1990 Fiscal Year Final Research Report Summary
Studies on the Human Chromosome Mapping
| 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)
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| Project Period (FY) |
1988 – 1990
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| Keywords | Rare cutter / Bsu E methylose / Dpn I / Taq I methylose / MHC / Linking library / X chromosome |
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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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