VIROLOGY 250, 7684 (1998)

ARTICLE NO. VY989347

Apoptosis Precedes Necrosis of Fish Cell Line with Infectious

Pancreatic Necrosis Virus Infection

Jiann-Ruey Hong,*, Tai-Lang Lin,* Ya-Li Hsu,* and Jen-Leih Wu*,1

*Laboratory of Marine Molecular Biology and Biotechnology, Institute of Zoology, Academia Sinica, Nankang, Taipei 115, Taiwan;
and Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 117, Taiwan

Received February 6, 1998; retuned to author for revision April 20, 1998; accepted July 22, 1998

The current view of infectious pancreatic necrosis virus (IPNV) infection includes a necrotic process that relies primarily
on the histological appearance of tissue after the degenerative process. We tested this view by examining the possibility that
apoptosis is a component of double-stranded RNA virus (IPNV) that induces fish embryonic cell death. Four kinds of assays
for apoptosis were used in analyzing IPNV-infected CHSE-214 cells: (1) assay with terminal deoxynucleotidyl transferase
(TdT)-mediated end-labeling of DNA in nuclei of intact cells during virus infection, (2) assay for procoagulant activity, (3) assay
for DNA ladders, and (4) electron microscopic assays for the ultrastructural changes in characteristic apoptotic cells. In all
p.i. samples, both low and high m.o.i. groups contained apoptotic nuclei, according to TdT-mediated dUTP labeling of intact
cells, but in control CHSE-214 cells, apoptotic nuclei were rare at all levels of incubation sampled by TdT-mediated dUTP
labeling. Prenecrotic or postnecrotic cells were found to express phosphatidylserine on the surface by annexin VFITC
labeling, but normal cells did not. DNAs from both 4 h p.i. of high m.o.i. and 8 h p.i. of low m.o.i. were found to be cleaved
into fragments indicative of preferential cleavage at internucleosomal sites. The IPNV-infected CHSE-214 cells were analyzed
with an electron microscope and showed a pattern of ultrastructural change, indicating that apoptosis appears before
pathological changes of necrosis, including condensed chromatin, fragmented nuclei, nuclei with chromatin marginations,
and secondary necrosis from prenecrotic cells in IPNV-infected CHSE-214 cells. Together, these findings show that apoptosis
precedes any detectable necrotic change in CHSE-214 cells that is currently viewed as necrosis. Thus, apoptosis charac-
terizes the onset of pathology in host cells and is followed by necrotic processes. 1998 Academic Press
Key Words: infectious pancreatic necrosis virus, apoptosis, fish cell, secondary necrosis.

INTRODUCTION Infectious pancreatic necrosis virus (IPNV) is the pro-

There are two major morphologically and biochemi-
cally distinct modes of death in eukaryotic cells: apopto-
sis and necrosis (Duvall and Wyllie, 1986; Kerr and Har-
mon, 1991; Wyllie et al., 1980). Apoptosis is characterized
morphologically by cell shrinkage and hyperchromatic
nuclear fragments and biochemically by chromatin
cleavage into nucleosomal oligomers (Wyllie et al., 1980).
Apoptosis is considered to be a physiological process
involved in normal tissue turnover that occurs during
embryogenesis, aging, and tumor regression (Wyllie et
al., 1980), but pathological stimuli, such as viral infec-
tions (Gougeon and Montagnier, 1993; Inoue et al., 1997;
Jacotot et al., 1997; Ohno et al., 1993; Noteborn et al.,
1994; Rojko et al., 1992; Vasconcelos and Lam, 1994), can
also be triggering factors. Necrosis is considered to be a
pathological reaction that occurs in response to pertur-
bations in the cellular environment such as complement
attack, severe hypoxia, or hyperthermia. These stimuli
increase the permeability of the plasma membrane, re-
sulting in irreversible swelling of the cells (Wyllie et al.,
1980).
1
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27858059. E-mail: ZOJLWU@ccvax.sinica.edu.tw.
0042-6822/98 $25.00
Copyright 1998 by Academic Press
All rights of reproduction in any form reserved.
totype of the group of viruses known as Birnaviridae
(Dobos et al., 1979). IPNV was discovered to be associ-
ated with a highly contagious disease of susceptible
hatchery-reared trout. As the name indicates, the infec-
tion among trout produces marked pancreatic necrosis,
but histopathological changes sometimes also occur in
adjacent adipose tissue, in renal hematopoietic tissue, in
the gut, and in the liver (Wolf et al., 1960). Histopatholog-
ical changes can also occur in renal excretory and he-
matopoietic tissues, as first reported by Yasutake et al.
(1965). Although renal damage is consistent with the
high titer of virus typically found in kidneys, at least in
carrier fish, confirmation was first reported by Wolf and
Quimby (1969). Sano et al. (1971, 1973) also described
and, in addition, illustrated renal damage from IPNV in
rainbow trout and in amago (Oncorhynchus rhodurus).
They found congestion or hemorrhage in glomeruli,
edema, and destruction or desquamation of tubule epi-
thelium. Swanson and Gillespie (1979) similarly noted
focal degeneration of liver parenchymal cells in yearling
Atlantic salmon that had been previously inoculated with
IPNV. Liver damage is consistent with virological find-
ings, and Kudo et al. (1973) showed that virions were
present in hepatocytes.
An alternative hypothesis, which is proposed here, is
76
 APOPTOSIS INDUCED BY IPNV
that the death of IPNV-infected host cells is initiated by
apoptosis because the criteria used to characterize the
death of IPNV-infected cells as an exclusively necrotic
process are not sufficient to eliminate the possibility that
apoptosis also occurs.
In the present investigation, to examine the possibility
that apoptosis may contribute to the death of IPNV-
infected cells, we assayed for DNA fragmentation and
changes in cell structure that indicate apoptotic cell
death. These findings indicated that IPNV induces cell
apoptosis and then induces postapoptotic necrosis (sec-
ondary necrosis) in fish embryonic cells in vitro.
RESULTS
Light microscopic identification of apoptotic nuclei
in prenecrotic CHSE-214 cells
Terminal deoxynucleotidyl transferase (TdT)-dUTP la-
beling of infected CHSE-214 cells without or with IPNV
showed that apoptosis, as indicated by the presence of
double-stranded DNA fragments in situ, occurred in vi-
rus-infected cells at all times sampled (Figs. 1 and 2).
Rarely, the apoptotic nuclei were observed in 1% FCS
serum CHSE-214 cells (1%; P , 0.05). This small con-
centration of apoptotic nuclei in control CHSE-214 cells
may reflect an apoptotic, developmental process under
low serum conditions. The concentration of apoptotic
nuclei observed in the m.o.i. 20 group at 4, 8, and 12 h p.i.
was 30%, 62%, and 57% (all P , 0.05), respectively. The
apoptotic nuclear population in the m.o.i. 1 group at 4, 8,
and 12 h p.i. was 4%, 39%, and 51% (all P , 0.05),
respectively.
Visualization of the morphological changes of
prenecrotic and postnecrotic cells in CHSE-214 cells
We used the CHSE-214 cell line to test the feasibility of
using TdT biotin-dUTP nick end labeling (TUNEL) assay
and annexin Vfluorescein labeling for the detection of
apoptotic cells. First, we used the in situ TUNEL assay to
identify prenecrotic and postnecrotic cells. The results,
as shown in Figs. 3A and 3B, can be clearly seen in the
prenecrotic cells (Fig. 3A) and postnecrotic cells (Fig.
3B). In a comparison of Fig. 3A with Fig. 3B, we found cell
volume increased ;3-fold, and the cell morphology
changed more dramatically in prenecrotic than in post-
necrotic cells. We then confirmed the cell morphology of
the prenecrotic and postnecrotic cells by annexin V in
CHSE-214 cells during IPNV infection under the condi-
tions described above. The results are shown in Figs. 3C
through 3F. Figures 3C and 3E show the infected m.o.i.
CHSE-214 cells double-stained with annexin V and pro-
pidium iodide at 8 and 12 h p.i., respectively. The double-
stained cells were observed under phase-contrast mi-
croscopy. The samples in Figs. 3D and 3F were exam-
ined by fluorescence microscopy. The prenecrotic cells
were annexin positive (Fig. 3D). At this same time, the
77
membrane integrity remained intact, and the propidium
iodide staining was negative. The postnecrotic cells
were double positive (Fig. 3F). Postnecrotic cells were
only weakly stained with annexin V, but because mem-
brane integrity was lost, they showed positive staining
with propidium iodide.
Induction of internucleosomal cleavage by IPNV
Because DNA fragmentation is a well-defined bio-
chemical marker of apoptosis (Schwartzman and Cid-
lowski, 1993), we first examined the abilities of IPNV
(E1-S) to induce DNA fragmentation in the fish embryonic
cell line. DNA extracted from CHSE-214 cells with virus
and CHSE-214 cells without virus as controls was exam-
ined for evidence of internucleosomal fragmentation,
which occurs in apoptosis. Intense internucleosomal
fragmentation of DNA, a pattern highly specific to apop-
tosis, was observed in CHSE-214 cells treated with IPNV
(Fig. 4). DNA from high and low m.o.i. infected cells were
found to be cleaved into fragments both at 4 h p.i. and at
8 p.i. On the other hand, the gel of the negative control at
0 and 12 h of incubation showed no DNA fragment
formation.
Electron microscopy shows that apoptosis precedes
necrosis in CHSE-214 cell
Ultrastructural analysis showed sequentially morpho-
logical patterns of apoptosis from the prenecrotic to
postnecrotic cells under IPNV (m.o.i. 1) infection. Figures
5A and 5B show uninfected CHSE-214 cells that were
incubated for 12 h and for 0 h in 1% serum as a negative
control. Condensated nuclei and nuclei with chromatin
margination in the early apoptotic cells (at 4 h p.i.) are
shown in Figs. 5C and 5D, respectively. At 8 h p.i., cells
showed uniformly electron dense micronuclei and small
micronuclei protruding from the nuclear periphery (Fig.
5E). Finally, at 12 h of p.i., cells also showed a swollen
morphology and exhibited small micronuclei but con-
tained the condensated chromatin and enclosed nuclear
membranes (Fig. 5F). At this time, other organelles, such
as mitochondria, in the cell appeared seriously swollen
and lost their morphologies.
Apoptotic cells phagocytosed by neighbor cells
Figure 6 shows the apoptotic body phagocytosed by a
neighbor cell. This apoptotic phagocytosed cell had the
morphology typical of apoptotic nuclei, which is charac-
terized by nuclear condensation and multimicronuclei.
The phagocytosed fish embryonic cell appeared intact,
suggesting that the fish embryonic cell recognized some
surface change (as in Figs. 3D and 3F) that promoted
their phagocytosis of apoptotic fish cells before lysis
(Fadok et al., 1992).
 APOPTOSIS INDUCED BY IPNV 79

FIG. 4. Agarose gel electrophoresis of the DNA fragment. There is no

DNA fragmentation in the negative control at 0 h of incubation (lane 2).
FIG. 2. Percentage of TdT-dUTP-labeled apoptotic CHSE-214 cells
Some background apoptosis is visible in the negative control at 12 h of
without or with m.o.i. 1, and m.o.i. 20 of IPNV infection at different time
incubation (lane 3); intense DNA fragmentation is apparent at 4, 8, and
points of p.i. At all time points, the number of apoptotic nuclei in
12 h p.i. in m.o.i. 1 infection (lanes 46, respectively) and with m.o.i. 20
IPNV-infected CHSE-214 cells was significantly greater than those in
infection at 4, 8, and 12 h p.i. (lanes 79, respectively). (Lane 1) DNA
mock-infected CHSE-214 cells.
size markers.

DISCUSSION
The results of the present investigation indicate that
the earliest fish cell line (CHSE-214) changes by IPNV are
apoptotic rather than necrotic. Specifically, the presence
of nuclei containing double-stranded DNA fragments and
abnormal chromatin-condensations before evidence of
cell necrosis is a strong indicator of apoptosis. Morpho-
logical assays for apoptosis are essential in this complex
at the intact cell instance of cell death because apopto-
sis is rapid and cell death in the degenerating CHSE-214
cell is not synchronized. Thus only a small fraction (1%)
of nuclei would be expected to be apoptotic at the same
time in control cells. The present findings indicate that
both m.o.i. 1 (4%) and m.o.i. 20 (30%) are recognizably
apoptotic cells at 4 h p.i. when assayed with TdT-medi-
ated dUTP labeling of virus-infected cells. However, nu-
clei are detectable apoptotic using this technique ;24
h after the onset of apoptosis, according to time course
studies on dexamethasone-stimulated thymocytes (Gav-
rieli et al., 1992).
A recently discovered family of proteins, the annexins,
has been found to have a high affinity for aminophos-
pholipids in the presence of Ca21 (Andree et al., 1990;
Raynal and Pollard, 1994; Tait et al., 1989). A member of
this family, annexin V, has been shown by several groups
to preferentially bind phosphatidylserine (PS) (Andree et
al., 1990; Tait et al., 1989; Thiagarajan and Tait, 1990).
Thus annexin V provides a convenient tool with which to
determine directly whether changes in membrane PS
distribution occur as a general feature of apoptosis and
to measure the kinetics of these changes on a single-cell
basis. In this regard, a recent report demonstrated in-
creased annexin V binding during serum withdrawal-
induced apoptosis of murine germinal center B cells as
well as a B cell line (Koopman et al., 1994). Here we
found that prenecrotic and postnecrotic cells (Figs. 3D
and 3F) induced by an m.o.i. 1 of IPNV were accompa-
nied by dramatic changes in PS distribution at the
plasma membrane (PM), as assessed by the increased
annexin V-binding properties of these cells. The obser-
vations of Fadok et al. (1992) suggest that apoptotic
lymphocytes lose membrane phospholipid asymmetry

FIG. 1. Apoptotic CHSE-214 cells (arrow) with or without IPNV infection labeled with TdT-dUTP. Apoptotic nuclei are labeled with an orange red
chromogen. (Top) Phase-contrast image of mock-infected CHSE-214 cell as a normal control with TdT-dUTP-labeled apoptotic nuclei. Magnification,
3100. (Middle) Phase-contrast image of infected CHSE-214 cells with m.o.i. 1 for at 8 h p.i., in which TdT-dUTP-labeled apoptotic nuclei (arrows) can
be clearly identified. Magnification, 3100. (Bottom) Phase-contrast image of IPNV-infected CHSE-214 cells with m.o.i. 20 at 8 h p.i. (Arrows)
TdT-dUTP-labeled apoptotic nuclei. Magnification, 3100.
FIG. 3. Phase-contrast micrographs of the morphological characterization of prenecrotic and postnecrotic IPNV-infected CHSE-214 cells with m.o.i. 1 at
8 h (A) and 12 h(B) p.i.; TdT-dUTP-labeled apoptotic cells are present. Cells are labeled with an orange red chromogen. (Arrows) Prenecrotic cell (A) and
postnecrotic cell (B). Phase-contrast micrographs of annexin Vfluorescein labeling of IPNV-infected CHSE-214 cells with m.o.i. 1 for at 8 h (C) and 12 h (E)
p.i. (D and F) Fluorescence images of anticoagulant annexin V from cells in C and E show the prenecrotic cell and postnecrotic cell, as indicated by arrows.
80 HONG ET AL.

FIG. 5. Electron micrographs of CHSE-214 cells. (A) CHSE-214 from 12 h of incubation as a negative control. (B) CHSE-214 from 0 h incubation as
the negative control. (C and D) Prenecrotic IPNV-infected CHSE-214 cells with m.o.i. 1 at 4 h p.i. showing the chromatin condensated (C) and chromatin
margination (D), indicated by arrows. (E) Middle necrotic CHSE-214 cell at 8 h p.i. that formed uniformly electron dense micronuclei indicated by
arrows. (F) Postnecrotic cell at 12 h p.i. (Arrow) The condensated chromatin enclosing the nuclear membrane. (Bar) 1 mm.

and expose PS on the outer leaflet of the plasma mem-
brane. Macrophages, then phagocytose apoptotic lym-
phocytes, specifically recognize exposed PS. CHSE-214
cells that had ingested IPNV-infected CHSE-214 cells
were examined by electron microscopy (Fig. 6). Phago-
cytosed virus-infected cells had the morphology typical
of apoptosis, which is characterized by the multimicro-
nuclei and cytoplasmic condensation. The phagocytosed
IPNV-infected CHSE-214 cells appeared intact, suggest-
ing that the CHSE-214 cells recognized some surface
change (as PS) that promoted their phagocytosis of
apoptotic IPNV-infected cells before lysis. These data
 APOPTOSIS INDUCED BY IPNV 81

FIG. 6. Electron micrograph of an intact apoptotic CHSE-214 engulfed by a neighbor cell. (Bar) 1 mm. CHSE-214 cells were infected with m.o.i. 1
of IPNV at 12 h p.i. Then the monolayers were washed, fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.2, and processed for electron
microscopy.

support the view that IPNV can induce CHSE-214 apo-
ptotic cell death.
Apoptosis is a morphologically distinct cell death that
spontaneously occurs in many different tissues under var-
ious conditions (Falcieri et al., 1994). It occurs in distinctly
separated cells and progresses very rapidly, never causing
exudative inflammation in tissues. No cell hydration takes
place, but nuclear and cytoplasmic condensation can ap-
pear (see Fig. 5C), followed by the formation of numerous
membrane-bound cell fragments termed apoptotic bod-
ies. In contrast to necrosis, the nuclear organization is
completely lost. Profound chromatin rearrangements take
place, followed by the formation of a variable number of
compact, electron-dense micronuclei (see Fig. 5E). Surpris-
ingly, despite the extensive nuclear changes, both cyto-
plasm and organellar components remain intact for some
time unless the cell undergoes secondary necrosis (see
Fig. 5F). Only in the final apoptotic stage is the whole cell
strongly involved and undergoes a secondary necrosis
(Falcieri et al., 1994).
82 HONG ET AL.
In the biochemical features of apoptosis when CHSE-
214 cells were infected with different m.o.i. of IPNV E1-S,
in the m.o.i. 20 treatment group the nucleosomal degra-
dation quickly happened at 30 min p.i., but in the m.o.i. 1
treatment group nucleosomal degradation did not occur
until 6 h p.i. (data not shown). We tested whether the
protein synthesis inhibitor cycloheximide (CHX) could
prevent cell death by IPNV infection (Ghibelli et al., 1992;
Martin, 1993). When treating the 10 mg/ml CHX with
CHSE-214 cells before m.o.i. 1 infection, we found that
cell death could be prevented, but in the m.o.i. 20 treat-
ment group it could not (data not shown). We proposed
that there might be some different mechanism involved
in inducing cell death by infection with different m.o.i. in
CHSE-214 cells. However, further experiments are re-
quired to evaluate the significance of our findings of how
different m.o.i. infections relate to the induction of host
cell death.
IPNV was discovered to be associated with a highly
contagious disease of susceptible hatchery-reared sal-
monids (Hedrick et al., 1983; Wolf, 1960; Wu et al., 1983)
and other nonsalmonid fish (Adair and Ferguson, 1981;
Chen et al., 1984, 1985; Hedrick et al., 1983; Ueno et al.,
1984). As the name indicates, the infection among trout
produces marked pancreatic necrosis, but histopatho-
logical changes sometimes also occur in adipose tissue,
in renal hematopoietic tissue, in the gut, and in the liver
(Sano, 1973). Pancreatic necrosis is typically evident in
acinar cells (Sano, 1973, 1971),and extensive areas can
be involved. Some affected acinar inclusions; the inclu-
sions, however, as in previous reports, were products of
cellular breakdown and not true viral inclusions. We have
shown that the exposure of a fish embryonic cell line to
IPNV can induce apoptosis with all its associated char-
acteristics: DNA fragmentation, nuclear and cellular seg-
mentation, multimicronuclei formation, and, finally, post-
apoptotic necrosis from early-stage apoptotic cells. Ne-
crotic cell death may occur during natural infections, but
these features support the hypothesis that IPNV causes
CHSE-214 cells to undergo apoptosis and then post-
apoptotic necrosis in vitro.
MATERIALS AND METHODS
Cell culture and virus infection
Chinook salmon embryo cells (CHSE-214) were ob-
tained from the American Type Culture Collection
(ATCC). Embryo cells grown at 18C as monolayers in
plastic tissue culture flasks (Nunc) using Eagle's mini-
mum essential medium (MEM) supplemented with 10%
(vol/vol) fetal calf serum (FCS) and 25 mg/ml gentamicin.
E1-S of IPNV Ab strain was isolated from Japanese eel
in Taiwan (Wu, 1987). E1-S was propagated in CHSE-214
cell monolayers at an m.o.i. of 0.01 cell. Infected cultures
were incubated at 18C until an extensive cytopatho-
genic effect (CPE) was observed. Virus plaque assay
was performed as previously described by Dobos et al.
(1977).
TdT-dUTP labeling
Cells were counted, and 103 cells/0.1 ml were seeded
onto the two wells of a chamber slide (Nunc) at 18C for
20 h. Before infection, the 10% FCS MEM was changed to
1% FCS MEM. The virus-infected group received m.o.i. 1
and m.o.i. 20 of IPNV serotype Ab (strain E1-S), which
had been propagated and titrated in CHSE-214 cells and
had a TCID50 of 106/0.1 ml. The negative control group
received 1.5 ml of 1% FCS MEM. The two treatment
groups were then incubated at 18C for 8 h in an incu-
bator.
At the end of various incubation times (4, 8, and 12 h),
each sample was removed from the medium and
washed with PBS, and then cell samples were fixed with
a freshly prepared paraformaldehyde solution (4% in
PBS, pH 7.4) for 30 min at room temperature. Slides were
washed with PBS and incubated with blocking solution
(0.3% H2O2 in methanol) for 30 min at room temperature.
Slides were rinsed with PBS and then incubated in per-
meabilization solution (0.1% Triton X-100 in 0.1% sodium
citrate) for 5 min on ice. Slides were rinsed twice with
PBS. Next, 50 ml TUNEL reaction mixture (as an in situ
cell death detection kit; Boehringer-Mannheim) was
added to the sample, and the slide was incubated in a
humidified chamber for 60 min at 37C. Samples were
either analyzed under a fluorescence microscope in this
state or 50 ml of anti-fluorescein antibody conjugated
with horseradish peroxidase (POD) was added to the
sample to signal conversion that was analyzed under a
phase-contrast microscope. The slide was incubated in
a humidified chamber for 60 min at 37C and rinsed with
PBS. Finally, 50100 ml of DAB substrate solution (Boeh-
ringer-Mannheim) was added for 10 min at room temper-
ature. The slide was then rinsed with PBS, mounted
under a glass coverslip, and analyzed under light micro-
scope.
Cell counts
The slides of both virus-infected CHSE-214 and unin-
fected control cells were labeled with TdT-dUTP and
examined by light microscopy using phase-contrast op-
tics. The number of TdT-dUTP-labeled nuclei in each
sample was counted per 200 cells.
Results were expressed as mean 6 SEM. Data were
analyzed using either paired or unpaired Student's t
tests, as appropriate. A value of P , 0.05 was taken to
represent a statistically significant difference between
group mean values.
Annexin VFITC labeling
An analysis of PS on the outer leaflet of apoptotic cell
membranes was performed using annexin Vfluorescein
and propidium iodide (PI) to determine differentiation of
 APOPTOSIS INDUCED BY IPNV
apoptotic from necrotic cells. The cell preparation and
virus infection conditions are described above. At the
end of the various incubation times (0, 4, 8, and 12 h),
each sample was removed from the medium and
washed with PBS, and then cells were incubated with
100 ml of staining solution (annexin Vfluorescein in a
HEPES buffer containing PI; Boehringer-Mannheim) for
1015 min. Evaluation was by fluorescence microscopy
using 488-nm excitation and a 515-nm long-pass filter for
detection.
DNA preparation and gel electrophoresis
About 105 cells/ml were seeded onto a 60-mm petri
dish for growth above 20 h. Monolayer cell growths were
rinsed twice with PBS. Then all control groups or infec-
tion groups were cultured in 1% FCS MEM. The high
m.o.i. group received m.o.i. 20 infected CHSE-214 and
was incubated for 0, 4, 8, and 12 h; the low m.o.i. group
received m.o.i. 1 CHSE-214 and was incubated over the
same time periods. The control and high m.o.i. and low
m.o.i. groups were also used for DNA fragmentation
studies. At the end of incubation, the cells were lysed
with lysis buffer (10 mM TrisHCl, 0.25% Triton X-100, 1
mM EDTA, pH 7.4). After treatment with phenolchloro-
formisoamyl alcohol (25:24:1), the DNA was precipi-
tated in the presence of 0.3 M sodium acetate and cold
absolute ethanol at 270C for 2 h and then resuspended
in 10 mM TrisHCl (pH 7.4) and 1 mM EDTA. Aliquots of
20 ml containing ;510 mg of DNA were then electro-
phoresed in 1.2% agarose gels for 3 h at 40 V. Gels were
stained with ethidium bromide and photographed under
UV transillumination.
Electron microscopy
The trypsinized cells were collected by centrifugation,
washed twice with PBS, and then fixed in 2.5% glutaral-
dehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h.
After a wash with the sodium cacodylate buffer, the cells
were postfixed in 1% aqueous osmium tetroxide for 2 h,
followed by a wash in the same buffer. They were then
dehydrated in a series of ethanol solutions of decreasing
dilution and embedded in a Spurr's mixture. Semithin
sections were stained with toluidine blue to count mor-
phological patterns under light microscopy (Nikon). Ul-
trathin sections were stained with lead citrate and uranyl
acetate and then observed using an electron microscope
(Hitachi H-7000).
ACKNOWLEDGMENTS
We thank Dr. Douglas Platt for reviewing and correcting the manu-
script. This work was supported by grants awarded to Dr. Jen-Leih Wu
from the National Science Council, Republic of China.
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