Copyright©1977 AmericanSocietyforMicrobiology Printed inU.S.A.
Evidence
for Recombination Between N- and
B-Tropic
Murine
Leukemia
Viruses:
Analysis
of Three Virion
Proteins
by Sodium
Dodecyl
Sulfate-Polyacrylamide
Gel
Electrophoresis
JOHNSCHINDLER, RICHARD HYNES,ANDNANCYHOPKINS*
Center for CancerResearch, Massachusetts Institute ofTechnology, Cambridge,Massachusetts 02139 Receivedfor publication 29March 1977
We have used sodium dodecyl sulfate-polyacrylamide gel electrophoresis to
analyze thevirionproteinsofanN-andaB-tropicC-typevirusderived from the
BALB/c mouse and 21 putative recombinants, designated XLP-N viruses,
ob-tained fromsevencrossesbetweenthese N- andB-tropic viruses. All the XLP-N
viruses are N-tropic but possess the XC plaque morphology of their B-tropic
virusparent. Threevirionproteins, p15,p30, and gp7O, of the parental viruses
each differ in electrophoretic mobility. Two recombinants were found that
possess a p15 that comigrates with p15 of the B virus; 19 possess ap15 that
comigrateswith N virusp15. Sixteen recombinantspossess agp7Othatmigrates
like thegp70oftheBvirus;fourhavegp70withanelectrophoreticmobilitylike
that ofthe N virusgp7O. All21recombinantspossess ap30 thatcomigrates with
p30 of theirN virus parent. Given the originandphenotype ofXLP-N viruses,
these results wouldseem toprovidegood evidence that these virusesare
recom-binants.
Naturally occurringecotropic murine
leuke-miavirusescanbe classified accordingtotheir
host range as either N-tropic or B-tropic
de-pendinguponwhethertheygrowpreferentially
oncells derived from NIH Swiss mice (N-type)
or BALB/c mice (B-type), respectively (8).
Inbredstrainsofmice canbeclassifiedaseither
N-orB-type, and thisphenotype is determined
byagenecalledFv-1 (22,23;reviewedin
refer-ence 19). Restriction toward a virus of the
"wrong" tropism is inherited as a dominant
trait. The restriction of viral growthon acellof
incompatible Fv-1 type is relative rather than
absolute and occurs after virus absorption and
penetration but before integration of proviral
DNA (13, 16, 17, 27).The viraldeterminants of
N- or B-tropism have not yet been identified
(11,24). The Fv-1 gene plays a role in
determin-ingsusceptibilitytoleukemogenesis by N- or
B-tropicmurineleukemiaviruses presumablyby
retarding the spreadandresulting viremiaofa
virus ofthe wrong tropism (see 19).
Using plaque morphology (9, 25) and N- and
B-tropism as genetic markers, Hopkins et al.
(12) havepresented biological evidence for
re-combination between an N-tropic virus that
makes smallXC-plaques (SP-N) and a B-tropic virus that makes large plaques (LP-B), both
derived fromthe BALB/c mouse: after
coinfec-tionwith SP-N andLP-Bthey obtainedviruses,
called XLP-N, thatareN-tropic butmakelarge
XC plaques. Hopkins etal. (10) have also
ob-tained evidence based on antigenicity, that
XLP-N viruses arerecombinants. We were
in-terested inextending this biochemical analysis,
since analysis of a number of recombinants
mightproveusefulinidentifying and mapping
the viral determinants of XC plaque
morphol-ogy and N- and B-tropism. Famulari,
O'Donnell,and Fleissner(personal
communica-tion;see 11)have shown that three virion
pro-teins of some N- and B-tropic virus isolates
derived fromBALB/c micecanbe distinguished
by their mobility on sodium dodecyl sulfate
(SDS)-polyacrylamide gels. These are p15, an
internal virion protein, p30, the major
struc-turalprotein of the capsid of thecoreshell,and
gp7O, the majorenvelopeglycoprotein which is
responsible forspecificabsorption of C-type
vi-ruses tocells. O'DonnellandStockert(20)have
shown that these viruses also differ in their
abilitytoinduce
G1Q,
anantigenicdeterminantbelievedto resideongp7O (28). These
observa-tionsprovideduswith ameans ofaskingifthe putativerecombinants obtainedby Hopkinset al. (12) possessed p15, p30, and gp7O proteins
withtheelectrophoreticmobilitycharacteristic
oftheir N- or B-tropic virus parent. Here we
present the results ofSDS-polyacrylamide gel electrophoresis analysis of virion proteins of 21 XLP-N viruses obtained from seven independ-ent crosses between SP-N and LP-B viruses.
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VIRION PROTEINS OF N- AND B-TROPIC VIRUSES
Analysis of these viruses for the ability to in-duce the Gx antigen has been reported previ-ously (10).
MATERIALS AND METHODS
Cells, viruses, and media. Sc-1 cells (7) were grown in Dulbecco modified Eagle medium with 10%inactivated fetal calf serum and antibiotics. The origin ofSP-N (9), LP-B (B-clone-18) (15), and XLP-N viruses (12) has been described. XLP-N viruses werepurified either by plaque purification (12) or by themicrotiter method (26).
Virus purification and gel electrophoresis. The preparation of virion proteins for gel electrophoresis has been described (11). Briefly, virus was purified by bandinginsucrose gradients and thenpelleted. Electrophoresis of virion proteins was on Laemmli (18)slab gels asdescribed (11). Immunoprecipitation of the p15 virion protein was performed with goat anti-AKRp15serumusingNonidet P-40-solubilized virus followed by precipitation with pig anti-goat immunoglobulin G. The antibodies were a gift from Nancy Famulari.
RESULTS
Parental viruses SP-N and LP-B possess p15's, p30's, and gp7O's that each differ in
electrophoretic mobility on
SDS-polyacryl-amide gels. Virus was purified from culture
fluids of Sc-1 cells (a cell line that does not
exhibit Fv-1 gene restriction) infected with either SP-N or LP-B virus and disrupted with
SDS, and the proteins were separated by gel
electrophoresis. Figure 1 shows a slab gel on
which thevirion proteins ofSP-N and LP-B are
visualizedbyCoomassie brilliant blue staining.
The identificationofp30 isbased onpublished
data (1) and on a comparison with molecular
weight markers (Fig. 1,track1). The
identifica-tion ofthe p15 band isbased on unpublished
observations ofN. Famulari and E. Fleissner
(personal communication), on published data
(1, 14), on acomparison with molecular weight
markers, andonthe factthatthis bandcan be
precipitated using antiserum specific for p15
(unpublished data).It canbeseen inFig.1that
thep15andp30proteinsof SP-NandLP-Beach
differinelectrophoretic mobility: the p30 of
SP-N runsslightly ahead of p30 ofLP-B whereas the N virus p15 migrates slightly behind the
p15 ofLP-B. These differences were observed
not only on the standard Laemmli gel system (18), but also on phosphate-buffered SDS gels
(6), 6 M ureaphosphate-buffered SDSgels,6M
urea Laemmli gels, and low-bis Laemmli gels
(5) (unpublisheddata).
gp7O was visualized by autoradiography of
gels containing virion proteins labeled with 14C-aminoacids(Fig. 2). Thegp70wasidentifiedby
comparison with molecular weight markers andbythe fact that thisband could be labeled
-p30
A:,;.::
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.
.
....
1
2
3
4
5
FIG. 1. Comparison ofp30 and p15 virion pro-teinsofparentalvirusesSP-N and LP-B.Figure1 is an 8to 24%slabgelstained with Coomassie brilliant blue. Samplesare asfollows: 1, Markermix:
phos-phorylase A, 90,000 daltons; catalase, 62,000 dal-tons;creatinekinase, 40,000daltons; carbonic anhy-drase, 29,000 daltons; cytochrome c, 12,700daltons; 2and 4, SP-N;3and 5, LP-B.
with[14C]glucosamine (unpublished data).The
gp7O of SP-N migratesahead of thegp7Oof
LP-B. The differences in electrophoretic mobility
betweenthep30's andthep15'sof SP-N and
LP-Bcan also be seenontheautoradiograminFig.
2, but the difference isnotas clearas inFig. 1
since the exposure time necessary to detect
gp70results in overexposure of thep15andp30
bands.
Virion proteins of recombinants between SP-N and LP-B. Having determinedthat the
pi5, p30, and gp70 proteins ofSP-N and LP-B,
theparentalvirusesusedtogenerate the
puta-tive recombinants, could be distinguished as
701
VOL. 23, 1977
.", --
N
...B
--.w
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[image:2.503.252.444.74.436.2]702 SCHINDLER, HYNES, AND HOPKINS
"O*
CIO_- w
B
N
B N
_"_"
a_
Id~
ing p30 bands. Thisgel shows the p30's ofsix
recombinants, and, for comparison, each is
flanked by the parentalviruses SP-N and
LP-B.All of therecombinants shownpossess ap30 thatcomigrateswith thep30 of SP-N.Figure5a
and b shows gelsonwhichvirionproteinswere
loadedat a concentrationoptimal for
visualiz-ingp15 bands. Of the recombinantsshown,two
(Fig. 5a, track9, andFig. 5b, tracks 2 and4)
possess a p15 that migrateslike thep15of
LP-B.
Figure 6 is an autoradiogram showing the gp7O's of six recombinants. Five of these
recom-_0 D
70
binants possess agp7O
thatmigrates
like thegp7O of LP-B; one (track 4) possesses a gp7O
whose electrophoretic mobility is like that of
SP-N gp7O. Some variation in the width ofgp7O
bands has beennoted, and sometimes the result
of this variationis to give the impression that
recombinant virus gp70's differ slightly from
those ofeither SP-N or LP-B; however, wedo
notknow ifthisdifference issignificant.
Table1is a summaryofdata from thistype of
analysis ofall 21 recombinants. Of the 21
vi-- p30 ruses, 16possess agp7O thatmigrateslike the
gp7O ofLP-Band2 outof21possess ap15that
comigrates with p15 of LP-B, whereas none
possesses a p30 that migrates with the p30 of
LP-B. All ofthe remaining proteins (with the
exceptionof thegp7O of recombinant5-1,whose
40
-0
-NW4NO
-
p15
1
2
3
4
FIG. 2. Comparison of gp7O protein of SP-N and LP-B viruses. Autoradiogram of '4C-labeled virion proteins; 8to20%slab gel. Samplesare asfollows:1 and3, LP-B;2and4,SP-N.
predicted,wecouldnowaskwhetherclonal
iso-lates of XLP-N virus possessed p15, p30, and gp7Oproteins with theelectrophoretic mobility characteristic of the corresponding proteins of their N-orB-tropic virusparent.Sc-1 cellswere
infected with each of21clonal isolates of XLP-N derived from seven independent crosses of
SP-N and LP-B. Much of the information
per-tainingto theorigin ofthese viruses has been reported (10, 12). It is summarized in Fig. 3.
Figure 4isagel onwhich virion proteinswere
loaded at aconcentrationoptimal for
visualiz-CoinfectSc-i cells (8) withSP-N andLP-B viruses
J,
Use progeny'toinfectNIH/3T3cells
Harvest progeny2 3 days later. Infect NI /3T3 cells
Harvest progeny3 3 days later. Determine end point titer by in-fection of
NIH/3T3 cells
Harvestprogeny4from end point infection. Cross (1)Plaque purify XLP-N from progeny4 to
obtain 1-Pl.
(2) Microtiter clone XLP-N fromprogeny3to obtain2-1, 2-2.
(3) MicrotitercloneXLP-Nfromprogeny3to obtain3-1, 3-2.
(4) Microtiter clone XLP-N fromprogeny3to obtain4-1.
(5)Microtiterclone XLP-N fromprogreny3to obtain5-1, 5-2, 5-3, 5-4, 5-5.
(6)MicrotitercloneXLP-N fromprogeny3to obtain6-1, 6-2, 6-3.
[image:3.503.68.256.78.481.2](7)Plaque purify XLP-N from progeny' to obtain 7-Pl, 7-P2, 7-P3, 7-P4, 7-P5, 7-P6, 7-P7.
FIG. 3. Origin of XLP-Nviruses.
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[image:3.503.272.464.393.653.2]VIRION PROTEINS OF N- AND B-TROPIC VIRUSES 703
B
R
N
R
B
R
N
R
B
R
N
R
- - - -_- .
--
-p30
1
2
3
4
5
6
7
8
9
10
11
12
FIG. 4. Comparison ofp30protein of SP-N, LP-B, andrecombinants. An 8 to 24% slab gel stained with Coomassie brilliant blue. Samples are as follows: 1, LP-B; 2, 2-1; 3, SP-N; 4, 2-2; 5, LP-B; 6,1-PJ;7, SP-N; 8, 4-1; 9, LP-B; 10, 7-P2; 11, SP-N; 12,5-4.
electrophoretic mobility has not been
deter-mined)appear to migratewith those ofSP-N.
The last column in the table shows the
re-sults ofanalysis ofthe parental and
recombi-nant viruses for the ability to induce the
G1x
antigen, an antigenic determinant believedto
reside on the gp7O ofsome murine leukemia
viruses and that is present oncells infectedby
the N-tropic (G+) but not the B-tropic (G- )
parent of the recombinants. These data are
taken fromHopkinsetal. (10). XLP-Nviruses
that are G& possess a gp7O that appears to migrate with SP-N virus gp7O, whereas the
gp7O of G1x XLP-N viruses migrates like the
gp7O of LP-B virus. This result is consistent
with evidence suggesting that
Gjx
resides ongp70 (28).
DISCUSSION
We have used SDS-polyacrylamide gel
elec-trophoresis toexaminethree virionproteins of
SP-N, an N-tropic virusthat makes small XC
plaques, LP-B, a B-tropic virus that makes
large XC plaques, and 21 large XC
plaque-forming N-tropic recombinants, designated
XLP-N viruses, betweenSP-N andLP-B. That
XLP-N viruses are recombinants was
sug-gested from their biological properties (12)and
from antigenic analysis (10). Since Famulari
and Fleissner (personal communication) had
shown, and we have confirmed here, that the
p15, p30, andgp7O proteins ofN- and B-tropic viruses of BALB/ceachdifferinelectrophoretic
mobility on SDS-polyacrylamide gels, we
ex-amined theseproteins in XLP-N viruses to
de-termine whether the recombinants inherited
both SP-N- and LP-B-like proteins. We have
found that some XLP-N viruses possess ap15
and/or gp70 with the electrophoretic mobility
characteristic of the corresponding proteins of
their B-tropicvirus parent.
A simple interpretation of these results is that XLP-N viruses that possessap15orgp7O
that comigrates withp15orgp7Oof LP-B inher-ited the corresponding genes from LP-B and,
similarly, that migration of an XLP-N virus
p15, p30, or gp70 with the corresponding pro-teins ofSP-Nimplies inheritance of the
corre-spondinggenes fromSP-N. However, sincep15
and p30 are cleaved from a precursor protein
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[image:4.503.50.445.84.367.2]C
M
mB
R
N
R
B
R
N
R
B
-~~~~~~~4--__~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~,.f
4
5
6
7
8
D
B
R
B
R
BR
B-_w_
...AIN..WI
_^0
a_
a
_a
a
.
0
DI
q _ | -I
2
3
4
5
6
FIG. 5. Comparisonofp15proteinofSP-N, LP-B,and recombinants.An 8 to24%slabgelstained with Coomassiebrilliant blue.Samplesareasfollows:Gela-1,marker mixasinFig. 1; 2,LP-B;3,5-2;4, SP-N; 5, 5-3; 6,LP-B; 7,5-4;8, SP-N; 9,5-5; 10, LP-B. Forgel b:1, LP-B (the band belowp30 inLP-B is a
contaminatingcellproteinpresent inthisparticular preparationof virus); 2, 5-5; 3,LP-B;4, 6-2; 5,LP-B; 6,
6-3; 7, LP-B.
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[image:5.503.109.411.57.632.2]VIRION PROTEINS OF N- AND B-TROPIC VIRUSES 705
. , , . ., o_ *f .
B
R
N
R
B
R
N
o a
e e e S
S
do
1 234 5
N
R
B
RN
B
S .4
"-
<io
e
a
- a
-gp7O
-p30
-p15
[image:6.503.49.447.79.438.2]6
7
8
9
10
11
12
13
14
FIG. 6. Comparison of thegp7O protein of SP-N, LP-B, and recombinants. Autoradiogram of'4C-labeled
virionproteins;8to 20%slab gel.Samples are as follows: 1, LP-B; 2,1-PZ;3,SP-N; 4, 5-4; 5, LP-B; 6, 7-6; 7, SP-N; 8, SP-N; 9, 6-1; 10, LP-B; 11, 3-2; 12, SP-N; 13, 6-3; 14, LP-B.
codedforbytheso-called gag gene (23, 29), and
since the "rules" governing this cleavage are
not known, otherinterpretations can be
imag-ined, and studies on the primary structure of
the proteins involved would be necessary to
confirm thissimple interpretation.Inany case,
that XLP-N viruses inherit proteins with the
electrophoretic mobility characteristic of the
corresponding proteins of LP-B virus would
seem to provide evidence that these viruses
have inherited portions of the LP-B virus
ge-nomethat determine the molecular parameters
of those proteins. Thus, the results presented
provide evidence that XLP-N viruses are
ge-neticrecombinants.
Of the 21recombinants studied, 16possess a
gp7O that comigrates with the gp70 ofLP-B.
Only2of the21recombinantspossessap15that
comigrates with the p15 of LP-B, and none
possessesap30 withtheelectrophoretic
mobil-ityof LP-B p30. Recombinants obtained from
any one crosscould have arisen from asingle recombination event, unless they have been
shown to differ in their virionproteins. Thus,
theminimumnumber ofindependent
recombi-nantsstudied is 11, ofwhich7have a gp7O with
the electrophoretic mobility characteristic of
LP-B virus gp7O and 2 have a p15 that
comi-grateswithp15 ofLP-B.
Ifone makes the assumption that
comigra-tionofXLP-N virusproteinswiththoseofSP-N
or LP-B implies inheritance of the
corre-sponding gene from the N- or B-tropic parent,
ifone assumes that recombination is equally
probable along the genome, and if one also
assumesthat theonlyselection pressures
oper-
---VOL. 23, 1977
Om
ilp -4
Owe am* one
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706 SCHINDLER, HYNES, AND HOPKINS
TABLE 1. Results of analysis of three virion proteins of parental and recombinant viruses by
SDS-polyacrylamidegelelectrophoresis
Electrophoretic mobility of:
Virus p15 p30 gp7O G,,a
SP-N Slowerb Faster Faster +
LP-B Faster Slower Slower
1-P1 Like N Like N Like B
2-1 N N N +
2-2 N N B
-3-1 N N B
-3-2 N N B
-4-1 N N N +
5-1 N N -c +
5-2 N N B
-5-3 N N B
-5-4 N N N +
5-5 B N B
-6-1 N N B
-6-2 B N N +
6-3 N N B
-7-P1 N N B
-7-P2 N N B
-7-P3 N N B
-7-P4 N N B
-7-P5 N N B
-7-P6 N N B
-7-P7 N N B
-a + or-referstotheabilityorinability, respectively, of
Sc-1cells infected by the particular virus to absorb cytotoxic antibody toGx.Thesedata are taken from Hopkins et al.
(10).
b"Slower"or"faster" refersto therelative electropho-retic mobilities of SP-Nand LP-B virus p15,p30,andgp7O proteins.
e- Electrophoretic mobility of the gp7O of recombinant 5-1has not yetbeen determined.
ating during isolation of the XLP-N viruses
wereforN-tropism and largeXC plaque
mor-phology, then the data presented above have
several implications of biological interest. (i)
That anN-tropic virus may possesap15orgp70
proteinlike that ofaB-tropic virusimpliesthat
tropism does not reside ineither of these
pro-teins (10, 13, 17).
(ii) That some XLP-N viruses, all of which
make large XC plaques, appear to possess a
gp7O derived from SP-N, which makes small
XC plaques, suggests that large XC plaque
morphology is not determined solely (ifatall)
by gp7O (10). However, thatsomany
recombi-nants inheritedgp70fromLP-Bmightsuggest
that thedeterminants(s)ofplaque morphology
lies nearthe genecoding for gp7O. (Note that
these results donotexcludethepossibility that
gp70 is involved in the formation of XC cell
syncytia.)
(iii) One interpretation of the observation
that all of the recombinants studied sofar
in-heritedap30thatcomigrateswithp30ofSP-N
is that N-tropism might be determined by a
gene that is closely linked to p30. We have
obtained other evidence consistent with the
possibility that p30 itself, or a gene closely
linkedtop30,might beadeterminant of N- and
B-tropism: murine leukemia virus variants
thatgrowequallywellonN-and B-typemouse
cells can be obtainedby forced passage ofa
B-tropic virusthrough N-type cells in vitro. The
resultingvirus iscalled NB-tropic(1, 11).Eight
NB-tropic viruses independently derived from
the B-tropic virusof BALB/c by serial passage
onNIH Swiss mouse cells have been found to
possess ap30 with altered electrophoretic
mo-bility on SDS gels (11). One of the eight
pos-sessedanaltered p15inaddition (unpublished
data). Both the apparent linkage of p30 and
N-tropism inthe present studyandthe
observa-tionof an altered- p30inNB-tropic viruses could
be explained in a number of ways that are
unrelated or only indirectly relatedto the
tro-pism of the viruses (see 21). However, it is
interesting to note that the possibility that a
protein present in virions may be a
determi-nantof N-orB-tropismis implied by the
stud-ies of Rein etal. (24) and Bassin et al. (4).
Since RNA tumorviruses arepolyploidand
canform heterozygotes (30), an alternative
ex-planation to recombination for the results we
have presented might be that XLP-N viruses
are stable heterozygotes of SP-N and LP-B
ge-nomes and selectively express, or package,
someproteins coded by one genome and some
codedby the other parentalgenome. Evidence
that XLP-N viruses are recombinants that
in-herited only some oftheir genes fromtheir N
virus parent andothers from their B virus
par-entcomes fromanalysisof theRNase
Ti-resist-antoligonucleotides derivedfrom theRNA
ge-nomes of parental and recombinant viruses
(Faller and Hopkins, unpublished data). This
analysis reveals great similarity between the
genomesof the N-andB-tropicvirusesderived
from BALB/c;ofapproximately35unique
oligo-nucleotides generated by RNase T1 cleavage
andtwo-dimensionalgel electrophoresis, about 30appear to besharedbetween the two viruses.
However, eachparental virus possesses sixor
seven distinctive oligonucleotide "spots," and
the XLP-Nviruses that havebeenanalyzedso
farhavebeenshowntopossessdifferent
combi-nations of N and B specific spots.Thesimplest
interpretationofthis result is thatXLP-N
vi-ruses aregeneticrecombinants assuggestedby
the biologicalevidence (12), antigenic analysis
(10), andthestudy described above.
ACKNOWLEDGMENTS
These studies are basedonobservations made byNancy Famulari and ErwinFleissner, Sloan Kettering Institute.
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VIRION PROTEINS OF N- AND B-TROPIC VIRUSES 707
Wethank Dr. Famulari for many helpful discussions and forcommunicatingunpublished data.
This work was supported by Public Health Service grants from the National Cancer Institute (CA 19308 to N.H., CA 17007 to R.H., and CA 14051 to theCenter for Cancer Research, MIT) and a PublicHealth Service grant from the National Institutes of Health,Biomedical Sciences Supportto MIT(RR 07047 toN.H.).
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2. Baltimore, D. 1974. Tumor viruses: 1974. Cold Spring HarborSymp. Quant. Biol. 34:1187-1200.
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4. Bassin, R. H., B. I. Gerwin, G. Duron-Troise, S. Gis-selbrecht, andA.Rein. 1975. Murine sarcoma virus pseudotypes acquire a determinant specifying N or B tropism fromleukemia virusduringrescue.Nature (London) 256:223-225.
5. Blattler, D. P., F.Garner, K. van Slyke, and A. Brad-ley. 1972. Quantitativeelectrophoresis in polyacryl-amidegelsof 2-40%. J.Chromatogr.64:147-155. 6. Fleissner. E. 1971. Chromatographic separation and
antigenicanalysis of proteins of the oncornaviruses. I. Avianleukemia-sarcoma viruses. J. Virol. 8:778-785.
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8. Hartley,J.W.,W. P.Rowe,and R.J. Huebner. 1970. Host-range restriction of murineleukemia virusesin
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N-tropic leukemia virusderived fromBALB/cmice. J. Virol. 16:991-999.
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NB-tropic virusesderived from a B-tropic virus ofBALB/c have alteredp30.J.Virol. 21:309-318.
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VOL. 23, 1977
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