| Project/Area Number |
02454492
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
Human genetics
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
IKEUCHI Tatsuro Med.Res.Inst., Tokyo Med.Dent.Univ., Assoc.Prof., 難治疾患研究所, 助教授 (90041839)
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| Co-Investigator(Kenkyū-buntansha) |
YOSHIDA Mitsuaki Med.Res.Inst., Tokyo Med.Dent.Univ., Assist.Prof., 難治疾患研究所, 助手 (60182789)
SAITO Fumiko Med.Res.Inst., Tokyo Med.Dent.Univ., Assist.Prof., 難治疾患研究所, 助手 (10158917)
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| Project Period (FY) |
1990 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥4,500,000 (Direct Cost: ¥4,500,000)
Fiscal Year 1992: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1991: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1990: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | High-resolution chromosome banding / Ethidium bromide method / Cell synchronization method / Hereditary disease loci / Lymphoblastoid cell lines / Fluorescent in situ hybridization / DNA segment clones / Human No.21 chromosome / 染色体構造異常 / ヒト#21番染色体 / 蛍光 in situ 分子雑種法 / 遺伝子マッピング / in situハイブダイゼ-ション / エチジウム・ブロマイド法 / 固形腫瘍 / ダウン症候群 / HMC症候群 / 神経線維腫症第2型 / イヌ染色体 / in situハイブリダイゼ-ション |
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| Research Abstract |
1. Various established methods for high-resolution chromosome banding were re-evaluated and in part improved for their application to different cell culture systems.
(1) The ethidium bromide (EB) method was found to be applicable, by improving the dose and duration of treatment with EB and Colcemid, to the well-grown tumor cells in culture.
(2) The method of cell synchronization with methotrexate (MTX) and subsequent BrdU treatment which was applied to lymphocyte cultures from the dog (Canis familiaris) yielded sufficient results for high-resolution replication R-banding.
(3) A reliable method to obtain high-resolution R-banded chromosomes from lymphoblastoid cell lines (LCL) was established by combination of EB addition (1-1.5 hr) and excess thymidine-induced cell synchronization followed by the BrdU treatment (6.5-7 hrs).
2. By applying the EB method to peripheral lymphocyte cultures, the break points of various structural chromosome abnormalities were precisely defined, leading to the regional mapping of the following disease loci : Down syndrome-related region 21q22.2->qter), HMC syndrome (1q31.2 or 7p15.1-p15.3), neurofibromatosis type 2 (22q12.2).
3. Fluorescent in situ hybridization was performed for molecular cytogenetic characterization of some structural chromosome abnormalities (rings, pseudodicentrics, etc.) by using DNA probes of repeated sequences (telomere'DNA, rDNA alpha satellite DNA and DNA segments containing Alu repeats). Furthermore, precise regional mapping of a total of 20 DNA segments derived from human No.21 chromosomes were performed by FISH analysis and high-resolution R-banding. They include 13 NotI- and 7 SfiI-linking clones.
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