J. gen. ViroL (I969), 4, 55-63
55
Printed in Great Britain
Quantum Yields for Inactivation of Tobacco Mosaic Virus
Nucleic Acid by Ultraviolet Radiation (254 rim.)
By N. A. EVANS AND A. D. M c L A R E N
College of Agricultural Sciences, University of California,
Berkeley, California, 9472o, U.S.A.
(Accepted 26 June I968)
SUMMARY
The quantum yield, under non-photoreactivating conditions, for inactivation of tobacco mosaic virus RNA by ultraviolet radiation (254 nm.) is
independent of the RNA concentration but varies appreciably with the ionic
strength of the irradiation solvent, being approximately three times greater
in water than in o-I M-potassium phosphate buffer. Photoreactivation of
inactivated tobacco mosaic virus RNA is dependent on RNA concentration,
ionic strength of solutions during irradiation, and on the light quality used
for photoreactivation. These data can be interpreted as evidence that pyrimidine hydrate is a lethal lesion at least in RNA irradiated at low ionic strength.
INTRODUCTION
Comparisons of the mechanisms of u.v. inactivation of tobacco mosaic virus (TMV)
and its ribonucleic acid (TMV-RNA) require reliable values for the quantum yields
for inactivation of TMV and TMV-RNA (Kleczkowski & McLaren, I967). Considerable uncertainty exists regarding the quantum yield for inactivation of TMVRNA, namely whether or not the quantum yield varies with RNA concentration
(Kleczkowski & McLaren, i967; Streeter & Gordon, I968). Some of this confusion
arises from the variety of irradiation and assay conditions used. In order to eliminate
this confusion, we have measured quantum yield for inactivation of TMV-RNA as a
function of concentration during irradiation and the amount of photoreactivation
of irradiated TMV-RNA under fixed conditions. We also report data on the influence
of buffer ion concentration (phosphate) on quantum yields and the influence of various
light sources on amounts of photoreactivation of irradiated TMV-RNA.
METHODS
Tobacco mosaic virus (TMV), COMMONstrain, was grown in Turkish tobacco plants
and isolated from harvested frozen leaves by the method of Knight (i962). Infectious
TMV-RNA was isolated from the virus by the phenol extraction method (FraenkelConrat, I966).
Irradiations of stirred solutions of TMV-RNA, in potassium phosphate buffer
(pH 7) or in water, were made in a cell of I cm. path length with a low-pressure mercury
lamp (Hanovia Sc-2537) which emits predominantly light of wavelength 253"7 nm.
(McLaren & Takahashi, 1957). Aqueous acetic acid (2o ~) was used to filter all light
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N. A. E V A N S A N D A. D. M C L A R E N
of wavelength below 24o rim. The incident intensity of the lamp, measured by uranyI
oxalate actinometry, was 30"8 x io -~ einstein./ml./min.
Infectivity assays for T M V - R N A were perfolTned with the primary leaves of
Phaseolus vulgaris L. var. Pinto. Unirradiated control and irradiated samples, both
in o.o 9 M-phosphate buffer containing bentonite, were compared on opposite primary
100~___.~=
i
i
I
I
i
50
10
2
1
I
0
l
20
I
I
l
40
I
60
Dose (nanoeinstein/ml.)
Fig. I. Survival of TMV-RNA as a function of the u.v. dose; I, plants were stored in the dark
for 8 hr after inoculation; 2, plants were stored under fluorescent lights. The RNA concentration was 31 #g.]ml. in o"I M-phosphatebuffer, pH 7"0. The points show average deviations
from the means.
leaves and the survival of the irradiated sample was expressed as a percentage of the
control. Dilutions were chosen such that there were approximately equal numbers of
lesions on both leaves and the number was in the range zo to 15o. To test for photoreactivation of irradiated T M V - R N A , plants were inoculated under red light. Half
the plants were transferred immediately to the light source being investigated, and the
other half were left in the dark. After 6 to 8 hr, both sets were placed in a greenhouse
until the lesions were ready to be counted (usually 3 to 5 days). In this manner,
survival curves were obtained. A typical result is shown in Fig. I, for which each
point is the average from six plants.
Photoreaetivation was studied under artificial light. Inoculated plants were illuminated either in a National Appliance Company controlled-environment growth
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UV-inactivation of T M V - R N A
57
chamber or in a small home-made growth chamber (Bradfute et aL 1962). Both chambers
gave equivalent results. These chambers were lighted by a combination of fluorescent
and incandescent lights, or by fluorescent light alone. Light was filtered through
either a layer of Teflon FEP fluorocarbon film (filter I) or a layer of Mylar polyester
film (filter 2). The transmission spectra of the films are shown in Fig. 2.
100
I
I
I
I
1
80
,-x
60
40
20
I
I
I
I
I
320
360
400
440
480
Wavelength (rim.)
Fig. 2. Transmission spectra of Teflon film I and Mylar film 2 used to filter
fluorescent light during photoreactivation studies.
Quantum yields for u.v. inactivation of TMV-RNA, in mole/einstein, were calculated from the survival curves as described previously (McLaren & Takahashi, I957)Percentage photoreactivation was calculated using the formula
PR
=
IOO (I -- ~bz/~b~),
where @z and ¢~ are the quantum yields for u.v.-inactivation of T M V - R N A under
photoreactivating and non-photoreactivating conditions, respectively (Werbin et al.
1966).
RESULTS
Inactivation of TMV-RNA by ultraviolet radiation
Quantum yields for u.v. inactivation of TMV-RNA under non-photoreactivating:
conditions at various concentrations are shown in Table I. The R N A was irradiated
in o. I M-potassium phosphate buffer, pH 7.o,at all concentrations, and dilutions were
made with the same buffer.
In the concentration range 13 to 115 #g./ml. the quantum yield was independent
of the concentration and the mean was I'I4 x IO-8 mole/einstein. The lower value
at 6/zg./ml. indicates that the quantum yield may be concentration-dependent at very
low concentrations.
We also observed that the quantum yield for u.v. inactivation of T M V - R N A was
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N.A. EVANS AND A.D. MCLAREN
dependent on the buffer concentration during irradiation, being lower at higher
concentration (Table 2).
Buffer concentration clearly had a m a r k e d effect on infectivity o f T M V - R N A
(Table 3)- This phosphate effect appears to be similar to that observed and m u c h
studied with the whole virus ( Y a r w o o d & Fulton, 1967). Thus, if one dilutes irradiated
solutions o f T M V - R N A with o-I M-phosphate buffer rather than to o.I M with m o r e
concentrated buffer, one will be measuring the resultant o f two effects--the buffer
effect and u.v. inactivation. I n our experiments, all inoculating solutions had a buffer
concentration o f o'09 U. Also the p H o f the solutions should be kept constant since
it has been shown that the infectivity o f T M V - R N A varies appreciably with p H
(Sarkar, I963).
Table I. Effect of TMV-RNA concentration on quantum yields of
inactivation and on photoreactivation
TMV-RNA
concentration
(/~g./ml.)
No. of
experiments
~
( x io 8)
6"3
I3"4
3I'5
5
3
5
0-8
i.I
1.2
<o.i
<o.I
<o.i
39
5
I'2
0"I
64
92
5
5
I'I
1.2
0"I
<o.I
2o
I4
4
I'2
O'I
IZ
II5
Standard
deviation
of ~D
Photoreactivation
(%)*
-
-
19
24
2I
* A combination of incandescent and fluorescent lamps was used.
Table 2. Effect of buffer concentration on quantum yields for u.v.-inactivation
of TMV-RNA ; concentration 40/zg./ml.
Phosphate buffer
concentration (M)
(pH 7"0)
~D
( x Io3)
o.I
o.ooi
o
1.2
2"3
3"2
% photoreactivation*
38I"
23
24
* Plants were illuminated with ' black light'.
t Data of Hidalgo-Salvatierra & McLaren 0968).
Table 3. Influence of potassium phosphate buffer concentration on the
infectivity of TMV-RNA ; concentration of RNA : o'5/zg./ml.
Phosphate buffer
concentration (M)
No. of lesions
per 6 leaves
o-o91
o'o46
o'oIo
947
440
39
The usual procedures for determining survival curves (Rushizky, Knight & McLaren,
I96o; Merriam & G o r d o n , I965) involve dilution o f irradiated solutions with o.i Mphosphate buffer. The dilution is dependent on the irradiation dose, being less for
higher doses. Hence, if a solution o f RNA, at lower buffer concentration, was diluted
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59
with o.i u-buffer, it is possible that, in earlier work, the buffer concentration in the
control solution was different from that in some of the irradiated solutions.
Photoreactivation of u.v.-inactivated TMV-RNA
Recent observations showed that the maximum amount of photoreactivation is
markedly affected by the wavelength of the photoreactivating light. This indicates
that the fight source is important in determining the percentage photoreactivation
under a given set of inactivating conditions (Hidalgo-Salvatierra & McLaren, I968).
We confirmed the importance of the light source in determining maximum photoreactivation (Table 4). The maximum percentage photoreactivation attainable from
T M V - R N A inactivated in o.I M-potassium phosphate buffer was 38, with either 'black
light' or new fluorescent lamps filtered through the Teflon film (filter I ; Fig. 2). The
decrease in photoreactivation on replacing filter I by Mylar (filter 2, Fig. 2) indicated
that low wavelength radiation was essential for one of the mechanisms of photoreactivation. Aged fluorescent lamps gave a reduced photoreactivation irrespective of
the filter used.
Table 4- Influence of light source on photoreactivation of
u. v.-inactivated T M V-RNA *
Light source
Light intensity
(ft.-candles)
Sunlight, I7. x. 67
Sunlight, i8. x. 67
New fluorescent and incandescent lamps
New fluorescent and incandescent lamps
Aged fluorescent and incandescent lamps
Aged fluorescent and incandescent lamps
3000
3000
500
5o0
2000
500
Aged fluorescent lamps
Black light (300 rim. to 400 nm.)
I4OO
...
Filter t
None
None
Teflon
Mylar
Teflon
Teflon or
Mylar
Teflon
None
% photoreactivation
22
33
36
26
24
25
27
38:1:
* The RNA concentration was 30 to 40/zg./ml. in o'I M-phosphate buffer, and plants were left
under the lights until maximum PR was obtained.
t Filter i is Teflon and filter 2 is Mylar film (see Methods section).
:~Data of Hidalgo-Salvatierra & McLaren (I968).
The over-all conclusion to be drawn from these experiments is that care must be
taken in choosing the light source for photoreactivation experiments. Fluorescent
lamps may not always be suitable, since the percentage photoreactivation depends on
the age of the lamp. Sunlight did not yield reproducible results even when care was
taken to keep the conditions as closely identical as possible. In our experiments
plants were inoculated at the same time of day and the plants were kept in the same
section of the greenhouse. The preferred source would appear to be a 'black light'
lamp, since the wavelength distribution of the radiation from this lamp is fairly
constant, and yields the m a x i m u m attainable photoreactivation of inactivated T M V R N A with Pinto bean as host.
Photoreactivation on bean showed a concentration effect (Table I). The percentage
photoreactivation was independent of concentration at lower concentrations but
decreased at R N A concentrations approaching I00 #g./ml.
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60
N.A. EVANS AND A. D. MCLAREN
DISCUSSION
The conditions and quantum yields published previously have varied considerably
(Tables 5, 6). Clearly there has been considerable difficulty in obtaining reproducible
values for the percentage photoreactivation of u.v.-inactivated T M V - R N A , both
within one laboratory and from one laboratory to another.
Table 5. Q u a n t u m yields f o r u.v.-inactivation o f T M V - R N A
under
non-photoreactivating conditions
Solution conditions
A
r
TMV-RNA
concentration
(/zg./ml.)
42
5o
Buffer
concerttration
(M)
o
o
6o
6o
6o
62
9I
Ioo
IOO
IO3
I42
o'o2
o.o2
o'o2
O
o
o'o/7
o-o17
o
o
660
o-oi7
¢Pz: Quantum yield, in
conditions.
Inactivating
wavelength
Assay
(rim.)
plant
254
Tobacco
254
Tobacco
~9
( × lO3)
Reference
3"8
Rushizky et al. (196o)
1"5
Merriam & Gordon
(I965)
7"3
254
Chenopodium
2"7
Werbin et al. (1966)
7"3
254
Tobacco
2"7
Werbin et al. 0966)
7"3
254
Bean
3"o
Werbin et al. (1966)
-248
Tobacco
3"4
Rushizky et al. (t96o)
-28o
Tobacco
3"8
Rushizky et al. (196o)
7"o
285
Tobacco
o'8o Kleczkowski (I967)
7"0
23o
Tobacco
o'99 Kleczkowski (I967)
-230
Tobacco
3"7
Rushizky et al. (196o)
-280
Tobacco
2-8
Merriam & Gordon
(I965)
7"0
254
Tobacco
0"64 Bawden &
Kleczkowski (I959)
mole/einstein, for inactivation of TMV-RNA under non-photoreactivating
pH
---
There are conflicting reports regarding the effect of buffer concentration on quantum
yields for u.v. inactivation of T M V - R N A . Lozeron (I967) observed that T M V - R N A
is inactivated 3"5 times slower in o. I M-sodium phosphate buffer, p H 6.o, than in water.
Rushizky et al. (I96o) observed no buffer effect. Our results (Tables 2, 3) show that
variations in buffer concentration can influence results both during irradiation and
during inoculation. We have nothing to say about the latter point. However, McMullen,
Jeskunas & Tinoco (i967) showed, with matrix rank analysis, that the optical rotatory
dispersion spectra of T M V - R N A consist of a superposition of two basic spectra
corresponding to single-stranded and double-stranded helical forms of the molecule.
Furthermore, these two forms are in equilibrium with the double-stranded form
being favoured at higher ionic strength. Since the quantum yield is lower at
higher buffer concentration, we conclude that the double-stranded form is more
resistant to u.v. radiation than the single-stranded form. That single-stranded R N A
is more sensitive to u.v. radiation is not surprising, since studies with D N A from
the bacteriophage ~X I74, which may be obtained in well defined single- and doublestranded infective forms, have shown that the singled-stranded form is the more
sensitive to u.v. radiation (Yarus & Sinsheimer, t967).
Perhaps the most suitable quantum yield of R N A for comparison with that of
T M V inactivation is that of the single-stranded form, since this is the form in the
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UV-inactivation o f T M V - R N A
61
whole virus (Franklin, Caspar & Klug, 1959). The nearest approach to these conditions
appears to obtain in distilled water. However, the hydration of R N A in water is
probably greater than in the virus, which adds another complication (McLaren, 1968).
Although in buffer the quantum yield, in the absence of photoreactivation, is
definitely lower in the highest dilution of RNA, we have no hypothesis to offer. Our
Table 6. Percentage photoreactivation o f u.v.-inactivated T M V - R N A
Solution conditions
t
TMV-RNA Buffer
concenconcentration
tration
(M)
(#g./ml.)
pH
Inactivating
wavelength
(rim.)
Assay
plant
Photoreactivating
light
Photoreactivation
References
(%)
4o
O"I
7"0
254
Bean
' Black light'
38
50
o
--
254
Tobacco
Fluorescent
33
60
o'o2
7'3
254
Tobacco
29
45
60
0"02
7"3
254
60
0"02
7"3
254
Fluorescent /
and
Chenopodium
incandescent
Bean
62
o
--
248
Tobacco
43
62
o-ol
7-1
248
Tobacco
43
142
o
--
254
Tobacco
28
I42
0
--
280
Tobacco
142
o
--
3o2
Tobacco
59
168
0
--
254
Tobacco
31
168
o
--
298
Tobacco
59
66o
o'017
7'o
254
Tobacco
Fluorescent
Daylight
3o
34
48
Hidalgo &
McLaren (I968)
Merriam&
Gordon (I965)
Werbinet al.
(I966)
Werbinet al.
(1966)
Werbinet aL
(1966)
Rushizkyet aL
(I96o)
Rushizky et aL
(I96o)
Merriam&
Gordon (I965)
Merriam&
Gordon (1965)
Merriam&
Gordon (I965)
Merriam&
Gordon (1965)
Merriam&
Gordon (I965)
Bawden&
Kleczkowski
(1959)
newest results confirm the differences observed in the reports of McLaren &Takahashi
(1957) and of Rushizky et al. 096o). To our knowledge the influence of dilution on the
structure of R N A in buffer has not been measured. In practice the quantum yield
seems to be constant over a wide range of concentration; the value observed (Table i),
namely about I q x lO-3, is about one-third that found by others at lower buffer salt
concentrations (Table 5). The quantum yield, at low ionic strength, has been reported
to be independent of the host plant (Chenopodium, bean and tobacco) and the quoted
values (Table 5) fall within the range found here (Table 2), all of which suggest that
at a given R N A concentration the quantum yield can decrease by a factor of about
three as phosphate is added to water as solvent. Perusal of Table 5 shows this for all
wavelengths at which published data can be compared.
The observed decrease in photoreactivation as the R N A concentration during
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N.A. EVANS AND A. D. MCLAREN
irradiation is increased indicates that the formation of a photoreactive lesion is suppressed at high concentration. These results contrast with those of Merriam & Gordon
(I967), who found no concentration effect for photoreactivation in tobacco and show
that care must be taken in comparing results obtained using bean as host with those
using tobacco as host. It would not be surprising if the mechanisms of photoreactivation
of inactivated TMV-RNA in bean were different from those in tobacco, since the
percentage photoreactivation depends to a marked degree on the host plant (Table 6).
Data in Table 6 also suggest that photo-recovery with a given host depends on the
wavelength of actinic u.v. (Merriam & Gordon, I965),
The percentage photoreactivation is decreased as the concentration of potassium
phosphate in the irradiation solvent is decreased (Table 2). Similar results have been
obtained with tobacco as host (Merriam & Gordon, I967). It is interesting that this
decrease in photoreactivation is also achieved by filtering out short-wavelength
radiation from the photoreactivating light (Table 4). That more than one kind of
inactivation damage can take place with differences in amounts of photoreactivation
has been discussed elsewhere (Hidalgo-Salvatierra & McLaren, I968). The nature of
these lesions is unknown.
It is tempting to compare results with synthetic polymers with our results with
RNA. Pearson & Johns (1966) have shown that production of hydrates and dimers
in irradiated poly A : U is less than in single-stranded poly U. Setlow, Carrier & Bollum
0965) showed that the principal photoproduct in poly d I : d C is a cytosine dimer and
that hydration occurs only in single-stranded poly C. If we assume that these polymers
are models for TMV-RNA, then the decrease in quantum yield for inactivation of
TMV-RNA with increased ordering (i.e. higher salt concentration) is evidence that
pyrimidine hydrate is a lethal lesion, at least in single-stranded RNA. This is consistent
with the evidence from deuterium isotope experiments (Tao, Gordon & Nester, I966).
Direct confirmation of this postulate would require measurement or pyrimidine
hydrates in u.v.-inactivated TMV-RNA. Possible methods might include a measurement of the uptake of tritium from tritiated water by uridine hydrates in TMV-RNA
(Chambers, I968) and enzymic hydrolysis of inactivated TMV-RNA using the reaction
of cytidine hydrate with hydroxylamine to increase its thermal stability (Small &
Gordon, I968). The photochemistry of TMV-RNA and TMV is reviewed elsewhere
(McLaren, I968).
Research was supported in part by U.S. Atomic Energy Commission, Contract
AT (I I-0-34, Project I I6; Photochemistry of Macromolecules, XXXI.
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