J. gen. Virol. 0972), x4, I65-~75
16 5
Printed in Great Britain
Virus Particles in
Aspergillusfoetidus:
a Multicomponent System
By G. R A T T I AND K. W. B U C K
Biochemistry Department, Imperial College of Science and Technology,
London, S.W. 7
(Accepted 5 October I97I)
SUMMARY
Two electrophoretically distinct classes of virus particles, designated fast (AfVF) and slow (AfV-S) according to their relative electrophoretic mobilities, were
isolated from the mycelium of Aspergillus foetidus, strain IMX4~87I. They were
almost completely separated by dialysis against o.o3M-phosphate buffer, pH 7"6,
in which F particles remained in suspension, while S particles precipitated, and were
further purified by caesium chloride density gradient centrifugation. Electron microscopy showed that both classes were composed of isometric particles of similar
diameter.
RNA, prepared from both AfV-F and AfV-S, was double-stranded. In polyacrylamide gel electrophoresis AfV-F RNA was resolved into four main components
with molecular weights of 2"3~, 1-87, ~'7o and I'44 x Io 6, while AfV-S RNA gave
two components with molecular weights of 2.76 and 2.24 x io •.
Both AfV-F and AfV-S could be separated by centrifugation in caesium chloride
gradients into a number of fractions containing particles with different buoyant
densities. Electrophoretic analysis of the RNA prepared from the virus fractions
indicated that the multiple RNA components are not fragments released from a
single virus particle, but are separately encapsidated in different particles.
INTRODUCTION
Isometric virus particles, approximately 4o nm. in diameter, have been isolated recently
from the filamentous fungus, Aspergillusfoetidus, strain 1MI 4187I (Banks et al. I97o), and
RNA isolated from the virus particles was shown to be double-stranded. One of the main
features of the virus isolates was the distribution into several discrete bands on isopycnic
centrifugation in caesium chloride density gradients. Moreover the virus double-stranded
RNA showed four main components when examined by polyacrylamide gel electrophoresis.
Although infectivity of the virus particles was not established, transmission of morphologically similar and serologically related particles in A. niger to a virus free strain through
heterokaryosis has been demonstrated (Lhoas, ~97o).
A modification of the original isolation procedure is now given which affords virus preparations, containing two distinct classes of virus particles designated fast (AfV-F) and slow
(AfV-S), according to their relative electrophoretic mobilities; the previously described
isolation method (Banks et al. ~97o) gave preparations containing predominantly AfV-F. The
heterogeneity of AfV-F and AfV-S has been examined by equilibrium centrifugation in
caesium chloride density gradients and evidence is presented that the multiple virus RNA
components are not fragments released from a single virus particle, as in the case of reovirus (Shatkin, Sipe & Loh, I968), but are separately encapsidated in different particles.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
I66
G. R A T T I
AND
K. W . B U C K
METHODS
Abbreviations for buffers. Phosphate buffer (0"03 M-sodium phosphate, pH 7"6); TAE
buffer (o.o4 M-tris + O-O2 M-sodium acetate + 0-o02 M-EDTA, pH 7"8); SSC (o-I 5 M-NaC1 +
o'oi5 M-Na Citrate, pH 7.o).
Isolation and purification of AfV-S and AfV-F. Aspergillus foetidus, strain IMI 418 7 I, was
grown in 6o 1. fermenters as described previously (Banks et al. 197o). A crude virus preparation was obtained from the mycelium, essentially as described for the PeniciIlium cyaneofulvum virus (Banks et al. 1969), but with a number of modifications: the homogenization
buffer was o"15 M-KCl+phosphate buffer; precipitation of the virus-yeast RNA complex
was carried out at pH 4"5; the virus-yeast RNA complex was resuspended in o.6 M-KCI+
phosphate buffer, and the final virus pellet was resuspended in o'15 M-KCl+phosphate
buffer.
Dialysis of the virus suspension against phosphate buffer resulted in precipitation of
AfV-S, leaving AfV-F in suspension. After centrifugation (I5,ooog, Io rain.), the supernatant was again centrifuged (I5,ooog, IO rain.) to remove traces of the precipitate. The
precipitate was washed several times with phosphate buffer and resuspended by dialysis,
first, against o-6 M-KC1 + phosphate buffer and finally against o' 15 M-KC1 + phosphate buffer.
Insoluble material was removed by centrifugation (I 5,ooog, IO rain.). Purification and further
separation of the two virus classes was effected by centrifugation in CsCI density gradients.
Virus preparations (I ml.) were layered on pre-formed gradients of CsC1 (33 ml., IO to
45 ~o (w/w) in phosphate buffer) and centrifuged for 3 hr at 24,ooo rev./min, at 5 ° in a Beckman SW 27 rotor. Fractions (o.z ml.) were collected using an ISCO Model D gradient
fractionator. The refractive index was measured using 5o #1. of each fraction; the remainder
of each fraction was diluted I : 3 with phosphate buffer for measurement of E26o. Densities
of CsC1 were calculated from refractive indices (Ifft, Voet & Vinograd, 196i ).
Analytical centrifugation. A Beckman Model E ultracentrifuge, equipped with a monochromator and a double beam ultraviolet absorption optical system with photoelectric
scanner (Hanlon et al. t962), was used to determine sedimentation coefficients and buoyant
densities of virus particles. Sedimentation coefficients were determined using a 12 ram.,
2.5 ° aluminium-filled Epon double sector centrepiece in the AN-H rotor at 16,ooo rev./
min. Low virus concentrations (E2~o0"5 to 0"7) in o'I5 M-KC1 +phosphate buffer were used
and detection of the moving boundary was made with the u.v. scanner at 265 nm. Sedimentation coefficients measured at these very low concentrations are close to S ° values (Schumaker & Schachman, 1957).
Equilibrium density gradient centrifugation (Meselson, Stahl & Vinograd, I957) was
used to determine the buoyant densities of the virus particles with the following conditions :
I 2 ram., 2-5 ° double sector charcoal-filled Epon centrepieces; CsC1 (average density 1-35 to
"43 g./ml.) containing o.oi M-phosphate buffer, pH 7'6; centrifugation for 24 hr at 4o,ooo
rev./min, at 25 ° in the AN-F rotor; profiles were obtained using the u.v. scanner and Multiplexer accessory. Densities of CsC1 solutions were calculated from refractive indices (Ifft
et al. I960, which were measured using a High Accuracy Abbe '60' refractometer (Bellingham & Stanley Ltd.). Buoyant densities of virus particles were calculated as described by
Szybalski (I 968).
Spectrophotometry. Ultraviolet spectra were measured with a Cary model 15 spectrophotometer using I cm. silica cells.
Preparation of virus RNA. Virus suspension was heated with sodium dodecyl sulphate at
I ~ concentration at 4 o° for I5 rain. and then extracted with phenol, once at 4 o° and twice
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
Virus particles in A. foetidus
(i)
(ii)
167
(iii)
Fig. I. Agarose gel electrophoresis of A. foetidus virus particles.
(i) Unseparated AfV; (ii) AfV-F; (iii) AfV-S.
at o °. Potassium acetate was added to 0.2 N concentration and RNA was precipitated by
addition of 2"5 vol. of ethanol at - 2 o °. After 18 hr the RNA precipitate was collected by
centrifugation, dissolved in SSC and dialysed against SSC, prior to storing at - 2 o ° in
sealed ampoules.
For analysis of RNA from small virus samples by polyacrylamide gel electrophoresis,
virus suspension was heated with I ~oo sodium dodecyl sulphate at 6o ° for ~5 min., and the
mixture was layered directly on to the gels; RNA prepared by this method gave electrophoretic patterns identical to those of R N A prepared by the first method.
Gel electrophoresis. 4 ~ polyacrylamide gels containing o'o4 ~ bis-acrylamide, were prepared in glass tubes, 5 mm. internal diameter, essentially as described by Loening (I967).
Running buffer was TAE or 2 x TAE and electrophoresis was carried out at 7 mA/tube for
3 to 8 hr. Gels were stained with acridine orange (Richards, C o l l & Gratzer, ~965) or
toluidine blue (Smith, I968). Gels stained with toluidine blue were scanned at 63o nm. using
a Unicam SP 5oo spectrophotometer with a Gilford gel scanning attachment.
Gels, containing o'5 ~ agarose in o.i 5 N-KC1 + o'o5 N-tris-HC1, pH 8.o, were prepared in
5 mm. internal diameter glass tubes, and supported with nylon gauze. Electrophoresis of
virus particles (IO #1., E~60 5 to ~o in oq5 N-KCl+phosphate buffer) was carried out for
~2
v~R
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
~4
I68
G. R A T T I
AND
K. W . B U C K
1.40 ~1,5
1.35
//Tr~
F4
(i)
u
1.30
1.0
l.o
0.5
1.5
-
(ii}
r
o 1.0 -
/
1~1
S
52
/~
0.5 -
S3
G
.....
..........
10
20
J ,
30
Fraction no.
,
",1%
40
50
Fig. 2. Purification o f A. foetidus virus preparations by centrifugation in Io to 45 % CsC1 gradients.
(i) Phosphate buffer supernatant; (ii) phosphate buffer precipitate.
2 hr at 7 mn/tube with o.15 M-KCI+o.o5 M-tris-HC1, pH 8.0 as running buffer. Gels were
stained with amido black (Smith, i968 ).
Molecular weights of virus RNA components. The molecular weights of virus RNA components were determined by comparing their electrophoretic mobilities on 4 % polyacrylamide gels with those of the ten double-stranded RNA components of reovirus type 3, as
described by Shatkin et al. (~968).
Electron microscopy. Samples were negatively stained with o.2 % potassium phosphotungstate, pH 7"o, and examined with a J.E.M. Model 7 electron microscope.
RESULTS
Separation and properties of AfV-F and AfV-S
Virus preparations, extracted from A. foetidus mycelium with o'15 M-KC1+ phosphate
buffer, contained two classes of virus particles which were designated fast (AfV-F) and
slow (AfV-S) to denote their relative mobilities in agarose gel electrophoresis (Fig. 1).
(In the original isolation procedure (Banks et al. I97o) phosphate buffer alone, in which the
S particles precipitated, was used for virus isolation; examination of such preparations by
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
I69
Virus particles in A. foetidus
Q
3
.7..
E
t~
4
c
c
s ~5
Fig. 3. Polyacrylamide gel electrophoresis of AfV-RNA. Electrophoresis was carried out for 5 hr in
TAE buffer. The gel was stained with toluidine blue.
T a b l e I.
Virus
component
Properties of virus particles and virus RNA's isolated from A. foetidus
Buoyant* density
(g./cm?)
S2o~;
E~6o/E28o
RNA
component
Mol. wt of
R N A x Io -6
I'44
I'70
t"87
F1
F2
F3
1'35I
I'362]
I'3671
N.D.
N.D.
i45:~
I'5~
6
5
4
F4
1.380
158
1'6
2
2'31
SI
.~(a) 1"396~
146
1.4
3
2'24
S2
j (a) 1-428 }
i72
I '7
1
2.76
[(b) I'4O3 !
t (b) I'435
* Mean value of three determinations. Mean deviation was o'oo3.
1" Sedimentation coefficients, determined in o. 15 M KCI + phosphate buffer, were calculated from the positions of the mid-points of the moving boundaries.
Determined with fractions corresponding to fraction 24 of Fig. 2i.
N.D. Not determined.
I2-2
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
I70
G. R A T T I
AND
K. W . B U C K
F3
F4
(i)
>~
o~
].35] 1.362 ] .367
1,380
Density CsCI (g./cm3.)
Fig. 4. Equilibrium centrifugation of AfV-F fractions in CsC1 density gradients : u.v. scanner tracings.
(i) Unfractionated AfV-F; (ii), (iii), (iv) and (v) fractions from preparative CsC1 gradients corresponding to fractions I9, 23, 25 & 29 respectively in Fig. 2(i).
agarose gel electrophoresis showed mainly AfV-F with only a trace of AfV-S.) Almost
complete separation of the two virus classes was achieved by dialysis against phosphate
buffer, when AfV-S precipitated, leaving AfV-F in suspension. Further purification of precipitate and supernatant was effected by centrifugation in CsC1 gradients. Typical profiles
are shown in Fig. 2. AfV-F was recovered from fractions ~8 to 3o (Fig. 2, i), while AfV-S was
obtained from fractions 25 to 53 (Fig. 2, ii). Both preparations gave a single band in agarose
gel electrophoresis (Fig. ~). The exclusion of fractions 3t to 45 (Fig. 2, i) from AfV-F preparations minimized possible contamination with the small amount of AfV-S which remained
in the supernatant. A lighter component, which was present in both gradients (Band G,
Fig. 2, i and ii), had a broad maximum extinction at 275 to 280 nm. and a minimum at
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
Virus particles in A. foetidus
I7I
(0
Sla
S2a
m
o~
(ii0
1.396 1.403
1.428 1.435
Density CsCI (g./cm3.)
Fig. 5- Equilibrium centrifugation of AfV-S fractions in CsC1 density gradients : u.v. scanner tracings.
(i) Unfractionated SI + $2 ; (ii) and (iii) fractions from preparative CsC1 gradients corresponding to
fractions 31 & 39 respectively in Fig. 2 0 0 .
252 nm. with E26o/E2so0"7 to 0.8; electron microscopy of this component showed mainly
empty particles.
Preparations of both AfV-F and AfV-S contained isometric particles of similar size
(33 to 37 nm., D. J. Border, unpublished results) when examined by electron microscopy and
had ultraviolet spectra typical of nucleoprotein with extinction maxima at 258 to z6o nm.
and minima at 242 to 245 nm. E26o/E2sowas I. 4 for AfV-F and 1.6 for AfV-S.
Properties of Virus RNA
On examination by polyacrylamide gel electrophoresis, RNA prepared from a mixture of
AfV-F and AfV-S showed six clear bands, designated I to 6 in order of increasing mobility
(Fig. 3). In some preparations a trace of a seventh faster moving band was detected; the
properties of this component have not been investigated.
The molecular weights of these six components are given in Table I. When RNA prepared from the separated virus classes was similarly examined it was found that AfV-F
R N A contained components 2, 4, 5 and 6 while AfV-S R N A contained components I and 3.
Gel profiles are shown in Fig. 6 and 7.
The four RNA components from AfV-F are those described previously by Banks et al.
(I97O) and have been shown to be double-stranded. In order to confirm that the nucleic acid
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
I72
G. R A T T I
AND
K. W. B U C K
I
I
I
I
0)
1
I---
3
k/
0ii)
I
1
2
3
4
Distance from top of gel (cm.)
Fig. 6
I
1
2
3
Distance from top of gel (cm.)
I
4
Fig. 7
Fig. 6. Polyacrylamide gel profiles of R N A released from A f V - F fractions. Gels were scanned at
63o nm. after staining with toluidine blue. (i) R N A from unfractionated AfV-F; (ii), (ii), (iv) and
(v) R N A released from virus fractions from preparative CsCI gradients corresponding to fractions
I9, 23, 25 and 29 in Fig. 2(0.
Fig. 7- Polyacrylamide gel profiles of R N A released from AfV-S fractions. Gels were scanned at
63o rim. after staining with toluidine blue. (i) R N A from unfractionated AfV-S ; (ii) and (iii) R N A
released from virus fractions from preparative CsC1 gradients corresponding to fractions 31 and 39
in Fig. 2 (ii).
components from AfV-S were also double-stranded RNA, AfV-S nucleic acid (E~,o 0'4) was
treated with pancreatic ribonuclease (o.2 #g./ml.) in SSC for 2 hr at 25 °. There was no
detectable hyperchromic effect and examination by polyacrylamide gel electrophoresis
revealed the same two bands as before treatment. When such treatment was carried out in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
Virus particles in A. foetidus
I73
o.~ × SSC the original RNA components could not be detected. The melting curve of
AfV-S RNA in SSC was very similar to that of AfV-F RNA with a Tm> IO0°.
Fractionation of AfV-F and AfV-S preparations by isopycnic eentrifugation
Both AfV-F and AfV-S gave rise to multiple bands when centrifuged in linear ro to 45
(w/w) CsC1 gradients. The efficiency of fractionation was assessed by dialysing fractions
against phosphate buffer, adjusting to the appropriate density with CsC1 and centrifuging to
equilibrium in the analytical centrifuge. The gradient profiles and the buoyant densities of
the four AfV-F components and the two major AfV-S components are shown in Fig. 4 and
5, and Table I.
In the case of AfV-S a complete separation of components S I and $2 was achieved; with
the higher resolution afforded by the analytical centrifuge each of these was further resolved
into two components, designated $I a, SI b and S2a, S2b respectively (Fig. 5). In fractionating AfV-F components a homogeneous sample of F4 particles was obtained (Fig. 4, ii),
while three fractions were obtained, containing FI, F2 and F3 particles respectively as the
principal component (Fig. 4, v, iv and iii). All the fractions contained particles of similar
morphology when examined with the electron microscope. Portions of the fractions were
dialysed against o.~ 5 ~-KC1 + phosphate buffer, prior to determination of sedimentation
coefficients and Ezoo/E28oratios. The results are given in Table I.
Eleetrophoresis of RNA from AfV-F and AfV-S fraetions
RNA from each virus fraction was released by heating with sodium dodecyl sulphate and
examined by polyacrylamide gel electrophoresis. The gel profiles are shown in Fig. 6 and 7.
It is clear that SI particles gave RNA-3, $2 particles gave RNA-~ (Fig. 7) and F4 particles
gave RNA-2 (Fig. 6, ii). The three fractions containing FI, F2 and F3 particles respectively
as the major components (Fig. 4, v, iv, and iii) gave rise to RNA-6, RNA-5 and RNA-4
respectively as the major components (Fig. 6, v, iv and iii). In each case the ratios of the areas
of the peaks FI, F2 and F3 in the CsCI gradient profiles (Fig. 4) were approximately equal
to the ratios of the areas of the peaks 6, 5 and 4 respectively in the gel profiles of the derived
RNA (Fig. 6). This suggests that FI, F2 and F3 particles each contain a single RNA component, namely RNA-6, RNA- 5 and RNA-4 respectively. The denser AfV-S fractions
(43 to 53, Fig. 2ii) gave RNA-~ and RNA-3, but were not examined further.
DISCUSSION
The population of virus particles, isolated from A. foetidus, can be separated into
two classes, designated fast (AfV-F) and slow (AfV-S), according to their relative electrophoretic mobilities; electron microscopy indicated that both classes contain isometric particles
of similar size. The low electrophoretic mobility of AfV-S in the o. I5 M-KC1 buffer and its
precipitation in phosphate buffer suggested that the S particles may consist of aggregates of
the F particles; this possibility was ruled out because the two virus classes contained different RNA components. Evidence that the slow and fast virus classes have differences in
their capsid proteins has been provided by serological tests in which AfV-F and AfV-S
preparations formed crossing precipitin lines when examined by agar double-diffusion with
an antiserum raised against a mixture of the two virus classes (G. Ratti, unpublished results).
Both AfV-F and AfV-S could be separated by centrifugation in caesium chloride density
gradients into a number of different particles having different buoyant densities. Electrophoretic analysis of the RNA from different virus fractions proved conclusively that SI, $2
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
~74
G. RATTI AND K. W. BUCK
and F4 particles each contained only one RNA component and suggested that the same was
true also for F~, F2 and F3 particles. These results indicated that the six RNA components
are separately encapsidated to form six different particles. It is of interest that both S~ and
$2 particles, which each contain only one RNA component, gave two bands (SI a, SI b and
S2a, S2b) of closely similar density on caesium chloride gradients (Fig. 5). The ratio between
the areas of the two peaks of each doublet did not change during centrifugation, but varied
slightly with different virus preparations; it is thus unlikely that the doublets are a result of
a direct degradative action of the caesium chloride. The individual components of the two
doublets have not been investigated, but could result from small differences in the virus
capsid. The minor components S3 and $4 (Fig. 2ii) accounted for about Io ~ of AfV-S and
contained no additional RNA components with respect to S~ and $2. It is possible that they
are derived from, or are variants of, S~ or $2.
Multiple double-stranded R N A components have been obtained from a number of ottier
viruses of fungi (Penicillium stoloniferum, Buck & Kempson-Jones, 197o; P. chrysogenum,
Buck, Chain & Himmelweit, ~97I; P. brevi-cornpacturn, Wood, Bozarth & Mislivec, ~97I;
P. cyaneo-fulvum, K. W. Buck, unpublished results) and also appear to be a general characteristic of double-stranded RNA viruses of animals and higher plants (Fuji-Kawata, Miura
& Fuke, ~97o). In the case of reovirus it has been demonstrated that the ten fragments of
double-stranded RNA are all enclosed in the same capsid and constitute different parts of
the genome (Shatkin et aL 1968); defective particles containing only nine of the ten RNA
fragments were not infective (Nonoyama, Watanabe & Graham, 197o). It is probable that the
genome of the other animal and higher plant viruses, containing double-stranded RNA, is
contained in single virus particles, since heterogeneity of virus preparations has not been
reported. In contrast, the particles in A. foetidus form a multicomponent virus system, in
which the several molecules of double stranded RNA are separately encapsidated. Whether
the two classes of particles, AfV-F and AfV-S, constitute different virus systems and how the
particles within each class are related, remain to be established.
Two immunologically distinct viruses have been isolated from P. stoloniferum (Buck &
Kempson-Jones, I97O). These viruses are also characterized by multiple types of particles,
each differing in its RNA content (G. F. Kempson-Jones & K. W. Buck, unpublished
results). This latter feature may well be common to all the known double-stranded RNA
viruses of fungi. The formation of multi-component virus systems may be favoured in fungi,
in which virus replication occurs in parallel with the growth of the organism (D. J. Border,
unpublished results). This situation, in which cell lysis and reinfection from without are
not required, may be propitious to the survival and accumulation of defective particles.
The work reported in this paper forms part of the research project on fungal viruses and
non-specific immunity under the direction of Professor Sir Ernst Chain, carried out in
collaboration with Beecham Research Laboratories, Brockham Park, Betchworth, Surrey.
it is supported by a grant of the Medical Research Council to Professor Chain, which is
gratefully acknowledged. We wish to thank Professor Chain for helpful discussions and
encouragement. We gratefully acknowledge the collaboration of Mr G. T. Banks for Pilot
Plant fermentations and Mrs J. E. Darbyshire and Dr D. J. Border for electron microscopy.
We thank Dr A. J. Shatkin for a sample of reovirus type 3 RNA and Dr H. F. S. King for
the use of a Gilford gel Scanner.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27
Virus particles in A. foetidus
175
REFERENCES
BANKS, G. T., BUCK, K. W., CHAIN, E. B., DARBYSHIRE,J. E. & HIMMELWEIT, F. (I969). Penicillium cyaneo-fulvum
virus and interferon stimulation. Nature, London 223, 155.
BANKS, G. T., BUCK, K. W., CHAIN, E. B., DARBYSHIRE, J. E., HIMMELWEIT, F., RATTI, G., SHARPE, T. J. & PLANTEROSE, D.N. 0970). Antiviral activity of double-stranded R N A derived from a virus isolated f r o m
Aspergillus foetidus. Nature, London 227, 5o5.
BUCK, K. W., CHAIN, E. B. & HIMMELWEIT, F. (1971). Comparison of interferon induction in mice by purified
Penicillium chrysogenum virus and derived double-standed R N A . Journal of General Virology i2, 13 I.
BUCK, K. w. & KEMPSON-~ONES,C. ~. (I970). Three types of virus particle in Penicillium stoloniferum. Nature,
London 225, 945FUM-KAWATA, I., MIURA, K. & FUKE, M. (I970). Segments of genome of viruses containing double-stranded
ribonucleic acid. Journal of Molecular Biology 5I, 247.
HANLON, S.~ LAMER, K., LAUTERBACH, G., JOHNSON, a. & SCHACHMAN, H. K. (1962). Ultracentrifuge studies with
absorption optics. An automatic photoelectric scanning absorption system. Archives of Biochemistry and
Biophysics 99, 157.
IFFT, J. B., VOLT, D. n. & VINOGRAD,J. (I961). The determination of density distributions and density gradients
in binary solutions at equilibrium in the ultracentrifuge. Journal of Physical Chemistry 65, I 138.
LHOAS, P. (1970). Use of heterokaryosis to infect virus free strains of Aspergillus niger. Aapergillus News
Letter No. I I , 8.
LOENING, U. E. 0967)- The fractionation of high molecular weight ribonucleic acid by polyacrylamide gel
electrophoresis. Biochemical Journal lO2, 25I.
MESELSON, M., STAHL, F. W. & VINOGRAD, J. (1957). Equilibrium sedimentation of macromolecules in density
gradients. Proceedings of the National Academy of Sciences of the United States of America 43, 58t.
NONOYAMA,M., WATANABE,Y. & GRAHAM,A. F. 0970). Defective virions of reovirus. Journal of Virology 6, 226.
RICHARDS, r. G., COLE, J. A. & GRATZER, W. B. (I965). Disc electrophoresis of ribonucleic acid in polyacrylamide gels. Analytical Biochemistry, I2, 452.
SCHUMAKER, V. N. & SCHACHMAN, IJ. K. 0957). Ultracentrifugal analysis of dilute solutions. Biochimica et
biophysica Beta 23, 628.
SHArKIN, A. J., SlPE, J. D. & LOH, P. (I968). Separation of ten reovirus genome segments by polyacrylamide gel
electrophoresis. Journal of Virology 2, 986.
SMITH, L. (I968). Chromatographic and Electrophoretic Techniques, Vol. I1, p. 382. L o n d o n : Heinemann.
SZYBALSKI, W. (1968). Use of caesium sulphate for equilibrium density gradient centrifugation. In Methods in
Enzymology, vol. xtlB, p. 33o. Ed. by Grossman, L. & Moldare, K. New York: Academic Press Inc.
WOOD, H. A., BOZARTH, R. r. & MISLIVEC,P. B. (i97I). Virus like particles associated with an isolate of Penicillium brevicompactum. Virology, 44, 592.
(Received 17 August 1971)
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 19:14:27