Mouse mammary tumor virus-related inducible sequences in mouse lymphocytes are by 12-O-tetradecanoyl phorbol-13-acetate.

Mouse mammary tumor virus-related
sequences in mouse lymphocytes are inducible
by 12-O-tetradecanoyl phorbol-13-acetate.
B S Kwon and S M Weissman
J. Virol. 1984, 52(3):1000.
These include:
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Vol. 52, No. 3
JOURNAL OF VIROLOGY, Dec. 1984, p. 1000-1004
0022-538X/84/121000-05$02.00/0
Copyright C 1984, American Society for Microbiology
Mouse Mammary Tumor Virus-Related Sequences in Mouse
Lymphocytes Are Inducible by 12-0-Tetradecanoyl phorbol-13acetate
BYOUNG S. KWON* AND SHERMAN M. WEISSMAN
Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510
Received 23 April 1984/Accepted 16 August 1984
The potent tumor promoter 12-O-tetradecanoyl phorbol13-acetate (TPA) accelerates development of tumors induced by primary carcinogens and has been shown to exert
pleiotropic effects in a number of tumor systems (13, 26). Its
effects seem to be mediated by specific cell membrane
receptors which are copurified with protein kinase C (5, 28).
However, the effect of TPA on the activation of gene
expression is unknown, and it is not known whether the
tumor promoter activity and the function of differentiation
induction are mediated by the same pathway. The murine
thymic leukemia T-cell line EL-4 produces T-cell growth
factor (11-2) constitutively in the absence of TPA but, upon
the addition of TPA, increases the production of 11-2 (7) and
other lymphokines such as colony-stimulating factor (11) and
B-cell growth factor (12). To obtain insight into the mechanisms of action of TPA, we screened cDNA libraries of TPAtreated EL-4 for TPA-inducible clones by a differential
hybridization procedure. Here, we report that the most
abundant inducible species of polyadenylated RNA contained mouse mammary tumor virus (MMTV)-related se-
Lambda phage BV-2 (16) was used as a vector to facilitate
the differential screening because plaques were uniform in
size and easier to manipulate than bacterial colonies. The
recombinant phages were plated and transferred to duplicate
filters (3). One set of filters was hybridized to a 32P-labeled
cDNA probe prepared with poly(A)+ membrane or cytoplasmic mRNA of induced cells and the other, to a 32P-labeled
cDNA probe prepared with poly(A)+ membrane or cytoplasmic mRNA of uninduced cells. The hybridization intensities
of each plaque with the two probes were compared for the
occurrence of TPA-inducible clones. Figure 1 shows an
example of the differential screening. The sensitivity of the
plaque hybridization for screening was estimated by two
reconstruction experiments (see the legend to Fig. 1), which
indicated that an input probe of ca. 1 x 103 cpm of 32p_
labeled cDNA was necessary to detect a specific sequence,
and under the conditions used for screening, a sequence
corresponding to as little as 0.02% of total mRNA could be
detected. In initial screening of ca. 20,000 cytoplasmic and
10,000 membrane-associated cDNA inserts, 200 potential
induction-specific clones were selected. The clones were
plaque purified and rescreened twice. Finally, phage DNAs
were prepared, fixed on to two copies of nitrocellulose filters
(14), and hybridized separately to the induced and uninduced
probes. Only 15 clones from membrane-cDNA libraries and
2 clones from cytoplasmic cDNA libraries were consistently
scored as inducible. The inserts from 10 of these clones were
excised and subcloned into pBR322. By cross-hybridization
among the clones, the clones were classified into six different groups. One group of clones appeared five times: four
times from membrane libraries and once from a cytoplasmic
library, thus representing the most abundant inducible species. In initial RNA blotting analysis, inserts from these
clones hybridized strongly to a 24S RNA species and weakly
to a 35S RNA species, suggesting to us that the insert
quences.
EL-4 cells were grown in RPMI 1640 medium containing
penicillin and streptomycin (100 ,ug/ml each) supplemented
with 100 mM sodium pyruvate, 10 mM nonessential amino
acids, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2ethanesulfonic acid [pH 7.2]), 2 mM glutamine, and 5% fetal
calf serum to a cell density of 1.5 x 106 cells per ml. The cells
were collected, washed twice with phosphate-buffered saline, and treated with TPA (10 ng/ml) for various times. The
stimulation was monitored by I1-2 assay (8) of the culture
supernatant with I1-2-dependent CT-6 cells which had been
maintained in RPMI 1640 medium supplemented with rat Il2.
cDNA libraries were prepared with the membrane-associated and cytoplasmic polyadenylic acid-containing
[poly(A)+] mRNA fraction of TPA-treated EL-4 cells.
*
sequences were related to a retrovirus genome. We chose
the longest insert of the clones (pKR1101) for further analysis. Cytoplasmic RNA of EL-4 cells was extracted at the
Corresponding author.
1000
Downloaded from http://jvi.asm.org/ on June 9, 2014 by guest
cDNA libraries from EL-4 cells treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA) were screened for
TPA-inducible sequences by differential hybridization. The most abundant inducible species was a sequence
similar to that of mouse mammary tumor virus (MMTV). Induction of the mRNA corresponding to the
MMTV-related sequences was already evident 30 min after TPA treatment, whereas the maximum accumulation occurred after 20 h of exposure to TPA. TPA also increased levels of MMTV-related RNA in the normal
spleen cells of BALB/c and C57BL/6 mice. The level of RNA expression corresponding to MMTV-related
sequences, however, was markedly elevated in EL-4 cells as compared with normal spleen cells. Southern blots
of EL-4 cell DNA showed that the MMTV-related sequences were inserted into multiple locations of the EL-4
genome. Sequence analysis revealed that the MMTV-related cDNA clones included a part of the env gene and
the right long terminal repeat of MMTV. However, the cDNA sequences were substantially different from
published MMTV proviral sequences, most notably because of a contiguous deletion of 491 base pairs in the
open reading frame within the U3 region.
NOTES
VOL. 52, 1984
B.
1001
..
.9
S.
*
0
S.
S
*'o *C
*..*
- .
I;
0
A
.5
Vb
..
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*.
0.
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*
.'
-
.5
various times after treatment of the cells with TPA, fractionated on a 1.4% formaldehyde gel, transferred to a nitrocellulose filter, and hybridized to nick-translated, 32P-labeled
pKR11O1 (Fig. 2). An increase in 24S RNA was already
evident at 30 min after TPA treatment. The maximum
92
72 48 40 26
20
1)
14
6
2
'2 ° oIter
TPA+~
Hours
-O----rigin
accumulation of pKR1101 cRNA occurred after 20 h of
exposure to TPA, and levels of RNA remained at an induced
level for at least 92 h. The enhancement of expression was
up to eightfold over the level of uninduced control (Fig. 2,
inset). Similar sequences were also inducible in the normal
lymphoid cells. As shown in Fig. 3, TPA induced pKR1101
cRNA expression in normal spleen cells of BALB/c and
C57BL/6 mice (parental strain of EL-4 cells). The level of
cRNA expression, however, was markedly elevated in EL-4
cells as compared with that in normal spleen cells. The level
of pKR1101 cRNA expression after TPA treatment in
BW5147 cells, another thymoma T-cell line, was similar to
-
-
- 28 S
- 18 S
92
X
4
4C
26
'4
E X PeSu* Trn T PA,
FIG. 2. Time course of TPA effect on pKR1101 cRNA induction.
Total RNA of EL-4 was extracted at the various times after
treatment with TPA. Total RNAs (50 ,ug) from TPA-treated or
untreated EL-4 were fractionated on 1.4% formaldehyde denaturing
gel and transferred to nitrocellulose filter (30). 32P-labeled, nicktranslated pKR1101 (specific activity, 5 x 108 cpm/Lg) was hybridized to the filter at 42°C in a solution containing 5 x SSC, 50o
formamide, 50 mM sodium phosphate buffer (pH 7.4), 1 x Denhardt
solution, 0.1% sodium dodecyl sulfate, and salmon sperm DNA (250
,ug/ml) for 24 h. Filters were then washed three times for 5 min each
in 2x SSC and 0.1% sodium dodecyl sulfate at room temperature
and three times at 42°C in 0.1x SSC and 0.1% sodium dodecyl
sulfate. The filter was exposed to X-ray film (Fuji RX) without an
intensifying screen for 36 h at -70°C. Inset: relative intensities of
the bands at the 24S position, as measured by a Beckman DU-8B
UVNVis spectrophotometer at 500 nm. The numbers were normalized with the 0-h control.
Downloaded from http://jvi.asm.org/ on June 9, 2014 by guest
FIG. 1. Differential screening of cDNA libraries: hybridization pattern of plaques containing cloned cDNAs with either induced (A) or
uninduced (B) 32P-labeled cDNA probes. Double-stranded cDNA from membrane-associated or cytoplasmic poly(A)+ mRNA of TPA-treated
EL-4 was prepared by standard procedure (17, 31). BamHI linkers were ligated to both ends of the cDNA. The cDNAs were inserted into a
unique BamHI site of lambda phage, BV-2 (16). The recombinant phage DNA was packaged in vitro. Escherichia coli LE392 cells were
infected by the recombinant phages and plated such that ca. 400 phages containing cDNA inserts were present per 150-mm petri dish. Phage
DNAs were transferred to two nitrocellulose filters and fixed to the filters. The sensitivity of the plaque hybridization for screening was
estimated by two reconstruction experiments with XKT137 (K. Takeshida, personal communication), which contains a human ,3-globin gene.
XKT-137 plaques were mixed with charon 4A plaques and plated such that 4 to 30 XKT137 plaques appeared among 1,000 charon 4A plaques
in a plate. The phage DNAs were transferred to duplicate filters. One set offilters was hybridized to a 32P-labeled 3-globin intron and the other
was hybridized to 10-fold serial dilutions of 32P-labeled 3-globin cDNA (ranging from 106 to 10 cpm per filter). An input of 1 x 103 cpm of ,Bglobin cDNA could detect XKT137 plaques. In other experiments, P-globin mRNA was mixed with EL-4 mRNA such that ,B-globin mRNA
represented 2, 0.2, 0.02, or 0.002% of the total RNA. 32P-labeled cDNA probes from the mixture were prepared and hybridized to the filters as
above, with an input probe of 5 x 106 cpm per filter. When 0.02% of ,-globin mRNA was present, the cDNA probe could detect XKT137
plaques. 32P-labeled cDNA probes (specific activity, 2 x 108 cpm/,Lg) were prepared from induced and uninduced EL-4 cells with
oligodeoxythymidylic acid 12-18 as a primer. One set of filters was hybridized to 5 x 106 cpm of the induced [32P]cDNA per filter. The
duplicate set of filters was hybridized to 5 x 106 cpm of the uninduced [32P]cDNA per filter. cDNA probes from the membrane-associated and
cytoplasmic poly(A)+ mRNA were used to screen membrane and cytoplasmic cDNA libraries, respectively. Filters were hybridized in a
solution containing 3 x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 1x Denhardt solution, 10%o dextran sulfate, and herring
sperm DNA (250 p.g/ml) at 68°C for 24 h. Filters were washed extensively at 68°C in each of 3 x SSC, 2 x SSC, and 1 x SSC and exposed to Xray film (Fuji RX) with an intensifying screen for 4 days at -70°C. An arrow indicates a clone that hybridized only to the induced cDNA
probe. X's indicate registration marks.
NOTES
1002
J. VIROL.
hrs.
BW5147
after
72 20 0
Bulb/C C57 BL/6
20 0 20 0
EL- 4
20 0
Burm HI
EL-4 (57IBI Balb/C
-. . .
_
_
Eco RI
EL-4 C57/BI Bulb/C
-- .......
---------- _
-
-
Origin
23.1 Kb
- 12.4 Kb
1-i
-
70 Kb
-
295 KD
.f
Control
20 0
u 6 Kr
. i.!
28S _%S
e
E
18S-
FIG. 3. Effect of TPA on pKR1101 cRNA induction in normal
spleen cells. Spleens were removed from BALB/c and
C57BL/6 mice. Splenocytes were squeezed out under a stainless
steel net and washed twice with RPMI 1640 medium supplemented
with antibiotics and 5% fetal calf serum. Cells were suspended in the
same medium as above. Cells were incubated with TPA (10 ng/ml)
up to 72 h. Untreated control was incubated the same length of time
in the same medium without TPA. Total RNA was extracted from
both TPA-treated and untreated cells at 20 and 72 h of incubation.
Total RNAs (50 ,ug) from TPA-treated or untreated normal mouse
splenocytes, BW5147, or EL-4 cells were fractioned on 1.4% formaldehyde gel, transferred to nitrocellulose filters, and hybridized to
32P-labeled, nick-translated pKR1101 as described in the legend to
Fig. 2. The filter was exposed to X-ray film (Fuji RX) with an
intensifying screen for 48 h at -70°C. Positions of the ribosomal
RNAs are indicated. A clone which appeared to be uninducible was
selected arbitrarily and used as a control for an amount of RNA
loaded (control lanes). A darkening at the top of 72-h lane of
BW5147 was an artifact which did not appear in repeated experimouse
ments.
FIG. 4. High-molelcular-weight DNAs
were
isolated from sple-
nocytes of normal C57BL/6 and BALB/c mice or from EL-4 cells by
a method described by Gross-Bellard et al. (9). The DNA samples
were digested to completion with either EcoRI or BamHI. Digested
DNA samples (10 jig) were run on horizontal, submerged 0.8%
agarose gels (0.7 cm thick) in standard Tris-EDTA-acetate buffer
(pH 7.8) at 1.5 V/cm for ca. 16 h. Molecular weight markers were
prepared by mixing an Aval- and KpnI-digested phage charon 28
DNA and a BamHI-digested phage charon 28 DNA. The DNA was
transferred to the nitrocellulose filter, hybridized to 32P-labeled
pKR1101, and washed essentially as described by Southern (29) and
Herr and Gilbert (10). The filters were autoradiographed at -70°C
with an intensifying screen for 48 h. The labeled DNA had a specific
activity of 5 x 10' cpm/,ug.
tion within the U3 sequence. The deletion spanned from the
base position -180 to -671, from the end of R sequence
which corresponded to the 3' end of the cDNA. Two other
clones which spanned this region had the same deletion,
indicating that the deletion might be a consistent pattern of
the MMTV-related sequences expressed in EL-4. The open
reading frame (ORF) of U3 sequence had a coding potential
of 216 amino acids, spanning from the beginning of the U3
sequence to 21 nucleotides upstream of the TATA box,
followed by three stop codons in phase. The amino-terminal
181 codons of the ORF were the same as those of the other
MMTV (18) except for 11 amino acids differences due to
single-base changes. The carboxy-terminal 35 amino acids
were completely different from the reported sequence (4,
18). Two out of five putative steroid-binding sites within the
LTR (22) were present, but three other regions were missing.
However, the deletion might not affect the sequences involved in the glucocorticoid regulation of downstream genes
(19). In addition, there were 63 single-base changes in the
pKR1101 coding region of the env gene and the ORF of the
LTR when compared with the MMTV proviral sequence of
strain C3H (1, 4): 38 changes were silent substitutions, and
25 resulted in 22 amino acid changes, of which 13 were
Downloaded from http://jvi.asm.org/ on June 9, 2014 by guest
that in normal spleen cells, although the maximum accumulation occurred after 72 h of exposure. Figure 4 shows
Southern blot analysis of genomic DNA of EL-4 and spleen
cells of C57BL/6 and BALB/c. The gene for pKR1101 was
inserted into multiple locations of the EL-4 genome and was
inserted in normal spleen cells in four to five locations.
The nucleotide sequence of the five cDNA clones related
to pKR1101 was determined by the dideoxynucleotide chain
termination method (27) after the fragments were subcloned
into the M13 vector mp8 (20). Figure 5 shows the nucleotide
sequence of the insert from clone pKR1101, with the positions at which MMTV proviral sequences of C3H (4, 18), GR
(15, 23), or both differ. The cDNA started at the end of the
R sequence of the right long terminal repeat (LTR) and
extended to the BamHI site of the env gene of MMTV.
The cDNA contained putative polyadenylation signals of
MMTV and the TATA box, which read AGTAAA and
TATAAAAGA, respectively. The cDNA also contained the
entire coding region for viral gp36 and part of the gp52
sequence. The cDNA, however, had a 491-nucleotide dele-
VOL. 52, 1984
._X Z&*^wbs_%W*
NOTES
Not1
50
CCC
CC
D P N D R D F T A L V P N T E L F R L V A A 8 R H L I L K K P G F
EL-4: GGATCCCAATGATAGAATG ACTOCTCTAGlcCCCAGA2Cl1GCTAGTTCAOCCTCACATTTCTCA
Am
T
0
C3H:
00*
G
c
GR
Stratus
C3H:
GR
E2-4:
C3H
G
C
260
T F H I S C 8 S C R L T N C L D S S A Y D Y A A I I V K R P PY V L
C I I I rCATATrCCrGrrCTTCTTGTAGATTGACTAATTG rTAGATTCTTs CGCCTACGACrATGCAGCGATCATAGTCAAGAGGCCGCCATATGTGC
C
OR
EL-4:
C3H
C
C
C
I
A
I
L G I
8 A L
I A I
C
460
T 8 F A V A T T A L
I
C
C
A
A
K E N Q T A T F V
I
GR
C
N Y L H Rt N V T
L A L
8
E Q R
T O
T A
T G
C O
A
A
GR
C
C
A
A
G
A
T
C
GR
T
E2-4:
GAGAGCTGGGAAAGAACCAGAGCACAT-TATTGGGCATTTGGAATGACAATAGATTTCATATAACATACAGGAGTTMACCMCCrGATTAGTGATATGA
M T
G
AG
O
C
C
T
CCC
C3H:
GA
GR
L
L G
GG C
S K Q H
C
T
C T
A
I
CCC
V N D N K I S Y N I
C
T
A
Q E L T N L I S D N
G
G
A A
A A
G
A
T
D A V D L S S L A Q S F A N G V K A L N P L D W T Q I F I
GGCTTTTAA
TCCATTGGATTGGCATTrCAT
G
A
C
G
A
A
G
G
G
T
A
T
960
F I G V G A L L L V I V L tt I F P I V F QC
L A K 8 L D Q V Q 8 D
Q C lA
I
T
C
GR
EL-4:
C3H:
TTATAGGAGTTGGAGCCCTALlLVIVAIV
T
T
GR
L
EL-4:
C3H
GR
C3H:
GR:
EL-4:
C3H
GR
C3H:
OR
K
G
C3H:
GR
U3H:
GR
L G L
C
C
C
L GKV A V R A
G
A
P
D K
S
D
F
I
(50)
L C C K L G
I
G
G
1260
R R A
G
G
A
N Y I
L A K
L T L D S
CCC
CCC
F N Y 8
I
s V
1460
I S R
Q K L E N G I
I
(160)
8 L S F
8 T I
*CC
S
I
CC E T 8 B F L
E E
G t4 bp Del
G
End orf
(200)
1660
K C F D L S 8 E C S ILLCSFCT Y P 8 L 14
CC
AA
I
C
C
G
G
G G
TG
T
T
T T
T C
1600
0
G
GACrTGGTTTGGTATCAMTGTTTTGATCTMGCTCTGAATGTTCTATTCTCCTATGTTCTTGGAACTTATCCAACTCTTATGTAAATGCrATGTAA
G
G
1600
T
C
TTACTCAAATTCAMGTTAGMATGGGAATAGAAAMTAGAAMAGACGCTCAACCTCMTTGMAACAGATCGCA 'GMTGAGACAATAGMCCT
G
1400
C
T
T
A
L L V T M L R N E
G A
G C
1660
E N R K R R 8 T
1300
CAM
ATGCTTGCTAAAAATTATATTTTTACCMTAAGACCAATCCAATAAGTAGATTATTAGTrACTATGTTAOAAATGAATCATrATCrAGTACTA1TT
T
G
1200
Q D Y N L
T' AG
G T
G T
C*C
F T N K T N P
1100
T
C
C
T
C A
T G
G
G
G
1000
R G E A
rAGGGGAGAAGCA
CCC
1360
(100)
N D S E N S T F L L G Q G P Q P T S 8 Y K P H R L C P S E I E I R
AAACGATTCGGAGMCTCMCCTTCCTCCTGGGGCMGGACCACAGCCMCTTCCTCTTACAAGCCACACCGACTTTGTCCTTCAGAAATAGAAATAAGA
T W F
EL-4:
CCC
11160
F P T K D D L S A H K R M S
L
00
gp6-
T
G N A A P A A E M V E L P R VS
M P R L Q Q K V L N S R E C P T
G
CCC
L
L
F T Q
EL-4:
L L K K KK
L
End
1060
CTCTGCTTTGCCTGGGGCTATTGGGGAAGTTGCGGTTCGTGCTCGCAGGGCTCTCACCCTTGATTCTfl MTTACTCTTCTGTGCMGATTACAATCT
M
EL-4:
A(1)
GCCAAGGGGTTGTTTCCCACCAAGGACGACCTGTCTGCGCACAACGGATGAGCCCATCAGACAAAGACATATTCATTCTCTGCTGCAAACTTGGCATAG
A
EL-4:
V
G
A
A
CTTAACGTGCTTCrTTTAAAAAAGAAAAAAGGGGGAMTGCCGCGCCTGCAGCAGAAATGGTTGAACTCCCGAGAGTGTCCTACA
C
Polypurin. track tCegln orf of LTR
T
Begin U3 C**
A
EL-4:
C3H
GR
N
600
A
EL-4: GCAAACAACATATTGACGCAG;TGGACCTCAGTAGCTTGGC TCAGTCCTTTGCCAATGGAGTAA
C3H:
700
A
760 ***
E S W E R T RAH
600
6560
L G Q D V A N L K T R N 8 T R C N A N H D F I C V T P L P Y N A 8
E2-4: GrrGGGTCMAGATGTGGCAAAMAAGCCTAGACCTGTTCCACTAGGTGTCATGCAAT
T
C
C3H: T
C
A
T
T
C T
A
T
A
500
1700
A
G
TATA'
Poly(A)+s1gual 1760
EL-4: ACCATGATATAAMAGACrGCTGAT;1TTGTGAGTMACTTGCMCAGTCCTMCAAAAAAAM
AG
L
j
T
TA
GR:
-
C3H:
FIG. 5. Nucleotide sequence and deduced amino acid sequence of pKR1101 cDNA insert and comparison of the sequence with published
of other strains, C3H and GR. Numbers below each line refer to nucleotide position from the BamHI site of the enm' gene of
MMTV. Numbers above each line of the LTR ORF refer to amino acid position from the first methionine residue. Functional landmarks such
as the polypurine tract, the TATA box, and polyadenylation signals are indicated. Positions at which the pKR1101 MMTV sequence of EL-4
varies from the proviral sequence of MMTV GR and C3H are shown under the pKR1101 sequence. Boundaries for U3 and R sequences of
LTR and for gp52 and gp36 of viral glycoprotein are indicated by referring to prior publications (1, 4, 18). Single asterisks (*) refer to stop codons. Sets of three asterisks (***) indicate that the amino acids below the marks are different between pKR1101 and C3H. AGGA appeared at
the other end of the deleted sequence.
sequences
conservative and 9 were nonconservative (five in env and
four in the LTR ORF). It appeared that the coding functions
of glycoproteins and the first 181 amino acids of the LTR
ORF were well maintained, even though multiple cycles of
amplification and reintegration had occurred in EL-4 cells.
The functional significance of the altered coding potential of
the LTR ORF in pKR1101 requires further investigation.
When the pKR1101 sequence was compared with that of
strain GR (15, 23), the divergence of env sequences was
similar to that found for C3H: 48 single-base changes resulted in 13 amino acid changes, of which 5 were nonconservative. However, there was a single-base addition and deletion
Downloaded from http://jvi.asm.org/ on June 9, 2014 by guest
*
400
**
55660
I D L It L E A R L N A L E E V V L E
I
EL-4: ATTATCTTCATAGGAATGTTACrTrAGCGTrATCrGAACAAAGMTAATAGATTTAAAATTAGAAGCTAGACTAATTrTA
C3N:
300
**C
C G T
C OT
T
E2-4: GCCATrA1-CTGGGCATATCT GCrGATTGCTATTATTCC
C3H:
200
G A
360
DeogI PM
gp 62
L P V D I 0 D E P V F D D S A I Q T F R Y A T D L I R A K RIP V P
GCTACCTGTAGATATTGGTGATGMCCATGGTTTGATGATTCTGCCATTCAACTFTTAGGTATGCCACAGATrMTTCGAGCTAAGCOATnOTTGC
OR
100
160
*CC
Q E H K N I P T 8 A C V T Y P Y A I L L G L P Q L I D I E K 11 G 8
TGCCATATTATTAGGATTACCTCAGCTAATAGAWTATAGGAAC_ATCTA
G
GO
T
E2-4:
1003
1004
NOTES
J. VIROL.
We thank G. Fathman and J. J. Farrar for suggestions and
providing EL-4 and 11-2-dependent T-cells, B. Forget and J. Eisenstadt for providing human ,-globin mRNA and a Beckman DU-8B
spectrophotometer, P. Lebowitz for a critical reading, J. Kim for
technical assistance, and Ann M. Mulvey for preparing this manuscript.
This research was supported by Public Health Service grant CA
30938-03 from the National Institutes of Health. B.S.K. acknowledges financial support from the Swebilius Cancer Research Award.
LITERATURE CITED
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2. Arya, S. K. 1980. Phorbol ester-mediated stimulation of the
synthesis of mouse mammary tumor virus. Nature (London)
284:71-72.
3. Benton, W. D., and R. W. Davis. 1977. Screening Agt recombinant clones by hybridization to single plaques in situ. Science
196:180-182.
4. Donehower, L. A., A. L. Huang, and G. L. Hager. 1981.
Regulatory and coding potential of the mouse mammary tumor
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5. Driedger, P. E., and P. M. Blumberg. 1980. Specific binding of
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in addition to 31 single-base changes in the LTR which
would result in a long frame shift.
The rapid induction of MMTV-related mRNA expression
by TPA indicated that its effect on the gene expression might
be directed via the TPA-receptor complex as in the case of
steroid stimulation. However, the half-time for the maximal
accumulation of MMTV RNA by TPA was considerably
longer than that reported for steroid induction (24). This
might indicate that the TPA induction process required a
recruitment of other inducers or that the half-life of induced
RNA was longer in lymphoid cells than in the mouse
mammary cells (25). The increase of MMTV mRNA expression after TPA treatment in EL-4 was very similar to that in
Mm5mt/c cells reported by Arya (2). Michalides et al. (21)
have noticed that acquisition of MMTV coincides with the
onset of leukemogenesis in GR mice. Very recently, Dudley
and Risser (6) reported that MMTV genomes were amplified
in several transplantable T-cell lymphomas of C57BL/6, A/J,
and BALB/c origin. If the multiple insertions of the pKR1101
sequences accounted for the elevated expression of the
mRNA, many of the MMTV sequences inserted in the EL-4
genome might be transcriptionally active. This might be a
factor involved in the aberrant regulation of growth or
lymphokine production by EL-4 cells.