[CANCER RESEARCH 34, 109-1U, January 1974]
Polyadenylate-containing RNA of Polyribosomes Isolated from
Rat Liver and Morris Hepatoma 78001
John W. Tweedie2 and Henry C. P¡tot3
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706
SUMMARY
The fraction of polyribosomal RNA that contains polyadenylate sequences was examined in rat liver and the
Morris hepatoma 7800. Sequential sodium dodecyl sulfatephénol
extraction at pH 7.6 and pH 9.0 followed by polythymidylate cellulose chromatography of the fraction
extracted at pH 9.0 was used to isolate the polyadenylatecontaining fraction of polyribosomal RNA. This fraction
from either tissue appeared to be the same with respect to
mobility on acrylamide gels, sensitivity to ribonuclease di
gestion, and 32P base composition analysis. There was no
difference in the absolute amount of polyadenylate-containing RNA in polyribosomes from either liver or hepatoma.
The time course of the decay in specific activity of the
polyadenylate-containing RNA was determined following
the administration of a single dose of 32P. From these data,
a half-life of approximately 9 to 12 hr could be calculated
for the polyadenylate-containing RNA fraction from both
liver and the hepatoma polyribosomes.
INTRODUCTION
laminated by other classes of RNA. The determination of
the specific activity of polyadenylate-containing RNA at
various times after the administration of a labeled RNA
precursor should permit an estimation of the rate of turn
over of this presumptive mRNA fraction. Previous
methods for the investigation of mRNA in eukaryotic cells
have involved indirect methods for the determination of the
amount of label present in mRNA (7, 22 -25).
In view of the probability that the heterogeneous, rapidly
labeled RNA fraction in the nucleus is the precursor of
mRNA in the cytoplasm (4, 19), several investigators have
suggested that, since polyadenylate sequences are found in
both types of RNA, these sequences may be involved in the
maturation of heterogeneous nRNA in the nucleus or in its
subsequent transport to the cytoplasm. Any derangement
of these processes would necessarily be manifested by a loss
of, or an alteration in, the expression of those functions that
were specified by the mRNA involved. In most of the transplantable hepatomas there is a loss of several enzymatic
functions expressed in normal liver (16). For this reason we
undertook an investigation of the characteristics of the poly
adenylate-containing RNA from polyribosomes of normal
rat liver and of the transplantable Morris hepatoma 7800.
Recent reports (2, 5, 6) from other laboratories have
demonstrated that some mRNA's from eukaryotic cells MATERIALS AND METHODS
contain a terminal sequence of polyadenylate homopolymer.
These sequences are also found in these cells in the hetero
Animals. Livers were obtained from male Holtzman al
geneous nRNA fraction which is believed to be the precur
bino rats fed a standard laboratory chow diet ad libitum
sor of cytoplasmic mRNA (4, 18, 19). A previous report and fasted for 18 hr prior to sacrifice. The Morris 7800
from this laboratory (26) has shown that 5-fluoroorotic acid tumor was maintained by transplantation, at 20-day inter
inhibits the formation of mature cytoplasmic rRN A but has vals, into the hind legs of male Buffalo rats (Texas Inbred
little effect on the synthesis of a class of RNA that has the Rat Co., Houston, Texas), which were also maintained on
characteristics of mRNA. Further studies (8) have revealed the standard chow diet. Administration of radioisotope was
that, while 5-fluoroorotic acid strongly inhibits the forma
by i.p. injection in the case of rats not bearing tumors.
tion of ribosomal 28 S and 18 S RNA, it has very little Radioisotope was given to tumor-bearing rats by i.v. in
effect on the incorporation of "P, or orotate-SH into poly jection into the penile vein under light ether anesthesia.
adenylate-containing RNA.
Preparation of Total Liver and Hepatoma Polyribosomes.
The occurrence of a sequence of polyadenylate in a frac
Total liver and hepatoma polyribosomes were prepared by a
tion of RNA that exhibits the properties expected of mRNA modification of the procedure of Blobel and Potter (1).
has facilitated the direct isolation of this material uncon- Livers and hepatomas were rapidly excised from decapi
tated and exsanguinated rats. In the case of hepatoma tis
'The work described was supported in part by Grant CA-07175 from sue, all necrotic and hemorrhagic areas were trimmed away.
the National Cancer Institute and Grant E-588 from the American Cancer The tissues were rinsed in ice-cold TKM 4buffer containing
Society.
0.44 Msucrose. After being minced with scissors, the tissues
2Present address: Department of Chemistry, Biochemistry and Bio
physics, Massey University, Palmerston North, New Zealand.
3To whom correspondence should be addressed.
Received July 16, 1973; accepted October 2, 1973.
JANUARY
'The
abbreviation
used is: TKM.
50 mM Tris, pH 7.5; 25 mM KC1;
and 5 mM MgCI2.
1974
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109
John W. Tweedie and Henry C. Pilot
¡sÃ-
II
2
Distane« («•")
Distance
4
(cm)
Chart 1. Polyacrylamide-agarose gel electrophoresis of RNA fractions obtained from 32P-labeled polyribosomes from rat liver (A and B) and from
the Morris hepatoma 7800 (C and D). Rats were given 1 mCi of 32Pi and were sacrificed 6 hr later. The various RNA fractions were prepared as de
scribed in "Materials and Methods." The approximate S values for rRNA's and RNA's are given at the lop of the charts and the numbers along the
ordinale are in hundreds of counts. A and C, radioactivity profile of RNA extracted from polyribosomes at pH 9.0 and fractionated on polythymidylate-cellulose. B and D, RNA fractions as in A, but after treatment with pancreatic RNase A (2.0^g/ml, 37°for 10 min).
were homogenized in 2 volumes of the same buffer with 5
strokes of a loose-fitting Teflon-glass Potter-Elvehjem
homogenizer. The homogenate was centrifuged at 12,000
rpm for 10 min in the Sorvall SS-34 rotor, and 1 volume of
20% Triton X-100 was added to 10 volumes of the resulting
postmitochondrial supernatant. Seven ml of Triton-treated
supernatant were layered over a discontinuous sucrose gra
dient consisting of 3 ml of 2.0 M sucrose in TKM buffer
overlayered with 2 ml of 1.4 M sucrose in TKM. The gra
dients were centrifuged at 50,000 rpm for 5 hr in a Spinco
type 50 Ti rotor. Polyribosome pellets were either used im
mediately or stored at -70°for up to 3 days before use.
Extraction of Polyadenylate-containing RNA. Polyadenylate-containing RNA was obtained from polyribo
somes by a sequential sodium dodecyl sulfate-phénolex
traction, first at pH 7.6 and then at pH 9.0, essentially as
described by Lee et al. (15). Both extractions were per
formed at 0°.Polyadenylate-containing RNA was found
exclusively in the aqueous phase from the pH 9.0 extraction
(8).
Polythymidylate-cellulose Column Chromatography of
RNA. Polythymidylate-cellulose was prepared by the pro
cedure of Gilham (9). Small columns (0.8 cm high: 1 sq cm
cross-sectional area) of polythymidylate cellulose were
poured in water-jacketed Pyrex columns in the cold room
110
and washed with "high-salt" buffer (10 mM Tris, pH 7.5
0.1 M NaCl) until the A2go of the eluate was below 0.01
absorbance unit. RNA samples (up to 12 A26o units) were
diluted with at least 5 volumes of high-salt buffer and ap
plied to the column. The column was washed with 10 ml of
high-salt buffer. The material not retained by the column
was eluted in the 1st 3 ml of eluate. The temperature of the
column was raised to 30°by means of a circulating water
bath, and elution was continued with 10 ml of deaerated
high-salt buffer. A small amount of material absorbing at
260 nm was eluted in this fraction but it was found not to be
enriched in adenylate. Polyadenylate-rich RNA emerged
in the 1st 3 ml of eluate after elution was started with 10
mvi Tris, pH 7.5, at 30°(8).
Gel Electrophoresis of RNA. RNA samples were applied
to gels consisting of 2.6% acrylamide 0.6% agarose pre
pared and run according to previously described procedures
(26). The RNA concentration in the gels was determined by
scanning at 260 nm in a Gilford 2400 spectrophotometer
with a linear transport attachment. For determination of the
distribution of radioactivity, the gels were cut into 2-mm
slices, the slices were solubilized overnight in 30% H2O2 at
80°,and their radioactivity was determined by liquid scin
tillation counting.
32P-Labeled Base Composition Analysis. Samples of
CANCER
RESEARCH
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Polyadenylate in Rat Liver and Hepatoma
C
SS*
V4S
SS*
*4S
II
•¿9
i
a
Distance
(cm)
Distance
Chart I, C and D
32P-labeled RNA were hydrolyzed in 0.3 M KOH and thè
base composition of the hydrolysate was determined by a
modification of the procedure of Greenman et al. (12) as
described previously (8). The determination of 32P radio
activity was made by utilizing the Cherenkov radiation
effect of this isotope. The 3H channel of a Packard TriCarb liquid scintillation spectrometer was used for this, as
described by Haviland and Bieber (14).
Materials. Carrier-free 32PO4 was obtained from
Schwarz/Mann, Orangeburg, N. Y. Sucrose, RNase-free,
was obtained from the Mann Research Laboratories, New
York, N. Y. Powdered cellulose was obtained from H.
Reeve Angel Co., Clifton, N. J., and Tris was purchased
from the Sigma Chemical Co., St. Louis, Mo.
The RNA extracted from polyribosomes at pH 9.0 was
first separated into polyadenylate-containing and nonpolyadenylate-containing fractions by polythymidylate-cellulose
chromatography. The radioactivity in the nonpolyadenylate-containing fraction (Chart I, A and C) was located
largely in the 28 S and 18 S ribosomal subunits that were
present in this fraction. In addition there was a considerable
portion of the label in a heterogeneous fraction of RNA with
mobilities corresponding to S values of 7 S to 30 S. The 32P
in the polyadenylate-containing fraction (Chart 1, A and C)
was located only in the heterogeneous RNA of 7 to 30 S size
distribution.
When the 2 fractions obtained by polythymidylate-cellu
lose chromatography were treated with pancreatic RNase
A before application to the gels, the results shown in Chart
l, B and D, were obtained. In the polyadenylate-containing
RESULTS
fraction, a substantial proportion of the radioactivity was
Characterization of Polyadenylate-containing RNA from located in a fraction that was resistant to RNase A and had
Liver and Hepatoma Polyribosomes. RNA fractions from a mobility that suggested a size of 10 to 12 S, which was
normal rat liver and from hepatoma polyribosomes were comparable to that described by Hadjivassiliou and Brawerexamined by gel electrophoresis on polyacrylamide-agarose
man (13). This fraction of RNase-resistant RNA was not
gels. The animals were each given injections of 1 mCi of observed in any other fraction after enzymatic hydrolysis.
32P¡,either i.v. (hepatoma) or i.p. (liver), 6 hr prior to A similar nuclease-resistant fraction has been observed by
sacrifice. Results described earlier (8) demonstrated that 6 several groups in HeLa cell polyribosomes and has been
hr after administration of 32PO4 almost all of the radio
shown to consist almost exclusively of adenylate residues
activity in the pH 7.6 RNA fraction was located in the 28 S (2, 4, 5).
The 32P base composition of the various RNA fractions
and 18 S ribosomal subunits, with a small amount at a posi
tion corresponding to 4 S and 5 S RNA.
were reported previously (8). The fraction extracted at pH
JANUARY
1974
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111
John W. Tweedie and Henry C. Pilot
7.6 had a low (A + U/G + C) ratio characteristic of rRNA.
The material extracted at pH 9.0 had an (A + U/G + C)
ratio of 1.0. This value is similar to that obtained from rat
liver polyribosomes labeled with 32P in the presence of
either actinomycin D or 5-fluoroorotic acid (8). After sep
aration on a column of polythymidylate-cellulose, this latter
RNA was shown to consist of a fraction high in adenylate
[(A + U/G + C) ratio of 1.4 to 1.5] and a fraction that had
a base composition similar to that of the RNA extracted at
pH 7.6.
The data of Chart 1 show that there were no major differerences between the corresponding RNA fractions from
normal liver and hepatoma polyribosomes. The possibility
remains, however, that minor differences do occur but were
not revealed by the methods used in this investigation.
Amount of Polyadenylate-containing RNA in Polyribosomes from Liver and Hepatoma. Polyribosomes were ob
tained from 10 normal livers and Morris 7800 hepatomas
as described in "Materials and Methods." The various
RNA fractions were separated and the amount of RNA in
the polyadenylate-containing fraction from the polythy
midylate-cellulose column was determined by its absorbance
at 260 nm. Preliminary experiments, in which polyadenylate-3H (Schwarz/Mann) was added to a total liver polyribosome suspension before extraction revealed that over
90% of the added polyadenylate was found in the fraction
bound by polythymidylate-cellulose.
Table 1 shows that, while there were differences in the
amount of polyadenylate-containing RNA in the 2 tissues on
a total tissue-weight basis, there was no difference when the
data was expressed as /¿gpolyadenylate RNA per mg of
total polyribosomal RNA. The difference found on a totalweight basis probably represents differences in the amount
of polyribosomes obtained from the 2 tissues by the proce
dure used.
Turnover of Polyadenylate-containing RNA in Liver and
Hepatoma Polyribosomes. The time course of incorpora
tion of 32P into the various RNA fractions in both liver and
hepatoma is shown in Chart 2. A single dose of isoto_pewas
administered at zero time. Animals were sacrificed at the
times shown and the RNA fractions were prepared. The
polyadenylate-rich fraction eluted from the polythymi
dylate-cellulose column was rapidly labeled in both normal
liver and hepatoma, although the rate of incorporation into
polyadenylate-rich material was slightly faster in liver than
in hepatoma. However, this difference may be a reflection
of the relative availability of 32P-labeled nucleotides due
to the different route of administration in each case (i.p.
versus i.v.) or the different vasculature of the 2 tissues, or
both.
In liver and hepatoma the specific activity of the poly
adenylate-rich fraction reached a maximum at 3 to 6 hr
after administration of the label. The pH 7.6 RNA fraction
(rRNA) and the nonpolyadenylate-containing fraction of
the pH 9.0 RNA were labeled to a far lesser degree and
their specific activity had still not reached a peak 15hr after
administration of the isotope.
In order to investigate the turnover of the various RNA
fractions, the labeling period was extended and the loss of
112
Table 1
Polyadenylate-containing RNA content of polyribosomes from rat liver
and Morris hepatoma 7800
Polyadenylate-containing RNA
Liver
Hepatoma
/ig/g tissue-
>»g/mg
polyribosomal
RNA
23.12 ±0.54"
11.72 ±0.87
10.40 ±0.45
9.56 ±0.62
°Based on wet weight of tissue. Polyadenylate-containing RNA was
prepared as described in the text.
"Mean ±S.E. for 10 determinations.
32P from the pH 9.0 fraction was plotted in a semilogarithmic manner (Chart 3). In this experiment there was con
siderable variation between animals in the absolute values
for the specific activities of corresponding RNA fractions.
For this reason the data have been plotted as the ratio of the
specific activity of each pH 9.0 RNA fraction to the specific
activity of the pH 7.6 RNA fraction at each time point. As
Chart 3 shows, there appears to be an exponential decay of
the activity of the polyadenylate-rich RNA in both liver
and hepatoma. The half-life calculated from the slopes of
these lines differed for liver and hepatoma (11.6 and 9.6
hr, respectively). This small difference in half-life may not
be significant due to possible differences in reutilization of
the isotope in each tissue. If there were greater reutilization
of 32P into RNA in liver, this could account for the longer
half-life of the polyadenylate-rich fraction in this tissue.
The relative specific activity of the nonpolyadenylatecontaining pH 9.0 RNA remained fairly constant through
out the time period shown. However, gel electrophoretic
examination of this RNA fraction at each time point showed
that there was an increase in the proportion of radioactivity
in rRNA subunits and a decrease in the proportion in het
erogeneous "messenger-like" material as the length of time
after labeling increased. Thus the decay curve observed for
this fraction is likely to represent a composite of the slow
increase in label into rRNA, together with a more rapid
decay of activity in those messenger-like RNA molecules
that do not have a substantial polyadenylate sequence and
that are not retained by polythymidylate-cellulose.
DISCUSSION
The data presented in this paper show that much of the
messenger-like RNA from polyribosomes of rat liver and
M orris hepatoma 7800 contains a segment of polyadenylate.
Similar sequences have been demonstrated previously in
the polydisperse RNA component of polyribosomes from
HeLa cells (6), mouse sarcoma ascites cells (15), and in the
RNA of several of the RNA tumor viruses (10, 11). Poly
adenylate sequences have also been reported in rat liver
cytoplasm (13), but their location in polyribosomal RNA
was not established in that investigation.
CANCER RESEARCH VOL. 34
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Polyadenylate in Rat Liver and Hepatoma
IO
-Liver
Polysomal
RNA
>>
<£
'>
.
<
o
u
x
tfÃ-o.
o
25 - Hepatomo Polysomal
RNA
20
A. A pH 7.6 RNA
O, •¿
Non poly-A containing
15
O, •¿
Poly-A containing
pH 9.0
RNA
IO
IO
Hours
in vivo. More recently, an attempt has been made to use the
sensitivity to RNase action of mRNA in polyribosomes to
examine mRNA turnover (22, 23).
The finding that polyadenylate-containing mRNA was
rapidly labeled after a single dose of >2P, (Chart 2) sug
gested a rapid rate of turnover for this fraction. In liver, the
maximum specific activity of the polyadenylate-containing
RNA was reached at 3 to 5 hr after labeling. This phase
was followed by a slow decline in specific activity. The de
cay portion of the labeling curve (Chart 3) showed an expo
nential decrease in activity with a calculated half-life of 10
to 12 hr. This was slower than expected considering the
rapid increase in specific activity shown by this fraction in
the early stages after administration of the label. A possible
explanation for this is that a proportion of the mRNA may
have been turning over more rapidly and that the turnover
of this material was not apparent in the experiment shown
in Chart 3 in which the early time points (0.5 to 6 hr) were
not examined. The half-life for rat liver polyribosomal
mRNA found in these studies (9 to 11 hr) is longer than the
value of 5 hr obtained for this tissue by Endo et al. (7).
These authors used a procedure in which the RNA from
polyribosomes was examined on sucrose gradients before
and after RNase treatment during which mRNA was pre-
15
2P Lobel
Liver Polysomal RNA
Chart 2. Incorporation of 32Plabel into RNA fractions of rat liver and
Morris hepatoma 7800 polyribosomes. Rats were given I mCi of 32Pand
were sacrificed at the times indicated. Total polyribosomes and the various
RNA fractions were isolated as described in "Materials and Methods."
Tj_«
11.6hr
'2
The numbers along the ordinale are in thousands of counts.
O , •¿
Poly-A containing
RNA
Polyadenylate sequences have also been found in the het
erogeneous nRNA fraction of eukaryotic cells (4). Since this
fraction of nRNA has been postulated to be the precursor
of cytoplasmic mRNA (4), it has been suggested that cytoplasmic RNA which contains a polyadenylate sequence
represents a large proportion of the cytoplasmic mRNA
(2). Sheiness and Darnell (21) have recently shown that, in
HeLa cells in culture, the polyadenylate sequences in the
cytoplasm shorten with increasing age of the cell. However,
to date the only class of mRNA shown to have no poly
adenylate sequences is the presumptive mRNA for histone
synthesis (20).
Trakatellis et al. (24) studied the turnover of mRNA in
mouse liver by determining the increase in specific activity
of the 18 S to 5 S RNA region isolated from a sucrose gra
dient after a single pulse of label. However, this region of
the gradient includes 18 S rRNA as well as some 4 S and
5 S RNA, and it is difficult to determine the contribution
of these types of RNA to the total radioactivity in this re
gion. These workers (25) have used a short pulse of label
followed by administration of actinomycin D to inhibit
further RNA synthesis. If the time between the administra
tion of label and actinomycin D is short, very little labeled
rRNA appears in the cytoplasm. However, the use of ac
tinomycin D has been criticized because of its effects other
than on RNA synthesis. Endo et al. (7) have suggested that
actinomycin D prolongs the lifetime of mRNA in rat liver
a , »NonPoly-A containing
RNA
o»
S
•¿*e
fl
B
o.
I Hepatoma Polysomal RNA
g
I
,T| -9.6 hr
en
I
10
20
30
Hours 32P Label
Chart 3. Relative specific activity of the polyadenylate-containing (O)
and nonpolyadenylate-containing (D) fractions of the RNA extracted at
pH 9.0 from polyribosomes from rat liver and Morris hepatoma 7800.
There was considerable variation in absolute specific activity of corre
sponding fractions between rats in this experiment. For this reason the
results are expressed as the ratio of the specific activity of the RNA frac
tion examined to the specific activity of the RNA fraction extracted at
pH 7.6. Other experimental details are the same as described for Chart 2.
JANUARY 1974
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113
John W. Tweedie and Henry C. Pilot
sumed to have been degraded. Recently, Murphy and At
tardi (17) have used polythymidylate-cellulose to isolate
an mRNA fraction from HeLa cells and showed that the
half-life of this RNA was 2 to 3 days. This is much longer
than the value of 3 hr obtained for mRNA from mouse Lcells by means of a procedure that involved the examination
of the protein synthetic capacity of polyribosomes isolated
at various times after actinomycin D treatment (3).
There were no significant differences observed between
the polyadenylate-containing RNA fraction from liver or
Morris hepatoma 7800 in any of the parameters studied.
There were slight differences in the time course of the label
ing pattern from each tissue, but it was not possible to at
tach any significance to them. It is almost certain that there
are differences in mRNA between the 2 tissues in view of
the obvious differences in growth rates and enzyme patterns
observed ( 16). These differences do not appear to be related
to the metabolism of the polyadenylate-containing mRNA
under the conditions of our experiments.
ACKNOWLEDGMENTS
The authors wish to thank Paul Hinderaker for his skilled technical
assistance.
8. Garre«, C. T., Wilkinson, D. S., Tweedie, J. W., and Pitot, H. C.
Effect of 5-Fluoroorotic Acid Administration on the 32P Base Com
position, DNA-RNA Hybridization Properties and Labeling of Polyadenylate-rich RNA in the Cytoplasm of Rat Liver Cells. Arch. Biochem. Biophys., 155: 342 354, 1973.
9. Gilham, P. T. The Synthesis of Polynucleotide-Celluloses and Their
Use in the Fractionation of Polynucleotides. J. Am. Chem. Soc., 86:
4982-4985, 1964.
10. Gillespie, D., Marshall, S., and Gallo, R. C. RNA of RNA Tumor
Viruses Contains polyA. Nature New Biol., 236: 227 231, 1972.
11. Green, M., and Cartas, M. The Genome of RNA Tumor Viruses Con
tains Polyadenylic Acid Sequences. Proc. Nati. Acad. Sei. U. S., 69:
791 794, 1972.
12. Greenman, D. L.. Huang, R. C., Smith, M., and Furrow, L. M. ThinLayer Separation and Quantitative Elution of Nucleosides and Nucleotides. Anal. Biochem., 31: 348-359, 1969.
13. Hadjivassiliou, A., and Brawerman, G. Polyadenylic Acid in the Cy
toplasm of Rat Liver. J. Mol. Biol., 20: 17, 1966.
14. Haviland, R. T., and Bieber, L. L. Scintillation Counting of 3ÃŽP
with
out Added Scintillator in Aqueous Solutions and Organic Solvents and
on Dry Chromatographie Media. Anal. Biochem., 33: 323 334, 1970.
15. Lee, S. Y., Mendecki, J., and Brawerman, G. A Polynucleotide Seg
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RNA Component of Mouse Sarcoma 180 Ascites Cells. Proc. Nati.
Acad. Sei. U. S., 68: 1331-1335, 1971.
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Rat Hepatomas with Different Growth Rate (Including "Minimal
Deviation" Hepatomas). In: H. Busch (ed.). Methods in Cancer Re
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114
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VOL. 34
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Polyadenylate-containing RNA of Polyribosomes Isolated from
Rat Liver and Morris Hepatoma 7800
John W. Tweedie and Henry C. Pitot
Cancer Res 1974;34:109-114.
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