@phdthesis{oai:nagoya.repo.nii.ac.jp:00005181,
 author = {Masumoto, Hiroshi and 舛本, 寛},
 month = {Oct},
 note = {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., 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},
 school = {名古屋大学, Nagoya University},
 title = {Molecular Analyses of Centromere in Human Chromosome},
 year = {1989}
}