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  1. B100 理学部/理学研究科
  2. B100d 学位論文
  3. 博士論文(理博・論理博)

Molecular Analyses of Centromere in Human Chromosome

http://hdl.handle.net/2237/6765
http://hdl.handle.net/2237/6765
c7b99306-5e86-4259-a077-285460b08729
名前 / ファイル ライセンス アクション
ko2253_abstr.pdf ko2253_abstr.pdf (332.1 kB)
ko2253.pdf ko2253.pdf (9.3 MB)
Item type 学位論文 / Thesis or Dissertation(1)
公開日 2006-07-31
タイトル
タイトル Molecular Analyses of Centromere in Human Chromosome
言語 en
その他のタイトル
その他のタイトル ヒト染色体セントロメアの分子構造解析
言語 ja
著者 Masumoto, Hiroshi

× Masumoto, Hiroshi

WEKO 12998

en Masumoto, Hiroshi

Search repository
舛本, 寛

× 舛本, 寛

WEKO 12999

ja 舛本, 寛

Search repository
アクセス権
アクセス権 open access
アクセス権URI http://purl.org/coar/access_right/c_abf2
抄録
内容記述 The aim of this study is to establish bases for understanding molecular mechanism of chromosome segregation. This has been achieved by isolating a DNA element specific to the centramere of human chromosome which is responsible for chromosome segregation, and by developing an in vitro system to bemonstrate the interaction between the DNA element and aprotein which is recognized by anticentramere (kinetochore) antibodies in sera from scleroderma patients and know to be a component of kinetochore structure. This is the first evidence showing a molecular interaction between the DNA element and a specific protein both of which reside at centramere region of chromatin. In the first part of the study, cloning of a DNA element specific to the centromere region was carried out. Purified HeLa chromosomes were digested with the restriction enzymes Sau3AI and fractionated by sedimentation through a sucrose gradient. Franctions showing antigenicity to anticentromere antibody were used to construct a DNA library. From this library one clone which specifically hybridized to the centromere domain of metaphase chromosomes was found using a biotinylated probe DNA and FITC conjugated avidin for its detection. The clone contained a stretch of alphoid DNA dimer, a centromeric satellite DNA, which consists of 170 bp long repeating units. TO determine precisely the relative location of the alphoid DNA stretch and the centramere antigen, a method was developed to carry out in situ hybridization of DNA and indirect immunofluorescent staining of antigen on the same cell preparation. Using this method, perfect overlapping of the slphoid DNA sites with the centromere antigen sites has been found in both metaphase chromosomes and nuclei at various stages in the cell cycle. This exact correlation was also observed at the attachment sites of artificially extended sister chromatids. These results suggest the possibility that alphoid DNA repeat is a monponent of kinetochore structure. IN the second part of the study, the interaction between a human centromere antigen and an alphoid DNA has been examined in molecular level. A cloned alphoid DNA fragment incubated with a HeLa cell nuclear extract was selectively immunoprecipitated by the anticentromere sera from scleroderma patients. Immunoprecipitation of the DNA made by primer extension on the alphoid DNA defined the 17 bp segment on the alphoid DNA that was required for formation of DNA-antigen complex. On the other hand, when proteins bound to the biotinylated alphoid DNA carrying the 17 bp motif were recovered by streptavidin agarose and immunoblotted, the 80 kd centromere antigen (CENP-B) was betected. DNA binding experiments for gelelectrophoresed and membrans-bound proteins showed that the polypeptide at the 80 kd mobility specifically bound to the DNA fragment with the 17 bp motif. Thus, the 17 bp motif was termed as CENP-B box. Alphoid monomers with the CWNP-B box art found in all the known alploid subclasses with varying frequencies, except in the one derived from the Y-chromosome so far cloned. These results imply that the interaction of the 80 kd centromere antigen with the CENP-B box in the alphoid repeats may play some crucial role in the formation of specified structure and/or function of human centromere.
言語 en
内容記述タイプ Abstract
内容記述
内容記述 名古屋大学博士学位論文 学位の種類:理学博士 (課程) 学位授与年月日:平成1年10月11日
言語 ja
内容記述タイプ Other
言語
言語 eng
資源タイプ
資源 http://purl.org/coar/resource_type/c_db06
タイプ doctoral thesis
書誌情報
発行日 1989-10-11
学位名
言語 ja
学位名 理学博士
学位授与機関
学位授与機関識別子Scheme kakenhi
学位授与機関識別子 13901
言語 ja
学位授与機関名 名古屋大学
言語 en
学位授与機関名 Nagoya University
学位授与年度
学位授与年度 1989
学位授与年月日
学位授与年月日 1989-10-11
学位授与番号
学位授与番号 甲第2253号
フォーマット
application/pdf
フォーマット
application/pdf
著者版フラグ
値 publisher
URI
識別子 http://hdl.handle.net/2237/6765
識別子タイプ HDL
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