Download Amino Acid Incorporation by in Vitro Tumor and

Amino Acid Incorporation
by in Vitro Tumor and Liver
Systems and Their Response to Exogenous
Ribonucleic Acid
M. A. O'NEAL* AND A. C. GRIFFINt
(Deparimeni
of Biochemistry,
The Univertity
of Texas M. D. Anderson Hoipital
and Tumor Institute,
Houston,
Texa.@)
SUMMARY
A procedure is described for the isolation of amino acid-incorporating systems from
Novikoff
ascites
tumor
and from rat liver.
Under
the conditions
employed
the tumor
system will incorporate approximately
80—70M@imolesof amino acid/mg ribosomal
protein when C'4-labeled valine, phenylalanine,
or lysine is added to the incubation
medium. Addition of ribonucleic acid preparations from tumor and liver nuclei, tumor
ribosomes, or tobacco mosaic virus to the tumor amino acid-incorporating
system
caused
a 16-45 per cent increase
in amino
acid incorporation.
Addition
of polyuridylic
acid to the tumor system with C'4-i@-phenylalanine caused a seven- to tenfold increase
in the incorporation of this amino acid. These findings indicate that this mammalian
system responds to the same nucleotide coding sequence for phenylalanine
as E. coli
and provide evidence that the tumor-incorporating
system will respond to a limited
extent to natural ribonucleic acids and to synthetic polynucleotides.
Extensive
progress
has been made
In all these systems,
in establish
ing the function of an informational or messenger
ribonucleic acid (RNA) in protein formation, with
the use of normal and phage-infected bacterial sys
tems (6, 8, Q4) as well as mammalian tissues (9. 16,
@O).The observation of Nirenberg and Matthaei
(18) that addition of polyuridylic acid to activat
ing components and ribosomes isolated from E.
coli causes the formation of a polypeptide
posed
entirely
stimulus
for
of phenylalanine
identification
com
provided
of a number
the
of the
.
although
cate that
discrepancies
and
code
degeneracy
it may be more complex
first considered.
A variety of mammalian
than
ity
Division
of Biology,
California
acid
Insti
November
bearing
minutes
t American Cancer Society Professorof Biochemistry.
for publication
their
capacity
RNA's,
to
polyuridylic,
respond
and
to
various
polycytidylic
7-day-old
transplants.
‘Thefluid was
immediately
chilled, and all subsequent
opera
tions were carried out at O@—5@
C. The cells were
centrifuged at 750 r.p.m. for 5 minutes and were
washed 5 times with a solution containing .O@M
glucose, .14 M NaCl, and .04 M Tris, pH 8.5 (11).
After a final centrifugation
at 1600 r.p.m. for 5
tute of Technology, Pasadena, California.
Reeived
and
rats
was at
incorporating
systems has been investigated,
in
eluding liver (7), reticulocytes (@1), Ehrlich ascites
tumor cells (11), pancreas (@5), and cell nuclei (1).
S Present address:
the amino acid in
MATERIALS
AND METHODS
Novikoff ascites fluid was aspirated from albino
mdi
in vitro amino
however,
may be attributed
to the inherent
RNA associated
with ribosomes.
Re
cently, several groups have demonstrated
a strik
ing effect of polyuridylic acid on phenylalanine
uptake by mammalian incorporating
systems (@,
3, 5, 16, @8).
In the present paper ribosomal fractions iso
lated from Novikoff ascites cells and from rat liver
are compared with respect to their inherent activ
natural
acids.
specific polynueleotide coding units corresponding
to most of the amino acids (14, @8).These findings,
along with other observations
(@, @7, @9),sup
port the concept of a more or less universal code,
.
corporation
messenger
the ascites
were stirred
water. The
5, 1962.
cells, now free of erythrocytes,
with 6 volumes of cold, deionized
cells were allowed to stand for 5
628
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‘O'NEALAND GRIFFIN—Amino
minutes
before
being disrupted
in a Potter-Elveh
jem homogenizer. The course of the homogeniza
tion was followed under a phase-contrast
micro
scope to achieve the maximum
cell rupture
without damage to the nuclei. In all preparations
more than 90 per cent of the cells were ruptured.
After the homogenate
was adjusted to .005 M
MgCl2, .O2@5M KC1, and .25 M sucrose, it was
centrifuged at 15,000X g for 20 minutes in the
Spinco preparative
ultracentrifuge,
Model L,
rotor #21. The resulting supernatant solution, free
of cell debris, nuclei, and mitochondria, was care
fully aspirated from under a lipide layer and was
centrifuged for 2 hours at 105,000X g (rotor #40).
The supernatant
solution (5100) was aspirated,
and the pellets were resuspended in a 2.5 per cent
deoxycholate solution in 0.2 Mglycyiglycine buffer,
pH 8.0 (11). After gentle homogenization
in a
Potter-Elvehjem
homogenizer, the solution was
diluted with 28 volumes of standard buffer (.005
M MgC12,
.025
M KC1,
.@5 M sucrose,
and
pH 7.6) and again centrifuged
@
M Tris,
pH
5. Both
fractions
were
frozen
and
stored
at —15°C. The yield of ribosomes from 185 ml.
of the ascites fluid, averaging 50 X 1O@cells per
ml., was approximately 100 mg. of protein. In a few
cases, the 15,000 X g supernatant fraction was used
in the incubation without separation of the ribo
somes and the activating system. Both the ribo
somes
and the 5100, pH 5, fraction
retained
their
activity for 1—2weeks when stored at —15°C.,
with only slight loss. Protein was determined by
a modification of the method of Lowry et at. (1%).
U@C'&.z,.Va1ine (25.9 mc/mmole) was obtained
from Nuclear-Chicago
Corp., U-C'4-i@-phenylal
anine (30 mc/mmole),
and U-C―-frlysine (8.3
mc/m.mole)
were obtained
from Yolk Radio
chemical Company. The crystalline ribonuclease
came from Worthington Biochemical Corporation.
The synthetic polynucleotides were obtained from
Miles Chemical Co., and the TMV-RNA
was a
gift of Dr. George Cochran,
Department
of
Botany, Utah State University. The solutions of
629
in Vitro
cold amino acids used in the incubations
contained
1 @tmole/mlof each of the following framino
acids : alanine, arginine, asparagine, aspartic acid,
cysteine, cystine, glutamic acid, glutamine, gly
cine, histidine, isoleucine, leucine, lysine, methio
nine, phenylalanine,
proline, serine, threonine,
tryptophan,
tyrosine, and valine. One or more
TABLE
LIVER
AND ASCITES
1
TUMOR
AMINO
ACm
INCORPORATING
SYSTEM
proteinComplete
Tumor system@qsmoIes
valine-C'4/mg
.
ribosomal
system5
—S100, pH 5
3@
0.6
26
4
—ATP,GTP
—AlT, GTP, PEP, PEP kinase
—Aminoacids
1@I@Cl2
+Ribonuclease
1.5
(10 pg.)
+Liver S100, pH 5 for Tumor S100,
pH5
@40t
21Liver
at 105,000X g
for 2 hours. The deoxycholate treatment was re
peated, and the final ribosomal pellets were resus
pended in a minimal amount of the standard
buffer. Ribosomal ‘preparations from liver ‘
tissue
were prepared by the same method but were
treated only once with deoxycholate.
The 5100
fraction was brought to pH 5.0 with 1 M acetic
acid, allowed to stand for 15 minutes in the cold,
and the flocculent precipitate
was collected by
centrifugation.
This precipitate,
redissolved
in
standard buffer to give a protein concentration
of
approximately
10—iS mg/ml,
constituted
the
amino acid-activating
system and was designated
5100,
@
.05
Acid Incorporation
system
. 8@
0.6
Complete system:5
@-‘--s$100,'
p'H@5
—ATP, GTP
—AlP,
—MgCl2
1
1
1.4
PEP, PEP kinase
1
+Ribonuclease
+Tumor S100, pH 5 for Liver S100,
16
pHS9
S The incubation medium consisted of the following: Tumor
System S100, pH 5 = 0.3 mg. protein; ribosomes, —1.0 mg.
protein; U-C'4-L-valine, 4.4 X 10@ pmole; 20 L-amino acids
minus valine, .09-5 smole each; AlP, 0.25 zmole; GTP,
7.5 X 10—i .amole; phosphoenolpyruvate
(Na), 2.5 @imole;
phosphoenolpyruvate kinase, 5.0 pg., MgC12,S smole. Buffer
was added to a total volume of 0.5 ml., and the reaction mix
tures were incubated at 37°C. for 30 minutes. Samples of 0.05
ml. were removed, precipitated with cold TCA, extracted with
hot TCA, and counted. Liver system was identical with the
above incubation medium,
S100, pH 5, and ribosomes
substituting
the same amounts
were prepared from liver.
of
t Average value, obtained from fifteen separate ribosomal
preparations
(range, 27-60).
Average value, obtainedjrom
preparations
fifteen separate ribosomal
(range, 0.6—5).
§
Average value, obtained from four separate ribosomal
preparations
â€C̃'4-labeled
(range, 4—19).
amino
acids
were
added
to
each
incu
bation tube, and the corresponding
unlabeled
amino acids were deleted from the mixture.
The complete reaction mixtures and the incuba
tion procedure are described in the legend to Table
1. After incubation
the mixtures
were chilled,
and
0.05-ml. samples of each were pipetted in dupli
cate onto 2.3-cm. paper discs, cut from Whatman
#3 MM filter paper. The discs were immediately
immersed in a large volume of cold 5 per cent
TCA (at least 15 ml/disc) and swirled for 10
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1963 American Association for Cancer Research.
@
@
@
T@1
630
.
Cancer
minutes. The discs were washed once more with
cold TCA, then extracted with hot 5 per cent TCA
at 90@C. for 7 minutes, followed by one cold TCA
wash and two ethanol washes to remove the TCA.
An additional ether wash did not affect the final
count, and this step was eliminated from the routine
procedure. The discs were dried with heat, and
the radioactivity
was determined
in a Packard
Tricarb liquid scintillation counter (Model EX).
The paper-disc method outlined is a modification
of the method described by Mans and Novelli
(13). The counting efficiency of the system varied
from 50 to 55 per cent.
The ribonucleic acid of Novikoff ascites and
liver cell fractions was prepared by phenol extrac
tion from the ribosomes and Si®, or from nuclei
isolated from the 15,000X g pellet. An equal vol
TABLE
2
COMPARISON OF AMINO ACID INcoRPoRATION
AUU@O ACID/MG
[email protected]
corporated approximately
40 s@moles of U-Ct4-r.@valine/mg of ribosomal protein. Omission of the
5100, pH 5, fraction from the incubation medium
reduced the valine incorporation to 2—3 @smoles,
indicating that the ribosomal preparations
were
essentially free of the amino acid-@activating corn
ponents. The over-all amino acid activation, as de
termined from the radioactivity of the cold TCA
precipitable material, was in the range of 75—100
,@moles/mg
ribosomal
protein for valine-C'4.
Omission of the energy or energy-regenerating
components greatly reduced the amino acid in
corporation
(Table 1). Ribonuclease
also de
stroyed the capacity of the system to incorporate
valine-C'4. Deletion of the cold amino acids from
the incubation medium caused a 40 per cent de
crease in incorporation similar to that reported by
Matthaei and Nirenberg for their E. coli system
(15). Overnight.dialysis
of the ribosomal and S100,
pH 5, fractions did not reduce this value further,
BY TUMORAND Lxvr@R
in Vifro SYSTEMS
psorzixtTumorLiver+U-C'4.L-valine
0
%O1. 2@, iviay 1963
Research
RIDOSOMAL
iNCUBATION SYSTEMIi,LM0LE$
+U-C―.L-phenylalanine
30
6
+U-C1'-L-lysine
+Above three amino acids43
70
10
1467
26
S Incubation medium and proceduresas described in “Meth.
ods―and in footnotes to Table 1. In above studies mixture of
cold amino acids, exclusive of labeled amino acid(s) was add
ed to system.
t Average of three or more comparable assays.
nine of redistilled phenol (80 per cent) was added
to the tissue fraction suspended in dilute phos
phate buffer, pH 7.0, and the mixture was stirred
at 5°C. for 15 minutes. After centrifugation,
the
aqueous layer was washed twice with 80 per cent
phenol. The final aqueous solution was made to 1
per cent with respect to sodium acetate, and the
RNA was precipitated
by the addition of 2 vol
umes of chilled ethanol. The precipitated
RNA
was redissolved in standard buffer and extracted
5 times with at least 2 volumes of ether each time.
The ether was removed by nitrogen, the RNA con
centration was determined by absorption at 260
mis, and the solutions were frozen and stored at
—15°C.
RESULTS
The characteristics
of the Novikoff ascites tu
mor and rat liver amino acid-incorporating
sys
tems are shown in Table 1. With optimal ratios of
s100, pH 5, to ribosomes, the tumor system in
and, owing to a slight loss of amino acid-incorpo
rating activity, dialysis was generally omitted.
Both the tumor and liver incorporating
systems
were dependent upon Mg+ + for activity.
The liver system, utilizing valine-C'4, incorpo
rated an average of 9.8 @pmoles/mg ribosomal pro
tein. Omission of S100, pH 5, ATP-GTP, or the
energy-regenerating
system reduced this incorpo
ration to low levels. Addition of ribonuclease to the
incubation medium also inhibited incorporation
(Table 1).
Rate studies showed that, for both the tumor
and liver systems, the over-all reaction proceeded
in a linear fashion for 10—iSminutes and reached
equilibrium at 30-40 minutes. Centrifugation
of
these 30-minute incubation mixtures at 105,000X
9 demonstrated that 90 per cent of the radioactiv
ity remains associated •
with the sedimentable,
ribosomal fraction, This evidence suggests that,
whatever the mechanism of polypeptide. release
from the ribosomes may be, it is either not present
or inactive in this system, and that the equilibrium
which is@reached after 40 minutes may be due to
this fact. Lamborg (10) has presented some new
findings recently regarding the release of protein
from ribosoines.
â€ẫ€¢
The S100, pH 5, and ribosomal fractions of tu
mor and liver are interchangeable
to some extent
(Table 1), although further studies will be re
quired to establish whether the transfer factors of
liver will operate with highly purified tumor ribo
somes.
The abilities
of li@rer and tumor
systems
to
incorporate specific amino acids are compared in
Table 2. Tumor ribosomes incorporated 4—Stimes
more valine and phenylalar,ine
than did liver
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1963 American Association for Cancer Research.
O'Na&L
ribosomes,
and
an
even
AND GRIFFIN—Am@nO Acid Incorporathn
higher
proportion
of
lysine.
The effects of exogenous natural RNA and
synthetic
polynucleotides
on the amino acid
incorporating
systems are shown in Table 3. Ad
dition to the tumor incubation systems of RNA
preparations
which may contain template infor
mation has resulted in consistent
increases in
amino acid incorporation,
ranging from @0to 100
per cent. Nuclear and ribosomal RNA prepared
from ascites tumor cells Stimulated the incorpora
631
in Vitro
The addition of polyuridylic acid to the tumor
system markedly stimulated the incorporation of
phenylalanine-C'4,
and as little as 50 @ig.of the
synthetic polynucleotide
resulted in a seven-fold
average increase in the radioactivity
(Table 3). In
some instances, the incorporation was raised from
27 to approximately
350 M@moles phenylalanine
C'4/mg ribosomal protein. The addition of poly
uridylic acid to the chilled systems after incuba
tion was without effect. The specificity of this
synthetic messenger as a template for the forma
TABLE
S
EFFECT OP ADDITION 0@ RIB0NucLEIc
ACID PREPARATIONS AND POLYNUCLEOTIDES
TO TUMOR AND lAYER in Vitro AMINO ACID-INCORPORATING SYSTEMS
PROWSC'4-i-ValineC'4-a-Phenylal&nineC'4-LysineC'4-L-Valine
AMINOACIDmCOIPO*AflONVALUES,pi@iMOL5$/MV
@iPOW@AL
SYSTEM5Avxn.&ox
C'4-Phenylal.
C'4-n-LysineComplete
+Aseites
+Ascites
@
tumor:
nuclear RNA
rib. RNA
47 (+85%)
48 (+23%
+Liver nuclear RNA
22 (—86@'
+Liver nuclear RNAt
+Liver RNA
+TMV.RNA
(50 pg.)
±Polyuridylic acid
46
21
42
37
(100
pg.)
acid
(1004)
(+30
(—38
(+90%
(+ 6%
81 (+
15%)
+Polycytidylie
26 (— 4%)
+Polyinosinic
acid
(100 @Lg.)
22 (— 18%)
Completeliver:
+Ascites
6 .5
7 (+ 8%)
7 (+ 8%)27
nuclear RNA
+TMV-RNA
+Polyuridylic
acid
91 (+45%)129
174 (+34%)
190 (+710%)
6
26 (+430%)63
(100
pg.)85
S Incubation
medium
and
procedure
are
described
in
“Methods―
and
in
footnote
to
Table
1.
In
above studies a mixture of cold amino acids, exclusive of C'4.labeled amino acid(s), was added. Ap.
proximately 600 pg. of RNA preparations were added to incubation mixtures unless otherwise noted.
t Livernuclear
RNwas added
15-20minutes
afterthestartofincubation.
@
tion ofeach
of the three labeled amino acids, and
collectively
the effect
was shown
to be additive.
It is possible, however, that some of the radio
activity attributed
to incorporation
of lysine-C'4
may be caused by other combinations
of this
amino acid through its epsilon amino group (26).
RNA prepared from total liver inhibited amino
acid incorporation
but stimulated
amino acid
activation.
The proportion
of messenger
RNA
in
such a preparation is undoubtedly
low, and con
tamination by proteolytic enzymes or ribonuclease
is a distinct possibility. To a variable extent RNA
added to an incubation in progress caused a greater
amino acid incorporation than was obtained when
the RNA was added at the start of incubation.
tion of a polypeptide composed solely of phenyl
alanine was evidenced by its incapacity to stimu
late the incorporation
of either valine or lysine.
Polycytidylic
acid and polyinosinic
acid were with
out effect on the incorporation
of phenylalanine.
The response of the tumor-phenylalanine
system
to polyuridylic acid was dependent upon ATP
and GTP,
the energy regeneration
system,
Mg+ +, and was inactivated by ribonuclease.
The liver system responded only slightly to the
addition of either TMV-RNA or ascites nuclear
RNA. Polyuridylic acid, however, did stimulate
the incorporation
of phenylalanine-C'4
to some
extent. Further work will be necessary to assess
the' effect of exogenous RNA on liver ribosomes.
Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1963 American Association for Cancer Research.
632
Cancer Research
The tumor
nents
resemble
DISCUSSION
amino acid-incorporating
in many
respects
Studies on the nature of RNA attachment
compo
comparable
sys
tems obtained from E. coli (15, 17), although it has
been impossible thus far to remove the inherent
messenger RNA of the tumor ribosomes. In pre
liminary studies, preincubation
of tumor ribo
somes reduced their amino acid-incorporating
abil
ity by 30 per cent,
a reduction
which
might
mdi
cate the removal of ribosomal messenger RNA.
These preincubated
ribosomes would not respond
to exogenous RNA, although the addition of
polyuridylic
acid and phenylalanine-C14
in a seven-fold
tion
(5). This
did result
increase in amino acid incorpora
level of incorporation
did not
ap
proach that obtained when polyuridylic acid and
labeled phenylalanine
were added to nonpreincu
bated ribosomes.
With this tumor cell system it has been possible
to produce
a small
but
consistent
increase
in
amino acid incorporation by the addition of most
natural RNA preparations.
With an incorporation
of 30—60 @molesof valine, phenylalanine,
or ly
sine per mg. of ribosomal protein, one could pre
dict an over-all incorporation,
based on twenty
amino acids, of the order of 800
@mo1es.The
natural RNA's which have been shown to increase
the incorporation
of individual amino acids by
16—45per cent would raise the theoretical total
incorporation
to as much as 1150 j@moles. The
addition of polyuridylic acid produces a seven-fold
increase in phenylalanine incorporation. However,
if we assume that this polynucleotide
synthesis
of a protein
composed
directs
entirely
the
of
phenylalanine,
a direct comparison
with the
natural RNA's must be made on the basis of the
theoretical total incorporation.
Calculated in this
manner, the polyuridylic acid stimulates amino
acid incorporation by 35 per cent, a figure which
compares favorably with those for the natural
RNA's. It appears, then, that under the conditions
defined here all the exogenous messenger polyribo
nucleotides stimulated amino acid incorporation
to roughly the same extent. Arnstein et a]. (3) have
observed that RNA isolated from rabbit reticu
locyte ribosomes stimulated
amino acid incorpora
tion by 30—SOper cent, which is also in agreement
with our findings. Maxwell (16) has recently re
ported that the addition of liver microsomal RNA
to a liver cell-free system stimulated the incorpo
rationof all amino acids tested. The increased in
corporation
indicated for phenylalanine
ranged
from 30 to 90 per cent. Further extensive investi
gation is thus required with mammalian systems
to produce a messenger RNA stimulation of the
magnitude
reported
for
microbial
Vol. 23, May
ribosomes.
ribosomes,
amino
acid polymerization,
1963
to the
protein
re
lease mechanisms, or concentration
of the 100 S
ribosomes (19) from tumor cells may result in
more active incorporating systems.
It is apparent
from the polyuridylic
acid
phenylalanine
experiments that this mammalian
system behaves like E. coli with respect to the cod
ing unit (UT,IU) reported for this amino acid (18).
Recently, we have noted that polycytidylic acid
will stimulate proline incorporation by the tumor
system. At least one of the coding units for proline
in the tumor system is (CCC), and this same code
is inherent in some microbial systems (4). There is
further indication from Maxwell's investigations
that the RNA coding units are the same for six
amino acids in liver and E. coli systems (16). To
what further extent tumor and other mammalian
ribosomes will correspond to the codes and the
degree of degeneracy in the codes still remains to
be established.
Although we will continue our efforts to produce
tumor ribosomes with a lowered inherent mes
senger RNA, we believe that the present system
may be useful in the study of many factors that
influence protein biosynthesis in mammalian sys
tems. From the observations made thus far it ap
pears that protein synthesis
in the Novikoff
ascites
tumor
cells closely
resembles
that
seen in
other mammalian and microbial systems. Further
comparative studies with regard to chemical and
physical characteristics
of specific-transfer
ribo
nucleic acids, specificity of transfer factor(s), and
mechanisms
of release
should reveal whether
of peptides from ribosomes
basic differences
do exist
between normal
forming systems.
malignant
and
tissue
protein
ACKNOWLEDGMENTS
The authors wish to acknowledge the contribution of Mrs.
Virginia Ward to this study. The research was supported in
part by grants from the American Cancer Society and the
Robert A. Welch Foundation.
REFERENCES
1. ALLFREY, V. G., and MInsKY, A. E. In: R. J. C. HARRiS
(ed.), Protein Biosynthesis, pp. 49-81. New York: Aca
demic Press, 1961.
2. ARNSTEIN, H. R. V.; Cox, R. A.; and Hu@v, J. A. Function
of Polyuridylic Acid and Ribonucleic Acid in Protein Bio
synthesis by Ribosomes from Mammalian
Reticulocytes.
Nature, 194: 1042—44,1962.
3.
. The Effect of Ribosomal Ribonucleic Acid and
Polyuridylic Acid on the Amino Acid Incorporation by
Rabbit-Reticulocyte
Ribosomes.
Proc. Biochem. Soc., pp.
91—92,
1962.
4. BRETSCHER,
M. S., and GRUNBERO-MANAGO,
M. Polyri
bonucleotide-directed
Protein Synthesis Using an E: coli
Cell-Free System. Nature, 195:283—84, 1962.
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O'NEAL
AND GRIFFIN—Amino
5. GRIFFIN,A. C., and O'Nz@u@,
M. A. Effect of Polyuridylic
Acid
Incorporation
17. NATHANS, D.; VON EHRENSTEIN, G.; MONRO, R.; and
Acid upon Incorporation in vitroof C'4 Phenylalanine by
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Evidence for a Messenger Ribonucleic Acid Active in the
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M. R. The in Vitro Release of Protein from
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in Vitro
F.
Protein
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18. NmmrnsmG, M. W., and M@trra&xz,J. H. The Dejend
ence of Cell-Free Protein Synthesis in E. coti upon Natural
ly Occurring or Synthetic Polyribonucleotides
Proc. Natl.
Aced. Sd.,47:1558—1602,
1961.
19.
ILISEBROUGH,
R.
W.;
TISSIEBES,
A.;
and
Messenger-RNA
Attachment
to Active
Nat!. Acad. Sci., 48:430—36, 1962.
WATSON,
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1963 American Association for Cancer Research.
Amino Acid Incorporation by in Vitro Tumor and Liver Systems
and Their Response to Exogenous Ribonucleic Acid
M. A. O'Neal and A. C. Griffin
Cancer Res 1963;23:628-633.
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