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Cell free transcription and translation of rotavirus RNA
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
June 13, 1979
Pages 791-796
CELL FREE TRANSCRIPTION
AND TRANSLATION
J. Cohen* *
Station
de Recherches
Grignon,
Received
and P. Dobos**
de Virologie
et d'Immunologie
INRA 78850,
Thiverval-
France
** Department Guelph,
OF ROTAVIRUS RNA
of Microbiology, Guelph,
April
College
Ontario
of Biological
Science,
University
of
NlG 2Wl
3,1979
SUMMARY ____ Rotavirus single stranded RNA synthesized in vitro by endogenous RNA transcriptase consists of 11 segments that hybr??ii%-i?with genomic RNA. It displays messenger activity when incubated with rabbit reticulocyte lysate. The products of translation were precipitable by anti-rotavirus serum, but not by normal serum. INTRODUCTION Rotavirus
RNA polymerase
when the outer
capsid
calcium
chelation
(1,2).
exhibit
a highly
active
stranded RNA.
is
layer
is
rotavirus
with
polypeptide
MATERIALS
AND METHODS
a) Preparation
from
that
single
reovirus might
the rotavirus
stimulate
removed
polymerase
enzyme product
characterize
within
Dense (D) particles
genome as template, By analogy
found
the
virion
fully
synthesizes,
stranded
serve
as messenger.
double
hybridizes
with
product
in a rabbit
that
The data
its
viral
the --in vitro
presented
and demonstrate reticulocyte
by
treatment
the
can be hypothesized
active
particles
by this using
RNA that
it
and becomes infectious
obtained
(3,4)
polymerase
synthesis
the
here
ability
to
lysate.
of SS RNA
The isolation and purification of calf rotavirus, as well as the in vitro production of rotavirus SS RNA have been described previously (1). For mass production of SS RNA 500 pg of D particles were suspended in 5 ml of reaction mixture (containing the four nucleoside triphosphates, 2 mM each; MgC12, 10 mM; phosphoenol pyruvate, 5 mg/ml; pyruvate kinase, 0.5 mg/ml; tris pH 8, 100 mM). The reaction mixture was incubated at 370C for 6 hours, followed by ultracentrifugation at 35,000 rpm for 60 minutes to remove rotavirus particles. The supernatant was extracted twice with phenol and 0006-291X/79/110791-06$01.00/0 791
Copyright @ 1979 by Academic Press. Inc. rights of reproduction in any form reserved
All
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the RNA precipitated with 66% ethanol at -200C. The dense precipitate was dissolved in sterile distilled water and an equal volume of 4 M LiCl solution was added and SS RNA was allowed to precipitate overnight at -2ooc. This procedure was repeated twice in order to remove trace amounts At this step the yield, of DS RNA which remained in the supernatant. estimated by spectrophotometry (O.D. 260) was 1.2 mg of RNA. The transcription product was analyzed by electrophoresis either in 5% polyacrylamide slab gels or using composite agarose-acrylamide (0.6% vs 2%, respectively) slab gels as described by Loening (5). The RNA was stained with ethidium bromide, or in case of isotopically labeled RNA, it was detected by fluorography as described by Laskey and Mills (6). b) Cell
free
protein
synthesis
Rotavirus SS RNA was translated in a rabbit reticulocyte lysate prepared essentially as described by Pelham and Jackson (7). Incorporation [35S]-LMethionine into protein was measured by spotting 1 ~1 aliquots of the 25 ul incubation mixture on Whattman 3 MM paper. After precipitation in cold, followed by hot 5% TCA (containing 5% casamino acids), the papers were washed in ethanol, dried, and the radioactivity counted in a liquid scintillation counter using a toluene based scintillation cocktail. All values were corrected for 0 time control. The translation products were analyzed by electrophoresis in 12.5% polyacrylamide slab gels as described by Laemmli (8) followed by autoradiography. Immunoprecipitation was performed as described by Kerr --et al. (9). RESULTS AND DISCUSSION a) Characterization
of the
When tritiated profile
polymerase
SS RNA was hybridized
obtained
to the
rotavirus
after
pattern
with
fluorography
obtained
with
(Fig.
purified
product an excess
viral
DS RNA (Fig.
that
each of the viral
genome segments
vitro
into
SS RNA.
the
did
not
correspond
particle. large the
virion
size
class
RNA's.
would segment
class
were
(Fig.
1B) indicating
RNA is
reduced
less
1C).
be expected (Mol.
wt.
than
gel
the
stranded
2.2 x 106)(10,11).
of each
in the
segment virus of the
to that
present
with
copy of the
and small
bands
no precise
size by
of SS RNA were
23s ribosomal largest
in
of large
was analyzed
eight
indi-in
hybrid
transcription
polymerase
However,
792
transcribed
of the middle
band co-migrated
This
the dsRNA hybrids
the --in vitro
electrophoresis,
single
fully
when compared
those
identical
16).
observed
lA,
of the endogenous
The slowest for
of those
in quantity that
were
amounts
in Figure
efficient
agaros-acrylamide (Fig.
ratio
as shown
When the product
composite resolved
to the molar
For example, size
relative
RNA, the gel
1A) was qualitatively
cated
However,
of viral
RNA as
DS RNA genome
molecular
weight
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
t 16s
t 23s Figure
1:
Electrophoretic
pattern
of j&
RESEARCH COMMUNICATIONS
vitro
transcription
product
Acrylamide gel electrophoresis in 5% gel of 3H-uridine labeled transcription product (SS RNA) annealed with excess unlabeled, denatured virion DS RNA (line A). Approximately 5 ug of purified DS RNA was annealed with 15,000 cpm of transcriptase product as described previously (1). The preparation was treated with RNase A (10 ug/ml in 0.3 M NaCl) precipitated with 66% ethanol and resuspended in Loening's buffer. After electrophoresis, the gel was processed for fluorography. On line B and C the genome template and the transcriptase product are analyzed in composite agarose - 2% E. coli 23s and acrylamide gels and stained with ethidium bromide. 16s rRNA's were run in parallel wells to serve as molecular weight markers. L, M, and S indicates the location of large, medium, and small size class virion dsRNA's in lines A and B. Migration was from left to right.
determination
could
denaturing
be done as electrophoresis Traces
conditions.
--in vitro
transcription
product
When used in protein
synthesis,
tation
since
b) Translation
of --in vitro
lysates
resulted
molecular
weight,
at a linear
rate
lower
was analyzed
for
30 minutes
however,
the
initial it
stopped
rate
at 37'C
precipita
removed
by LiCl
protein
synthes is
precipi-
and for
synthesis
incorporation
2 hours acid
continued
at 22'C
incorporation
large
(Fig.
was higher
2).
was
15 fold above the control.
20 minutes
(12).
lysates
into
This
the
tion.
[35S]-L-Methionine
of amino
793
1C where
reticulocyte
RNA about
after
LiCl
under
SS RNA to rabbit
product.
of protein
completely
free
performed
in Fig.
reticulocyte
of
the extent
increased by the added rotavirus 37'C,
cell
rotavirus
precipitable
temperature,
before
rabbit
the incorporation
acid
visible
DS RNA was always
SS RNA in produced
in
of DS RNA are
known to inhibit
of rotavirus
Addition
At the
DS RNA is
was not
than
at 2Z°C,
of incubation.
The
At
Vol. 88, No. 3, 1979
8lOCHEMlCAL
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
6 6
40
hf’l mM Figure
2:
Time
course
of protein
Mg2+ concentration lation of rotavirus
3:
120
160200
IRNA1
pg/ml
synthesis
Control lysates (open symbols) and reticulocyte mented with 24 ug/ml of rotavirus SS RNA (filled [35S]-L-Methionine incubated at 370C and 220C. assayed by taking 1 ~1 aliquots at various times lysate 220C (0) and 370C (A); and lysate with 24 220C (0) and 370C (A). Figure
80
optimum and RNA saturation SS RNA
lysates supplesymbols) were incorporation was as shown. Control rig/ml of SS RNA at
curve
for
the
trans-
assays were performed In the rabbit reticulocyte system, translation Samples of 1 ~1 were removed at at 22OC in a total volume of 25 ~1. and processed to measure the incorpora20, 40, 835 and 120 minutes, tion of [ S]-L-Methionine into TCA precipitable material. The Mgtt concentration optimum was determined using 80 mM Kt and 24 pg/ml of SS RNA. The RNA saturation curve was measured using a Kt of 80 mM and a Mgtt of 0.7 mM.
optimal
magnesium
concentration
ion
was 0.6
DS RNA could
of exogenous
100 mM) potassium
shown
a 25 ~1 assay for
inhibition
RNA.
concentration
concentration
synthesized
of added in
was 1 mM and the
Figure
messenger
(Fig.
In the limited
of 1 mM (data
in the RNA after
pattern cell
free
794
3).
not
SS RNA
Residual
traces
synthesis
range influence
tested
of
at high (from
on protein
50 to syn-
shown).
on SDS polyacrylamide system
a two hour
4.
optimal
of protein
had little
of the electrophoretic
of protein
absence is
ion
at a Mg"
A comparison gels
ng for
be responsible
concentrations
thesis
concentration
in the presence
incubation
period
slab or at 22'C
0
Vol. 88, No. 3, 1979
BIOCHEMICAL
DISTANCE Figure
4:
Analysis lysate
of the
AND BIOPHYSICAL
OF
protein
MIGRATION
products
synthesized
Purified rotavirus and --in vitro 12.5% polyacrylamide slab gels. proteins stained with coomassie radiograms of labeled products and analyzed in the same gel. the polypeptides produced with represents the immune precipitate B. Curves below are densitometric Track C (lower). Direction of
Without
added
reticulocyte products
lysate shows
to 12,000. protein. serum
SS RNA, little
several
4C).
proteins
(Fig.
polypeptide
4D).
of glycoprotein
in
molecular
weight
two bands
are also
for
the
synthesized
the molecular
precipitated
to the unglycosylated
by the
incubated
SS RNA directed
weight
range
as the major
a little
capsid
glycosylated
reticulocyte
from
rotavirus
by anti-rota-hyperimmune
migrate
outer
rabbit
in nuclease
of the rotavirus
is precipitated
in the
in
translation products analyzed in Track A shows rotavirus structural blue. Track B, C, and D are automade --in vitro (22OC, 2 hr incubation) Track B and C respectively represents or without rotavirus SS RNA. Track D of the material analyzed in Track tracings of Track B (upper) and migration is from left to right.
same mobility
Two heavy bands present
is
Analysis
One of them has the This
correspond
(Fig.
protein
RESEARCH COMMUNICATIONS
peptides,
faster
layer
of rotavirus 33,000
hyperimmune
form of the
795
than
structural
capsid rabbit
the doublet (apparent
and 31,000).
serum,
44,000
These
and could polypeptides
(Fig.
Vol. 88, No. 3, 1979
4D).
It
is
reticulocyte the
noteworthy
that
product
in Track
C (also
product
(Track
immunoprecipitated
smaller
polypeptides
termination class,
which
into in
BIOCHEMICAL
high the
the
compared very
fidelity
conditions
weight
polypeptides. control.
of labeled
labeled
results
messenger
(3,4).
Some of the
virion
culture, is
the
not
provide
evidence
activity
as it
studies
because
were
could
rotavirus
at present.
synthesized
by a specific
directed
not
protein
[35S]-L-
For this
reason
immune serum.
SS RNA obtained for
The results synthesis
be
of
seem to be virus
immune serum.
maps of
replicates
anti-rotavirus
rotavirus
detectable
peptide
and purification
using that
system
size
be translated
proteins
has been shown previously
polypeptides
on virus
free
eight
large
not
tryptic
growth
feasible
was established
are precipitated
used could
the cell
in
to premature
SS RNA of the
No labeled
proteins
thus
rotavirus
of translation
in
not present
immune precipitate
Unfortunately,
produced
as an endogenous
B) is
correspond
the experimental
in tissue
use in further
could
under
exhibits
they
In the
D).
they
present
in Track
due to degraded
to those
The above
present
or products
polypeptides
Methionine
polypeptide
products
immune precipitate
poorly
single
are visible;
molecular
labeled
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
--in vitro reovirus
specific will
in the
as be of
infected
cell. REFERENCES Cohen, J. (1977) J. Gen. Virol. 36, 395-402. Cohen, J., Laporte, J., Charpilienne, A., and Scherrer, R. (1978) Archiv. of Virology (In Press). 3. Levin, D.H., Kyner, D., and Acs, G. (1971) Biochem. Biophys. Res. Commun. 42, 454-461. 4. McDowell, M.J. and Joklik, W.K. (1971) Virology 45, 724-733. 5. Loening, V.E. (1967) Biochem. Journal 102, 251-257. 6. Laskey, R.A. and Mills, A.D. (1975) Eur. J. Biochem. 56, 335-341. Pelham, H.R.B. and Jackson, R.J. (1976) Eur. J. Biochem. 67, 247-256. ;: Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685. 9. Kerr, I.M., Olshevsky, U., Lodish, H.F.,and Baltimore, D. (1976) J. of Virology 18, 627-635. 10. Verly, E. and Cohen, J. (1977) J. Gen. Virol. 35, 583-586. 11. Bellamy, A.R. and Joklok, W.K. (1967) 3. Mol. Biol. 29, 19-26. 12. Baglioni, C., Lenz, J.R., Maromey, P.A., and Weber, L.A. (1978) Biochemistry 17, 3257-3262. ::
796
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
June 13, 1979
Pages 791-796
CELL FREE TRANSCRIPTION
AND TRANSLATION
J. Cohen* *
Station
de Recherches
Grignon,
Received
and P. Dobos**
de Virologie
et d'Immunologie
INRA 78850,
Thiverval-
France
** Department Guelph,
OF ROTAVIRUS RNA
of Microbiology, Guelph,
April
College
Ontario
of Biological
Science,
University
of
NlG 2Wl
3,1979
SUMMARY ____ Rotavirus single stranded RNA synthesized in vitro by endogenous RNA transcriptase consists of 11 segments that hybr??ii%-i?with genomic RNA. It displays messenger activity when incubated with rabbit reticulocyte lysate. The products of translation were precipitable by anti-rotavirus serum, but not by normal serum. INTRODUCTION Rotavirus
RNA polymerase
when the outer
capsid
calcium
chelation
(1,2).
exhibit
a highly
active
stranded RNA.
is
layer
is
rotavirus
with
polypeptide
MATERIALS
AND METHODS
a) Preparation
from
that
single
reovirus might
the rotavirus
stimulate
removed
polymerase
enzyme product
characterize
within
Dense (D) particles
genome as template, By analogy
found
the
virion
fully
synthesizes,
stranded
serve
as messenger.
double
hybridizes
with
product
in a rabbit
that
The data
its
viral
the --in vitro
presented
and demonstrate reticulocyte
by
treatment
the
can be hypothesized
active
particles
by this using
RNA that
it
and becomes infectious
obtained
(3,4)
polymerase
synthesis
the
here
ability
to
lysate.
of SS RNA
The isolation and purification of calf rotavirus, as well as the in vitro production of rotavirus SS RNA have been described previously (1). For mass production of SS RNA 500 pg of D particles were suspended in 5 ml of reaction mixture (containing the four nucleoside triphosphates, 2 mM each; MgC12, 10 mM; phosphoenol pyruvate, 5 mg/ml; pyruvate kinase, 0.5 mg/ml; tris pH 8, 100 mM). The reaction mixture was incubated at 370C for 6 hours, followed by ultracentrifugation at 35,000 rpm for 60 minutes to remove rotavirus particles. The supernatant was extracted twice with phenol and 0006-291X/79/110791-06$01.00/0 791
Copyright @ 1979 by Academic Press. Inc. rights of reproduction in any form reserved
All
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
the RNA precipitated with 66% ethanol at -200C. The dense precipitate was dissolved in sterile distilled water and an equal volume of 4 M LiCl solution was added and SS RNA was allowed to precipitate overnight at -2ooc. This procedure was repeated twice in order to remove trace amounts At this step the yield, of DS RNA which remained in the supernatant. estimated by spectrophotometry (O.D. 260) was 1.2 mg of RNA. The transcription product was analyzed by electrophoresis either in 5% polyacrylamide slab gels or using composite agarose-acrylamide (0.6% vs 2%, respectively) slab gels as described by Loening (5). The RNA was stained with ethidium bromide, or in case of isotopically labeled RNA, it was detected by fluorography as described by Laskey and Mills (6). b) Cell
free
protein
synthesis
Rotavirus SS RNA was translated in a rabbit reticulocyte lysate prepared essentially as described by Pelham and Jackson (7). Incorporation [35S]-LMethionine into protein was measured by spotting 1 ~1 aliquots of the 25 ul incubation mixture on Whattman 3 MM paper. After precipitation in cold, followed by hot 5% TCA (containing 5% casamino acids), the papers were washed in ethanol, dried, and the radioactivity counted in a liquid scintillation counter using a toluene based scintillation cocktail. All values were corrected for 0 time control. The translation products were analyzed by electrophoresis in 12.5% polyacrylamide slab gels as described by Laemmli (8) followed by autoradiography. Immunoprecipitation was performed as described by Kerr --et al. (9). RESULTS AND DISCUSSION a) Characterization
of the
When tritiated profile
polymerase
SS RNA was hybridized
obtained
to the
rotavirus
after
pattern
with
fluorography
obtained
with
(Fig.
purified
product an excess
viral
DS RNA (Fig.
that
each of the viral
genome segments
vitro
into
SS RNA.
the
did
not
correspond
particle. large the
virion
size
class
RNA's.
would segment
class
were
(Fig.
1B) indicating
RNA is
reduced
less
1C).
be expected (Mol.
wt.
than
gel
the
stranded
2.2 x 106)(10,11).
of each
in the
segment virus of the
to that
present
with
copy of the
and small
bands
no precise
size by
of SS RNA were
23s ribosomal largest
in
of large
was analyzed
eight
indi-in
hybrid
transcription
polymerase
However,
792
transcribed
of the middle
band co-migrated
This
the dsRNA hybrids
the --in vitro
electrophoresis,
single
fully
when compared
those
identical
16).
observed
lA,
of the endogenous
The slowest for
of those
in quantity that
were
amounts
in Figure
efficient
agaros-acrylamide (Fig.
ratio
as shown
When the product
composite resolved
to the molar
For example, size
relative
RNA, the gel
1A) was qualitatively
cated
However,
of viral
RNA as
DS RNA genome
molecular
weight
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
t 16s
t 23s Figure
1:
Electrophoretic
pattern
of j&
RESEARCH COMMUNICATIONS
vitro
transcription
product
Acrylamide gel electrophoresis in 5% gel of 3H-uridine labeled transcription product (SS RNA) annealed with excess unlabeled, denatured virion DS RNA (line A). Approximately 5 ug of purified DS RNA was annealed with 15,000 cpm of transcriptase product as described previously (1). The preparation was treated with RNase A (10 ug/ml in 0.3 M NaCl) precipitated with 66% ethanol and resuspended in Loening's buffer. After electrophoresis, the gel was processed for fluorography. On line B and C the genome template and the transcriptase product are analyzed in composite agarose - 2% E. coli 23s and acrylamide gels and stained with ethidium bromide. 16s rRNA's were run in parallel wells to serve as molecular weight markers. L, M, and S indicates the location of large, medium, and small size class virion dsRNA's in lines A and B. Migration was from left to right.
determination
could
denaturing
be done as electrophoresis Traces
conditions.
--in vitro
transcription
product
When used in protein
synthesis,
tation
since
b) Translation
of --in vitro
lysates
resulted
molecular
weight,
at a linear
rate
lower
was analyzed
for
30 minutes
however,
the
initial it
stopped
rate
at 37'C
precipita
removed
by LiCl
protein
synthes is
precipi-
and for
synthesis
incorporation
2 hours acid
continued
at 22'C
incorporation
large
(Fig.
was higher
2).
was
15 fold above the control.
20 minutes
(12).
lysates
into
This
the
tion.
[35S]-L-Methionine
of amino
793
1C where
reticulocyte
RNA about
after
LiCl
under
SS RNA to rabbit
product.
of protein
completely
free
performed
in Fig.
reticulocyte
of
the extent
increased by the added rotavirus 37'C,
cell
rotavirus
precipitable
temperature,
before
rabbit
the incorporation
acid
visible
DS RNA was always
SS RNA in produced
in
of DS RNA are
known to inhibit
of rotavirus
Addition
At the
DS RNA is
was not
than
at 2Z°C,
of incubation.
The
At
Vol. 88, No. 3, 1979
8lOCHEMlCAL
AND BIOPHYSICAL
RESEARCH COMMUNlCATlONS
6 6
40
hf’l mM Figure
2:
Time
course
of protein
Mg2+ concentration lation of rotavirus
3:
120
160200
IRNA1
pg/ml
synthesis
Control lysates (open symbols) and reticulocyte mented with 24 ug/ml of rotavirus SS RNA (filled [35S]-L-Methionine incubated at 370C and 220C. assayed by taking 1 ~1 aliquots at various times lysate 220C (0) and 370C (A); and lysate with 24 220C (0) and 370C (A). Figure
80
optimum and RNA saturation SS RNA
lysates supplesymbols) were incorporation was as shown. Control rig/ml of SS RNA at
curve
for
the
trans-
assays were performed In the rabbit reticulocyte system, translation Samples of 1 ~1 were removed at at 22OC in a total volume of 25 ~1. and processed to measure the incorpora20, 40, 835 and 120 minutes, tion of [ S]-L-Methionine into TCA precipitable material. The Mgtt concentration optimum was determined using 80 mM Kt and 24 pg/ml of SS RNA. The RNA saturation curve was measured using a Kt of 80 mM and a Mgtt of 0.7 mM.
optimal
magnesium
concentration
ion
was 0.6
DS RNA could
of exogenous
100 mM) potassium
shown
a 25 ~1 assay for
inhibition
RNA.
concentration
concentration
synthesized
of added in
was 1 mM and the
Figure
messenger
(Fig.
In the limited
of 1 mM (data
in the RNA after
pattern cell
free
794
3).
not
SS RNA
Residual
traces
synthesis
range influence
tested
of
at high (from
on protein
50 to syn-
shown).
on SDS polyacrylamide system
a two hour
4.
optimal
of protein
had little
of the electrophoretic
of protein
absence is
ion
at a Mg"
A comparison gels
ng for
be responsible
concentrations
thesis
concentration
in the presence
incubation
period
slab or at 22'C
0
Vol. 88, No. 3, 1979
BIOCHEMICAL
DISTANCE Figure
4:
Analysis lysate
of the
AND BIOPHYSICAL
OF
protein
MIGRATION
products
synthesized
Purified rotavirus and --in vitro 12.5% polyacrylamide slab gels. proteins stained with coomassie radiograms of labeled products and analyzed in the same gel. the polypeptides produced with represents the immune precipitate B. Curves below are densitometric Track C (lower). Direction of
Without
added
reticulocyte products
lysate shows
to 12,000. protein. serum
SS RNA, little
several
4C).
proteins
(Fig.
polypeptide
4D).
of glycoprotein
in
molecular
weight
two bands
are also
for
the
synthesized
the molecular
precipitated
to the unglycosylated
by the
incubated
SS RNA directed
weight
range
as the major
a little
capsid
glycosylated
reticulocyte
from
rotavirus
by anti-rota-hyperimmune
migrate
outer
rabbit
in nuclease
of the rotavirus
is precipitated
in the
in
translation products analyzed in Track A shows rotavirus structural blue. Track B, C, and D are automade --in vitro (22OC, 2 hr incubation) Track B and C respectively represents or without rotavirus SS RNA. Track D of the material analyzed in Track tracings of Track B (upper) and migration is from left to right.
same mobility
Two heavy bands present
is
Analysis
One of them has the This
correspond
(Fig.
protein
RESEARCH COMMUNICATIONS
peptides,
faster
layer
of rotavirus 33,000
hyperimmune
form of the
795
than
structural
capsid rabbit
the doublet (apparent
and 31,000).
serum,
44,000
These
and could polypeptides
(Fig.
Vol. 88, No. 3, 1979
4D).
It
is
reticulocyte the
noteworthy
that
product
in Track
C (also
product
(Track
immunoprecipitated
smaller
polypeptides
termination class,
which
into in
BIOCHEMICAL
high the
the
compared very
fidelity
conditions
weight
polypeptides. control.
of labeled
labeled
results
messenger
(3,4).
Some of the
virion
culture, is
the
not
provide
evidence
activity
as it
studies
because
were
could
rotavirus
at present.
synthesized
by a specific
directed
not
protein
[35S]-L-
For this
reason
immune serum.
SS RNA obtained for
The results synthesis
be
of
seem to be virus
immune serum.
maps of
replicates
anti-rotavirus
rotavirus
detectable
peptide
and purification
using that
system
size
be translated
proteins
has been shown previously
polypeptides
on virus
free
eight
large
not
tryptic
growth
feasible
was established
are precipitated
used could
the cell
in
to premature
SS RNA of the
No labeled
proteins
thus
rotavirus
of translation
in
not present
immune precipitate
Unfortunately,
produced
as an endogenous
B) is
correspond
the experimental
in tissue
use in further
could
under
exhibits
they
In the
D).
they
present
in Track
due to degraded
to those
The above
present
or products
polypeptides
Methionine
polypeptide
products
immune precipitate
poorly
single
are visible;
molecular
labeled
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
--in vitro reovirus
specific will
in the
as be of
infected
cell. REFERENCES Cohen, J. (1977) J. Gen. Virol. 36, 395-402. Cohen, J., Laporte, J., Charpilienne, A., and Scherrer, R. (1978) Archiv. of Virology (In Press). 3. Levin, D.H., Kyner, D., and Acs, G. (1971) Biochem. Biophys. Res. Commun. 42, 454-461. 4. McDowell, M.J. and Joklik, W.K. (1971) Virology 45, 724-733. 5. Loening, V.E. (1967) Biochem. Journal 102, 251-257. 6. Laskey, R.A. and Mills, A.D. (1975) Eur. J. Biochem. 56, 335-341. Pelham, H.R.B. and Jackson, R.J. (1976) Eur. J. Biochem. 67, 247-256. ;: Laemmli, U.K. (1970) Nature (Lond.) 227, 680-685. 9. Kerr, I.M., Olshevsky, U., Lodish, H.F.,and Baltimore, D. (1976) J. of Virology 18, 627-635. 10. Verly, E. and Cohen, J. (1977) J. Gen. Virol. 35, 583-586. 11. Bellamy, A.R. and Joklok, W.K. (1967) 3. Mol. Biol. 29, 19-26. 12. Baglioni, C., Lenz, J.R., Maromey, P.A., and Weber, L.A. (1978) Biochemistry 17, 3257-3262. ::
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