Nonradioactive in Situ Nick Translation : A Useful Molecular Histochemical Tool to Detect Single-Stranded DNA Breaks

この論文にアクセスする

この論文をさがす

著者

    • KOJI Takehiko
    • Department of Anatomy (III), Nagasaki University School of Medicine

抄録

At various stages of the life cycle of cells, the occurrence of DNA single-strand breaks (SSB) is a common event. In order to understand better the relationship of SSB to physiological states of cells, one requires the analysis of SSB at the level of individual cells by in situ nick translation (ISNT). In the principle of ISNT, the DNA strand with breaks is elongated in the presence of biotin-11-dUTP by E. coil DNA polymerase I at the nicked sites. The biotin moieties incorporated into the newly synthesized strand are visualized enzyme-immunohistochemically with horseradish peroxidase-labeled antibiotin antibody. In this article, I will firstly describe the details of optimization of the ISNT reaction for its full implementation, using nicked λ phage DNA, which was fixed onto nitrocellulose filters, as well as using fresh frozen sections of rat testis and small intestine. Subsequently, I would like to demonstrate the presence of two types of SSB, which can be practically distinguished by protease-dependency of the staining; one is readily detected by ISNT without any deproteination steps (protease-independent type), and the other requires the protease treatment to be detected by ISNT (protease-dependent type). In our case, the single-strand breaks of DNA observed in terminally differentiated cells as well as the cells undergoing necrosis were protease-independent type. On the other hand, the DNA breaks in the cells undergoing replicative DNA synthesis and apoptosis were regarded as the latter type. Consequently, ISNT should be regarded as a useful molecular histochemical tool to categorize naturally occurring SSB.

At various stages of the life cycle of cells, the occurrence of DNA single-strand breaks (SSB) is a common event. In order to understand better the relationship of SSB to physiological states of cells, one requires the analysis of SSB at the level of individual cells by <I>in situ</I> nick translation (ISNT). In the principle of ISNT, the DNA strand with breaks is elongated in the presence of biotin-11-dUTP by <I>E. coil</I> DNA polymerase I at the nicked sites. The biotin moieties incorporated into the newly synthesized strand are visualized enzyme-immunohistochemically with horseradish peroxidase-labeled antibiotin antibody. In this article, I will firstly describe the details of optimization of the ISNT reaction for its full implementation, using nicked λ phage DNA, which was fixed onto nitrocellulose filters, as well as using fresh frozen sections of rat testis and small intestine. Subsequently, I would like to demonstrate the presence of two types of SSB, which can be practically distinguished by protease-dependency of the staining; one is readily detected by ISNT without any deproteination steps (protease-independent type), and the other requires the protease treatment to be detected by ISNT (protease-dependent type). In our case, the single-strand breaks of DNA observed in terminally differentiated cells as well as the cells undergoing necrosis were protease-independent type. On the other hand, the DNA breaks in the cells undergoing replicative DNA synthesis and apoptosis were regarded as the latter type. Consequently, ISNT should be regarded as a useful molecular histochemical tool to categorize naturally occurring SSB.

収録刊行物

  • Acta histochemica et cytochemica

    Acta histochemica et cytochemica 29(1), 71-79, 1996-02-01

    JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY

参考文献:  43件中 1-43件 を表示

被引用文献:  8件中 1-8件 を表示

各種コード

  • NII論文ID(NAID)
    10008605215
  • NII書誌ID(NCID)
    AA00508022
  • 本文言語コード
    ENG
  • 資料種別
    ART
  • ISSN
    00445991
  • データ提供元
    CJP書誌  CJP引用  IR  J-STAGE 
ページトップへ