J. gen. Virol. (I97I), io, I I I - I t 4
III
Printed in Great Britain
Nucleic Acids in the Potato Virus X Group
and in some Other Plant Viruses: Comparison of the Molecular
Weights by Electrophoresis in Acrylamide-agarose Composite Gels
(Accepted I4 October I97o )
The molecular weight of the nucleic acid is a basic property of a virus, and has been shown
to be of great value for classifying animal viruses (Bellett, 1967). However, this property is
known for few plant viruses. In the potato virus X group, for example, only the RNA of
potato virus X (PVX) itself has a known molecular weight. The reported value (2.1 x 106) has
been calculated from the total particle weight of the virus and its phosphorus content
(Knight, ~963).
Recently electrophoresis in acrylamide gels of low concentration has been successfully
used to estimate the molecular weights of large RNA molecules (Loening, I968; I969).
Studies including the RNAs of five plant viruses have been reported by Bishop, Claybrook
& Spiegelman 0967), and the RNA of tobacco mosaic virus (TMV) has been used as a
marker in the re-examination of the molecular weight of poliovirus RNA (Tannock, Gibbs
& Cooper, I97O). Gels prepared with less than 3"5 % acrylamide are difficult to handle as
vertical slabs, but acrylamide-agarose composite gels are more stable and have increased
the usefulness of the technique (Peacock & Dingman, I968). With slight modifications this
technique was used in the work reported here to compare the RNA molecular weights of
viruses in the PVX group, namely PVX, cactus virus X (CaVX), white clover mosaic
(WCMV), clover yellow mosaic (CYMV) and pawpaw mosaic (PMV) viruses, as well as
of some other plant viruses, namely TMV, sowbane mosaic (SMV), belladonna mottle
(BMV) and brome mosaic (BrMV) viruses.
The method used to extract infectious RNAs from purified viruses or ribosomal marker
RNAs from plant, animal or microbial cells was similar to that described by Bockstahler &
Kaesberg (I965). It was essential to free all equipment from nucleases using the treatments
suggested by these authors, and deionized water had to be autoclaved. With most viruses
more RNA was obtained when 0.02 M-tris-borate buffer, pH 8"8 containing o'o89 M-EDTA,
1 % sodium dodecylsulphate (SDS) and 1 % Bentonite was used instead of the acetate buffer
used by Bockstahler & Kaesberg. In some experiments ~ % Macaloid or 5 % diethylpyrocarbonate (DEPC) were used instead of Bentonite. After phenol extraction the RNAs were
precipitated with ethanol at - 2 o ° overnight, the precipitates were washed with 70 %
ethanol and dissolved in an electrophoresis buffer of tris-EDTA-boric acid, pH 8"3 (Peacock
& Dingman, I968). To remove any intact virus particles which might have interfered in
infectivity tests, the samples were ultracentifuged for I hr at ~3o,ooo g. Most virus RNAs
were unstable in solution and had to be used as soon as possible. The infectivity of the various
RNAs was tested on the following host plants : Gomphrena globosa for PVX, CaVX, CYMV
and PMV, Chenopodium quinoa for CYMV, Phaseohts vulgaris cv. Saxa for WCMV and
Nicotiana glutinosa for BMV. The infectivity of all the virus nucleic acids was destroyed
completely when they were incubated for 30 min. at 37 ° with I #g./ml. pancreatic
ribonuclease.
Electrophoresis experiments were done in the vertical apparatus of Stegemann (~97o)
using either the standard buffer of Loening (1969) or that of Peacock & Dingman (I968)
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with o.2 ~ SDS with gels containing I ' z 5 ~ agarose and acrylamide at concentrations
ranging between I-5 ~ and 2"5 ~ . C u r r e n t was applied for i½ to 3 hr depending on the acrylamide c o n c e n t r a t i o n and the voltage used (between zoo and 400 v). After electrophoresis
the gels were placed in I M-acetic acid for 2o min., rinsed in r u n n i n g tap water for 3o min.,
stained with o-o2 ~ methylene blue in o'4 M-sodium acetate buffer, p H 4"7, for 3 to 4 h r
and destained with running tap water overnight. The m o l e c u l a r weights estimated by this
m e t h o d seemed to be i n d e p e n d e n t o f buffer, acrylamide c o n c e n t r a t i o n and voltage used.
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Fig. I
Fig. 2
Fig. I. Electrophoresis at 200 v and I6o ma for 3 hr of RNAs of the PVX group and of marker
RNAs in a 1.25 % agarose 2 ~ acrylamide gel with 36 mM-tris, 3o mM-NaHaPO4, i mM-EDTA
and o.2 ~ sodium dodecylsulphate at pH 7"9. Column a from top: RNA from TMV, 25 s and I8 s
RNA from carrot; columns b, e, d, e, l a n d g: RNAs from WCMV, PVX, CaVX strain K ~I,
CaVX strain B I, CYMV and PMV, respectively; column h from top: RNA from SMV, RNAs from
BrMV. The fourth band from top in column h is due to an unidentified component in the BrMV
preparation.
Fig. 2. Relationship between molecular weight and electrophoretic migration of marker RNAs.
The conditions of electrophoresis were the same as in Fig. I. V, PVX RNA; A, TMV RNA;
©, 28 s and 18 s ribosomal RNA from rabbit; • and II, 25 s and 18 s ribosomal RNA from
radish and carrot, respectively; ½, SMV RNA, z~, BrMV RNAs; V, ribosomal RNA from Euglena.
The following R N A s served as markers (mol. wts. and references in parentheses): ribosomal R N A s from carrot and radish (o.7 x Io 6 and 1.29 x Io6; Loening, I968), from
Euglena gracilis (o.85 x Io6; Loening, I968) and from rabbit liver (o'7 x io G and I'72 x lO6;
Loening, I968), R N A f r o m T M V (2.0 x 1oG; Boedtker, 196o), from PVX (2.I x io6; Knight,
I963), from S M V (I.27 x Io6; K a d o & Black, I968) and f r o m B r M V ( I ' o x io 6, o'7 x lO 6
and o. 3 x IoG; Bockstahler & Kaesberg, I965).
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Fig. I shows an electropherogram of nucleic acids of viruses of the PVX group together
with virus and ribosomal marker RNAs. With all plant viruses studied with the exception
of BrMV (Bockstahler & Kaesberg, ~965) only one strong R N A band was formed. The
faint smear of stain in front of the R N A bands (Fig. I, WCMV) indicates a degradation of
the RNAs which was probably due to traces of nucleases.
Plant nucleases have been found to be strongly inhibited by DEPC (Solymosy et al. ~968).
In our experiments with some viruses (PVX, WCMV) a stronger band was obtained with
D E P C in the extraction medium, whereas with others the band was not affected (BMV) or
was almost completely absent (PMV, 3 strains of CaVX, SMV). The position of the bands
of the RNAs of these viruses was not changed by DEPC. The infectivity of all RNAs, however, was lost completely or almost completely (PVX, CaVX) with D E P C in the extraction
medium. Similar observations have recently been made by Oberg 0970) with poliovirus
RNA.
The distances that the marker RNAs migrated were plotted against the log values of their
reported molecular weights (Fig. 2). The molecular weights of the PVX and SMV RNAs,
which so far have not been studied by acrylamide electrophoresis, fit well into this straight
line relationship. The size of the R N A of BMV was estimated to be 2.o x io 6 on the basis of
this relationship. This estimate also agrees well with a value of 1.8 to 2.o x io 6 obtained from
the total particle weight and its phosphorus content (Paul et al. ~968).
For the RNAs of the other viruses in the PVX group the following sizes were estimated
(number of observations in parentheses): CaVX strain B~ 2.I )< Io 6 daltons (I2), CaVX
strain K I I z ' l - x Io G04), C Y M V 2-4 × IO6 ( 1 7 ) , PMV 2-2 × I06 ( I 5 ) , W C M V 2. 4 x to 6 (i3).
The standard errors of each mean were less than r ~ .
The size of the nucleic acid of a virus is one of the properties encoded in the virus cryptograms suggested by Gibbs et al. (I966). The work reported here shows that the cryptograms
of viruses in the PVX group are, at present:
CaVX
CYMV
PMV
PVX
WCMV
R/I : 2.I/* : E/E: S/*
R/I : 2.4/* :E/E: S/*
R/1: 2.2/* : E/E :S/*
R / 1 : 2 . I / 6 : E / E : S / O (Fu)
R/I : 2"4/5 :E/E: S/O (Ap)
The author is very grateful to Dr H. Stegemann for helping this investigation both
practically and theoretically, to Miss Renate Buch for expert technical assistance, to the
Deutsche Forschungsgemeinschaft for financial aid and to Mr Schlobach for photography.
Institut fiir Virusserologie
der Biologischen Bundesanstalt
fiir Land- und Forstwirtschaft,
Messeweg I ~/I2
Braun~chweig, W. Germany
RENATE KOENIG
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(Received 14 September 1 9 7 o )
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