J. gen. ViroL (1967), 1, 115-124
115
Printed in Great Britain
D N A Synthesis in Scrapie-Affected Mouse Brain
By R. H. K I M B E R L I N AND G. D . H U N T E R
Institute for Research on Animal Diseases,
Compton, Newbury, Berkshire, England
(Accepted 19 September 1966)
SUMMARY
During the later stages of the development of scrapie, an enhanced
synthesis of DNA is detectable in the brains of affected mice. The synthesized
DNA is largely confined to the nuclear fraction of the cell whereas the
agent is mainly associated with mitochondria. There is evidence that the
synthesis of the scrapie-associated DNA can be depressed in vivo in the
presence of some specific inhibitors of DNA synthesis. An unsuccessful
attempt was made to interfere with thc normal development of scrapie by the
repeated injection of inhibitors throughout the incubation period of the
disease. It seems that the synthesis of DNA is not primarily involved in the
development of the disease.
INTRODUCTION
Scrapie is a naturally occurring disease of sheep characterized by a progressive
degeneration of the central nervous system which usually, if not always, terminates
fatally. Although the disease has been studied for many years and the causal agent
has been shown to be filtrable and transmissible, it has not been adequately characterized (Stamp, 1962). The early work using sheep and goats was hampered by the
long incubation period (sometimes more than a year) and the resistance of many
sheep to experimental infection. Successful transmission of scrapie to mice (Chandler,
1963), which are 100 ~ susceptible to the mouse-adapted agent, has enabled research
work on the disease to be intensified, but the incubation period in mice is still long
(about 4 months) and attempts to isolate the agent and grow it in tissue culture
systems have so far been unsuccessful.
Experiments in the mouse (Hunter & Millson, 1964a) confirmed earlier observations
in the goat and sheep that the scrapie agent is very resistant to heat, and brain extracts
lose very little activity when heated to 80 ° for 1 hr. At higher temperatures a progressive inactivation of the agent is observed, although some infectivity remains in
boilcd material. This property and the similar resistance of the agent to formalin
(Pattison, 1965) is reminiscent of the physicochemical stability of polyoma DNA
(Well, 1963) and would be consistent with the presence of a double-stranded nucleic
acid component in the scrapie agent. Although the agent is widely distributed among
the tissues of clinically affected mice the brain contains a particularly high titre of
infectivity (Hunter & Millson, 1964b) and most of the biochemical and histological
abnormalities of scrapie also occur in brain (Pattison & Smith, 1963; Slater, 1965;
Millson, 1965; Hunter & Millson, 1966; Mackenzie & Wilson, 1966). The present
paper therefore describes an investigation of D N A synthesis in the brains of scrapie8-2
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116
R.H. KIMBERLIN
A N D G. D. H U N T E R
affected mice with particular reference to the nature and mode of replication of the
scrapie agent. Part of this work has already been published in a preliminary form
(Kimberlin & Hunter, 1965).
METHODS
Mice. B.S.V.S. mice (bacteria susceptible, virus susceptible; Schneider, 1959) were
used in this work. The mice were infected by intracerebral inoculation with 0.02 ml.
of a 10-3 dilution in saline of a scrapie mouse brain extract. Diagnosis of clinically
affected scrapie mice was made as described previously (Chandler, 1963; Pattison &
Smith, 1963). Titrations of infective material were made in mice (Hunter, Millson &
Chandler, 1963) and all ID50 values refer to 0-02 ml. inocula and the wet weight of
brain from which the relevant material was originally derived.
Cellularfractionation. A relatively simple method was used. A 10 % homogenate of
mouse brain in 0.32 M-sucrose was centrifuged at 850g for 10 min. The pellet was
washed twice by gently resuspending in 0.32 M-sucrose and resedimenting at 850g for
10 min. to give a nuclear fraction. The combined supernatant fractions were centrifuged
at 15,000g (av.) for 15 min. The pellet obtained was resuspended in 0-32 M-sucrose
and centrifuged at 4000 g for 45 rain. to give a mitochondrial fraction. The combined
supernatant solutions were pooled to give a soluble fraction which here includes
microsomal and ribosomal material.
Extraction of DNA. A6 % homogenate of mouse brain was prepared in 0.02 M-potassium phosphate buffer p H 7.3, containing 1.0 M-NaC1 and 0.5 % Na deoxycholate.
This homogenate was extracted at 4 ° for 30 min. with an equal volume of a
mixture containing phenol+m-cresol+8-hydroxyquinoline+water (500:70:0.5:55).
After centrifuging at 35,000g (av.) for 20 rain. the clear upper phase was carefully
removed and an equal vol. of absolute alcohol added while stirring with a glass rod
on which the precipitated D N A was collected. The D N A was washed with ethanol
and ether and digested overnight with 0.3N-KOH at 37 ° to hydrolyse contaminating
RNA. Normal HC1 was added to bring the p H to 1.0 and the solution was cooled
to 4 ° for 1 hr. The precipitated D N A was sedimented at 1000g for 10 rain. and washed
twice with 0.5N-HC104.
In many of the experiments involving the use of labelled thymidine, D N A was
obtained as an acid-soluble extract of brain nucleic acids. The material containing
D N A was precipitated with an equal volume of 10 % trichloracetic acid at 4 ° and
centrifuged. The pellet was washed once with 5 % trichloracetic acid and extracted
successively once with ethanol (70 %, v/v) at 50 ° for 30 min. and twice with chloroform + methanol (2: 1, by vol.) at 70 ° for 20 min. The pellet was then washed twice more
with cold 5 % trichloracetic acid. A nucleic acid hydrolysate was then prepared by
treating the pellet with the minimum convenient volume of 7.5 % trichloracetic acid
at 90 to 100 ° for 30 rain. The pelleted residue was further extracted with a small volume
of 5 ~ trichloracetic acid at 90 to 100 ° for 5 min. and trichloracetic acid was removed
from the combined extracts with ether.
Tracer experiments. [Methyl-~H] and [2-14C]thymidine supplied by the Radiochemical Centre, Amersham, were dissolved in physiological saline before use. Mice
were injected intracerebrally (0.01 to 0.03 ml.) using an Agla syringe. At appropriate
times after injection of the isotopically labelled compounds, the mice were killed by
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DNA synthesis in scrapie-affected mouse brain
117
decapitation and the brains rapidly removed and either homogenized in 0.32 M-sucrose
for cellular fractionation as described above, or treated with 10 ~ trichloracetic acid
or phenol for nucleic acid extraction. Phenol extracts of whole D N A were prepared
for radioactive assay by heating for 15 min. at 98 ° in the minimum convenient volume
of 0.75N-HC104. The tubes were cooled, the solutions filtered and the filter washed
with water to give a total volume of 5 ml. After determining the amount of deoxynucleotide present spectrophotometrically at 260 m # the solution was concentrated
to exactly 0.8 ml. Samples of mixed D N A + R N A hydrolysates obtained when the
trichloracetic acid method was used were also reduced to a volume of 0.8 ml. and
250
<
Z 200
~ 150
o
~ 100
50
0
I
I
20
40
I
60
80
100
120
140
Days
Fig. 1. Incorporation of [2-14C]thymidine into the DNA of normal (shaded histogram)
and scrapie mouse brain (open histogram) at different times after inoculation of the scruple
agent. The mice were killed by decapitation 65 to 67 hr after intracerebral inoculation of
[2-14C]thymidine (1.5/zc. per mouse). Whole DNA was extracted from the brains by a
phenol method as described in Methods.
the samples were mixed with 10 ml. of scintillation fluid and counted in an Echo
N 644B liquid scintillation counter. The scintillation fluid consisted of 5 g. of 2,5diphenyloxazole, 50 mg. of 1,4-bis(5-phenyloxazol-2-yl)benzene and 80 g. of naphthalene dissolved in 1 1. of a toluene + dioxan + ethanol mixture (5: 5: 3, by vol.). Under
these conditions [2-z4C]thymidine was counted with an efficiency of 16-18 ~ against
a background of 60 to 70 counts/min. Samples containing [methyl-3H]thymidine were
concentrated to a volume of 0-3 ml. and mixed with 2 ml. of 1M-methanolic hyamine
10-X. Scintillation fluid (10 ml.) was added containing the same solids used above
dissolved in 1 1. of toluene + ethanol (770: 230, by vol.). Tritium-labelled samples were
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118
R.H. KIMBERLIN
A N D G. D. H U N T E R
counted at - 1 0 ° to - 2 0 ° with an efficiency of 6 to 8 ~ against a background of
200 counts/min. Corrections for quenching were found to be unnecessary. In all cases
the background count was subtracted from the experimental data.
Experiments with inhibitors of DNA synthesis. Monohydroxyurea (15 mg./ml.) and
5-fluorodeoxyuridine (2.5 mg./ml.) were dissolved in physiological saline and 0.02 ml.
per mouse was used in intracerebral injections. 5-Iododeoxyuridine (2.5 mg./ml.) was
suspended in the appropriate volume of physiological saline and brought into solutionby
raising the pH value to about 8.5 with 0.1N-NaOH, before injecting 0.02 ml. per mouse
intracerebraUy. The effects of the inhibitor on brain DNA synthesis were measured
by subsequent intracerebral inoculation of labelled thymidine as described above.
1500
.EE 1000
o
.o
500
!
0
20
40
I
60
80
Days
!
100
!
120
140
Fig. 2. Incorporation of [2-x4C]thymidine into the D N A of control (filled histogram)
and scrapie mouse brain (open histogram). Control mice were inoculated with a suspension
of normal mouse brain equivalent to the scrapie-affected inoculum. D N A was isolated as
a nucleic acid hydrolysate according to the procedure described in Methods. Other experimental details were as for Fig. 1.
RESULTS
Synthesis of DNA in scrapie-affected mouse brain. An increased incorporation of
[2-1~C]thymidine into DNA has been repeatedly observed in the brains of scrapieaffected mice. On the assumption that the size of the thymidine pool in scrapie-affected
mouse brain is not appreciably different from the normal, this finding indicates an
increased rate of DNA synthesis. Figure 1 shows the result of an experiment in which
DNA was isolated by the extraction of tissue homogenates with phenol. A low level
of DNA turnover was observed in normal mice but [2-14C]thymidine incorporation
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D N A synthesis in scrapie-affected mouse brain
119
appeared to be increased 2- or 3-fold in mice clinically affected with scrapie (16 to 20
weeks post-inoculation). The experiment was repeated (Fig. 2) using controls inoculated
with normal mouse brain. In order to overcome the incomplete extraction of brain
D N A with phenol, a nucleic acid extract was prepared containing both D N A and
RNA. Although the increased D N A synthesis was particularly evident in the clinical
stages of the disease it would appear that an increased turnover of D N A also occurs
during incubation. However, the observed increase is small and an approximate
estimate suggests that the rate of D N A turnover in scrapie mouse brain may only
be 0.1 ~ per week.
Table 1. The distribution of radioactivity in cell fractions from normal and
scrapie-affected mouse brain 4 days after the injection of [2-14C]thymidine
Groups of 3 mice (17 weeks post-inoculation) received 1 pz./mouse of [2-14C]thymidine intracerebrally and were killed 4 days later. Cellular fractionation was carried out and a nucleic acid
hydrolysate was prepared as described in Methods.
Total radioactivity (couuts/min./group
of 3 mice after 4 days)
t
Cellular fraction
Normal
Scrapie
29
24
22
181
34
18
Nuclei
Mitochondria
Soluble
Table 2. Location of radioactivity in normal and scrapie mouse brain
4 days and 28 days after injection of [methyl-ZH]thymidine
Total radioactivity: (counts/min./group of 3 mice)
x
4 days
_
_
^
Cellular fraction
Normal
Nuclei
Mitochondria
Soluble
2001
56
97
_
28 days
_
A
Scrapie
7168
94
113
Normal
Scrapie
1011
125
85
2224
134
92
Groups of 3 mice received 5/zc. per mouse of [methyl-3H]thymidineintracerebrally 16½ weeks
after they had received the scrapie agent, and were killed by decapitation either 4 or 28 days later.
Cellular fractionation was carried out and a hydrolysate of DNA+RNA obtained as described in
Methods.
Site of the increased DNA synthesis. The mouse agent was recently shown to be
associated with the large-particle fraction of the cell (Hunter, Millson & Meek, 1964;
Mould, Dawson & Smith, 1964). In particular the agent was associated with mitochondria and less than 10 ~o of the infectivity was found in a nuclear fraction from
brains of clinically affected mice. There is little doubt that most o f the labelled D N A
is associated with the cell nucleus (Table 1). This finding was confirmed in a second
experiment (Table 2) which also indicated that 50 ~o of the nuclear label of both
normal and scrapie-affected mouse brain was unstable. Since there was very little
change in the amount of radioactivity in the mitochondrial and soluble fractions it
would appear that the nuclear material was degraded to acid-soluble components.
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120
R . H . KIMBERLIN AND G. D. HUNTER
An experiment was made to investigate the possible enclosure of multiple infective
units within the nuclear membrane. Such a phenomenon could account for the
relatively low titre of nuclei prepared from scrapie brain. Ultrasonic vibration of
a whole brain extract was previously shown to have very little effect on the infective
titre of the scrapie agent (Hunter & Millson, 1964a). However, treatment with ultrasonic vibration did not alter the titre of cell fractions and most of the infectivity was
still found in the supernatant solution which contained mitochondria (Table 3). The
observed increase in D N A synthesis in scrapie presumably does not represent the
synthesis of the causal agent.
Table 3. Infective titre of the scrapie agent within nuclei and supernatant
fractions of affected mouse brain before and after ultrasonic treatment
A nuclear fraction was prepared and washed as described in Methods. The combined supernatant
solutions from the nuclear preparation were centrifuged twice at 800g for 10 min. to remove residual
nuclei. Ultrasonic treatment was at 0 to 10° for 15 rain., using an M.S.E. ultrasonic power unit:
the supernatant fraction was treated intact and the nuclear fraction suspended in a small volume of
physiological saline.
Infective titre
Fraction
1/logzo ID 50
Whole extract
7-2
Whole nuclei
5-9
Ultrasonic-treated nuclei
5-9
Whole supematant
6-8
Ultrasonic-treated supernatant
7.2
The effect of specific inhibitors on DNA synthesis. In the absence of detailed information on the nature of the scrapie agent it is difficult to assess the significance of
an increased D N A turnover. It was considered that the D N A might represent some
sort of intermediate or provirus of the kind which has been reported to occur in the
replication of Rous sarcoma virus (Temin, 1964); or perhaps the synthesized D N A
plays some other role in the clinical development of the disease. An attempt was
therefore made to investigate the functional significance of the increased D N A turnover in scrapie by the use of specific inhibitors of D N A synthesis. Both 5-fluorodeoxyuridine and 5-iododeoxyuridine produced an effect on the synthesis of D N A in
scrapie-affected mouse brain. A more pronounced stimulation of [2-14C]thymidine
incorporation was obtained after a single intraperitoneal or intracerebral dose of
monohydroxyurea (Fig. 3). This observation is consistent with the inhibitory action
of hydroxyurea on the conversion of ribonucleotides to deoxyribonucleotides ( y o u n g
& Hodas, 1964). However, a complicating feature of these experiments was the
increased uptake of [2-14C]thymidine following a control injection of saline. Since
this phenomenon occurred in scrapie mice after an intraperitoneal injection it may
represent a nonspecific response to stress.
Effect of DNA inhibitors on the development of scrapie. Two experiments were made
to study the effect of 5-iododeoxyuridine and monohydroxyurea on the development
of scrapie. Since intracerebral injections repeated over a period of 4 months may
have serious traumatic effects on mice, the mice received an intracerebral injection of
0.02 ml. of the inhibitor solution weekly and an intraperitoneal injection of 0.1 ml. on
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DNA synthesis in scrapie-affected mouse brain
121
alternate days. G r o u p s o f 6 scrapie-affected mice were selected which had been experimentally infected 3 months, 2 m o n t h s and 1 m o n t h earlier. A f o u r t h g r o u p received
the first intracerebral dose o f inhibitor 24 hr before experimental infection and the
injections were then continued until the first clinical signs o f mouse scrapie appeared
in the control (non-injected) mice. It was f o u n d that all groups o f mice developed
scrapie at the same time as their corresponding controls and there was no significant
alteration in the length o f the incubation period as a result o f the inhibitor treatment.
Histological examinations o f mouse brains were made but there was no difference
between the lesions o f treated and control mice and it was concluded that the inhibitor
treatment had no significant effect on the normal development o f the disease.
A
6000
II
m
I
I
I
t
I
1
I
4000
I
/
I
t
I
tt
l
t
t
t
l
l
lII
2000
• i
Q
~
°~
0
,/,
!
,
,,
B
O
4000
0
i
I
l
k
,
2000
t
i-- I
1½
3
I
5
24
II ' ~ t
48
72
168
Hours
Fig. 3. The effect of monohydroxyurea on the incorporation of [2-14C]thymidine into
the DNA of scrapie mouse brain. Each mouse (16 to 18 weeks post-inoculation) received
a single injection of inhibitor solution or of physiological saline. At varying intervals,
groups of three mice received a further injection of [2-1~C]tbymidine (1/zc./mouse) intracerebrally and were killed 24 hr later. A DNA and RNA hydrolysate was prepared as
described in Methods. A, Monobydroxyurea (0-1 ml. of 15mg./ml. solution in saline)
administered intraperitoneally. B, Monohydroxyurea (0.02 ml. of a 15 mg./ml, solution in
saline) administered intracerebrally. 0 - - 0 , Saline; O--©, monohydroxyurea.
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122
R. H. K I M B E R L I N
AND
G.D. HUNTER
DISCUSSION
In view of the stable cell population in adult mouse brain it was of interest to find
a small but significant turnover of normal brain DNA. A similar finding was reported
by Pelc (1964), who showed that labelled thymidine was incorporated into brain DNA
in the absence of cell division. Pelc suggested that the slow turnover of DNA may
represent the replacement of molecules which have lost their biological activity as
a result of wear and tear. Alternatively it is possible that the low incorporation of
thymidine may reflect the repair of DNA strands which are broken during the process
of transcription to messenger RNA (Jones & Truman, 1964). The earliest detectable
increase in DNA synthesis occurred about 50 to 60 days after inoculation, corresponding
to the time when the earliest histological (Pattison & Smith, 1963) and biochemical
(Millson, 1965) abnormalities of mouse scrapie occur. The marked association of
the scrapie agent with the cytoplasmic fraction of the cell contrasts with the nuclear
site of the increased DNA synthesis. Since the decline in nuclear label with time was
not associated with an increase in the radioactivity of mitochondrial or soluble
fractions the increased DNA synthesis in scrapie probably does not represent the
synthesis of the agent. However a DNA-containing agent is not excluded by these
tracer experiments. In the first place there is no conclusive evidence that the agent is
actually synthesized in brain and secondly the rate of DNA synthesis may be too
slow to be detected by the present techniques.
The observed increase in DNA synthesis in scrapie, therefore, seems to be a
secondary response of the host cell to the presence of the agent. There is no reported
evidence of an abnormal cell species within affected brain which would account for
the observed DNA synthesis, and mitotic figures appear to be totally absent from
both normal and diseased mouse brain. In view of the suggestion by Pelc (1964), the
DNA synthesis in scruple-affected mouse brain may represent the increased activity
of a normal process which gradually replaces unusable DNA. This possibility is
certainly consistent with the appearance of clinically affected mice, which suggests
that scrapie may accelerate the normal ageing process. Alternatively the DNA repair
mechanism postulated by Jones & Truman (1964) could explain the increased turnover
of DNA if there is also an enhanced rate of messenger RNA production in scrapie.
An increased rate of host-cell DNA synthesis has been reported after polyoma
infection of mouse kidney cells and it has been suggested that this phenomenon is
important in the process of cell transformation (Dulbecco, Hartwell & Vogt, 1965).
Although there is little justification for pressing an analogy between scrapie and
polyoma infection, it is possible that the scrapie agent may uncouple the normal
regulatory processes in a manner which results in an increased DNA synthesis. An
interesting histological feature of scrapie is the pronounced hypertrophy of the
astroglia (Chandler, 1961; Pattison & Jones, 1967). This feature is reflected in vitro
by the greater glial growth in scrapie brain explants than in normal brain explants
(Field & Windsor, 1965; Haig & Pattison, 1967). It would therefore be of considerable
interest to investigate DNA synthesis within the astroglia since metabolism within
them may be altered.
The experiments with specific inhibitors showed that an appreciable effect could
be obtained on the synthesis of DNA in scrapie-affected mouse brain. Although the
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DNA
s y n t h e s i s in s c r a p i e - a f f e c t e d m o u s e b r a i n
123
synthesis of D N A was not completely inhibited during the two trials, the doses o f
inhibitors used in these experiments were adequate to ensure a certain level of
inhibition throughout the 4-month period. Hence it is concluded that the synthesis of
D N A is not a primary feature of the development of scrapie. The possible existence
of a DNA-containing scrapie agent was not excluded by the tracer experiments but
this point may be reconsidered in the light of the inhibitor experiments. Bauer (1966)
suggested that the m a x i m u m activity of an inhibitor would be obtained when one
molecule is present in a volume of the cytoplasm equal to the volume of the virus. On
this assumption one can calculate the minimum concentration of inhibitor which
would totally inhibit the maturation of a virus of known size. The intraperitoneal
dose of monohydroxyurea used in these experiments would be adequate to depress
the synthesis of an agent 150 A in diameter, and the concentration of monohydroxyurea in brain (after an intracerebral injection) would be enough to interfere with the
replication of an agent 70 A in diameter. A certain margin must of course be allowed
for the instability and non-uniform distribution of the inhibitor in vivo. Nevertheless,
from the results of the inhibitor experiments we would predict a very small size for
a DNA-containing scrapie agent. Alper, Haig & Clarke (1966) suggested that the
particle size of the agent may be approximately 70 A in diameter with an estimated
molecular weight of 2.0 x 105. Alper et aL (1966) also found that the scrapie agent
was highly resistant to the action of ultraviolet irradiation and they consider the
possibility that the nucleic acid content of the agent may be extremely small.
We should like to acknowledge the skilled technical assistance of Mr G. C. Millson,
Mr M. D. Vockins, Miss A. Davis and Miss S. Langford. We are indebted to Mr C. F.
Grigg, of Roche Products Ltd., Welwyn Garden City, for a gift of 5-fluorodeoxyuridine; and to D r G. Gale of the Veterans Administration Hospital, Durham, N.C.,
for a gift of monohydroxyurea. The research was financed in part by a grant made
by the United States Department o f Agriculture under P.L. 480.
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