Copyright©1976 American Society for Microbiology Printed in U-SA.
Translation
of Murine Leukemia Virus RNA in Cell-Free
Systems
from Animal Cells
IAN M. KERRl* UDY OLSHEVSKY, HARVEY F. LODISH, AND DAVID BALTIMORE Department ofBiology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139
Received for publication 8 December 1975
The
virion RNA of Moloney murine leukemia virus (MuLV) has beentrans-lated
ineukaryotic
cell-free systemsderived from
mouse L-and human
HeLacells. In both
systems atleast three polypeptides, approximately 60,000, 70,000,
and
180,000 in apparentmolecular weight,
wereformed
inresponse to the added35S
MuLV
RNA. All three
polypeptides
wereprecipitable
with antiserum
todetergent-disrupted
MuLV. Fingerprint
analysis of tryptic digests indicated
that all three
containaminoacid
sequences in commonwith each other and with
the major methionine-containing structural proteins of the virion.
The
genomeof the RNA
tumor viruses ismade
up of two to threeapparently
identicalRNA
molecules, each of
molecular weight
2.5x106
to 3x 106,sufficient
toencode
polypeptide(s)
of total molecular weight
250,000 to300,000.
The RNA
carriesthe
geneticinformation
for
the
major virion antigens,glycoprotein(s), and
reversetranscriptase,
which
amounts toabout
200,000 inmolecular
weight.
It is not yetclear,
however, if
this
isall that is
coded
(reviewed
inref.
2).As
adirect approach
todetermining
the
in-formation
contentof the
genome, thevirion
RNAs have been
isolated from
anumber of
oncornaviruses
and
translated
in avariety of
cell-free
systems(9, 19, 23,
25, 28). One
major
Rous
sarcomavirus
(RSV)-specific
polypeptide
of molecular
weight 70,000
to80,000
wassyn-thesized
inresponse toRSV RNA
in acell-free
system
from Krebs II ascites
tumorcells
(28).
Inaddition,
Naso
etal. (19) have
reported
that the
translation of Rauscher
murineleukemia
virus(RLV) RNA
in extractsof
starved RLV-infected
cells
yielded
atleast
twoclasses of
polypeptides
of 50,000
to70,000
and 140,000
to185,000
inmolecular
weight.
Here
wereport aninvestigation
of the
trans-lation of
Moloney
murineleukemia
virus(MuLV) RNA
incell-free
systemsfrom
mouseand human cells. At least three
major
polypep-tides of
apparentmolecular
weight 60,000,
70,000,
and 180,000
onelectrophoresis
inso-dium
dodecyl sulfate
(SDS)-polyacrylamide
gels
wereformed
in response to theadded
MuLVRNA.
Aninitial
immunological
charac-' Presentaddress: The National Institute forMedical Research, LondonN.W.71AA,England.
terization
and
tryptic
fingerprint analysis
of
these products
ispresented.
MATERIALS AND METHODS
Chemicals for useinthe cell-free system and for the isolationof cell fractionsand viral RNA were as described previously (8, 10-13). Spermidine (neu-tralized withHCl)was from SigmaChemical Co., St. Louis, Mo.; [35S]methionine (100 to 450 Ci/mmol)
was from New England Nuclear Corp., Boston, Mass.;encephalomyocarditis virus (EMC) was from G. D. Searle andCo., Ltd., HighWycombe, Bucks,
England; TPCK trypsin was fromWorthington Bio-chemicalCorp., Freehold, N.J.;and rabbitanti-goat
yglobulin was fromMicrobiologicalAssociates, Be-thesda, Md. Goat antiserum to detergent
(tween-ether)-disrupted MoloneyMuLVwasobtained from
the National CancerInstitute, Bethesda,Md. (NCI
ID1-S-0157).
Cells, virus, and viral RNA. Clone 1 Moloney
MuLV was derived and maintained as previously
(7). Clone2wasobtained from clone 1byendpoint
dilution of the virusonNIH-3T3cells with
immedi-atecloningof the cells after infection(M. Paskind,
D. Smotkin, and D. Baltimore, unpublished data). For large-scale production of rapid-harvest virus,
productively infected cells were grown in roller
bottles in Dulbecco modified Eagle medium (DME) with10% calf serum. Virus was purified from me-dium collected at 4-h intervals. This was clarified
by centifugationat1,000xgfor15minat4 C,and
the viruswaspelleted at27,000 xg for2.5 h. The viruswasresuspendedinTNE[50mM
tris(hydroxy-methyl)aminomethane (Tris)-hydrochloride (pH
7.2), 100 mM NaCl, 1 mM ethylenediaminetetraa-cetic acid] and centrifuged through a 25 to 45% (wt/wt)sucrose gradient in TNE for 17 h at 82,000
x g at 4 C. The banded virus was pooled, diluted threefold with TNE,pelletedby further centrifuga-tionfor30min at160,000 xg, andresuspendedin TNE to a protein concentration of approximately
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20 mg/ml. SDSwas added toafinal concentration of2%priortocentrifugation for 2.5 h at160,000 x
gat22Cthrougha15 to 30% (wt/wt)sucrose gradi-ent in TNE, 1% SDS. The 70S virion RNA was
pooled andprecipitated bythe addition of two
vol-umesofethanol. Itwastakenup in 2 mMsodium acetate, 2 mM EDTA,4 mM Tris-acetate (pH 7.3),
0.2% SDS, and denaturedbyheating for 2min at
78C (5). The heat-denatured virion RNAwas
cen-trifuged for 5 h at 160,000 x g at 22 Cthrough a 15to30% (wt/wt)sucrose gradientin TNE and 1% SDS. The 35S RNA was pooled and precipitated with ethanol. The precipitate was washed three times with a mixture of two parts ethanol to one part 100 mM sodium acetate (pH 6), dissolved in water toafinalconcentration of 0.5to1mg/ml, and
storedat -70C.
The growth and purificationofEMC virus and the preparation of EMC RNA have already been de-scribed (11, 13).
['5Slmethionine-labeled MuLV. Clone 1 cells
werelabeledat37 C in DME with 10%dialyzedcalf serum,0.75mgofcold methionineperliter, and10 to15,uCiof[35S]methionine perliter. After 2 hthe mediumwascollected, and thecellswereincubated
forafurther 2 h in DME and 10% calfserum. The
twobatches of mediumwerepooled, and the radio-active viruswaspurifiedasabove.
Preparation of cell extracts and the assay of
amino acid incorporation in the cell-free system. Thepreparationandpreincubationof postmitochon-drialsupernatantfractions frommouseL-cellswere as described (8). The post-mitochondrial
superna-tant fractions weredialyzedfor 6 h at 4C against threechangesof 10 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid (HEPES) (pH 7.5), 90mM
KCl, 1.5 mM magnesium acetate, and 7 mM
2-mercaptoethanol (one hundred volumes in all) to
reduce the endogenous poolof amino acids. Assay
of amino acid incorporation was in 15-/il reaction mixtures containing: 20 mM HEPES (pH 7.5); 75 mM (for MuLV RNA) or110 mM (for EMC RNA)
KCl; mM (for MuLV RNA), or 2 mM (for EMC
RNA) magnesium acetate; 0.15 mM spermidine;
8 mM 2-mercaptoethanol; 1 mM ATP; 0.15 mM
GTP;0.6 mMCTP;10 mMcreatinephosphate; 0.16
mg of creatine kinase per ml; 0.2 to 0.9 mCi of
[F3S]methionineperml;unlabeled amino acids(each
100 ,M) minus methionine; 3
Al
ofL-cell extract (equivalenttoapproximately50,tgofprotein);and 1 A1 ofreticulocyte ribosome wash-initiation factors(16,22).Theamountsofdifferentpreparations ofthe L-cellextractandreticulocytefactors usedwere
ad-justed foroptimum aminoacidincorporationineach
case. Theaddition of thereticulocyte initiation
fac-tors had no detectable effect on the nature ofthe
polypeptide products synthesized butincreasedthe level ofbothendogenousand MuLVorEMC
RNA-stimulated aminoacid incorporation two- to
three-fold. MuLVorEMCRNAswereaddedtofinal
con-centrations of 20 to 60 or 12 to 20 ,ug/ml,
respec-tively. Incubation was for 120 min at30C unless otherwise stated. Preincubated and dialyzed
postmi-tochondrial supernatant fractions from HeLa and Krebs II mouse ascites tumor cells wereprepared
andassayedasfor theL-cellextracts.
Immuneprecipitation.Allserawereclarifiedby
centifugation
for 10 min at 2,000 x g before use.Samples
(10 /Ll)of thecell-free systemweredilutedwith an equal volume ofTNE, 1% NP40 and 1%
sodium
deoxycholate
andallowedtointeract with 5 ,4lofgoat anti-MuLVorcontrol serumfor10minat room temperature andthen overnight at 4C. The mixturewasdilutedto0.5 ml withTNEcontaining1% NP40 and 40
Aul
ofrabbit anti-goat y globulin serumandincubated asabove. The precipitatewaspelleted
at2,000
xgfor10min,
washedthree times with TNE-0.5% NP40 and once with acetone, andanalyzed
by
electrophoresisonpolyacrylamidegels.Polyacrylamide gelelectrophoresis. The
polypep-tidessynthesized inthecell-free systems were
pre-cipitated
with acetone, dissolved by boiling in 2% SDS, 70 mM 2-mercaptoethanol, 62.5 mMTris-hy-drochloride(pH 6.8),20%glycerol, and analyzedby
electrophoresis
on10%polyacrylamide slab gelscon-taining
0.1%SDS(14).Upto25samples, each equiv-alentto3 to5Al
ofcell-free system, couldconven-iently be run on a single gel. Alternatively, the
acetone-precipitated polypeptide products were
eitherredissolved in8M urea, 1%SDS, 170mM
2-mercaptoethanol,5mMTris-glycine,orin 1%
SDS,
100 mM 2-mercaptoethanol, 50 mM sodium
phos-phate (pH 7), boiled for2 to3
min,
andanalyzedoncylindrical
7.5% polyacrylamide gels containing0.1%SDS in the presenceorabsence of 5 M ureaas described
by
McDowell and Joklik (15) and Weber andOsborn(29),respectively.Analysis
oftryptic peptides. Tryptic digestionof35S-labeledpurifiedMuLV and of thetotal
polypep-tide products synthesizedinthe cell-free systemin
responsetoEMC RNAwascarriedoutafter precipi-tationandperformicacidoxidationasdescribed
pre-viously (4), exceptthatTPCK trypsin equivalentto
atotal 20%by weightoftheproducttobe
digested
was used. Trypsin digestion ofindividual
polypep-tideswascarriedout onindividualbandscutfrom thestaineddriedgels after exposure for
autoradiog-raphy (17). The dried gel slices were swollenin a
slight excess by volume of TPCK trypsin (50 ,g/
ml) and 0.2 M NH4HCO3 (pH 8.5) and incubated
overnightat37C and forafurther4h with afresh
batch oftrypsin. Recoveryofradioactivityfrom the
gel slices was greater than or equal to 70%. The
peptide digestswerelyophilized and resuspended in
10to20 ulof 0.3% NH40H, and 0.2- to1.5-,ul sam-ples were subjected to electrophoresis and chro-matographyonprecoatedthin-layer (0.1 mm) cellu-lose plates (Brinkman Instruments Inc.,
Westbury,
N.Y.) asdescribed previously (4), except that
elec-trophoresis at pH 3.5 was in pyridine-acetic
acid-water(1:10:100).
Autoradiographyofthedriedpolyacrylamide gels
andthin-layer plateswaswithKodak XOmatfilm
RP R54.
RESULTS
Electrophoretic
analysis of polypeptideproducts.
To investigate whether MuLV RNAcan stimulate the synthesis of
characteristic
polypeptides,
35S RNAs of clone 1 and clone 2MuLV derived by heat
denaturation
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CELL-FREE SYNTHESIS OF MuLV POLYPEPTIDES
629
RNA
wereadded
tocell-free
systemsfrom
mouse
L-cells. After electrophoretic separation
of
the P15S]methionine-labeled products, three
major
MuLV
RNA-stimulated polypeptides
were evident on
autoradiography
of the driedgels.
These had
apparentmolecular weights
of60,000, 70,000,
and
180,000 (60K, 70K,and
180K,
arrowed
Fig.lA). Occasionally
the 60Kand 70K
products migrated
aspoorly
resolved
doublets. Essentially identical results
wereob-tained
irrespectiveof whether the
electropho-retic
analysis
wascarried
out inslabgels
afterdenaturation
inSDS
and mercaptoethanol
(Fig. 1A) or in
cylindrical gels
inthe presence
or
absence
of additional
8 M urea (15, 29;data
notshown).
Atthe
relatively
low K+ ioncon-centration
optimumfor the
translation of
MuLV
RNA
inthe
L-cell-free
system (70mM
compared with
110 mMfor
EMCRNA), there
is arelatively high level of
endogenous
aminoacid incorporation (slot
1,Fig.
1A) and
only
atwo- to
threefold stimulation
inresponse tothe
added
MuLV
RNA. Under all of the conditions
tested here, however, the L-cell-free
systemyielded
moreMuLV-specific polypeptide
ofhigher molecular
weight thandid
the wheatgerm (21),
Krebs
ascites tumor cell, rabbitre-ticulocyte
(26), orthe
partially purified
Krebs-reticulocyte (1) cell-free
systems
(data notpre-sented). The
HeLacell-free
systemyielded the
sameproducts
asdid the
L-cell-free
system,but
insmaller
amounts.Thevirion RNA of MuLV already has at its
5'-end
a7-methylguanosine residue
in 5'-5'triphosphate linkage
to theremainder
of theRNA
(J.Rose,
personal communication).
Inac-cord with
this, the addition of
S-adenosylme-thionine
orof
S-adenosylhomocysteine
waswithout
significant effect
on aminoacid
incor-poration or
the
spectrumof
polypeptides formed
inresponse to MuLV RNA in
these
systems.Kinetics
offormation
of theMuLV-specific
polypeptides
inthe L-cell-free
system.To
de-termine the time course
of appearance
of theB
A
_ ~ .t
_ z
-155
140
--
210
= 155
140
--
72
-
72
-
34
P30--
34
-
P30
1
2
3
4
FIG. 1. Electrophoretic analysisof cell-freesystem
products
and thepolypeptides ofpurified
MuLV.(A)L-cell-freesystemproductsin thepresenceandabsenceofMuLVRNAorEMC RNA. Slot1, minus MuLV RNA (42,000counts/min); slots2and3,plusMuLVRNA clone1 and clone2(95,000 and70,000counts/min,
respectively); slot4,plus EMC RNA (130,000counts/min);minusEMC RNA (notshown, 3,000counts/min). Each RNA was assayed under optimum conditions for that RNA (Materials and Methods). Material equivalentto3
kd
ofeachcell-freesystemwasanalyzed byelectrophoresison a10%acrylamideslabgel. (B)[35S]methionine-labeled MuLVwassubjectedtoelectrophoresison agradient(8to20%acrylamide)slab
gel.
ForbothAandBanautoradiograph ofpartofthestained, driedgelisshown, and the numberstothe
right
give themolecular weightsin thousandsofchickmyosin and the majorreovirionpolypeptides A1, A2, p2,
and o-3 (24), which were included as nonradioactive markers. The 60K, 70K, and 180K MuLV RNA-programmed products in(A)areindicatedby thearrows totheleft.
VOL. 18, 1976
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[image:3.503.108.389.302.559.2]three
newpolypeptides, samples of
extractsin-cubated for
various times weresubmitted
toelectrophoresis (Fig. 2). The
60Kproduct
wasfirst
evident
at 30 minand the
180K at 90min,
as
would be expected for newly initiated
poly-peptides of these sizes
inthis
typeof
system.The
higher-molecular-weight
EMCand
polio-virus
RNA-directed
polypeptide products, for
example, require comparable times for their
synthesis
invitro
(Fig.
3 inreference 6;
I.M.
Kerr,
unpublished data).
Translation
invivo is
considerably faster,
but
complete
translation of
these latter RNAs takes
12 min even at 37C
inthe intact
infected cell (3, 20).
Immunological
characterization. The
pre-cipitability
by
aMuLV-specific antiserum of
the
polypeptide products stimulated by
MuLVRNA
wastested
todetermine if they
werevi-rus-specific. Using
aHeLa
cell-free system,
[35S]methionine-labeled products
weremade
withand without
added
MuLVRNA.
They
wereexposed
togoat antiserum againstdeter-gent-disrupted MuLV, and material reacting
with
the
serumwasprecipitated by rabbit
anti-goat
immunoglobulin and
analyzed by
electro-phoresis
onpolyacrylamide gels. Whereas
afew
background polypeptides
werecarried down
by
both immune and non-immune
sera,all three
NO
MLV
RNA
30'
60
120'
1801
45
90'
180
of the
MuLV
RNA-stimulated
polypeptides
wereprecipitated only by
theanti-MuLV
se-rum
and
notby
the
corresponding
controlmate-rial
(Fig. 3). Essentially identical
resultswereobtained with L-cell extracts on similar
immu-nological analysis
of theproducts formed
inresponse
tothe MuLV RNA(data
notshown).
Fingerprint analysis of tryptic peptides.
Foranalysis
of thetryptic
digests
of theMuLV-specific
products,
the60K, 70K,
and180K,
[P5S]methionine-labeled
polypeptide
bandswereeluted from stained dried
polyacrylamide
gels by digestion
withtrypsin (17; Materials
andMethods). Autoradiographs
oftwo-dimen-sional
thin-layer fingerprints
ofthesedigests
were
compared
with thatobtained with
a simi-lardigest
ofthe 130K to 140Kpolypeptide
prod-uctformed
inresponse
toEMCRNA
inthe
L-cell-free system and with
those for
digests of
thetotal
virionpolypeptides
ofpurified MuLV,
themajor
MuLVgroup-specific antigen
(P30) and themajor
small[:15S]methionine-labeled
poly-peptide
of MuLV(presumable P15, Fig.
1B;
Fig. 4, 5).
The
products made
inresponse
to MuLVRNA were
clearly different from those
pro-grammed
by
EMC RNA(Fig. 4). The
finger-prints obtained
fromthe 60K and
70Kproducts,
-?-M ~ .,
30
6C
1 5
45'
?o 8t
4-
4--2
3
4
5
6
7
8
9
10
112
:3 4 95FIG. 2. Synthesis of MuLV-specific polypeptides with time in the
cell-free
system.L-cell-free
systems were incubatedinthe presence andabsence ofMuLV RNA. Samples (5pid)
were taken at thetimes(min)indicatedatthe topof the figure and analyzed on a10% acrylamide slab gel.
[3,5]methionine
incorporation at 180minwas: minusMuLVRNA30,000 and plus MuLV RNA 86,000counts/minper 5piu. The numbers to the left give the molecularweights in thousands of markerpolypeptides as in Fig. 1. The arrows to the right indicate the positionof the 60K, 70K, and 180K MuLVRNA-programmed products.
1;
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[image:4.503.115.413.367.592.2]CELL-FREE SYNTHESIS OF MuLV
POLYPEPTIDES
631
12
3
4
-4
-
210
- 155
140
.
-
72
- 34
FIG. 3. Immuneprecipitation of thecell-free sys-tem productssynthesized in response to MuLV RNA. HeLa cell extracts were incubated in the cell-free system in the presence and absence of MuLV RNA andsubjected to immune precipitation. Plus MuLV RNA: slot 1, control serum; slot 2, anti-MuLV se-rum.No addedRNA: slot 3, control serum; slot 4, anti-MuLV serum. Electrophoresis was on a 10% acrylamide slab gel. An autoradiograph ofpart of the stained, dried gel is shown. The numbers to the right and the arrows to the left are as described in the legendtoFig.1.
on
the other hand,
wereindistinguishable and
bore
noobvious relation
tothe background
ma-terial evident
indigests of
extracts towhich
noRNA
had been added (Fig.
4).All of
the
trypticpeptides
present inthe
digests of the
60Kand
70Kproducts (Fig.
4A, 4B, 5D)
werealso
evi-dent
inthe digest of whole
virus(Fig. 5A),
many
of
whichcould
be accounted for by thosepresent in
the
digests of the
P30and
P15 (Fig.5C and E). The
fingerprint of the 180K product,
although closely related
tothat of whole
virus(Fig. 5A),
wassurprisingly similar
tothose for
the
60Kand
70Kproducts
(Fig.
4A,
4B, and
5D).
Afew
additional MuLV RNA-stimulated
peptides
have been
tentatively identified
inthe
basic
region of the 180K
map,and there could
be
additional material
inthe
congested
band of
neutral peptides. The fact
remains,however,
that for
anincreaseof
over100,000
inapparentmolecular
weight of the
polypeptide
from which
it was
derived, the 180K fingerprint shows
re-markably little
greater apparentcomplexity
than those for the 60 and 70Kproducts.
The results in
Fig.
4 and 5 wereobtained
using
electrophoresis
atpH
6.5.Despite
pooreroverall
resolution,
thegeneral
similaritiesand
differences observed
withthe
differentdigests
were essentially confirmed
by
afurther seriesof
fingerprints
employing electrophoresis
atpH
3.5 inthe first dimension.
DISCUSSION
At
least three polypeptides have been
demon-strated
tobe synthesized
in response toMuLV
35S RNA added
tocell-free
systemsfrom
both human and mouse cells. That thesepolypep-tides
aretruly MuLV-specific has
been shownby
their specific immunoprecipitability and by
fingerprint analysis
of their trypticpeptides.
Whereas
it isconceivable that the results
ob-tained with
mouseL-cell
extractscould reflect
anincreased translation of endogenous mRNA
inresponse to the added RNA, it is extremely
improbable that this could be the
case withthe
human
HeLacell
extracts.The results
obtained
withthe latter, therefore, clearly show that
itisthe
added
MuLVRNA that
isdirecting the
synthesis of the three
MuLV-specific
polypep-tides
inthe
cell-free
systems.This,
in turn,confirms that the
virionRNA
isthe
messengerstrand for the RNA
tumorviruses(2,
19,38).The
nearidentity of the fingerprints of the
60K,
70K, and
particularly the
180Kproducts
with that for MuLV is
striking (Fig.
4,5).All of
the
majorPjS]methionine
peptides
present inthe
60Kproduct
arealso
present in the 70K,and
virtually all
are present in the 180K. Itmay
be,
therefore, that these are overlappingpolypeptides extending different distances from
the
same initiation site onthe
RNA.The
pep-tide
sequences common toall three
products(those
inthe
60K) are those of the majorstructural
virion corepolypeptides (Fig. 1B,
5).In this
sensethe
60Kproduct
maybe analogous
to
the
precursor(Pre6O)
tothese latter
poly-peptides found
inthe infected cell
(18, 27; M.Paskind and David Baltimore,
unpublisheddata).
Similarly, that
at180K
could correspond
to
the
140,000- to185,000-molecular-weight
pre-cursor
reported
tobe
present in intactRLV-infected cells and cell-free
systems (18, 19).There
areseveral
possible explanations for
the
apparently small
increase(if
any)
inthe
complexity of the 180K
mapoverthose
for the
60Kand
70Kproducts. Of these the
simplest
are
(i)
aggregation; (ii) tandem
repetition
of the
information for the 60K
sequences in atleast
one
of
the
virionRNAs; and (iii) the fact that
the
sequencesadditional
tothe
60K sequence inthe
180Kpolypeptide
arerelatively
poor inmethionine.
The time of
appearanceof
the
180K product after addition ofMuLV RNAto
the cell-free system is in
accord
with itssyn-thesis
as aunique
polypeptide (Fig. 2).
Inaddi-tion we
have
no evidencefor
aggregation
inany
of the
three types ofhighly
denaturing
gel
system used
for the
electrophoretic
analysis
ofthe
products,
andpreliminary
results suggest thatcomparable
amountsof the60K,
butnotof18, 1976
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[image:5.503.77.212.70.222.2]9,
6
PR*
*0
*
9t
0 ...
a
PR4
*0
¶
tI
9
PR#
a
4.
+ PR4 to
FIG. 4. Fingerprint analysis of tryptic digests of polypeptide products formedin the cell-free system in
responsetoMuLVorEMC RNAs. (A) and (B) Polypeptides(60and 70K) synthesizedinthe L-cell-freesystem
inresponse toMuLVRNA. (C) 130,000- to140,000-molecular-weightpolypeptidesynthesizedin thesame
632
A
+
B
4ja
+
C
+
9
D
4
0
44M.
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[image:6.503.69.456.39.653.2]CELL-FREE SYNTHESIS OF MuLV POLYPEPTIDES 633
A
+
PRt
tO
C
+
PR!
to-E
+
PR!
to
FIG. 5. Fingerprint analysis of trypticdigests of [35S]methionine-labeled MuLV, of virionpolypeptides, andofMuLV-specificproducts synthesizedintheL-cell-freesystem.(A) Total MuLV virionpolypeptides. (C) and(E) MuLV-virionpolypeptides P30 andP15, respectively. (B) and(D) Polypeptides (180K and 60K)
synthesizedinresponsetothe MuLV RNAintheL-cell-freesystem.(F) Endogenous polypeptide(s) of180,000
in molecularweight synthesized inthe absenceof MuLV RNA under the sameconditions asfor B and D. Loads in counts per minute were 12,000, 12,000, 7,000, 14,000, 2,200, and 6,000for A, B, C,D, E, andF, respectively.Autoradiographywasfor21 daysinallcases.Thematerial used was that describedinthe legend
toFig.4.
systemin responsetoEMC RNA. (D) endogenous polypeptide(s) of 60,000 molecular weightsynthesizedin the absenceofMuLV RNAunder the same cell-free system conditions as for A andB.After electrophoresisof
thecell-freesystemproducts as in Fig. 1, individual polypeptide bands were elated by digestion with trypsin.
Sampleswereappliedtothe bottomcenter(0)asshownof thin-layer celluloseplates.Electrophoresiswas at
pH6.5with the anode to the left. PR indicates the position of a phenol red marker. Chromatographywas
to-wards the top of the sheet.Fordirect comparison, twosampleswereelectrophoresedinparallelonthesame
thin-layer plate which was cut in half for subsequent chromatography. Loads in counts perminute were
18,000,18,000, 40,000, and 6,000 for A,B, C, and D, respectively.Autoradiography ofA,B, andD wasfor
10days.Inthis experiment and thatshown in Fig. 5,300-pdreaction mixtures were used in thecell-free
sys-temassays. Total recoveries in thefinal tryptic digests werefor plus MuLV RNA, 60K, 70K, and 180K, 440,000, 270,000, and 96,000 counts/min, respectively. In the absence of added RNA thecorresponding
figureswere120,000, 80,000, and 40,000counts/min.
VOL. 18, 1976
on November 10, 2019 by guest
http://jvi.asm.org/
[image:7.503.85.406.70.473.2]B
p
PR.
4
D
S
.+.
F
PR.
J. VIROL.
4
4
I
9
to
FIG. 5 B, D, F
the
180K,
product can be detected in thesesystems when smaller
fragments
of MuLVRNA are used asmessenger. Tandemrepeats
remainan
intriguing possibility,
butthey
seema priori an unnecessary complication.
Accord-ingly,
we would currently favor explanation(iii),
particularly
asthe presenceofadditionalnon-methionine-containing
MuLV-specific
amino acidsequences inthe 180K product can
by no means be ruled out at this stage.
Which,
ifany,oftheseexplanationsiscorrect,however,
will have to await the results ofa moredetailed
analysis and the identificationof all of the sequences present in this
high-molecular-weight polypeptide.
S
ACKNOWLEDGMENTS
This workwassupportedbyanAmerican CancerSociety
Eleanor Roosevelt Cancer ResearchFellowshiptoI.M.K.,
byU.S.Public Health ServiceInternationalFellowship FO-5-TW-2135toU.O.,andbyPublic HealthServicegrantsCA 14051 andAI 08814from theNational Cancer Institute and from theNational Institute ofAllergyandInfectious
Dis-eases, respectively. D.B. and H.F.L. were recipients of grantsVC-4G and NP-180 from theAmericanCancer Soci-ety. D.B. is anAmerican Cancer Society Professor, and H.F.L. is therecipientofN.I.H.Research Career Develop-mentaward5-K04-GM50175.
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