4
TAKEHIKO KO]I
conformation were analyzed on a sucrose-density gradient and
the nucleoid sedimentation rate was determined as an indicator
of the degree of SSB (Johnstone and Williams 1982). Although
the presence of SSB in resting human peripheral blood lymphocytes was discovered by means of this method (Johnstone and
Williams 1982; Greer and Kaplan 1984), the finding led to a big
debate. In a short episode of whether the SSB are present in the
nuclei of resting human peripheral blood lymphocytes or not,
rightness of the nucleoid sedimentation technique was a major
point in many conflicting reports. In fact, Jostes et al. (1989) and
Boerrigter (1991) did not detect SSB in the DNA of the lymphocytes by means of alkaline elution method.
Besides, biochemical methods are generally not sufficient to
correlate the occurrence of SSB with the changes in physiological
states of each cell. Therefore, to understand better the biological
significance of SSB and to get more direct evidence of a causeand-effect relationship between SSB and cellular changes, a histochemical approach to localize the sites of SSB would be indispensable.
In the case of DSB detection, a ladder formation of DNA in
agarose gel-electrophoresis is a most popular biochemical method and used especially often to show the presence of apoptotic
cells. If about 4% of cell population are undergoing apoptosis
(Collins et al. 1992), we can detect discrete ladder bands consisting of multiples of 180-200 base pairs. However, the method will
not provide information on how many cells are apoptotic.
Histochemical methods provide useful information on the
sites of DNA strand breaks at individual nuclear level and so
we can get evidence of how many cells are haboring the strand
breaks and where the positive cells are located. The combination
with other histochemical methods to demonstrate the presence
of various marker molecules may permit us to investigate the
relation between occurrence of SSB or DSB of DNA and physiological changes at individual cell level.
In situ nick translation as a specific method to detect SSB
In order to analyze the occurrence of SSB directly at a level of
individual cells, in situ nick translation (ISNT) could be a powerful tool (Koji 1996), which was originally developed to investigate
TAKEHIKO KO]I
conformation were analyzed on a sucrose-density gradient and
the nucleoid sedimentation rate was determined as an indicator
of the degree of SSB (Johnstone and Williams 1982). Although
the presence of SSB in resting human peripheral blood lymphocytes was discovered by means of this method (Johnstone and
Williams 1982; Greer and Kaplan 1984), the finding led to a big
debate. In a short episode of whether the SSB are present in the
nuclei of resting human peripheral blood lymphocytes or not,
rightness of the nucleoid sedimentation technique was a major
point in many conflicting reports. In fact, Jostes et al. (1989) and
Boerrigter (1991) did not detect SSB in the DNA of the lymphocytes by means of alkaline elution method.
Besides, biochemical methods are generally not sufficient to
correlate the occurrence of SSB with the changes in physiological
states of each cell. Therefore, to understand better the biological
significance of SSB and to get more direct evidence of a causeand-effect relationship between SSB and cellular changes, a histochemical approach to localize the sites of SSB would be indispensable.
In the case of DSB detection, a ladder formation of DNA in
agarose gel-electrophoresis is a most popular biochemical method and used especially often to show the presence of apoptotic
cells. If about 4% of cell population are undergoing apoptosis
(Collins et al. 1992), we can detect discrete ladder bands consisting of multiples of 180-200 base pairs. However, the method will
not provide information on how many cells are apoptotic.
Histochemical methods provide useful information on the
sites of DNA strand breaks at individual nuclear level and so
we can get evidence of how many cells are haboring the strand
breaks and where the positive cells are located. The combination
with other histochemical methods to demonstrate the presence
of various marker molecules may permit us to investigate the
relation between occurrence of SSB or DSB of DNA and physiological changes at individual cell level.
In situ nick translation as a specific method to detect SSB
In order to analyze the occurrence of SSB directly at a level of
individual cells, in situ nick translation (ISNT) could be a powerful tool (Koji 1996), which was originally developed to investigate