The Presence and Expression of RNA Tumor
Virus Genes in Normal and Infected Cells:
Detection by Molecular Hybridization
J. M. BISHOP, M.D.,
N. JACKSON, M.A.,
W. E. LEVINSON, M.D.,
PH.D.,
E . MliDlilROS, M . A . , N . QUINTRELL, M . A . , AND H . E . VARMUS, M . D .
Department
of Microbiology,
University of California, San Francisco, California 94122
ABSTRACT
Bishop, J. M., Jackson, N., Levinson, W. E., Medeiros, E., Quintrell, N., and
Varmus, H. E.: The presence and expression of RNA tumor virus genes in
normal and infected cells: Detection by molecular hybridization. Am. J.
Clin. Pathol. 60: 31-43, 1973. Three molecular probes have been used to
detect genes of the Rous sarcoma virus and mouse mammary tumor virus in
normal and infected cells: the 70S ribonucleic acid (RNA) of the viral genomes, and the single- and double-stranded deoxyribonucleic acids (DNA)
transcribed from viral RNA by RNA-directed DNA polymerase. The findings
indicate that multiple copies of viral genes may be present in the DNA of
ostensibly normal cells. However, infection and transformation of mammalian (mouse and rat) cells by Rous sarcoma virus is accompanied by the
appearance of virus-specific nucleotide sequences in nuclear DNA of the
host cells. The expression of Rous sarcoma virus and mouse mammary tumor
virus genes in normal and infected cells has been measured by RNA-DNA
hybridization. Virus-specific RNA is present in both normal and transformed
cells which produce neither virus nor detectable viral proteins. These data
present the possibility that the expression of viral genes is not controlled
exclusively by regulation of transcripton.
the viral genome is deoxyribonucleic acid
(DNA) or ribonucleic acid (RNA), and
has led to two hypotheses which attempt
to account for certain features of cellular
transformation by RNA tumor viruses.
(1) The "provirus hypothesis" holds that
replication of and cellular transformation
by RNA tumor viruses occurs through the
agency of a DNA copy of the viral RNA
genome,31 synthesized early in infection by
the RNA-directed DNA polymerase found
in virions of RNA tumor viruses,32 and
then integrated into the DNA of host
chromosome(s) in a manner analogous to
that of DNA tumor viruses and bacterial
lysogenization.
that viral oncogenesis
involves the expression of viral genes
which have been inserted into the genetic
apparatus of the host cell. This axiom is
thought to apply irrespective of whether
I T IS NOW AXIOMATIC
Received December 19, 1972.
Supported by grants from the U. S. Public
Health Service (AI 088G4, CA 12380, CA 12705,
AI 06862, AI 00299, ACS VC-70); contract 71-2147
within the Special Virus-Cancer Program of the
National Cancer Institute, National Institutes of
Health, Public Health Service; H. E. Varmus is
recipient of the Senior Dernham Fellowship D-164
of the American Cancer Society, California Division, and the U. S. Public Health Service Research
Career Development Award No. CA 70193.
Reprints of this entire Research Symposium are
available from the ASCP Convention Department,
2100 West Harrison Street, Chicago, Illinois 60612,
for §3.00 per copy.
31
32
A.J.C.P.—Vol.
BISHOP ET AL.
Table 1. Manifestations of Viral Genes
in Normal Avian Cells
1. Avian leukosis virus antigen26
2. Complementation of defective sarcoma virus10'41
3. Release of virus
a. Induced
Chemical-physical1'21'31
Helper virus-rescue10'41
b. Spontaneous release28'29
(2) The "oncogene hypothesis" endows
all forms of neoplastic transformation with
a final common pathway. 33 Oncogenic
genes homologous to those carried by RNA
tumor viruses are held to be quiescent constituents of the genome of normal cells.
Any factor capable of activating the expression of these "oncogenes" may be oncogenic. This view is based primarily on
the fact that normal cells of at least several
species harbor the potential to produce
C-type viruses. 1 - 10 ' 21 - 28 ' 40 ' 42 Expression of
this potential (when it occurs at all) may
be either partial or complete, as summarized in Table 1, and will be discussed by
Friis elsewhere in this Symposium. What
is not clear at present is whether any of
the viral genetic information indigenous
to normal cells is actually oncogenic.
These two hypotheses are not mutually
exclusive, and at least portions of each are
now accessible to test by molecular technics. This communication summarizes the
present status of our efforts to perform
such tests using two model systems: Rous
sarcoma virus (RSV) and mouse mammary
tumor virus (MMTV).
Infection of chick fibroblasts with Rous
sarcoma virus results in coincident cellular
transformation and production of virus.31
As noted above (Table 1), viral genes introduced into the avian cell by infection
are superimposed on a considerable background of potentially similar or even identical genetic information. This fact can,
and does, obscure molecular tests of the
provirus hypothesis (Table 2). However,
60
Rous sarcoma virus will also infect cells
of other species, such as mice and rats. 31
No virus is produced, but at least a small
portion of the cell population is permanently transformed. This system has provided the opportunity for a relatively decisive test of the provirus hypothesis to be
described below (Table 3).
MMTV occurs spontaneously very frequently in lactating females of certain
mouse strains.24 The viruses are usually
transmitted by way of maternal milk.
However, certain strains of mice also efficiently transmit the viruses through the
gamete as an autosomal dominant trait 24
(Table 4). Thus, female progeny of these
strains, foster-nursed on virus-free mice,
have a high incidence of MMTV and
virus-induced carcinoma of the breast.24
This remarkable example of genetic transmission of an oncogenic virus is now serving as a major analog for the study of
human carcinoma of the breast 3 ' -° and
merits extensive biologic and molecular
characterization.
Table 2. Detection of Rous Sarcoma Virusspecific Nucleotide Sequences in Chicken
Deoxyribonucleic Acid
Cell
CEFf
CEF
CEF(RSV)t
CEF-nuclei
(RSV)
Avian tumor
(RSV)
HeLa
Calf thymus
Salmon sperm
Viral
Virus
Production Antigen
0
0
0
Copies of
RSV-DNA
per Cell*
10-15
10-15
10-15
+
+
+
+
+
10-15
+
+
10-15
0
0
0
* RSV-specific DNA was detected with the assay developed
by Gelb and associates,14 using double-stranded DNA synthesized by RSV as probe."•' 8 .
t CEF denotes normal chick embryo fibroblasts.
% CEF (RSV) = the same cells infected with and transformed by RSV. DNA's from nuclei of CEF(RSV). from RSVinduced sarcomas in chickens, from HeLa cells, and from calf
thymus and salmon sperm were also tested. Details have been
described.*'-**
July
1973
33
MOLECULAR HYBRIDIZATION AND RNA TUMOR VIRUSES
Materials and Methods
Cells and Viruses. We previously described the source and preparation of all
materials for the Schmidt-Ruppin strain
of RSV 7 and the RIII strain of MMTV. 35
Preparation of Nucleic Acids. Viral RNA
(70S) was extracted with SDS-phenol and
purified by rate-zonal centrifugation.7 Cellular DNA was extracted as described previously and sheared at 50,000 p.s.i.30 Virusspecific DNA was synthesized with detergent-disrupted virions, extracted, and purified as before.12 Single- and double-stranded
fractions were prepared by elution from
hydroxyapatite. 10 RNA was extracted from
cells and tissues with phenol at 60 C ,
treated with DNase, and fractionated by
rate-zonal centrifugation.
Molecular Hybridization. The following
technics have been described previously:
(a) detection of DNA-RNA hybridization
by hydrolysis with the single strand-specific
endonuclease of Aspergillus oryzae,2" and
(b) measurement of virus-specific nucleotide sequences in cellular DNA using reassociation kinetics.38 Hybridization of radioactive RNA with vast excesses of DNA
was modified from the procedure of Melli
and associates.23 Details in all instances
are given in the legends to the figures.
Table 3. Detection of Rous Sarcoma Virusspecific Nucleotide Sequences in Mammalian Deoxyribonuleic Acid
Cell
Production
3T3
3T3(RSV)
3T3-nuclei
(RSV)
NRK
NRK(RSV)
HeLa
Calf
thymus
Salmon
sperm
0
0
0
+
or-
0
2
0
0
0
0
+ or0
+ or —
0
2
0
2
0
0
0
0
0
0
0
Antigen
per C
t The procedures and nomenclature are the same as for
Table 2. NRK denotes normal rat fibroblasts.
Table 4. Incidence and Transmission of
Mouse Mammary Tumor Virus
Mouse Strain
Incidence in
Lactating
Females
C3H
GR
C57JB1/6
C58
Balb/c
129
100%
100%
0
0
0
0
Transmission
Milk Gamete
+
+
0
0
0
0
±*
+
0
0
0
0
Results
*The symbol ± denotes transmission of an MMTV strain
with delayed onset and relatively low oncogenicity."
The Probes for Molecular Hybridization.
The specificity and sensitivity of molecular
hybridization have facilitated the identification of RNA tumor virus-specific nucleic
acids in normal and infected cells. Table 5
summarizes the nature and pertinent characteristics of the available reagents. (1)
The 70S RNA of the viral genome can be
labeled to reasonably high specific activities with presently available radioisotopes,
and has been widely used in assays to detect viral DNA. 4 ' 2T It is far less useful in
studies of virus-specific RNA because it
facilitates detection of RNA complementary to the viral genome only. This fact
constitutes a serious limitation, because
either the bulk or all of the viral RNA
in cells is composed of nucleotide sequences identical to those of the viral genome.20 (2) The DNA products of RNAdirected DNA synthesis by virions of RNA
tumor viruses contain nucleotide sequences
both complementary and identical to the
viral genome, and can be radioisotopically
labeled to exceptionally high specific activities. These DNA's therefore constitute
specific and sensitive reagents for the detection of virus-specific nucleic acids. Un-
34
BISHOP ET
Table 5. Molecular Probes for Virusspecific Nucleic Acids
Virus
RSV
MMTV
Probe
70S RNA
ssDNAf (reverse
transcriptase &
actinomycin)
dsDNAJ (reverse
transcriptase)
70S RNA
ssDNAf (reverse
transcriptase &
actinomycin)
dsDNAJ (reverse
transcriptase)
Proportion of
Viral Genome
Represented*
100%
100%
30-50%
100%
?
5%
* The proportion of viral genomes represented in doublestranded DNA synthesized by detergent-activated virions
was
estimated by measurement of reassociation kinetics.18 The
single-stranded DNA synthesized by Rous sarcoma virus in
the presence of actinomycin D (100 ng. per ml.) contains
nucleotide sequences representing the entire viral genome.11
A similar determination has not been made to date for mouse
mammary tumor virus single-stranded DNA.
t ssDNA = single-stranded DNA.
t dsDNA = double-stranded DNA.
A.J.C.P.—Vol.
AL.
60
of the latter, and summarize our findings
to date with both avian sarcoma virus and
mouse mammary tumor virus.
Radioactive RNA can be extensively hybridized to complementary cellular DNA
in solution if the DNA is used in vast excess of RNA in order to permit R N A DNA interactions to occur in the face
of competing DNA-DNA interactions. 15 ' 23
The hybridization of 70S RSV RNA to
vast excesses of DNA from normal and
RSV-infected chicken cells is illustrated in
Figure 1. There is extensive interaction in
both cases, but the reaction with DNA
from infected cells occurs at a lower C0t
and is more complete (65 vs. 40%) than
that with DNA from uninfected cells.
o
LU
fortunately, most of the enzymatic products prepared to date do not contain nucleotide sequences representative of the
entire viral genome (Table 5). This problem has now been circumvented in at
least one instance. The single-stranded
DNA synthesized by several avian sarcoma
viruses (Schmidt-Ruppin and B77 strains of
Rous sarcoma virus) in the presence of
actinomycin D contains a complete and
relatively uniform representation of the
entire viral genome.13 It is not presently
known whether the same phenomenon also
occurs with other RNA tumor viruses.
Virus-specific DNA in Normal and Infected Cells. The most widely used assay
for virus-specific nucleotide sequences in
cellular DNA involves the hybridization of
radiolabeled RNA to DNA immobilized on
membrane filters.4'27 This procedure is subject to serious limitations in the study of
eukaryotic DNA, 6 and is being supplanted
by several more recently developed technics. We illustrate here the use of each
Q
>X
0.5
Z
Q
i—
v
<
• 70S RNA WITH CAIF THYMUS DNA
A AVIAN cRNA WITH AVIAN DNA
O 7 0 S RNA WITH N O R M A ! AVIAN DNA
• 70S RNA WITH TRANSFORMED AVIAN DNA
A A V I A N rRNA WITH NORMA! AVIAN DNA
1.0
I
10'
I
10 2
I
10 3
I
104
105
C01 (mole-sec/liter
FIG. 1. Hybridization of RNA with a vast excess
of cellular DNA. RNA's were labeled with S Hnucleosides to a specific activity of approximately
5 X 10" c.p.m. per ng. and annealed with sheared
and denatured cellular DNA in 0.4 M sodium
phosphate, p H 6.8, at 68 C. T h e ratio of DNA
to RNA was at least 1 X 10" in every instance.
Hybridization was measured by hydrolysis with
pancreatic ribonuclease A (RNase) in 0.3 M NaCl
(37 C , 60 minutes). Ribosomal RNA (28S) and
70S RNA's were purified by rate-zonal centrifugation. "Complementary RNA" (cRNA) was transcribed from native chick DNA using DNA-dependent RNA polymerase of Escherichia coli° and
3
H-GTP as the radioactive precursor. RNA prepared in this manner is probably transcribed
principally from unique nucleotide sequences in
the DNA.23 T h e results of hybridization are expressed as a function of the convention C„t (concentration of DNA times the time of incubation)
first proposed by Britten and Kohne."
July 1973
MOLECULAR HYBRIDIZATION AND RNA T U M O R VIRUSES
"*
" \
1
*
1 1
<
u
O
z
o
-D--0-
--D—
"v-
35
0.5
\
\ \ \\
0
MOUSE DNA(3T3 Balb C)
•
MMTV DNA WITH MOUSE DNA|3T3 Balb C|
a
MMTV DNA WITH CALF THYMUS DNA
r1
_
<
\\ \\
\ \
1.0
1
10"
1
10u
1
10'
1
10^
1
10 J
10"
CQt (mole-sec liter)
FIG. 2. Hybridization of mouse mammary tumor virus (MMTV) single-stranded deoxyribonucleic acid (DNA) to normal mouse DNA. 3 H-labeled single-stranded DNA was synthesized
with detergent-activated virions of MMTV in the presence of actinomycin D (100 fig. per ml.),
purified as described previously,20 incubated with either denatured mouse DNA (extracted
from ISalb/c 3T3 cells) or denatured calf thymus DNA in 0.6 M NaCl at 68 C. T h e ratio of
cellular DNA to MMTV DNA was 2 X 10'. Hybridization of the viral DNA was measured by
hydrolysis with single strand-specific endonuclease from Aspergillus oryiae.--3' Reassociation of
cellular DNA was monitored by fractionation on hydroxyapatite and measuring the absorbance
(260 nm.) of the eluates. 38
Comparison of these results with those obtained for chick 28S ribosomal RNA and
cRNA (thought to be mainly RNA transcribed from unique nucleotide sequences
of DNA) indicates that the virus-specific
nucleotide sequences are present in normal
cells at an extremely low concentration—
perhaps only one copy per cell. There are
apparently multiple copies of viral DNA
sequences per transformed cell, and a
greater portion of the viral genome may
be represented here than in normal cells.
However, it is not possible to determine
from these data whether the entire RSV
genome is represented in the DNA of
transformed cells. Neiman 2 5 has obtained
similar results with the Prague strain of
Rous sarcoma virus.
Single-stranded DNA transcribed from
viral RNA by RNA-directed DNA polymerase in the presence of actinomycin is
a highly specific reagent (Table 2) which
reacts efficiently with complementary DNA
in solution. This reaction is not limited
by a disadvantageous reaction constant, as
is that between RNA and DNA, 23 and is
readily measured by using a single strandspecific endonuclease. 2 ' 20 The results illustrated in Figure 2 demonstrate the potential of this technic. MMTV-specinc singlestranded DNA reacts completely with
DNA from normal Balb/c 3T3 cells, but
not with calf thymus DNA. This result
indicates that whatever portion (presently
unknown) of the MMTV genome is represented by the probe also is present in normal mouse DNA. The data are too preliminary to permit calculation of the number of copies of MMTV DNA per cell.
The interaction between virus-specific
double-stranded DNA and cellular DNA
provides a sensitive assay for the presence
of virus-specific nucleotide sequences in
the cellular DNA and permits a reasonably
precise estimate of the number of copies
of viral DNA per cell.14 This procedure
has been well validated for use in the
study of both DNA 1 4 and RNA 3 7 tumor
36
BISHOP ET AL.
Table 6. Specificity of Single-stranded
Enzymatic Product*
3
RNA
RSV 70S
MMTV 70S
MuLV 70S
Poly A
H-ssDNA: Resistance
to Endonucleaset
RSV MMTV MuLV PolyT
100%
0
0
0
0
100%
0
0
0
0
100%
0
100%
100%
100%,
100%
* K e y : RSV = Rous sarcoma virus; MMTV — mouse
mammary tumor virus; MuLV = murine leukemia virus;
Poly A = polyadenylic acid; Poly T = polythymidylic acid.
T >H TMP-labeled single-stranded DNA was prepared with
detergent-activated virions in the presence of actinomycin D
(100 jig. per ml.) and hybridized to the indicated RNA's. Hybridization was detected by hydrolysis with a single strandspecific endonuclease from Aspergillus (oryzaefo The singlestranded DNA's were all 5 to 10% resistant to hydrolysis prior
to hybridization, and the data have been corrected for these
backgrounds. High molecular weight poly A was purchased
from P. L. Biochemicals. High molecular
weight poly A was
purchased from P. L. Biochemicals. BH-labeled poly T (approximately 100 nucleotides) was synthesized with DNA polymerase I of Escherichia coli, using the poly A of poliovirus RNA
as template. 30
viruses. In the latter instance, doublestranded DNA synthesized with RNA-directed DNA polymerase serves as the virusspecific probe. This DNA does not represent the entire viral genome (Table 5), a
fact which to some extent limits the significance of the results obtained to date.
Nevertheless, the precision and sensitivity
of the procedure make it a presently indispensable tool for the molecular study of
RNA tumor viruses.
Normal chick cells contain 10 to 15
copies per cell of RSV-specific DNA, irrespective of the presence or absence of
viral antigen (Table 2). There is no perceptible increase in virus-specific DNA
consequent to infection with and transformation by RSV, although a change of less
than twofold might go undetected by the
present assay.88 These results substantiate
the presence of viral genetic information
in the DNA of uninfected cells, but leave
unresolved the events that follow infection. More informative data regarding the
latter issue were obtained with mammalian
cells (Table 3). Normal rat and mouse
cells contain no RSV-specific DNA detectable by either t'ne present technic or hy-
A.J.CP.—Vol.
60
bridization of 70S RNA to vast excesses of
cellular DNA (unpublished observation).
Following transformation by RSV, both
mouse and rat cells contain two copies per
cell of RSV-specific DNA, representing 30
to 50% of the RSV genome (i.e., the extent to which the RSV genome is represented in the hybridization probe). The
results of unpublished experiments indicate that the virus-specific DNA is covalently linked with (i.e., "integrated into")
high molecular weight DNA of the host
chromosome(s).
The DNA from several mouse strains
has been tested for the presence of MMTVspecific nucleotide sequences, using a double-stranded probe representing approximately 5% of the viral genome (Table 5).
These strains have very different genotypes vis-a-vis the incidence and transmission of MMTV (Table 4). Nevertheless,
the tissues of all strains tested contain approximately 90 copies per cell of MMTVspecific DNA (Table 8). Mammary glands
and liver of the GR mouse contain the
same amount of MMTV DNA (Table 6),
suggesting that mechanisms other than
gene amplification are responsible for production of viruses by one tissue but not
the other.
Expression of Viral Genes: Virus-specific
RNA in Normal and Infected Cells. The
apparently universal distribution of C-type
viral genes (i.e., virus-specific DNA) in ostensibly normal cells connotes the existence of mechanisms which regulate the
expression of these genes. Control of gene
expression is commonly accomplished, at
least in part, by regulating transcription
of RNA from chromosomal DNA. We
have therefore tested RNA from a variety
of normal and virus-transformed cells for
nucleotide sequences homologous to those
of C-type viral genomes. Virus-specific single-stranded DNA synthesized by RNAdirected DNA polymerase in the presence
of actinomycin D was annealed with cellu-
July
1973
37
MOLECULAR HYBRIDIZATION AND RNA TUMOR VIRUSES
O
05
i
Q---I-Q
m-
C r l (mole-sec liter)
FIG. 3. Rous sarcoma virus (RSV)-specific RNA in normal and infected cells. 0H-labeled singlestranded DNA was synthesized with detergent-activated virions of RSV in the presence of
actinomycin D (100 /ig. per ml.), purified as described previously, and hybridized (0.3 M NaCl,
68 C.) with the indicated RNA's in vast excess (1,000:1 in every instance). Details of the cells
and viruses used are given in Table 7. Hybridization was measured as in Figure 2, and the
results expressed
as a function of Crt (concentration of RNA X time of incubation), as described
elsewhere.20 Each of uthe hybridizations illustrated here was also tested by equilibrium centrifugation in CsaS04 and the formation of DNA-RNA hybrids confirmed.
lar RNA. Hybridization was routinely detected by hydrolysis with a single strandspecific endonuclease,20 and confirmed by
equilibrium centrifugation in CsoSOj.11
The specificity of hybridization is illustrated in Table 6. The probes were tested
for cross-reactions with heterologous viral
RNA's, and for polythymidylic acid (poly
T) which could give spurious interactions
with polyadenylic acid (poly A) in cellular
RNA. Both tests were negative under circumstances which permitted interaction
between poly T and either poly A or 70S
viral RNA's which contain regions of poly
A.18
RNA of RSV in Chick Cells. RNA from
normal chick fibroblasts has been tested
for nucleotide sequences homologous to
the genome of RSV. T h e data are presented in terms of C r t (concentration of
RNA multiplied by the time of incubation 6 - 20 ), a convention which permits direct comparison of results obtained under
quite different hybridization conditions.
If the cells contain group-specific antigen
of avian leukosis viruses, at least 80% of
the DNA probe can be hybridized to the
cellular RNA (Fig. 3). This suggests that
a major portion of the viral genome is
represented in cellular RNA species. By
comparison with the results for 70S viral
RNA and RSV-infected chick cells (Fig. 3),
we compute that antigen-positive uninfected cells contain approximately 30 to
50 viral genome equivalents of RSV-specific RNA per cell (Table 7). Chick cells
free of viral antigen contain no detectable
RSV RNA (Fig. 3 and Table 7). These
Table 7. RSV-specific RNA in Normal
and Infected Cells*
Cell
CEF
CEF
CEF (RSV)
3T3
3T3(SR)
3T3(B-77)
NRK
NRK(SR)
Virus
Viral
Production Rescue
0
0
0
+
+
+
0
0
0
0
0
Viral
Antigen
0
0
+
+
0
+
Viral
RNAf
<0.04
30-50
3,000-5,000
<0.04
<0.04
ca. 10
<0.04
30-50
* K e y : CEF = chick embryo fiberblasts; CEF(RSV) ~
check embryo fibroblasts infected with and transformed by
Rous sarcoma virus; NRK = normal rat fibroblasts; NRK(SR)
=» rat fibroblasts transformed by Schmidt-Ruppin RSV.
t Genome equivalents of viral RNA per cell were measured
and computed as described previously.20 Viral antigen (rtroup-0
specific avian leukosis) was detected by complement fixation.*
3T3 (Balb/c) cells were infected and transformed with either
the Schmidt-Ruppin (SR) or the B77 strain of RSV.
38
BISHOP ET
Table 8. Detection of Mouse Mammary Tumor
Virus-specific Nucleotide Sequences in
Cellular Deoxyribonucleic Acid*
Virus
Production
C57B1 liver
GR liver
GR
lactating
breast
HeLa
Calf thymus
Salmon sperm
Viral
Antigen
Copies of
Sponta- MMTV
neous
DNA
Tumor per Cellf
90
90
+
+
+
90
0
0
0
* The assay was the same as in Tables 2 and 3, except that
double-stranded DNA synthesized with MMTV served as
probe.
t MMTV = mouse mammary tumor virus; DNA = deoxyribonucleic acid.
are preliminary results which may have to
be revised following additional assays using
both sarcoma and leukosis virus probes,
but the present data suffice to indicate the
existence of strong and variable controls
over transcription of C-type viral genes in
normal avian cells.
Normal rat and mouse cells contain
neither RSV-specific DNA (Table 3) nor
RSV RNA (Fig. 3). Following transformation by RSV these cells contain RSV-specific DNA (Table 3) yet produce no viruses.31 We have tested two RSV-transformed lines of mouse cells for viral RNA.
A.J.C.P.—Vol.
AL.
60
Table 9. Mouse Mammary Tumor Virusspecific Ribonucleic Acid in Mouse
Cells and Tissues*
Viral
Antigen
Tissues producing
MMTV
C3H lactating
mammary gland
GR lactating
mammary gland
C3H mammary
tumor
GR mammary
tumor
Tissues not producing MMTV
BALB/c
mammary tumor
BALB/c lactating
mammary gland
C57B1/6 lactating
mammary gland
129 lactating
mammary gland
C3H liver (male)
GR liver (male)
C57B1/6 liver (male)
C3H spleen (male)
GR spleen (male)
Cells not producing
MMTV
BALB/c 3T3
BALB/c 3T3 (RSV)
Viral
RNAf
+
300
+
5000
+
2200
+
500
0
4
0
5
0
100
0
430
0
0
0
0.4
0.7
0.8
+
0
8
2
0
0
<0.04
<0.04
" Key: MMTV = mouse mammary tumor virus; RSV =
Rous sarcoma virus; RNA = ribonucleic acid.
T Genome equivalents per cell were measured and computed as described elsewhere for RSV RNA.»° Viral antigens
were measured by the method of Hilgers and colleagues."
FIG. 4. Mouse mammary tumor
virus (MMTV)-specific RNA in
mouse cells and tissues. 8 H-labeled
MMTV single-stranded DNA was
prepared as described for Figure 2
and hybridization carried out as
in Figure 3. Details of the cells
and tissues used are given in Table
9. All hybridizations were also
tested by equilibrium centrifugation as noted for Figure 3.
C r t (mole-sec/liler)
July 1973
MOLECULAR HYBRIDIZATION AND RNA T U M O R VIRUSES
One cell line yields viruses when cocultivated with permissive avian cells 39 and
contains approximately 10 genome equivalents per cell of RNA which hybridizes
with essentially all of the single-stranded
DNA probe (Fig. 3 and Table 7). It follows that virtually the entire RSV genome
is represented in cellular RNA. The other
RSV-transformed mouse cell line has not
produced viruses when cocultivated with
permissive cells, and contains no detectable RSV-specific RNA (Fig. 3 and Table
5). The only line of RSV-transformed rat
cells examined to date contains RSV RNA,
whereas normal rat cells do not (Fig. 3
and Table 5). These data again indicate
the existence of controls over transcription
which may vary in their extent, and an
apparent correlation between this molecular parameter and the tendency of the cell
to yield viruses when subjected to conditions permitting viral rescue.
RNA of MMTV
in Mouse Tissues.
There are major genotypic variations
among mouse strains with respect to the
spontaneous incidence and transmission of
MMTV (Table 4). Moreover, the production of virus occurs only in lactating mammary glands and mammary carcinomas.
These strain and tissue specificities contrast sharply with the universal distribution of MMTV structural genomes 35
(Table 8), and indicate the existence of
control mechanisms determined by both
genetic and hormonal factors. RNA capable of hybridizing with MMTV-specific
DNA is present in a variety of tissues in
every strain of mouse examined to date,
irrespective of the mouse genotype vis a vis
incidence and transmission of MMTV
(Fig. 4 and Table 9). The amounts of
MMTV RNA vary in a reasonable manner,
i.e., virus-producing tissues contain more
viral RNA than virus-free tissues. Nevertheless, even livers and spleens from males
of low incidence mouse strains contain
39
1.0
<
z
Q
0.5
Z
o
u
<
O
•
O
•
*•'
•tTvft'gn -rWv
50
60
70
MMTV
MMTV
MMTV
MMTV
d*0NA
DNA, 70S RNA
DNA. GR TUMOR RNA
DNA: C57 Bl LMG RNA *
_L
80
JL
90
100
TEMPERATURE (°C)
Fie. 5. Thermal denaturation of mouse mammary tumor virus (MMTV) DNA and DNA-RNA
hybrids. Hybrids were formed as in Figure 4, recovered by ethanol precipitation, and dissolved in
0.02 M tris:HCl, p H 7.4. Samples were heated to
the indicated temperatures for 15 minutes, quenched
in ice, and tested by hydrolysis with the single
strand-specific endonuclease of Aspergillus oryzae."-a
Details of the tissues used are given in Table 9.
T h e double-stranded DNA synthesized by detergent-disrupted MMTV was purified by el u tion
from hydroxyapatite and denatured in 0.02 M
tris:HCl as described above.
MMTV RNA. Normal and RSV-transformed Balb/c 3T3 cells are the only
mouse materials tested to date which are
free of detectable MMTV RNA.
The specificity of hybridization was examined by thermal denaturation of representative hybrids (Figs. 5 and 6). All of
the hybrids tested displayed a high level
of thermal stability, but there were reproducible differences (manifest in T m 's) between the hybrids formed with RNA from
virus-producing tissues and those formed
with RNA from nonproductive tissues
(Table 10). These differences in T m (4 to
8 C.) are indicative of approximately 5%
mismatching of nucleotide sequences in
the hybrids with the lower T m 's, 19 and
suggest that the reactive RNA in tissues
producing no virus is not entirely homologous to the genome of MMTV (RIII
strain).
40
A.J.C.P.—Vol.
BISHOP ET AL.
1.0
o C57 Bl LMG
•
fifL
60
1*
GR Tumor
0.5
I—
<
z
z
o
1.0
I—
u
<
0.5
50
60
70
80
TEMPERATURE ( ° C )
FIG. 6. Thermal denaturation of hybrids between mouse mammary tumor virus (MMTV)
ssDNA and RNA from mouse tissues. Hybrids were formed and denatured as described for
Figure 5. LMG denotes lactating mammary glands.
Discussion
The "Provirus" of Rous Sarcoma Virus.
Results of experiments with mammalian
cells (Table 3) clearly suggest that infection with and transformation by RSV are
accompanied by the appearance of virusspecific nucleotide sequences in nuclear
DNA of the host cell. Others have reported
similar findings with RSV and mammalian
cells using hybridization of RNA to DNA
immobilized on membrane filters,* but this
technic provides no indication as to what
fraction of die total viral genome reacts
with the DNA. Our results, obtained by
measuring the effect of cellular DNA on
the reassociation of virus-specific doublestranded DNA, indicate that all of the
nucleotide sequences contained in the molecular probe (30 to 50% of the entire viral
genome) are represented in cellular DNA.
Unpublished data indicate that the viral
DNA is integrated into chromosomal DNA
of the host cell.
At present, the best physicochemical indication that the entire genome of RSV
July 1973
41
MOLECULAR HYBRIDIZATION AND RNA TUMOR VIRUSES
might be represented in the DNA of transformed cells comes from the results of
RNA-DNA hybridization with DNA in
vast excess2B (Fig. 1). However, the data
are inconclusive and pertain only to avian
cells which also contain an appreciable
portion of the RSV genome in their DNA
prior to infection. It is now possible to prepare highly radioactive single-stranded
DNA representative of the entire RSV genome (Table 5). This material can be used
in hybridization assays with single strandspecific endonuclease, as described above
for MMTV (Fig. 2), and probably constitutes the most promising reagent for testing to determine whether nucleotide sequences of the entire RSV genome are
present in various cellular DNA's.
Viral Genes in Normal Cells. The RSVspecific DNA present in normal avian cells
may not represent the entire RSV genome 26 (Fig. 1) and might therefore lack
oncogenic (transforming) genes. The latter
would be introduced by infection with
RSV, either as a supplement to the portion of the RSV genome already present
in the cell or as part of entire genome
complements newly inserted into host
DNA. The latter alternative is suggested
by the general quantitative increase in
RSV DNA per cell following infection and
transformation of avian cells by RSV (Fig.
1). The RSV-specific DNA in normal avian
cells may represent nucleotide sequences
shared by RSV and the leukosis virus(es)
indigenous to these cells.16-40 This is a
likely possibility because we have unpublished evidence that approximately 70%
of the nucleotide sequences of RAV-O 40
genome are also present in RSV genome.
We have shown that MMTV-specific
DNA is widely distributed among different
mouse strains, irrespective of the incidence
of MMTV (Table 8). It is not possible
at present to state what fraction of the
MMTV genome is involved, but the data
suggest that genotypic variation among
Table 10. Thermal Stability of Ribonucleic
Acid: Deoxyribonucleic Acid Hybrids*
RNAf
Temperature (C.)
MMTVt 70S RXA
68
Tissues producing MMTV
C3II lactating mammary gland
GR lactating mammary gland
C3H mammary tumor
GR mammary tumor
68
68
68
68
Tissues not producing MMTV
Balb/c mammary tumor
C57BI/6 lactating mammary gland
Balb/c lactating mammary gland
129 lactating mammary gland
C3H spleen (male)
64
64
61
61
61
*' Hybrids were formed between single-stranded DNA of
MMTV and the indicated RNA's, and denatured as illustrated
in Figures 5 and 6. The midpoints (Tm) of each denaturation
were estimated by visual inspection, and are based on at least
two separate determinations. Hybrids with tumor RNA were
denatured in tandem with each determination to provide a
standard. These standards never varied by more than ±0.5 C.
t RNA ~ ribonucleic acid.
% MMTV = mouse mammary tumor virus.
mouse strains with respect to the spontaneous incidence of MMTV represents
changes in a regulator function rather than
in structural viral genes. This system,
therefore, appears to be suitable for further
study of the means by which the expression of potentially oncogenic genes is inhibited in normal cells.88
Expression of Viral Genes in Normal
and Transformed Cells. We have found
RNA representative of either most or all
of the RSV genome in two types of cells
which are not producing virus: normal
chick cells containing avian leukosis virus
group-specific antigen, and RSV-transformed mammalian cells (Fig. 3 and Table
7). Similarly, MMTV-specific RNA is
widely distributed in normal mouse tissues which contain neither virus nor viral
antigen (Table 9). These data raise the
possibility that the expression of viral
genes in normal and transformed cells is
not controlled exclusively by regulation of
transcription. This suggestion conforms to
42
BISHOP ET
one widely discussed view of gene regulation in eukaryotic cells.34
T h e MMTV-specific RNA found in
various tissues of mice which are free of
virus may not be entirely homologous to
the viral RNA of RIII MMTV (Table 10).
We cannot presently explain this observation, but it is possible that the mouse
strains in question harbor genomes of
MMTV strains which are not identical to
the virus present in RIII, C3H, and GR
mice.
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