J. gen. Virol. (1981), 56, 153-162.
Printed in Great Britain
153
Key words: interferon/encephalomyocarditis virus~protein synthesis~membrane integrity
Protein Synthesis and Membrane Integrity in Interferon-treated HeLa
Cells Infected with Encephalomyocarditis Virus
By A L B E R T O
MU/~/OZ AND L U I S C A R R A S C O *
Department of Microbiology, Centro de Biologfa Molecular, Universidad Aut6noma, Canto
Blanco, Madrid-34, Spain
(Accepted 1 May 1981)
SUMMARY
The survival of interferon (IFN)-treated cells after encephalomyocarditis (EMC)
virus infection depends on both the concentration of interferon and the multiplicity of
infection (m.o.i.) used. The cell survived EMC infection if a high IFN/m.o.i. ratio
was used in the experiment, whereas cell death took place at low IFN/m.o.i. ratios,
even if IFN is also present during infection. Analysis by polyacrylamide gel
electrophoresis of the proteins synthesized in IFN-treated cells subsequently infected
with EMC indicated that no virus proteins were detected at either low or high
multiplicities of infection. However, at low m.o.i, the cell survived and continued
synthesizing cellular proteins exclusively, whereas at high m.o.i, a drastic shut-off of
host protein synthesis took place. Virus which had been inactivated by u.v.
irradiation was unable to cause the shut-off of host protein synthesis, either in control
or in IFN-treated cells. This result suggests that some virus gene expression occurs in
cells treated with IFN, although no virus protein synthesis was detected. The
synthesis of virus RNA was also strongly inhibited after treatment of cells with IFN.
The integrity of the cell membrane in control and in IFN-treated cells was studied
by analysing the S~Rb+ ion leakage, the thymidine pool, the choline uptake and the
entry of the translation inhibitor hygromycin B, to which ceils are impermeable, at
different times after EMC infection. The results obtained indicate that the early
membrane leakiness observed after virus infection is not prevented by IFN
treatment. However, the development of late leakiness to S~Rb+ ions, thymidine and
hygromycin B was not observed in IFN-treated cells.
INTRODUCTION
Picornavirus infection of cultured cells leads to a profound inhibition of host protein
synthesis (Carrasco & Smith, 1976, 1980; Lucas-Lenard, 1979). This inhibition is not
prevented by treatment of cells with interferon (Levy, 1964; Gauntt & Lockart, 1968).
Treatment of L cells with homologous interferon and subsequent infection with EMC virus
produces a strong inhibition of both cellular and virus protein synthesis (Kerr et aL, 1973).
Similarly, infection of interferon-treated cells with vaccinia virus (Joklik & Merigan, 1966) or
vesicular stomatitis virus (Yamazaki & Wagner, 1970) produces an indiscriminate inhibition
of both cellular and virus protein synthesis, which leads to cell death. However, under some
conditions, an early shut-off of host protein synthesis is observed in interferon-treated L cells
after mengovirus infection. This is followed by recovery of the cellular translation capacity
(Falcoff & Sanceau, 1979). In some of these virus-cell systems it is not clear whether the
inhibition of virus production by interferon, thought to be a consequence of the interference
with virus translation, leads to cell death or whether the cell recovers its translational capacity
0022-1317/81/0000-4641 $02.00 @ 1981 SGM
154
A. MUI~OZ AND L. CARRASCO
and survives after virus infection. We report here an analysis of this inhibition and of the
cellular functions which are altered after virus infection.
METHODS
Cells and virus. HeLa cells and L929 cells were grown in Dulbecco's modified Eagle's
medium (E4D) supplemented with 10% newborn calf serum (Difco) (E4D 10 medium) and
incubated at 37 °C in a 5 % CO2 atmosphere. Encephalomyocarditis (EMC)virus was grown
on L929 cells in a mixture of Eagle's medium, phosphate-buffered saline (PBS) solution and
E4D medium (80:15:5) supplemented with 1% newborn calf serum. The fraction obtained
after removal of cell debris by low-speed centrifugation was used as source of virus.
Virus infection and measurement of protein synthesis. HeLa cells grown on 16 mm diam.
24-well plates (Falcon Plastics) were infected with EMC virus at the m.o.i, described in each
experiment. After 1 h incubation at 37 °C the medium was removed and 1 ml E4D2 was
added. Time of virus addition was considered - 1 h and 0 time was taken when the virus was
removed. The incubation at 37 °C was continued until the labelling period. For this purpose
0.5 ml methionine-free E4D1 medium and 0.11 pCi [35S]methionine (1100 Ci/mmol; 5-4
mCi/ml, The Radiochemical Centre, Amersham) were added to the cells for 1 h pulse. The
medium was then removed, the cells were washed with PBS solution and precipitated with
5 % trichloroacetic acid (TCA). After 5 rain the TCA was removed and the cell monolayer
washed three times with ethanol, dried under an infrared lamp and dissolved with 250 pl 0.1
M-NaOH plus 1% SDS. 125 pl were counted in an Intertechnique scintillation spectrometer.
Analysis of the proteins synthesized in virus-infected cells. HeLa cells were grown on 30
mm Petri dishes and pulsed with 5.4 pCi [35S]methionine. At the end of the pulse period the
cells were washed with 1 ml PBS solution and dissolved in 200/11 0.02 M-NaOH plus 1%
SDS and 200 pl sample buffer (62-5 mM-tris pH 6-8; 2% SDS; 0-1 M-dithiothreitol; 17%
glycerol and 0.024 % bromophenol blue as indicator). Each sample was sonicated to reduce
viscosity and heated to 90 °C for 5 min. Ten pl were applied to a 15% polyacrylamide gel
and run overnight at 30 V. Fluorography of the gel was carried out with 2,5diphenyloxazole/dimethyl sulphoxide (20%, w/w). The dried gels were exposed using XS-5 X
ray films (Kodak).
Interferons. Human lymphoblastoid interferon [HuIFN-ct (Ly); 1-6 x 106 IU/mg protein]
was a generous gift of Drs Finter, Fantes and Johnston, Wellcome Research Laboratories,
Beckenham, U.K.
Nucleic acid synthesis and thymidine pool in EMC virus-infected HeLa cells. Cellular
DNA synthesis was measured by estimating the incorporation of methyl-[3H]thymidine (40
to 60 Ci/mmol, 1 mCi/ml; The Radiochemical Centre, Amersham) into TCA precipitate
material. At the times indicated after infection 1 pCi/ml of the isotope was added and after a
1 h pulse the cells were processed as described for protein synthesis. The thymidine pool was
measured by estimating the cell radioactivity soluble in 5 % TCA, after incubation of cells
with 1 pCi/ml methyl-[3Hlthymidine for 1 h at 37 °C, in a culture medium supplemented with
2 % calf serum.
Measurement ofS6Rb + content. HeLa cells grown in E4D 10 medium were placed in 280 gl
of a mixture of methionine-free E4D1 medium:E4D10 medium (3:1); 0.2 pCi S6Rb+ (1
mCi/ml; The Radiochemical Centre, Amersham) was added, and the cells were incubated for
17 h at 37 °C. Virus infection was then carried out while maintaining constant the 86Rb+
concentration. At the times indicated the cells were pulsed with 0.14 /~Ci [aSS]methionine.
After 1 h incubation, the medium was removed and the cells were washed three times with 1
ml PBS, and 0.5 ml 5 % TCA was added to extract the 86Rb+ from the cells. The radioactivity
of 0.4 ml of the TCA extract was determined by estimating the Cerenkov radiation in a liquid
155
Translation in interferon-treated cells
(a) 1
0
(b)'
/-_::.2o.
I00
//1°
50
0
10
100
(c) :
'
0
10
100
Interferon (IU/ml)
'
ilOO
0
10
100
Fig. 1. Influence of interferon concentration, multiplicity of infection and cell confluence on the antiviral
state induced by h u m a n interferon in HeLa cells. Interferon was added 18 h before E M C virus infection
and the level of protein synthesis at 24 h after infection was measured by a 1 h pulse of [35S]methionine.
Control were uninfected cells treated with the corresponding interferon concentration. Numbers indicate
the m.o.i, employed. (a) 4.4 × l0 s cells/16 m m diam. well; (b) 5.3 × 105 cells/16 m m well; (c) 6-1 ×
10 ~ cells/16 m m well.
scintillation counter. The level of protein synthesis was estimated in parallel cultures as
described above.
Choline uptake. Choline uptake by HeLa cells was measured by estimating the
incorporation of methyl-[3H]choline (84 Ci/mmol; 1 mCi/ml; The Radiochemical Centre,
Amersham) into TCA-precipitable material. After virus adsorption for 1 h at 37 °C, the cells
were placed in E4D 1 medium containing only 5 % of the normal vitamin concentration and 2
gCi/ml methyl-[3H]choline. At the times indicated the medium was removed, the cells were
washed twice with PBS and precipitated with 5 % TCA. The radioactivity incorporated into
insoluble material was determined as in the case of protein synthesis measurement.
RESULTS
We first analysed protein synthesis after treatment of HeLa cells with different interferon
concentrations followed by infection with different multiplicities of EMC virus. Fig. 1 shows
the analysis of protein synthesis in HeLa cells 24 h after infection with EMC virus. A high
m.o.i, led to cell death as assessed by their appearance under the microscope and loss of the
capacity to synthesize proteins, whereas if the cells were treated with HulFN-a (Ly) and
infected at low multiplicities, they survived virus infection. The percentage of cells that survive
at a given multiplicity depended on the concentration of interferon used. The prevention of
cell death depends on the ratio of interferon to m.o.i. The higher this ratio the better the
protection achieved. The influence of cell confluence is also noted in Fig. 1. An increased cell
survival after EMC virus infection is observed in the very confluent cell cultures (6-1 x 105
cells/16 mm diam. well; Fig. 1 c). We next analysed the pattern of the proteins synthesized at
different times after infection after treatment with 200 IU/ml HulFN-a (Ly) and infection
with EMC virus at either 20 or 200 p.f.u./cell. Inhibition of protein synthesis was apparent in
control cells not treated with interferon using either m.o.i. (Fig. 2), whereas if cells pretreated
with IFN were infected with 20 p.f.u./cell only a slight decrease in protein synthesis 3 to 4 h
after infection was observed, followed by a recovery, the infected cells continuing to
synthesize proteins at control levels. When the IFN-treated cells were infected at 200
p.f.u./ceU the inhibition of protein synthesis was pronounced and irreversible. Analysis by
polyacrylamide gel electrophoresis of the proteins synthesized showed that no virus proteins
could be detected in IFN-treated cells (Fig. 3). These results indicate that two different
156
A. MUNOZ AND L. CARRASCO
(a) I
I
I
I
I
(b)
I
I
I
I
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120
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o - - o-,
xx
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6
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I
l0 0
2 4 6 8 l0
Time (h)
Fig. 2. Kinetics of protein synthesis in human HeLa cells infected with EMC virus at high and low
multiplicity,untreated (O) or treated (0) with human interferon(200 IU/ml) 18 h beforeinfection.The
cell monolayerswerelabelledand processedas indicatedin Methods. (a) M.o.i. 20; (b) m.o.i. 200.
2
4
situations could result after EMC virus infection of IFN-treated HeLa cells. If the IFN/m.o.i.
ratio was high, no inhibition of host protein synthesis was observed, the cells synthesizing
cellular proteins exclusively and surviving the viral infection. At low IFN/m.o.i. ratios
inhibition of host protein synthesis occurred but still no virus proteins were detected.
However, the cytopathic effect developed and the cell died, as did control cells not treated
with interferon. Similar results were obtained with mouse L cells infected with EMC virus and
mouse IFN (data not shown), suggesting that these observations were not specific for human
cells and HuIFN-ct (Ly). The results can be reconciled with the previous findings by Kerr et
al. (1973) and Esteban (1975) who found that EMC virus infection oflFN-treated L cells led
to the total inhibition of protein synthesis because they used a low IFN concentration (40
IU/ml). On the other hand, the results reported by Falcoff & Sanceau (1979) in L cells
treated with 500 IU/ml IFN and infected with mengovirus at 20 p.f.u./cell are more similar to
those observed by us in Fig. 3 (a). In conclusion, the effect of virus infection on protein
synthesis varies considerably and depends on the IFN/m.o.i. ratio.
In order to establish whether the continuous presence of IFN could prevent cell death at
low IFN/m.o.i. ratios, HuIFN-a (Ly) was added either 22 h before infection and then
removed from the medium, or was continuously present before and after EMC virus infection.
The level of protein synthesis in both instances was similar at 8 h and at 30 h after infection,
suggesting that cell death does, in fact, take place if a low IFN/m.o.i. ratio is used and the
continuous presence of IFN does not influence this result (results not shown).
In IFN-treated RK cells infected with vesicular stomatitis virus the inhibition of protein
synthesis is due to a component of the virion (Yamazaki & Wagner, 1970), while in
mengovirus-infected cells it has been suggested that the inhibition of protein synthesis
observed in IFN-treated cells was a consequence of virus double-stranded RNA production
(Faleoff & Sanceau, 1979). It was of interest to determine whether virus gene expression was
necessary for the inhibition of protein synthesis observed at low IFN/m.o.i. ratios. When
EMC virus was inactivated by more than two log units using u.v. irradiation, no inhibition of
protein synthesis was observed in IFN-treated HeLa cells (Fig. 4). This result suggests that
although no virus proteins could be detected, virus gene expression was necessary for the
shut-off to occur, both in control and IFN-treated HeLa cells.
In a number of virus-cell systems, the cellular membrane becomes permeable to ions some
time after infection, when the bulk of virion proteins are synthesized (Carrasco & Smith,
1976; Egberts et al., 1977; Nair et al., 1978; Garry et al., 1979). The effect o f l F N treatment
Translation in interferon-treated cells
(a)
1
2
3
4
5
157
(b)
6
7
8
9
1
2
3
4
5
7
6
(a)
(c)
1
2
3
4
5
6
8
7
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9 1011
1
2
3
4
5
6
7
ct
F
Fig. 3. Analysis by polyacrylamide gel electrophoresis of the polypeptides synthesized in HeLa cells
infected with EMC virus at (c, d) high (200 p.f.u./cell) and (a, b) low (4 p.f.u./cell) multiplicities of
infection, (b, d) untreated or (a, c) treated with human interferon (200 IU/ml). Cellular actin (Act) and
polypeptides of virus origin are indicated (Carrasco & Smith, 1980). Numbers above the tracks indicate
time after infection (h).
on S6Rb+ leakage after E M C virus infection is shown in Fig. 5. If a low multiplicity o f E M C
virus was used, there was a correlation between the inhibition of protein synthesis and the
decrease in86Rb+ content in control cells not treated with interferon (Fig. 5 a, b). If the cells
were infected with a higher multiplicity of infection, there was an earlier inhibition of host
protein synthesis but 86Rb+ leakage did not occur until 5 h after infection when the bulk o f
virus protein synthesis takes place (Fig. 5 c, d). If the cells were treated with IFN, the S6Rb+
158
A. MUI~OZ AND L. C A R R A S C O
I
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8
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x
=
--
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t
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4
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Fig. 4. Kinetics of protein synthesis in HeLa cellstreated 18 h before infectionwith 50 IU/ml interferon
(
) or untreated ( - - - ) . The cells were infected with normal (O), heat-inactivated (IS/) or
u.v.-inactivated (&) EMC virus (100 p.f.u./cell). The heat inactivation was carried out by heating the
virus preparation at 60 °C for 10 rain. The u.v. inactivation was carried out by exposingthe EMC virus
for 3 rain to a G-E Germicidal GST5 General Electric lamp at a distance of 3 cm.
(a)
I
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-(b)
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6
8
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10 0
2
Time (h)
4
6
8
I
10
Fig. 5. Simultaneous kinetics of S~Rb÷content and protein synthesis in EMC virus-infectedHeLa cells
untreated or treated with 50 IU/ml IFN. (a) IFN-treated cells, m.o.i. 5; (b) untreated cells, m.o.i. 5; (c)
IFN-treated cells, m.o.i. 100; (d) untreated cells, m.o.i. 100. 0 , [35S]methionineincorporation; O,
86Rb+content.
leakage was prevented, both when inhibition of host protein synthesis did not occur because a
low multiplicity was used and also when the inhibition of host protein synthesis took place at
the higher multiplicity of infection.
M e m b r a n e integrity was also analysed by means of the translation inhibitor h y g r o m y c i n B,
which does not enter normal cells, but which readily penetrates virus-infected cells both early
after infection and also late in virus infection. Fig. 6 indicates that the early m e m b r a n e
leakiness to hygromycin B developed in both interferon-treated and control ceils, but the
modification of m e m b r a n e permeability late in infection is prevented if the cells are pretreated
with interferon. These results are in agreement with those illustrated in Fig. 5 for 86Rb+
leakage.
Translation in interferon-treated cells
(C/)
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(b)
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6
8
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Time (h)
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4
6
8
i0
Fig. 6. Effect of interferon treatment on EMC virus-induced permeability to hygromycin B (H.B.).
Permeability to H.B. of HeLa cells untreated (O) or treated (O) with 50 IU/ml IFN 18 h before
infection was estimated by measurement of the level of protein synthesis in ceils treated with
[35S]methionine (including 1 mM-H.B.) for 1 h. The controls were cells not treated with the inhibitor and
infected at the indicated multiplicity. (a) M.o.i. 5; (b) m.o.i. 100. The radioactivity incorporated in
uninfected IFN- and H.B.-untreated cells was 44 835 ct/min, in uninfected IFN-untreated H.B.-treated
cells 38441 ct/min, in uninfected IFN-treated H.B.-untreated cells 36 854 ct/min and in uninfected
IFN- and H.B.-treated cells 31 116 ct/min.
.(a)
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20
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, _ _ , J ~ x ~ - , , _ ~ , , , ~ - - , ---~---•
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2
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Time (h)
%0
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8
10
Fig. 7. Time course of (a) DNA synthesis and (b) entry of radioactivity into the thymidine pool in
HeLa cells infected with the EMC virus at high and low multiplicities and untreated or treated with IFN
(50 IU/ml) 18 h before infection. A, Untreated cells, m.o.i. 5; A, IFN-treated cells, m.o.i. 5; O ,
untreated cells, m.o.i. 100; O , IFN-treated cells, m.o.i. 100.
T h e synthesis o f cellular D N A and the t h y m i d i n e c o n t e n t o f the cells was a n a l y s e d b o t h in
c o n t r o l and I F N - t r e a t e d cells (Fig. 7). D N A synthesis is drastically inhibited in E M C - i n f e c t e d
cells p e r h a p s as a c o n s e q u e n c e o f inhibition o f protein synthesis. I f the cells w e r e p r e t r e a t e d
160
A. MUI'qOZ AND
(a)
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L. CARRASCO
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(b)
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2
4
6
8
Time (h)
Fig. 8. Kinetics of methyl-[3H]choline uptake in HeLa cells. IFN (50 IU/ml) was added 18 h before
infection. I-7, Control untreated, uninfected cells; ~ , untreated infected cells; A, IFN-treated infected
cells. (a) M.o.i. 5; (b) m.o.i. 100.
with IFN, a lower level of DNA synthesis occurred. However, the inhibition only occurred if
a high EMC virus input was used. The thymidine pool decreased in EMC virus-infected cells
from 5 h after infection, which is about the time when membrane integrity is damaged as
measured by 86Rb+ content and hygromycin B entry. However, in IFN-treated EMC virusinfected cells the thymidine pool stayed constant, except at a long time after infection with a
high multiplicity of EMC virus.
Finally, the capacity of the cells to take up choline was analysed. In several virus-infected
cells there is an increased membrane proliferation, accompanied by an increase in the uptake
of choline, which is used in the synthesis of several membrane constituents (Penman, 1965;
Plagemann et al., 1970). Such an increase was observed in EMC virus-infected HeLa cells
(Fig. 8). Surprisingly enough, if the cells were pretreated with IFN and infected with EMC
virus a similar increase is observed, suggesting that membrane proliferation probably also
takes place in interferon-treated EMC virus-infected cells.
DISCUSSION
Human cells treated with IFN do not support the growth of a number of animal viruses
(Friedman, 1977; Burke, 1979). The failure of picornaviruses to multiply in IFN-treated cells
is believed to lie in an impairment of protein synthesis. However, it was not clear from
previous studies whether the IFN-treated cells were able to overcome viral infection and
survive (Falcoff & Sanceau, 1979), or whether the cell dies (Esteban, 1975; Metz, 1975). Our
results indicate that both situations can take place, and that the cellular response depends on
the IFN/m.o.i. ratio used. When treated with a high concentration of IFN and infected at a
low multiplicity, the cell survived virus infection, at least for the first 24 h, whereas when they
were treated with a low concentration of interferon and later infected at a high multiplicity
with EMC virus, the cells died, although the production of new infectious virus was strongly
inhibited in both situations.
In our system, no virus protein synthesis was detected in IFN-treated cells, whereas in L
cells treated with mouse IFN and infected with mengovirus (Falcoff & Sanceau, 1979) and
also in IFN-treated HeLa cells infected with VSV (Simili et al., 1980) the synthesis of some
virus proteins was apparent. Since u.v.-inactivated virus was unable to induce the inhibition of
host protein synthesis, it seems reasonable to suggest that some translation of the EMC RNA
Translation in interferon-treated cells
161
takes place, and that a virus product is responsible for the inhibition. It remains to be
established whether the inhibition of host protein synthesis at high multiplicities is triggered
off by the formation of double-stranded RNA. In any case, this system provides a good
model to test whether the blockade of translation is mediated by the degradation of cellular
mRNA via the 2,5 A system, or whether the modification of a protein factor involved in
translation is the cause of that inhibition.
Modification of membrane permeability takes place after virus infection (Carrasco &
Smith, 1980; Imprain et al., 1980; Foster et al., 1980). A correlation between such
modification and the shut-off of protein synthesis exists in Sindbis virus-infected cells (Garry
et al., 1979) and also in EMC virus-infected cells at low multiplicities of infection (Carrasco
& Smith, 1976; Fig. 5b). However, at high multiplicities the membrane becomes modified
when the bulk of viral proteins are synthesized, but the inhibition of host protein synthesis
occurred earlier than the release of 86Rb+ ions from the cells (Fig. 5 d). In IFN-treated HeLa
cells, the inhibition of host protein synthesis observed at high multiplicities, took place in a cell
with an integral membrane, as measured by means of the unpermeant translation inhibitor
hygromycin B and by the release of 86Rb+ ions. These results suggest that the shut-off of
translation in IFN-treated cells is not a consequence of the modification of the ionic
composition in the cell cytoplasm.
The expert technical assistance of Ms M. A. Ramos is acknowledged. We also acknowledge
CAICYT, Asociaci6n Espafiola Contra el Chncer and Plan Concertado de Investigaci6n no. 5/78 for
financial support.
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(Received 9 March 1981)