JUly 82-87
Copyright©1972 AmericanSociety forMicrobiology PrinitedinU.S.A.
Cell
Cycle-Dependent Activation of Rous
Sarcoma
Virus-Infected Stationary Chicken Cells: Avian
Leukosis
Virus
Group-Specific
Antigens
and
Ribonucleic Acid
ERIC H. HUMPHRIES AND HOWARD M. TEMIN
McArdle Laboratory, UniiversityofWisconsin, Madisoni, Wisconsil53706
Receivedforpublication 24 April 1972
Stationary chicken embryo fibroblasts exposed to Rous sarcoma virus (RSV) remained stably infected for at least 5 days, but they did not release infectious virus or become transformed until after cell division. These infected stationary
cells did not contain avian leukosis virus group-specific antigens or ribonucleic
acid (RNA) hybridizable to deoxyribonucleic acid (DNA) made by the RSV
endogenous RNA-directed DNA polymerase activity.
Roussarcoma virus (RSV) appears to replicate through a deoxyribonucleic acid
(DNA)
inter-mediate,
the DNA provirus(14).
Initiationof
RSV production requires two types of early DNA synthesis, one viral and one cellular (12). In stationarychicken cells exposed to RSV, viral DNA synthesis occurs, whereas cellular DNA
synthesis
does not.Therefore,
there is no celldivision or virus
production. The
presentstudies
were undertaken to determine whether
either
avian
leukosis
virusgroup-specific (gs) antigens
or viral-specific
ribonucleic
acid(RNA)
were presentin
stationary RSV-infected
chickencells.
MATERIALS AND METHODS
Generalexperimental procedureswerethesame as
thosepreviouslydescribed for thislaboratory(12,13). Cell culture. Primary cultures of fibroblasts were prepared from 12-day-old White Leghorn chicken embryos (Sunnyside Hatchery Co., Oregon, Wis.) and weregrown inmodifiedEaglemedium with 20% tryptose phosphate broth (ET medium) and 5% calf
or fetal bovine serum. Secondary andlattercultures ofchickencells wereprepared at 5.0 X 105cells per
60-mm plastic dish (FalconPlastics, Division of B-D
Laboratories, Inc., LosAngeles, Calif.) and at 2.0 X 106 cells per100-mmdish.Cultures of stationarycells
were preparedat 1.0 X 106cells per 60-mm plastic dish or at 4.0 X 106 cells per 100-mm dish in ET mediumandincubated for 3 days at 38 C in a humidi-fiedCO2incubator.
Viruses. Schmidt-Ruppin (SRV)and B77virus were previously described (1). A heat-resistant strain of
SRV (SRV-HR), at approximately 1 X 108 focus-forming units (FFU) per ml, was obtained from D. Boettiger (Ph.D. thesis, Univ. of Wisconsin, Madison,
1972). Viruswas concentrated by centrifugation and
purified by equilibrium density gradient
centrifuga-tion in 15 to 65% sucrose gradients in 0.01 M tris
(hydroxymethyl)aminomethane-0.001M
ethylenedia-minetetraacetic acid (pH 7.5) buffer.
Antisera. Two antisera were used. For immuno-fluorescence, rabbit immunoglobulin G
(lgG)
anti-serum prepared against Tween-ether-disrupted avian
myeloblastosis virus (AMV) was supplied by R. Nowinski. This serum had a fluorescence titer of
1:150onuninfected chicken cells andatiterof 1:2000
on SRV-infected chicken cells. Since the rabbit anti-AMV antiserum contained antibodies that reacted with both normal cellular components and virus-specific antigens, in vivo absorption was carriedout to
eliminate nonviral fluorescence (8). Absorption was
carried out in 2-week-old quail by intraperitoneal injection of 0.5ml of antiserum followed by
ex-sanguination6hrlater. Serawerecollected and stored
at -70C. This procedurereduced the titer of
fluo-rescence on normal chicken cells to 1:8, whereas
viral-specific fluorescence had a titer of 1:500. (Ab-sorption in 1-week-old chickens resulted in a large
reduction offluorescence on both uninfected and
in-fected chickencells.) Fortesting cell preparationsfor avian leukosis virus gs antigens, cellswere incubated with a 1:500 dilution of rabbit anti-AMV (purified
IgG) or with a 1:128 dilution of rabbit anti-AMV
(purifiedIgG) absorbed inquail.
The antiserum used in complement fixation was
preparedagainst avianleukosis virus gs antigens ina
rabbit by using ether-Nonidet P-40 (Shell Chemical Co., New York, N.Y.)-disrupted SRV. The
anti-serum was absorbed twice withsheep redblood cells
andinactivatedat56Cfor30min. The
complement-fixing titer ofthe antiserum was 1:4 on 4 units of
antigenprepared from uninfectedquail,rat,ormouse
cells,and 1:16on4units ofantigenfrom uninfected
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VOL. 10, 1972 ACTIVATION OF VIRUS-INFECTED STATIONARY CELLS
chicken cells. Thetiteron4unitsof antigen prepared from chicken cells infected with SRV or from chicken cells found to release avianleukosis virus "spontan-eously" was 1:128. For testing antigen preparations, the antiserum was usedat 1:64 and 1: 128 dilutions. Complement fixation. Complement fixation was carried out by the microtechnique of Sarma (11). Fresh serumobtained from guinea pigs was absorbed twice with 0.3 ml of packed sheep red blood cells per1 ml ofcomplement and was frozen at -70 C in 0.2-ml fractions. Rabbitanti-sheep hemolysin (Miles Laboratories, Kanakee, Ill.) was inactivated at 56 C for 30 min. Antigens were prepared after two washes withVeronal buffer. Cells, 2 X 107 per ml, were frozen and thawed three times, extracted with an equal volume of ether, andcentrifuged for 1 hrat20,000 X g. Indirect immunofluorescence. Indirect immuno-fluorescence wasusedtotestcell preparationsforviral antigens by the methodofHilgersetal. (8).
Immunofluorescence absorption test. Cellswerealso
examined for the presence ofviral antigens bytheir
ability to absorb the positive fluorescence from a
known standard system by the method of Hilgers
etal. (8). Antigens were prepared from cells as de-scribed above for complement fixation. Antiserum
wasusedat afourfold dilutionbelowtheendpointof viral-specific fluorescence on standard positive cells.
Fifty
1liters
of antisera were absorbed overnight at4Cwithvariousamountsoflyophilized antigen pro-tein. Antiserum without antigen was included as a
positive control. These antisera preparations were
then used in indirect fluorescence tests on standard
positivecells.
Polymerase reaction. The standard polymerase
re-action of Temin and Mizutani (16) was used to
produce large amounts of radioactive virus DNA product.
Reactions were carried out in a total volume of 2.50 to 6.50 ml of reaction mixture containing 160
pg ofpurified virus per ml and 20,Ci each of
3H-thymidine-5'-triphosphate (13.4 Ci/mmole; Schwarz BioResearch, Inc.) and
3H-deoxycytidine-5'-triphos-phate (22.5 Ci/mmole; Schwarz BioResearch, Inc.)
perml. Thereaction mixturewasdistributed in 0.5-ml samples andincubated at40 Cfor 1 hr, after which the reaction mixtures were pooled for nucleic acid extraction. Approximately 6,000 counts/min of
tri-chloroacetic
acid-precipitable
productcorresponded
to 1ngofDNA.
Nucleic acids and hybridizations. The procedures
usedforextraction of nucleic acids from
polymerase
reactions, purified virus, and cells by using
diethyl-pyrocarbonate (Baycovin; Bayer, Leverkusen, Germany) andsodium
dodecyl
sulfate andfor RNA-DNA hybridizations have been described by Coffin and Temin (3, 4). Control hybridizations between uninfected chicken cell RNA and B77 virus productDNA contained a background of 3 to 5% of the
countsbandingat a density
equal
to orgreaterthan1.54 g/ml. Percent hybridization with virus-infected chicken cell RNA was defined as the fraction of
countsbanding at adensity
equal
to orgreaterthan1.54g/mlminus the
background
ofparallel
hybridi-zationswithuninfected chicken cellRNA.The
density
of 1.54 g/ml gave a sligbtly lower background com-pared with the density of 1.53 g/ml used earlier
(3, 4). Approximately45 to 50% oft'hetotal B77 virus product DNA used in these studies was shown to hybridize with a large excess of viral RNA. However, in the experiments reported here withcell RNA, lower levels of hybridization were achieved because there were lower concentrations of viral-specific RNA. Recoveryof labeled DNA from gradients was always greater than 65 to 70%. Previous reports (3, 6, 7) have established that hybrids between RSV product DNA andviral-specific RNA band in the RNA region ofCs2SO4 gradients after equilibrium centrifugation. DNAcomplementary to the RSV RNA, "minus" DNA, was prepared aspreviously described by Coffin andTemin(5), byhybridization of total virus product DNAwith excess viral RNA and alkali hydrolysis of the fractionof DNA banding with a density greater than 1.65 g/ml after Cs2SO4 equilibrium centrifuga-tion.
RESUTLTS
Absence of avian leukosis virus gs antigens in RSV-infected stationary chicken cells.
RSV-infected
stationary chicken cells contain thegenome of RSV, but do not release infectious virus
(12).
These cells were tested by using threeimmunological
techniques, indirectimmuno-fluorescence,
complement fixation, andimmuno-absorption fluorescence, to determine whether they contained avian leukosis virus gs antigens.
Stationary chicken embryo fibroblast cells
wereexposed
to SRV-HR, either ET medium or ETmedium with serum was added, and various times
after infection
the percentage ofcells
containing
gs
antigens
wasdetermined
by using indirectimmunofluorescence (Fig.
1). Whereas morethan
80% of
the cells
in theserum-containing culturesbecame
positive for gs antigen within 48 hr,cultures
of
stationary
cells were nevermorethan10%
positive
evenafter
130 hr. At 84 hrafter
infection, serum was added to cultures of sta-tionary cells, and the cells were examined for gs
antigens.
Thesecells
were50%
positive for gsantigen within
48 hrafter addition of
serum.The
low level
of positive cells in the cultures ofstationary
cells and the low levelof
viruspro-duced
inthese
cultures are mostprobably the
result
of division occurring inasmallproportion
of
the
population (15). This interpretation is
sup-ported
by
the observation that autoradiographyof cultures of stationary cells
labeled
with3H-thymidine
for 24 hr showedapproximately10%
ofthe nuclei were labeled (data not
shown).
Infected
stationary
cells
werealsotested for gsantigens by complement
fixation(Fig.
2).
Again
nogs
antigens
werefound.To demonstrate the specificity of the
immuno-logical
tests described above,immunoabsorption
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HUMPHRIES AND TEMIN
A
80/ / /(106)
40 I /
| /(104) ~~(le4) X (104)/
1
o5(lo2)
~-g
--- x--x0o 20 40 60 80 loo 120 140 160 180 HOURSAFTER INFECTION
I
C,)
w oI
0-'fo
8~--x _
8)2
~ ~ ~ ~ ~ ~
I L L
20 40 60 EC 100 120 140 160 180
HOURS AFTER INFECTION
FIG. 1. Absenice of avian leukosis virus
group-specificantigensfrom RSV-inifectedstationzarychicken cells. A,Stationarychickenembryofibroblasts, 1.0 X
106 cells per 60-mm culture dish, were exposed to
SR V-HR at a multiplicity of I FFU/cell, anid the inoculum was replaced with ET medium (X) or ET
medium with
4%lo
fetalbovine serum (0). At varioustimesafter infection,cellswereharvested and examined
for gsantigensby usinig indirectfluorescence. Rabbit
anti-AMV(purified IgG) wasused ata1:500dilutiont.
Eachpointrepresents300 cells. At 84 anid 130 hrafter
infection, serum-containing medium was added to
cultures without serum
(I
.). Culturefluids were nlotchanged during theexperiment. Virus titers (indicated
inparentheses)representFFU per mlofculturefluidat
the timeofcellharvest. Uninfectedcellswereincubated in medium with 4% fetal bovine serumfor48 hr and
lusedas a negative control (*). B, Cell niumbersper
culture dish were determinedJbr the infectedcultures
with ETmedium (X), for the infectedcultures with
ETmedium with4%.fetalbovineserum (0), atdfor
the uninfectedcultures withETmedium with4%fetal
bovine serum (U) described int A.
fluorescence was carried out with the antigen preparations from the experiment described in
Fig.2. Theantigenswereusedexactlyasprepared forcomplementfixation. Theimmunoabsorption
wascarriedoutby using overnightabsorption of z
O
g, 60~
o
Z0
X
--20
0 z
8 oo
0
A
--;L___~~~~~~~~~~a-
-24 48 72 96 120 144 168
HOURSAFTER INFECTION
| ~~~~~~~~~B
24j
o20L 16-'0
Cr 122-//
Z 8- //
U 4s/ ---J-.
0 24 48 72 96 120 144 168
[image:3.493.59.253.58.373.2]HOURS AFTER INFECTION
FIG. 2.Absetnce ofaviatn leukosis viruisgroup-specific
antigenis from RSV-inifected stationary cells. A,
Stationiary cells, 4.0 X 106 cells/100-mmculturedish,
wereexposedto SR V-HRatamultiplicity of1FFU/
cell, and the inoculum was replaced with ETmedium
(X) or ETmedium containinig 4%O fetal bovineserum
(0). At 24-hr intervals, cells were harvested and processedforcomplementfixationi.Anztigen preparations
werenormalizedto30 ug of proteiniper25ulitersand
assayed with complement fixation forgs antigenis by
using rabbit anti-SR Vata1:64dilutionz. Culture fluids
were changed on all cultures 72 hr after inifectionz.
Serum was added to ciultures with ETmedium (i)
120hrafter infection. B, Cell numbersperculture dish
weredeterminedfor the cultures with ETmedium (X)
and ET medium with 4%, fetal bovinie serum (0)
describedinA.
rabbit anti-AMV at4 Cwith variousamountsof
lyophilized protein from different antigen
prepara-tions(Table 1). Onlydividingcells at 72 and 120 hrafter infectioncontained enoughgsantigensto absorb specifically the capacity of the antiserum
to react with the positive controls. The fact that
equivalentamounts offetal bovineserumand of
84
J)
-J
-i
> 0
0.
z I)
LU
J. VIROL.
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[image:3.493.119.445.64.405.2]VOL. 10, 1972 ACTIVATION OF VIRUS-INFECTED STATIONARY CELLS
TABLE 1. Detectioni ofavian leukosis virus group-specific anitigensbyusingimmunoabsorption
fluorescence
Antigenpreparationa
RSV-infected cells Stationary Dividing Stationary Dividing Stationary Dividing Fetal bovine serum
Uninfected chicken cells
HAIb
24 24 72 72 120 120
Proteinc (mg)
500 500 500 50 500 50 500 500
Pres-ence of fluo- res-cenced
aAntigens prepared from cell extracts as
de-scribed in Fig. 2 for complement fixation. RSV,
Roussarcoma virus.
I Hoursafter infection.
cMilligrams of protein usedtoabsorb50 ,tliters
of 1:500 rabbit anti-avian myeloblastosis virus
(purified immunoglobulin G).
dObservedpresenceoffluorescenceonstandard
slides usingantisera afterabsorption.
ePresence ofgroup + specific (gs) antigens in
protein used for absorption (indicated bythe
ab-sence offluorescence).
uninfected chickencellprotein didnot absorbthe fluorescence illustrates the specificity of this test.
The results of these experiments demonstrate thatgsantigensarepresentonlyin low amounts in
culturesofRSV-infectedstationarycells,whereas
theyarepresent inlargeamountsindividingcells.
Absence of viral-specific RNA in stationary RSV-infected cells. To determine whether
viral-specific RNA was present in RSV-infected
stationary chicken cells, RNA-DNA hybridiza-tions were carried out with RNA from infected
stationary cells and DNA from the B77 virus
endogenous RNA-directed DNA polymerase
re-action. Sample gradients show the analysis of
hybridizations between total B77 virus product
DNA and RNA from stationary and dividing chickencells 120 hr after infection with SRV-HR
(Fig. 3). A controlhybridization withuninfected chicken cellRNAis also included.
Figure 4 summarizes the results of hybridiza-tions of RNA extracted 36 hr after infection from SRV-HR-infected stationary chicken
em-bryo fibroblasts with whole B77 virus product DNA (Fig. 4A) and with the "minus" DNA
isolated from whole B77 virus product DNA
(Fig. 4B). In both sets ofhybridizations, 150 ,g
of RNA from infectedstationarycellsshowedno
morethan
0.5%c,
hybridization above background values of 4.5 and 2.5%o hybridization with unin-fected chicken cell RNA. There was approx-imately a 100- to 150-fold difference between the amount of viral-specific RNA in RSV-infected dividing and RSV-infected stationary cells.Figure 5 shows the results ofhybridizations of B77 virus product DNA and various amounts of RNA extracted 72 and 120 hr after infection fromstationary and dividing cells. RNA prepara-tions from dividing cells 72 and 120 hr after
infection
gaveapproximately 21 to22%
hybridi-zation at saturation. A
150-,ug
amount of RNA from parallel stationary cells gave only 3 to4%
hybridization. Hybridization with uninfected chicken cell RNA gave a background value of 4%. There was at these times approximately 20-fold more RNA hybridizable with viral product DNA in RSV-infected dividing cells than in RSV-infected stationary cells.
The results of these experiments demonstrate
that
viral-specific
RNA is present only in lowamounts in cultures of RSV-infected stationary cells,whereas RSV-infected dividing cells contain 20-to 150-foldmoreviral-specificRNA.
DISCUSSION
Provirus formation occurs in stationary cells of RSV-infected chicken embryo fibroblasts, whereas cell cycle-dependent activation and release of
infectious
virus does not (12). The resultspre-sented above demonstrate that cultures of
stationary
cellscontain
only small amounts ofavian leukosis
virus gsantigens
andviral-specific
RNA. Although
90%
ofRSV-infected dividing cells contained gsantigens
48 hr afterinfection,
only
10%
of the cells in cultures without serumcontained gsantigens6 daysafterinfection. Since
a small proportion of cells in cultures without
serum
proceed through
celldivision,
it islikely
thatthe cells in cultures withoutserumthat were positive for gs antigens had proceeded through mitosis. In
parallel
experiments,
RSV-infectedstationary
cells containedonly
small amounts ofviral-specific RNA compared to the amounts
found in
parallel
dividing
cells. At 36 hr afterinfection,
dividing
cells contained 100-fold moreviral-specific RNA than found in
stationary
cells. Three and fourdays
afterinfection,
this ratiodecreasedto a 15to20-fold difference. It is
likely
that this decrease is the result ofcell division in
the cultureswithoutserum.Itappearsthat neither
viral-specific
RNA nor avian leukosis viral gsantigens are
produced
instationary
cells andthat, until cell
cycle-dependent
activation,
trans-cription of the
provirus
does not occur.85
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UNINFECTED CHICKEN A INFECTED STATIONARY CHICKEN INFECTED DIVIDING CHICKEN
- 17 7 17
Q16Qb,v8 8- 1:
662 20 2 4 6 2 4 6
Q. ~~~~~~~~~~~~0.0.
2- 2~ 2
4 8 12 14 20 24 4 8 12 16 20 24 4 8 12 16 20 24
FRACTION FRACTION FRACTION
FIG. 3. Hybridization withB77 virusproduct DNA and RNA extracted 120 hrafter infection fromculturesof
stationaryanddividingSRV-HR-infectedchickencells.Stationarychickenembryo fibroblasts,4.0X 106 per
100-mmdish, wereexposedto SR V-HR at amultiplicityof100FFU/cell, andthe inoculum was
replaced
withET mediumorET medium containing4%fetal calfserum.At 120 hrafterinfection,cellswereharvested,andthe RNAwasextracted. RNAwashybridizedwith6,000counts/minofB77virusendogenous
3H-labeled
DNAproduct
for
5hr at68 C,andthe mixturewasanalyzed by Cs2SO4 equilibrium density gradientcentrifugation.Thehybridizations
werecarried outwith RNAasfollows:A, 150,sg of uninfectedchickencellRNA;B, 150 ,&gof infected stationary
chicken cellRNA, 120 hrafterinfection;C, 75pg ofinfected dividingchicken cellRNA,120hr
after infection,
plus75ugofuninfected chicken cell RNA. Thearrowsindicatethedensityof1.54g/ml.
"MINUS STRAND"
WHOLE VIRUSPRODUCT DNA VIRUS PRODUCT DNA
8 A '
20I20 B
Al~~~~~~~~~~~~~~A
Al
2~~~~~~~~~~~~~~~~2
2z DIVIDING CELLm IIIGCL
2
w z
n
~~~~~~~~~~ID
Zlo
_
<
10_
/
z
8I-/ STATIONARY CELL ~~~~~~~z /
CL STATIONARY CELL
a.
I0 I0 IO Oc
&)50 100 150 0 50 100 150
jxg RNA 9ugRNA
FIG.4. Hybridizationbetween B77 virusproductDNAandRNAextracted 36 hrafter infectionfromculturesof
stationaryanddividing SRV-HR-infectedchickencells.StationarycellswereinfectedasdescribedinFig.3and
wereharvested36 hrafter infection. A, VaryingamountsofRNAfrom infected stationarycells(X)andinfected
dividing cells ( )werehybridized with 3,000counts/minofendogenous3H-labeledB77 virusDNAproduct for5hr
at68Candanalyzed by
Cs2S04
equilibrium density gradientcentrifugation. Varyingamountsofuninfected chickencellRNA were addedsothat allhybridizationswerecarried out in thepresenceof150
pg
ofchickencellRNA. A150-pg
amountof uninfectedchickencell RNAwasalsohybridized without addedinfected cell RNA(U).
B,Dupli-catehybridizations werecarriedoutas in Aexceptthat 3,000counts/minof"minus"strand B77 virusproduct DNA
wasusedineachhybridization.
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[image:5.493.57.453.50.238.2] [image:5.493.111.396.379.569.2]VOL. 10, 1972 ACTIVATION OF VIRUS-INFECTED STATIONARY CELLS
10, 17). Itispossible that activationof transcrip-tion isinvolved inallthesesystems.
ACKNOWLEDGMENTS
We thank R.Chen, R.Grimstad,V.Kassner,and A.Kato for technicalassistance.
DIVIDING CELL This investigation was supported by Public Health Service researchgrantCA-07175 from the National Cancer Institute atsd grant VC-7 from the American CancerSociety. E. Humphries wassupported by traininggrantTOI-CA-5002from the National CancerInstitute. H. M.Temin holds Research Career
Develop-mentAward1OK3-CA-8182 from the National Cancer Institute.
STATIONARY CELL
,ug RNA
FIG. 5. Hybridization between B77 virus product
DNAand RNAextracted72and 120hrafter infection
from SRV-HR-infected chicken cells, dividing and stationary. Cells were infected, and RNA was
ex-tracted and hybridized with whole B77 virusproduct
DNA as described in Fig. 3. Under these conditions,
1.5 ugofB77 virusRNAgaveapproximately25to27%
hybridization. The RNA species in the hybridization
mixtures were: uninfected chicken cell RNA (-);
infectedstationary (A) anddividing (A) chicken cell RNAat 72hrafterinfection; infectedstationary (a)
anddividing (0) chicken cell RNA at 120 hr after infection.
The RNA-DNA hybridizations were carried
out with an endogenous B77 viral DNA
polym-eraseproduct capableofdetecting approximately 20% ofthe viral RNA (5). Although the large
amounts ofviral-specific RNA found individing
cells were not present in stationary cells, it is
possible that some viral-specific RNA was in
stationary cells which was not detected by the DNAusedin thishybridizationprocedure.
Leong et al. (9) have reported that therate of
productionof RSV-RNAvarieswiththestageof
thecellcycleoftheinfectedcells.Thiswould bea second control of viral transcription once the
transcription was activated by the initial cell
division.
Cell cycle-dependent activation of differentia-tion hasbeenreportedinseveral othersystems(2,
LIERATURE CITED
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rat cells in vitro by avian sarcoma virus. Virology 40: 113-134.
2. Bischoff, R., and H. Holtzer. 1969. Mitosis and theprocesses ofdifferentiation of myogenic cells invitro. J. Cell. Biol. 41:188-200.
3. Coffin,J. M.,and H. M. Temin. 1971.Comparison of Rous sarcoma virus-specific deoxyribonucleic acid polymerases invirions of Roussarcomavirus andRoussarcoma virus-infected chicken cells. J. Virol. 7:625-634.
4. Coffin, J. M., and H.M.Temin.1971.Ribonuclease-sensitive
deoxyribonucleic acid polymerase activity in uninfected ratcells andratcellsinfected with Roussarcomavirus. J. Virol. 8:630-642.
5. Coffin, J. M.,andH.M. Temin.1972.Hybridization of Rous sarcoma virus deoxyribonucleic acid polymerase product and ribonucleic acids from chicken andratcellsinfected with Roussarcomavirus. J.Virol. 9:766-784.
6. DuLesberg, P. H., and E. Canaani. 1970. Complementarity between Rous sarcoma virus (RSV) RNA and the in
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7. Fanshier, L., A.-C. Garapin, J. McDonnell, A. Faras, W. Levinson, andJ. M. Bishop. 1971. Deoxyribonucleic acid polymerase associatedwith avian tumorviruses:secondary
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8.Hilgers, J., R. C. Nowinski, G. Geering, and W. Hardy. 1972. Detection of avian and mammalian oncogenic RNA viruses (oncornaviruses) by immunofluorescence. Cancer Res. 32:98-106.
9. Leong, J. A., W.Levinson, andJ.M.Bishop. 1972.
Synchroni-zation of Roussarcomavirusproductioninchickembryo
cells.Virology 47:133-141.
10.Pietsch,P., andS. B.McCallister. 1965.Replication and the activation of muscle differentiation. Nature (London)
208:1170-1173.
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avianleukosisgroup-specific complement-fixation reaction: applicationforthe detectionandassayof non-cytopatho-genic leukosisviruses.Virology23:313-321.
12. Temin. H. M. 1967. Studies on carcinogenesis by avian sarcomaviruses. V. RequirementfornewDNAsynthesis and forcell division. J. CellPhysiol. 69:53-63.
13. Temin, H. M. 1968. Studies on carcinogenesis by avian sarcoma viruses. VIII. Glycolysisand cellmultiplication. Int. J. Cancer 3:273-282.
14. Temin, H. M. 1971. Mechanism of cell transformation by RNAtumor viruses. Annu. Rev. Microbiol. 25:609-648. 15. Temin, H. M. 1971. Stimulation byserumofmultiplication ofstationarychicken cells. J.Cell Physiol. 78:161-170.
16.Temin, H. M., and S. Mizutani. 1970. RNA-dependent DNA polymerase invirions of Roussarcomavirus.Nature (London) 226:1211-1213.
17. Yamada,T.1966. Control of tissuespecificity:thepatternof cellularsyntheticacdvities in tissuetransformation. Amer.
Zool. 6:21-31. Q
0
Al
z m z
w H z
N C,,
z
0
0 z w
w
a-87