| Project/Area Number |
63870105
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| Research Category |
Grant-in-Aid for Developmental 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 Dental University |
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Principal Investigator |
YASUKOCHI Yukio Tokyo Med & Dent Univ, Med Res Inst, Professor, 難治疾患研究所, 教授 (60037398)
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| Co-Investigator(Kenkyū-buntansha) |
KOBAYASHI Yasushi Tokyo Med & Dent Univ, Med Res Inst, Assistant Professor, 難治疾患研究所, 助手 (70225548)
KITAJIMA Shigetaka Tokyo Med & Dent Univ, Med Res Inst, Associate Professor, 難治疾患研究所, 助教授 (30186241)
OTSUKA Tuyoshi Kyushu Univ Sch of Med, Assistant Professor, 医学部, 助手 (50213773)
沢田 育久 東京医科歯科大学, 難治疾患研究所, 助手 (30215909)
川口 達夫 東京医科学歯科大学, 難治疾患研究所, 助手 (70191997)
横田 英介 九州大学, 医学部, 助手 (60167723)
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| Project Period (FY) |
1988 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥11,500,000 (Direct Cost: ¥11,500,000)
Fiscal Year 1990: ¥2,500,000 (Direct Cost: ¥2,500,000)
Fiscal Year 1989: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 1988: ¥5,900,000 (Direct Cost: ¥5,900,000)
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| Keywords | Rare cutter / Bsu E methylose / Dpn I / Taq I methylose / MHC / Linking library / X chromosome / メチル化抵抗性NotIリンキングクロ-ン / Linking Library / Jumping Library / NanII / BsuEメチレース / 精製 |
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| Research Abstract |
1. Development of rare cutter enzyme system and its application to the human genome. We have developed two rare cutter enzyme systems which consist of Taq I methylose-Dpn I (Nan II) and Bsu E methylose-Not I systems. First we tried to purify Bsu E methylose and Nan II which were not available commercially. Purified Bsu E methylose showed an apparent molecular weight of 46 K on SDS-PAGE. However Nan II could not be purified to near homogeneity. To evaluate the usefullness of the Bsu E methylae-Not I system as a mapping procedure, we applied this system to analysis of the human MHC. Prior methylation of genomic DNA resulted in shifting up into fragments of size 2 million bp or more in all of the known MHC Not I fragments except one Not I site which did not contain a CGCG sequence overlapping the GCGGCCGC as determined by DNA sequencing. The Taq I methylose-Dpn I system gave distinct bands on PFGE when the E. coli genome was digested with the system.
2. Construction and characterization of linking libraries. We have constructed chromosome specific Not I or Not I-Bsu E linking libraries. The latter approach consists of methylating the X chromosome specific cloned DNA with Bsu E methylose, followed by selection of the methylation resistant Not I sites by insertion of the kanamycin gene in the clones cleavable by Not I. We have isolated, partially sequenced and characterized 114 Not I-Bsu E linking clones, and mapped 51 clones to various regions along the chromosomes. The Not I linking clones specific for the human chromosome 6 were prepared in the same method as the Not I-Bsu E linking clones except omitting methylation. At least 10 different clones were localized on the short arm and 29 clones on the long arm of the human chromosome 6.
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