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

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Author(s)

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

Abstract

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.

Journal

  • ACTA HISTOCHEMICA ET CYTOCHEMICA

    ACTA HISTOCHEMICA ET CYTOCHEMICA 29(1), 71-79, 1996-02-01

    The Japan Society of Histochemistry and Cytochemistry

References:  43

Cited by:  8

Codes

  • NII Article ID (NAID)
    10008605215
  • NII NACSIS-CAT ID (NCID)
    AA00508022
  • Text Lang
    ENG
  • Article Type
    Journal Article
  • ISSN
    00445991
  • Data Source
    CJP  CJPref  IR  J-STAGE 
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