部, 助教授 (10232696)
濱田 富美男 金沢医科大学, 医学部, 助手 (80240779)
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| Project Period (FY) |
2002 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2003: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2002: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Keywords | PKU-beta / TLK1 / 53BP1 / X-ray / DNA double-strand break / DNA repair / siRNA / chromosomal segregation / centrosome / TLK-2 / クロマチン / PKU / IRIF / RNAi / PKU結合タンパク質 |
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| Research Abstract |
1)X-ray irradiation induced foci (IRIF) was observed in nuclei of HeLa or MCF7 cells when cells were immunostained by polyclonal anti-PKU-β (TLK1) antibodies. However, IRIF was not detected by monoclonal antibody. There is no significant difference in reacted proteins by two antibodies. Two of three proteins that recognized by antibodies in MCF7 cell lysate were identified as splicing variants.
2)When exogenous HA-tagged PKU was introduced into cells, no IRIF was observed by anti-Ha antibody. However, we could not determine whether failure was due to overexpression.
3)The siRNA significantly reduced the expression of PKU-beta at 3 days after transfedction. These cells, howver, still formed IRIF.
4)MCF7 cells with low expression of PKU-beta mediated by siRNA was sensitive to either X-ray irradiation, UV-irradiation or genotoxic reagents such as mytomisin C.
5)When analyzed by FACS, PKU-knockdown with MCF 7 cells lead to aneuploidy or polyploidy. The number of chromosome of MCF7 which usually contains 62 increased to 62-120 at three days after the siRNA transfection.
6)RNAi-mediated disruption of PKU-beta in cells interferes with normal spindle assembly and positioning. SiRNA treatment blocked the separation and positioning of duplicated centrosomes, thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. These results suggests that PKU-beta involves G2-M transition and plays an important role on chromosomal segregation.
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