Journal of General Virology (1992), 73, 1025-1029.
Printed in Great Britain
1025
Further analysis of nucleic acids in purified scrapie prion preparations by
improved return refocusing gel electrophoresis
Klaus Kellings, ~ Norbert Meyer, 1 Carol Mirenda, 2 Stanley B. Prusiner 2,3 and Detlev Riesner ~*
1 lnstitut far Physikalische Biologic, Heinrich-Heine-Universitiit Di~sseldorf Universitiitsstrasse 1, D-4000 Di~sseldorf 1,
Germany and Departments of 2 Neurology and 3 Biochemistry and Biophysics, University of California, San Francisco,
California 93143, U.S.A.
Although increasingly unlikely, the possibility of a
scrapie-specific nucleic acid carried by infectious prion
particles is still unresolved. Return refocusing gel
electrophoresis was developed to detect homogeneous
and heterogeneous nucleic acids extracted from highly
purified scrapie prion preparations. This method was
improved with respect to the size range from 13 to 1100
nucleotides (nt) over which analyses could be performed. The yield of nucleic acid, particularly of small
DNA oligonucleotides and polyadenylated RNA, was
determined after deproteinization and two-phase extraction. Despite extensive nuclease digestions some
small polynucleotides remained. Although a scrapiespecific nucleic acid cannot be excluded, the results
further define the possible characteristics of a hypothetical molecule. If homogeneous in size, such a
molecule would be < 80 nt in length at a particle-toinfectivity ratio near unity, if heterogeneous, scrapiespecific nucleic acids would have to include molecules
smaller than 240 nt.
A wealth of data indicate that a protein, designated prion
protein (PrP), is required for scrapie infectivity. Protease
treatment and protein denaturing agents affect infectivity, whereas procedures that modify or hydrolyse nucleic
acids do not alter infectivity (Prusiner, 1982). In spite of
many attempts, no physical or chemical evidence for a
scrapie-specific nucleic acid has been found to date. The
existence of multiple isolates or 'strains' with different
biological properties (Bruce & Dickinson, 1987; Kimberlin et al., 1987) has offered the strongest argument for a
scrapie-specific nucleic acid. The failure to explain such
strain variation in terms of molecular variation in PrP
continues to stimulate the search for a scrapie-specific
nucleic acid (Prusiner, 1991; Weissmann, 1991).
A recent search for a putative scrapie genome in
purified prion preparations revealed a paucity of nucleic
acids (Meyer et al., 1991). Purified fractions were
extensively treated with nucleases and Zn 2+ ions,
dispersed into detergent-lipid-protein complexes
(DLPC), treated again with nucleases followed by
deproteinization, and extracted for nucleic acids. Nucleic acids were analysed by PAGE and detected by
silver staining. No well defined molecular species of
nucleic acid could be detected. The use of return
refocusing gel electrophoresis (RRGE) has made it
possible to analyse nucleic acids which are heterogeneous in length with the same sensitivity after silver
staining as is possible for nucleic acids of homogeneous
length. Based on the consideration that a hypothetical
scrapie-specific nucleic acid has to be present with at
least one copy per infectious unit (ID50), our results
suggest that such a nucleic acid would have to be quite
small [< 100 nucleotides (nt)], possess a particle-toinfectivity (P/I) ratio near unity or be heterogeneous in
size. Since such experiments have been restricted to
analyses of nucleic acids smaller than 200 nt and the
conclusions were based on semiquantitative estimates of
nucleic acids, we modified the procedures to extend the
size range of nucleic acids investigated, and improved
our quantification of nucleic acid molecules. The
inclusion of larger nucleic acid sizes into our experimental analysis was necessary as genomic segments of
conventional viruses and viroids are larger than 200 nt;
some recent reports (Aiken et al., 1990; Akowitz et al.,
1990; Narang, 1990) have suggested larger scrapiespecific nucleic acids.
R R G E was described earlier (Meyer et al., 1991) and
the improvements in R R G E are outlined briefly in the
legend of Fig. 1. We found that a 9 ~ polyacrylamide gel
matrix (acrylamide :bisacrylamide, 19:1) and a Trisacetate-EDTA buffer (8 mM-Tris, 4 mM-sodium acetate
and 0-4 M-EDTA pH 8-4) was the most suitable for
analysis of nucleic acids from 13 to 1100 nt in length.
Since the time of refocusing depends slightly upon the
0001-0512 © 1992 SGM
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1026
(a)
®
(b)
1
2
I
3
I
I
P
I
b : 1100-340 nt
I
F_
--
-
h: 19-13 nt
®
a
b--
(marker)
C
?
k
d
e
f
(c, d, e, f,g)
(marker)
®
ii:
[ ........................................................
I
®
i
1 2 3 - P -
t
1
2
3
-
P
-
t
Fig. 1. Scheme of the improved RRGE. The RRGE consists of a separation and a refocusing gel electrophoresis. The separation run
(100 min, 250 V) is equivalent to a normal PAGE (a). The nucleic acid from a prion sample yields an undetectable smear (P in a). The
gel is cut into different segments (a to h). These are repolymerized at the bottom of new gel matrices (b). The refocusing run (250 V) is
carried out in the opposite direction and in presence of SDS. The times of refocusing of different gel segments are given in Table 1. All
nucleic acids which were contained in one gel segment are concentrated into one band, thereby increasing the sensitivity of detection.
The unknown nucleic acid amount (? in b) of the prion sample is determined by comparison with described markers of known
concentrations (Table 1, markers 1, 2 and 3). Only two gel segments, b and h, are given as an example for the refocusing run; gel segment
a is not used for refocusing, but silver-stained after the separation run.
Table 1. R R G E analysis of prion preparation C A M 8
Gel
segment
a
b
c
d
e
f
g
h
Size
range
(nt)
Time of
refocusing
(min)*
> 1100
No refocusing
1100-340
48
339-141
44
140-80
42
79-54
42
53-40
42
39-20
42
19-13
42
Nucleic acid
marker (Pg)t
1
860
220
400
450
415
2100
2100
2
3
260
25
20
50
200 100
215 100
205 160
1200 600
1200 600
Prion
sample
(pg)
NI)~
75
50
300
450
600
8000
20000
* Taken after 100 rain in the separation run.
t The marker nucleic acids consisted of purified restriction fragments
from pTZ18U digested with HinfI (1075, 517, 465, 396, 201, 75, 65, 44
and 22 nt), PSTVd fragment (192 nO, pTZ18U digested with Sau3A
(105 nO, pTZI8U digested with AluI (64, 63 and 49 nO and synthesized
oligomeric DNA (28 and 16 nt). These nucleic acids were mixed in ratios
such that the markers designated 1, 2 and 3 contained the concentrations
as listed for the size range. The same markers were applied to the RRGE
of Fig. 2.
:[:ND, Not detectable.
size o f nucleic acids, specific t i m e s were c h o s e n for the
refocusing o f gel s e g m e n t s o f different sizes ( T a b l e 1).
A m o u n t s o f nucleic a c i d were d e t e r m i n e d b y c o m p a r i n g
intensities o f s i l v e r - s t a i n e d b a n d s to those o f nucleic
a c i d s t a n d a r d s used at t h r e e different c o n c e n t r a t i o n s
( T a b l e 1).
P r e p a r a t i o n o f purified p r i o n f r a c t i o n s for R R G E was
s i m i l a r to t h a t used for p r e v i o u s studies ( M e y e r et al.,
1991). I n o r d e r to i m p r o v e the r e c o v e r y o f p r i o n
i n f e c t i v i t y d u r i n g the collection o f p r i o n rods f r o m
sucrose g r a d i e n t fractions, u l t r a c e n t r i f u g a t i o n steps
(2 × 4 h , 1 0 0 0 0 0 g ) were s u b s t i t u t e d for the e t h a n o l
p r e c i p i t a t i o n p r e v i o u s l y used. F u r t h e r m o r e , D N a s e
d i g e s t i o n of t h e rods w a s o m i t t e d b e c a u s e the m a t e r i a l
was t r e a t e d w i t h Bal 31, m i c r o c o c c a l nuclease a n d
R N a s e A after D L P C f o r m a t i o n . T h r e e i n d e p e n d e n t
p r i o n p r e p a r a t i o n s ( C A M 6 , C A M 7 a n d C A M 8 ) were
a n a l y s e d , C A M 6 for nucleic a c i d sizes f r o m 40 to
1100 nt, a n d C A M 7 a n d C A M 8 o v e r a w i d e r r a n g e f r o m
13 to 1100 nt. Before the d e p r o t e i n i z a t i o n / n u c l e i c a c i d
e x t r a c t i o n step the infectivities o f these p r e p a r a t i o n s
were log l D s o 8 " 3 ( C A M 6 ) , 8.5 ( C A M 7 ) a n d 8.7
( C A M 8 ) . T h e b i o a s s a y s were c a r r i e d out w i t h 1 0 ~ o f one
s a m p l e a n d the r e m a i n i n g 9 0 ~ , for w h i c h the IDs0
values were calculated, were used in tot® for one gel
e l e c t r o p h o r e t i c analysis.
T h e yield o f nucleic a c i d after d e p r o t e i n i z a t i o n was
e s t i m a t e d q u a n t i t a t i v e l y b y three r a d i o l a b e l l e d m o d e l
nucleic a c i d s : an R N A t r a n s c r i p t o f 154 nt, the s a m e
R N A t r a n s c r i p t w i t h a p o l y a d e n y l a t e d tail o f 80 to 100 n t
a n d a short D N A o l i g o m e r o f 28 nt. N o significant loss
was o b s e r v e d d u r i n g the p h e n o l a n d p h e n o l : c h l o r o f o r m
e x t r a c t i o n s w i t h the R N A t r a n s c r i p t s . T h e yield o f the
D N A o l i g o m e r was, h o w e v e r , only 5 0 ~ ; therefore, t h e
p h e n o l e x t r a c t i o n o f f o r m e r p r i o n p r e p a r a t i o n s was
r e p l a c e d by a p h e n o l : c h l o r o f o r m : i s o a m y l alcohol
(25:24:1) extraction which permitted approximately
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1027
Segment
b
c
123456
d
123456
123456
e
123456
f
123456
g
123456
h
123456
Fig. 2. RRGE of a prion~sample. The refocused gel segments b to h are shown. Each gel segment consists of six lanes: sonicated calf
thymus DNA (ctDNA), 85 ng (total); nucleic acid markers 1, 2, 3 (Table 1); empty; prion sample CAM8 (lanes 1 to 6, respectively).The
estimation of the nucleic acid content of the prion sample was carried out on the original gels. The intensity as wellas the width of a band
were considered for comparison between prion and marker bands. Bands from adjacent gel segments were also included in the
comparison. The sonicated ctDNA was used as an internal control of refocusing and not for quantification. The silver-stained gel
segment a showed no nucleic acid or protein signal after the separation run (not shown).
9 0 ~ recovery (data not shown). The quantitative
determination of the recovery shows that the evaluation
as carried out by Meyer et al. (1991) was correct for
nucleic acids in the larger size range (64 to 200 nO,
whereas the number of molecules per IDs0 given for
smaller sizes needs correction by a factor of about two.
This correction does not affect the basic conclusion of
that work.
The results of experiments with prion preparation
CAM8 are given in Table 1 and Fig. 2. Gel segment a
( > 1100 nO was also silver-stained, but no stained band
or smear could be detected after the electrophoretic
separation in any of the prion preparations (data not
shown). I f a scrapie-specific nucleic acid larger than
l l 0 0 n t exists, a log IDso titre of 8.5 (CAM7) would
necessitate at least 3.2 x 108 molecules corresponding to
about 200 pg, an amount which would have been
detected easily in gel segment a.
From the amounts of nucleic acids as estimated from
gel segments b to h (Fig. 2) and from the infectivity of the
prion samples before deproteinization, the P/I ratio was
determined. This calculation is based on the assumption
of a scrapie-specific nucleic acid molecule which is
hidden in the heterogeneous background nucleic acid.
Fig. 3(a) is a double-logarithmic plot of P/I ratio as a
function of the length of the hypothetical nucleic acid
molecule. For the size range from 60 to 200 nt, the results
from earlier measurements (Meyer et al., 1991) were
confirmed, but more nucleic acid molecules of < 60 nt
were found than previously reported. Besides the twofold
increase due to the more effective extraction as men-
tioned above, we have to conclude that the omission o1
D N a s e digestion of prion rods yields more undigested
nucleic acids in this size range and thus results in higher
P/I ratios. Amounts of nucleic acids above 200 nt were
determined by R R G E for the first time. Since the P/I
ratio continues to drop several orders of magnitude
below unity, nucleic acids of this size range cannot be
scrapie-specific. The straight line (Fig. 3) is an interpolation of the experimental data by linear regression i n
order to determine an average nucleic acid size at a P/I
ratio of 1.
Fig. 3 (a) also contains an estimation of the m a x i m u m
error: it was assumed that the correct titre was one log
ID50 lower than the measured one (see Meyer et al., 1991)
and that the nucleic acid content was twofold higher than
estimated from R R G E . This calculation shifts the P/I
ratio of unity from 76 nt to 165 nt (Fig. 3, dotted line). It
is worth noting that the limit of one nucleic acid molecule
per ID5o (at a P/I ratio of 1) is an extreme assumption
(see Meyer et al., 1991), for comparison 104 to 105 PrP
molecules are necessary for one infectious unit (Prusiner
et al., 1982, 1983).
I f heterogeneous scrapie-specific nucleic acids were
assumed, all molecules of a certain heterogeneity class
would have to be added up to account for the
corresponding P/I ratio. Such heterogeneity would be an
intrinsic property of the scrapie genome and not an
artefact from nuclease digestion, because the bioassays
yielding the ID50 value were carried out after nuclease
digestion. In Fig. 3 (b) the P/I ratio is plotted against the
length of hypothetical heterogeneous scrapie-specific
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1028
105 !- (a)
I
I
I
I
I
I
I
~ I
I
~
I
I
I
I
I
l
(b)
10 4
~:'-::::::::::.....
10 3
102
H = l~
H = l0
/
""'~'-'..'.::......... /
100
H = 1000
10~
10°
10-~
i
A .............
10 2
10 3
l0
4
10 -5
10
(76 nt)
(165 nt)
1
,-t
A
1O0
,1000
100
10
1000
Minimal length (nt)
Length (nt)
Fig. 3. Particle-to-infectivity ratio (P/I); dependence upon the length of the hypothetical scrapie-specific nucleic acid. In (a) a distinct
molecular species among the heterogeneous background nucleic acids was assumed. The experimental values from three independent
prion preparations (CAM6, CAM7 and CAM8) were evaluated. The straight line is an interpolation of the experimental data by linear
regression in order to determine an average nucleic acid size at P/I = 1. The abcissa denotes the average of the size range of a gel
segment (see Table 1). The interpolating line is shown for the whole experimentally covered range of 13 to 1000 nt. The dotted lines
represent the maximum error, i.e. one log ID5o for the bioassay and a factor of two in the nucleic acid content. This calculation shifts the
P/I ratio of unity from 76 nt to 165 nt. In (b) heterogeneous scrapie-specific nucleic acids were assumed. The same experimental data as
in (a) were evaluated. P/I values for heterogeneity classes (H = 10, 100, 1000) are plotted against the minimum length of the class. H
denotes the number of heterogeneous molecules in one class and, because of the continuous size distribution, the maximum difference in
length. For example, P/I = 1 at 209 nt for H = 100 means that all molecules between 209 nt and 308 nt add up to a P/I ratio of one. In
the case of nearly total heterogeneity, i.e. H = 1000, nucleic acid molecules smaller than 240 nt are needed for a P/I ratio of at least
unity.
nucleic acids. The abcissa respresents the minimum
length of the heterogeneity class H; H denotes the
number of different lengths in one class and, because of
the continuous size distribution, it also denotes the
maximum difference in lengths of one class. If, for
example H is 100, at a P/I ratio of 1 the minimum length
is 209 nt, and all molecules between 209 nt and 308 nt
add up to a P/I ratio of 1, and each would represent the
essential component of a hypothetical scrapie genome.
Assuming nearly total heterogeneity (H of 1000), P/I is 1
for 239 nt. Thus, we have to conclude that even in such
an extreme hypothetical situation, i.e. total heterogeneity, nucleic acid molecules as small as 239 nt could act as
the scrapie genome.
ence between the purification protocol applied in our
work and those used by the other groups is the DLPC
formation and consecutive nuclease digestions. In a
previous report (Meyer et al., 1991) we described a
reduction in the P/I ratio of nearly two orders of
magnitude due to the additional steps.
Although detectable amounts of nucleic acids were
still found in highly purified prion preparations, it
appears at present inappropriate to discuss their possible
functional relevance; on the contrary, more rigorous
procedures for degrading nucleic acids are needed to
decide whether the small or heterogeneous nucleic acids
described in this work are essential for scrapie infectivity
or are non-specific background.
I n c o n t r a s t to o u r w o r k , A k o v i t z et al. (1990) d e s c r i b e d
infectious Creutzfeldt-Jakob disease prion samples that
c o n t a i n R N A a n d h o s t p o l y a d e n y l a t e d s e q u e n c e s o f 1 to
4 kb. F r o m t h e i r v a l u e s o f i n f e c t i v i t y a n d a m o u n t s o f
nucleic acid, a P/I ratio well above unity could be
c a l c u l a t e d . A i k e n et al. (1990) f o u n d a m i t o c h o n d r i a l D l o o p D N A o f 450 n t l e n g t h in i n f e c t i o u s s c r a p i e f r a c t i o n s
a n d a P / I r a t i o o f a p p r o x i m a t e l y 10. W e c o n s i d e r t h a t t h e
findings of these investigators resulted from incomplete
d e g r a d a t i o n o f c e l l u l a r n u c l e i c acids. T h e m a i n differ-
This work was supported by research grants from the National
Institutes of Health (NS14069, NS22786 and AG08967), from the
Minister ffir Wissenschaft und Forschung von Nordrhein-Westfalen,
Germany and the Fond der Chemischen Industrie.
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1029
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(Received 26 July 1991; Accepted 21 "_~ovember1991)
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