| Project/Area Number |
08044200
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Nagoya University |
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Principal Investigator |
OKAZAKI Tuneko Nagoya University, Graduate School of Science, Professor, 理学研究科, 教授 (10022584)
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| Co-Investigator(Kenkyū-buntansha) |
COOKE Howard MRC,Human Genetics Unit, Head of Chromosome Biology, 染色体生物学部門, 主任研究員
HIETER Philip A The Johns Hopkins University, School of Mcdicine, professor, 医学部, 教授
YODA Kinya Bioscience Center, Research Associate, 生物分子応答研究センター, 助手 (30126916)
MASUMOTO Hiroshi Graduate School of Science, Assistant Professor, 理学研究科, 講師 (70229384)
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| Project Period (FY) |
1996
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| Project Status |
Completed (Fiscal Year 1996)
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| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1996: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | cetromere / teloreme / alphoid DNA / YAC / MAC / CENP-B / CENP-B box |
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| Research Abstract |
Centromere is the chromosomal domain essential to the segregation of eukaryotic chromosomes. Although DNA is believed to be the primary determinant of the functional centromere structure in mammalian cells, no conclusive intormation on the essential DNA structure is available until now. The purpose of this work is to clone functional human centromere DNA using technology of the yeast artificial chromosome (YAC). We have found previously two megabase-sized domains of alphoid DNA,alpha21-I and alpha21-II,in the human chromosome 21. The alpha21-I contained many CENP-B boxes, binding sites of CENP-B,at high and regular intervals, while the alpha21-II contained very few, if any. We have cloned into YAC 100 kb alphoid DNA derived from alpha21-Iand alpha21-II domain using a recombination minus yeast strain. In collaboration with Dr.Cooke, we constructed YAC arm vectors containing human telomere sequences and selectable markers for mammalian cells and both arms of alphoid YAC were replaced with these arms by recombination in vivo. We transformed a cultured human cell line with these human telomere and alphoid containing YAC and obtained transformants with both YACs. FISH analyzes of the transformed cell lines showed that transformants of alpha21-I YAC contained a stably maintained mammalian artificial chromosome but those of alpha21-II YAC was integrated at telomere or centromere. These results indicated that the essential centromere DNA sequence resides in alpha21-I region.
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