J. gen. Virol. (I975), 27, 65-70
6I
Printed in Great Britain
Temperature-sensitive Mutants of Foot-and-Mouth
Disease Virus: the Isolation of Mutants and Observations on their
Properties and Genetic Recombination
By J. S. M A C K E N Z I E * , W. R. S L A D E , J. L A K E , R. A. J. P R I S T O N ,
J A N E B I S B Y , S Y L V I A L A I N G AND J. N E W M A N
Genetics Department, Animal Virus Research Imtitute,
Pirbright, Surrey, United Kingdom
(Accepted II December ~974)
SUMMARY
A number of temperature-sensitive mutants were isolated from two strains of
foot-and-mouth disease virus (FMDV). Various properties of the mutants were
examined including comparative growth curves at permissive and restrictive
temperatures, cut-off temperatures, thermal lability and pH sensitivity. Recombination was observed between various pairs of mutants of F M D V strain Pacheco. It
occurred early in the growth cycle and the proportion of recombinants remained
constant thereafter. Maximmn recombination was achieved if the input multiplicity
of each virus was 6 p.f.u./cell or greater, provided the ratio of the input multiplicities did not vary by more than a factor of two. Day-to-day variations could be
substantially reduced by normalizing recombination frequencies in terms of a
standard cross. The results suggested that genetic mapping was possible with twoor three-factor crosses.
INTRODUCTION
Temperature-sensitive (ts) mutants of foot-and-mouth disease virus (FMDV) induced by
5-fluorouracil have been reported previously with evidence of recombination in pair-wise
crosses (Pringle, I968), and with the elucidation of various physiological characteristics of
the mutants (Pringle et al. 197o).
This paper describes the isolation of ts mutants from two further strains of FMDV with
preliminary observations on their properties and their ability to undergo efficient and
reproducible recombination.
METHODS
Virus strains and the preparation of virus stocks. Two strains of FMDV were used: strain
Pacheco of immunological type O, and strain SAz/67 of immunological type SAT z. The
viruses were obtained from the World Reference Laboratory, Pirbright, as glycerinated
suspensions, and were cloned in B H K 2I monolayer cultures by three successive plaque
passages at 37 °C, followed by three successive plaque passages at 41 °C.
Tissue culture. B H K 2I clone I3 cells were employed throughout this study. Monolayers
for restrictive temperature assays (41"3 °C) were prepared in screw-capped I oz medical flat
bottles, and for other assays, in 60 mm plastic Petri dishes. Small monolayers for mixed
infections were grown in the bottom of 6 inch test-tubes which were incubated vertically.
* Present address: University Department of Microbiology, Perth Medical Centre, Shenton Park,
Western Australia 6oo8.
5
VIR 27
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J.S. MACKENZIE AND OTHERS
Small monolayers were also prepared in Flow Microtitre plastic plates. The liquid and solid
growth media have been described previously (Lake & MacKenzie, I973).
Temperature control. Monolayers were incubated at the restrictive temperature (41"3 °C)
by total immersion in a well-insulated waterbath controlled by a Braun 'Thermomix'
thermostat control unit coupled to a Foster 'Fostronic' recorder. Assays at the permissive
temperature (37 ° C) were incubated in a humidified incubator which was gassed with a
mixture of 5 ~ CO2 in air.
Mutagenesis. Monolayer cultures were infected with a virus at an input multiplicity of
I o p.f.u./cell in the presence of either IOO #g/ml or 2oo #g/ml of 5-fluorouracil, and incubated
for 4 h at 37 °C. The medium was removed, clarified by centrifuging, and stored at
-2o
°C in I ml samples as stock mutagenized virus.
Screening procedure for ts mutants. Monolayer cultures were inoculated with stock
mutagenized virus preparations at limiting dilutions, and incubated at 37 °C. Well-isolated
plaques were picked, replated and incubated at permissive and restrictive temperatures. If
plaques were only produced after incubation at the permissive temperature, a second plaque
was picked and rescreened. Stocks of plaque-purified putative mutants were then prepared
and assayed at restrictive and permissive temperatures, and if the ratio of the e.o.p, at
41"3 and 37 °C did not exceed IO-2"°, the stock was designated a mutant. The restrictive
temperature was chosen as the highest temperature at which normal yields of virus could
be attained compared to the yields at 37 °C, The e.o.p, of parent strains varied between
I.O and o"17.
Double infections. Double infection experiments were performed in monolayer cultures
in test tubes. Each mutant was diluted in PBS to give an input multiplicity of IO p.f.u./cell,
and pairs of mutants were pre-mixed to provide a total inoculum of 20 p.f.u/cell.
After 45 rain adsorption at 37 °C the cultures were washed with pH 6-2 buffer for IO min
to inactivate unadsorbed inoculum virus, followed by three washes with PBS and three
washes with growth medium. The cultures were then incubated at 37 °C for 4 h, frozen and
thawed twice, and titrated at permissive and restrictive temperatures.
Formulae. Recombination frequencies were calculated as the per cent of wild-type (ts +)
virus in the progeny from:
AB4v - (A + B)41o
AB37°
. I00,
where AB~lo was the titre of the cross at the restrictive temperature, ABa7o was the titre of
the cross at the permissive temperature, and ( A + B)41o was the sum of titres of the selfcrosses at the restrictive temperature.
To obviate day-to-day variations (see below), recombination frequencies were standardized against a 'standard' cross (ts PI × ts PI2) which was included in each experiment. The
standardization procedure was:
(A,,d__)(XBo~) (B~,~),
(XA0.~p)
where (A~) was the recombination frequency of the standard cross (ts PI × t s P I 2 ) ,
(XAox~) and (XBex~)were the mean recombination frequencies of the standard cross and of
cross B respectively in the ex0eriment, and (B~ed)was the standardized frequency of cross B.
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Mutants o f foot-and-mouth disease virus
T a b l e I. M u t a n t
cut-off
63
temperatures
(a) Under solid overlay
Temperature
39-40 °C
Below 39 °C
ts P~o, ts
ts P I8
ts P I , ts SA4, ts
ts SA6, ts SA7,
ts P12, ts P15,
ts P27, ts P28
PI3,
Temperature
40-40'9 °C
P5,
ts
ts
ts
ts
ts
P3, ts P8, ts
PII, ts PI4,
PI7, ts P2o,
P22, ts P23,
P25, ts P26,
Above
40"9 °C
P9,
ts PI6,
ts P2I,
ts P24,
ts P29
Nil
(b) Under fluid medium
Below
39 °C
Nil
39-40 °C
40-40"5 °C
40"5-41 °C
ts P I , ts SA4,
ts PIO, ts PI3,
ts PI8
ts P5, ts SA6,
t s P 9 , ts PI2,
ts P19, ts Pz7
SA7, ts PI I,
ts P I 4 , / s PI5,
ts P16, ts PI7,
ts P2o, ts P23,
ts P26, ts P28,
ts P29
ts
Greater than
41 °C*
ts P3, ts P8, ts P 2 r ,
ts P22, t s P 2 4 ,
ts P25
* The cut-off temperature was defined as a 99 ~ drop in infectivity compared to the titre at 37 °C. These
mutants could not be classified under the definition as the drop in titre was less than 99 ~ at 4I °C.
RESULTS
Isolation of mutants
Temperature-sensitive mutants were isolated from virus stocks grown in the presence of
either IOO #g/ml or 2oo #g/ml of 5-fluorouracil. The isolation frequencies were: Pacheco,
o.68 (ts PI, ts P3) at ioo/zg/ml and 2"75 (ts P5, ts P8-ts P28) at 2oo/zg/ml; SA2/67, I.o8
( ts SA4) at 2oo/zg/ml and 2"29 (ts SA6, ts SA7).
Properties of the mutants
Cut-off temperatures
The cut-off temperature of each mutant was defined as the temperature at which a 99
loss of infectivity occurred. Two series of experiments were performed, one under agar overlay and the other under liquid overlay. Plaque titrations of the mutants in I oz medical flat
bottles were incubated by total immersion in water baths at 37, 39"2, 4o and 40"9 °C. The
results are shown in Table I a. Under liquid overlay, replicate fitrations were prepared in
small monolayer cultures in Flow Microtitre plastic plates (Lake & MacKenzie, 1973) which
were also incubated by total immersion in controlled water baths at 37, 38, 40, 4o'5 and
41 °C. End points were plotted and the cut-off temperature indicated at the point showing
a 99 ~ loss of infectivity. The results are shown in Table I b.
All the mutants exhibited relatively high cut-off temperatures under both conditions, but
particularly under liquid medium where none were found with a cut-off temperature below
39 °C, and six mutants, ts P3, ts P8, ts P2I, ts P22, ts P24 and ts P25 were found to have
insufficient penetrance to provide a cut-off temperature below 41 °C. Under solid medium,
all the mutants had cut-off temperatures below 4o'9 °C, and three mutants, ts PIo, ts PI3
and ts P 18 had cut-off temperatures below 39 °C.
5-2
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J. S. M A C K E N Z I E A N D O T H E R S
Table 2. Sensitivity of the mutants to pH 6.8 and p H 6.6
Virus
Control titre
at pH 7"2
(log10 p.f.u.)
Pacheco t s +
t s P27
t s P22
t s P28
t s P26
t s PzI
t s PI8
t s P24
t s PI7
t s Pzo
t s PI3
t s PI
t s P19
tsPII
t s P15
t s P8
t s P25
t s PI2
t s PI6
t s P9
t s PIO
t s P23
t s PI4
t s P29
5'39
5'Ol
4.85
4'83
SA2/67 t s +
t s SA7
t s SA4
t s SA6
5"77
3"77
4"76
3"7o
5"24
5"30
5'48
5"92
5"o2
4"83
4"8o
5"o2
5"49
4'52
5"46
3.51
4"92
4'59
5"32
5'39
5"13
4"47
5'84
5.28
c
~ survival
~"
pH 6'8
pH 6'6
ioo
1oo
72
93
76
lOO
IOO
53
50
54
83
43
60
20
23
68
62
zo
5*
25
II
6*
4*
2*
29
o. I*
o'3*
o'o5*
2i
23
17
II
11
9
7
6
6
3
3
3
2
2
2
I*
o'7*
0"6*
o-2"
o'o9*
0"05*
o'o3*
o'o1"
o.oi*
2"5
o.o6*
o-o2*
Nil (< o.o2)*
* Mutants considered to be significantly acid labile with a survival of less than to ~ compared to the
parent viruses.
Thermostability of infectivity
The mutants were examined for their ability to withstand exposure to 45 and 53 °C for
I h. Under these conditions, the titre of the Pacheco parent virus strain dropped from
3"5 x IO7 p.f.u./o.2 ml to 6"9 x Io 6 p.f.u, and 2-2 × IOs p.f.u, respectively, and the titre of the
SA2/67 parent strain dropped from 1.7 x IO7 p.f.u./0"2 ml to 6-6 x Io 5 p.f.u, at 45 °C but
was totally thermolabile at 53 °C. Stock preparations of each mutant were diluted tenfold
in PBS and placed in waterbaths at 45 or 53 °C. After incubation for I h, the samples were
chilled in ice and immediately titrated in parallel with unexposed controls. None of the
Pacheco mutants were significantly thermolabile at either temperature compared to the
parent strains; the SA2/67 mutants were totally thermolabile at 53 °C, but did not exhibit
increased thermolability at 45 °C.
Sensitivity to acid p H
Foot-and-mouth disease virus is pH sensitive being rapidly inactivated below pH 6'4.
The mutants were compared to the parent virus strains for their degree of sensitivity to
acid conditions. Stock preparations of each mutant and tile parent viruses were dialysed
against o.I M-phosphate buffer, pH 7"2, overnight at 4 °C, and then diluted tenfold in a
range of phosphate buffers at pH 7"2, 7"o, 6.8, 6-6, 6"4, 6.2 and 5"9, and held overnight at
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Mutants o f foot-and-mouth disease virus
I
105
I
i
I
i
I
I
I
65
I
m
t"q
104
q
10s
1.25 1-75 2-25 2-75 3.25 3.75 4.25 4.75 5.25
Time (h)
Fig. I. Comparative single cycle growth curves at 41"3° C for Pacheco ts+, ts P8 and ts PIz. The
mutants were classified into three groups on the basis of their yields at 41'3 °C. The yields as a percentage of the parent virus were o-oo2 to o'oi5 ~ for group I, 0"I2 tO 0"6 ~oo for group 2; and 16 to
28 ~ for group 3. O--tl, Pacheco ts+; (3--0, ts P8 (group 3); 11--O, ts Plz (group 2).
4 °C. T h e samples were
in parallel. T h e results
T a b l e 2. Seven m u t a n t s
p H 6.6. N o lability was
survival b e l o w p H 6.2.
restored to p H 7.2 b y dilution in a p p r o p r i a t e buffer, a n d t i t r a t e d
o f titrations after exposure to p H 6.8 a n d p H 6.6 are shown in
were significantly p H sensitive at p H 6-8, a n d twelve m u t a n t s a t
observed f o r a n y o f the m u t a n t s a b o v e p H 6-8, a n d there was no
Comparative growth cycles at 37 and 41"3 °C
C o m p a r a t i v e g r o w t h cycles at 37 a n d 41"3 °C were p e r f o r m e d for twelve Pacheco m u t a n t s
a n d the p a r e n t virus. W a s h e d m o n o l a y e r cultures were i n o c u l a t e d with virus at a n i n p u t
multiplicity o f 5 p.f.u./cell a n d a d s o r b e d for 15 rain at 37 °C. U n a d s o r b e d virus was inactivated b y i o min exposure to p H 6.2 buffer, the cells w a s h e d twice, a n d g r o w t h m e d i u m
a d d e d which h a d been p r e - w a r m e d to 37 °C. The cultures were i n c u b a t e d in w a t e r b a t h s set
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66
J. S. MACKENZIE AND OTHERS
Table 3. Effect o f input multiplicity on recombination frequency
in cross ts P I x ts P I 2
Input multiplicity/cell
c
ts P1
ts PI2
12
I2
6
3
6
3
I "5
1 "5
o'75
[2
i2
12
I2
6
3
I'5
0"75
recombination*
(+ twice the standard
error)
o'75
6
3
1"5
0"75
12
I2
12
12
o'39 + o'07
0"35___0'05
0 " 2 2 --b 0 - 0 5
0.08 _ 0.02
0'05 + O'OI
o'44 + 0"o9
o'I3+o'o4
0"04+ 0'03
0 " 0 2 -I- 0 ' 0 I
0"37+o'o3
o-I9+o'o5
o-]o+o.o4
0'03 + 0"02
* Mean recombination frequency of four replicates.
at 37 or 4I'3 °C, and 2 ml samples withdrawn at 30 min intervals over 5.25 h. Each sample
removed was replaced with an equivalent amount of pre-warmed medium, and the samples
frozen until the end of the growth cycles. They were then titrated in parallel. No significant
differences between the growth cycles of the mutants and the parent virus were found at
37 °C. At 4I'3 °C however, the mutants fell into three clearly distinct groups: group I
(ts P t , ts PIo, ts PI3 and ts PI8) were virtually unable to produce infectious virus; group 2
(ts P9, ts PI2, ts P~4, ts Pr 5, ts PI6, ts P I 7 and ts PI9) produced low quantities of infectious
virus; and group 3 (ts P8 and ts P I I ) were able to produce relatively high quantities of
infectious virus. The growth curves at 4I'3 °C for representatives of group 2 (ts PI2) and
group 3 (ts PI8) are shown in Fig. I with the growth curve of the Pacheco parent virus.
Character&tics o f genetic recombination
Effect o f input multiplicity on recombination frequency
Preliminary recombination experiments had shown that recombination occurred readily
between certain pairs of Pacheco ts mutants. The effect of input multiplicity on recombination
frequencies was examined in pairwise crosses of two mutants, ts PI and ts PI2. Twofold
dilutions of each mutant were prepared in PBS, and various combinations of the dilutions
were mixed and inoculated onto small monolayer cultures with five replicates for each
combination, as described above. Mean recombination frequencies of the various combinations were calculated from the yields assayed at restrictive and permissive temperatures
(Table 3). The results indicated that for this pair of mutants, a maximum recombination
frequency required a minimum of 6 infectious particles (p.f.u.) of each mutant, and that the
input ratio had to be within a twofold margin. At lower multiplicities or larger input ratios,
the mean recombination frequencies were significantly reduced.
Effect o f time o f incubation on recombination frequency
The effect of length of incubation on the frequency of recombination was examined in the
cross ts PI x ts PI2. Small monolayer cultures were inoculated at an input multiplicity of
I2 p.f.u./cell with each mutant and incubated at 37 °C. Three replicate cultures were with-
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M u t a n t s o f f o o t - a n d m o u t h disease virus
I
i
'
'
'
'.A.-~
'
~. _ -e. ~ .~ ~....~
.~
67
'
105
104
'l
QI
I
I
103
I
I
'~" "1,
iI
~fJ
7
;
I
I
I
!
10 2
/?\
ZX
/
l0 t
I
• ^
1
,J/t
2
,
,
3
Time (h)
4
1,,
5
Fig, z. The time course of appearance of recombinant virus in the cross ts PI x ts Plz in relation to
the total progeny in the cross. © - - O, mixed infection titre at 37 °C (ts P t x ts P r z); • - - 0, mixed
infection titre at 4I'3 °C (ts P i × ts PI 2) ; O - - O, single infection titre of ts PI at 37 °C; 0 - - 0 , single
infection titre of ts PI2 at 37 °C; A--L~, single infection titre of ts PI at 4I'3 °C; A__A, single
infection titre of ts PI2 at 4I'3 °C.
d r a w n at v a r i o u s intervals d u r i n g the g r o w t h cycle a n d frozen a n d t h a w e d twice before the
yields were t i t r a t e d at restrictive a n d permissive temperatures. Single cultures o f self-cross
controls were w i t h d r a w n at the same intervals. R e c o m b i n a n t s could be detected f r o m
I ' 5 h post-infection a n d the g r o w t h cycles at b o t h t e m p e r a t u r e s reached a p e a k a b o u t 4 h
post-infection (Fig. 2). The r e c o m b i n a t i o n frequency d i d n o t v a r y significantly d u r i n g this
period.
Stability o f ts + recombinants
Twenty-seven ts+~recombinants were p i c k e d f r o m two crosses, ts P I x ts P I 2 a n d ts P I
× ts P I 8 , a n d subjected to three successive passages b y p l a q u e isolation with each passage
being screened at permissive a n d restrictive temperatures. A l l r e c o m b i n a n t s were f o u n d to
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68
J. S. M A C K E N Z I E A N D O T H E R S
Table 4. Effect o f standardization on variation in recombination frequency o f crosses ts P I
x ts P I z , ts P I x ts PI3, and ts P I 2 x ts P I 3
Experiment t* :
recombination frequency
Experiment 2t :
recombination frequency
Cross
Before
standardization
After
standardization
Before
standardization
After
standardization
PI x PI2
PI x PI3
PI2 x PI3
o.2I
o-I7
o'03
0-27
0"42
0"33
o'o9
0"27
o-2I
0.o6
o'22
0-o4
* Mean recombination frequencies of 6 replicate cultures.
t Mean recombination frequencies of 5 replicate cultures.
be stable for the ts + character, and no evidence was observed to indicate the presence of
complementing heterozygotes.
Standardization o f recombination frequencies
The recombination frequencies from replicate samples of a given cross in each experiment
were highly reproducible if the assays were performed in parallel, but a considerable variation was observed between assays of the same experiment performed on different days, or
between different experiments. For any two crosses, however, the ratio of the mean recombination frequencies in different experiments was found to be constant. It was possible,
therefore, that day-to-day variation could be obviated if the mean recombination frequency
of each cross in an experiment was related to a standard cross, which was included in each
experiment, and for which an accurate recombination frequency had been pre-determined
from a large number of replicate samples.
To obtain a standard cross, 22 replicate samples of the cross ts PI x ts PI2 were prepared
in small monolayer cultures, and after a 4 h incubation period, the yields were assayed in
parallel at the permissive and restrictive temperatures. The mean recombination frequency
was found to be o.27, with a standard deviation of o'o5 and a standard error of the mean o f
O'OI.
To determine the effectiveness of standardization, experiments were performed with
ts PI, ts PI2 and ts PI3 in small monolayer cultures, with six replicates of each cross in the
first experiment and five replicates in the second experiment. The mean recombination
frequencies were calculated and standardized against the ts P1 x ts PI2 standard (Table 4).
These results indicated that day-to-day variation could be substantially reduced in these
three crosses by standardization.
DISCUSSION
The isolation of a number of ts mutants has been described from two strains of F M D V
with observations on some of their properties and on the ability of some of the mutants to
undergo genetic recombination. The conditions employed for their isolation were chosen
to provide an optimum isolation frequency but at the same time to lessen the chance o f
isolating sister and double mutants. To minimize the occurrence of sister mutants, mutagenesis was carried out with cloned parental viruses over a single growth cycle. Two concentrations of 5-fluorouracil were used. At the higher concentration (2oo/zg/ml), the isolation frequency in the absence of selection pressure was sufficiently high to enable mutants
to be isolated relatively easily, yet was less than that reported in a previous study (Pringle,
1968).
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M u t a n t s o f f o o t - a n d - m o u t h disease virus
69
Comparative growth cycles of some of the mutants at permissive and restrictive temperatures permitted their classification into three clearly distinct groups by virtue of the yields
of infectious virus at the restrictive temperature. This grouping could be correlated with
the mutant cut-off temperatures under liquid overlay; the group yielding greater titres of
infectious virus at the restrictive temperature having the highest cut-off temperatures.
This would suggest that high yields of infectious virus were due to 'leakiness' rather than
growth of revertant virus.
The cut-off temperatures under liquid medium were, in general, higher than those under
solid overlay, a finding similar to that reported for ts mutants of influenza virus (MacKenzie & Dimmock, 1973). The reasons for this are not understood, but it is possible that
solid overlay restricts the spread of revertant or 'leak' virus, whereas under liquid overlay,
virus is able to spread to uninfected cells resulting in increased yields at that temperature.
Thermolability and acid 1ability reflect properties of the virus coat proteins, and a
mutation in a cistron responsible for a coat protein might, therefore, render the virus more
susceptible to inactivation by exposure to heat or acid conditions. No differences were found
between the mutants and their parent viruses to inactivation by exposure to 45 or 53 °C,
but considerable variation was observed in the susceptibility of the mutants to inactivation
by exposure to pH 6.8 and pH 6.6. This property, however, cannot be interpreted on the
basis of temperature-sensitivity without a careful analysis of ts + revertants since other
mutations must have been accumulated during mutagenesis.
The demonstration of genetic recombination between ts mutants isolated from the Kenya
3/57 strain of immunological type SAT2 by Pringle 0968) has been extended in this study
to ts mutants of immunological type O. The recombination frequencies observed were
similar in magnitude to those reported by Pringle, and to those by Cooper (1968) between
poliovirus ts mutants, but certain anomalies were found in the conditions required to
achieve maximum recombination frequencies. In the cross ts PI x ts PI2, the minimum
multiplicity of each of the two input viruses for maximum recombination was found to be
approx. 6 p.f.u./cell, and no difference was observed at higher multiplicities provided the
ratio of multiplicities did not exceed a twofold margin. These results suggested that multiplicity
was fairly critical, and also that non-infectious viruses of FMDV did not take an active part in
recombination. With poliovirus, however, recombination frequencies showed little or no
variation over a wider range of ratios (Cooper, 1968).
The recombination frequency for cross ts PI x ts PI2 was not found to vary significantly
during a single growth cycle. The growth curve of recombinant virus paralleled the curve
for total progeny virus in the exponential phase which indicated that recombination was an
early event, and must occur before replication. A similar result was reported by Pringle
(I968). A slight but significant increase in recombination frequency was found during the
growth cycle of a cross between poliovirus mutants which suggested to Cooper (1968) that
recombination events were not necessarily linked to replication. This was based on the
amount of R N A replication at the times during the growth cycle at which samples were
examined.
Considerable variation in recombination frequency was observed for any one cross
performed or assayed on different occasions. A similar variation was reported by Pringle
et al. (197o), and to a lesser extent, by Cooper (~968) in crosses between poliovirus ts
mutants. Within each experiment, however, the relative recombination frequencies of
different crosses assayed together were comparable, and so by including a standard cross in
each experiment and relating the recombination frequencies for other crosses to it, this
variation could be practically eliminated. Guanidine-resistant mutants have also been
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70
J.S. MACKENZIE
AND
OTHERS
isolated from the Pacheco strain o f F M D V (J. Lake, J. Bisby, J. S. MacKenzie, u n p u b l i s h e d
observations), a n d so by using conditions for m a x i m u m standardized r e c o m b i n a t i o n
described in this report, accurate genetic m a p p i n g should be feasible by means of threefactor crosses.
REFERENCES
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