[CANCER
RESEARCH
26, 2196-2201, October
1966]
Base Composition of RNA Fractions of Nuclei of
Walker Tumor Isolated with the Citric Acid Procedure1
KEN HIGASHI,
HELEN R. ADAMS,
AND
Department of Phai macology and Tumor By-Products
HARRIS BUSCH
Laboratory, Baylor University College of Medicine, Houston, Texas
Summary
RNA was extracted from tumor nuclei isolated with the citric
acid procedure. The RNA was subfractionated with phenol,
and fractions of differing sedimentation constants were separated
on sucrose density gradients. Each peak was refractionated by
repeated sucrose density gradient centrifugaron until a sym
metric peak was obtained. After phenol fractionation, the interphase or iRNA obtained by extraction at 65°Chad a higher
uptake of orthophosphate-32P and larger amounts of rapidly
sedimenting RNA than the aRNA fraction obtained after ex
traction at room temperature.
The individual peaks of RNA had characteristic base composi
tions. The 6 S peak of the iRNA had a high content of adenylic
and uridylic acids as determined both by optical density and
32Panalyses. It differed from the 6 S peak of the aRNA which
contained more guanylic and cytidylic acid. As has been found
for ribosomal RNA, nuclear 18 S RNA had a higher content of
both adenylic and uridylic acids than 28 S RNA. The 35 S and
45 S fractions of iRNA and aRNA had a high content of guanylic
and cytidylic acids as determined both by ultraviolet and MP
analysis. The finding that the content of adenylic and uridylic
acids of the 35 S and 45 S RNA of tumor nuclei was markedly
lower than that of nuclei of rat liver supports previous studies
which showed marked differences of the base compositions of
newly synthesized rapidly sedimenting nuclear RNA of the
Walker tumor and liver (19).
Introduction
In previous studies from this laboratory (16, 19), marked
differences have been found in the base composition of newly
synthesized RNA in nuclear and nucleolar preparations of the
Walker tumor and normal liver. Although the differences in the
base compositions of the "newly synthesized" RNA were the
greatest differences found, the UV2 base compositions also showed
a preponderance of guanylic and cytidylic acids in the RNA of
the nuclear preparations of the Walker tumor.
1These studies were supported in part by grants from the Amer
ican Cancer Society, the Jane Coffin Childs Fund, the National
Science Foundation and USPHS Cirant ÇA08182.
*The following abbreviations are used: aRNA, aqueous ribonucleic acid; iRNA, interphase ribonucleic acid; TJV,ultraviolet;
SDS, sodium dodecyl sulfate; A, adenylic acid; U, uridylic acid;
G, guanylic acid; C, cytidylic acid; EDTA, disodium ethylenediaminetetraacetate; PCA, perchloric acid.
Received for publication
2196
March 17, 19(if>.
One difficulty associated with preparation of nuclei from transpiantabile tumor tissues by the technic of Chauveau et al. (3)
has been the presence of a perinuclear cytoplasmic halo which wasnot removed when transplantable cells were homogenized in
media containing divalent cations. Accordingly, it was difficult
to interpret the data on UV base composition of the nuclear
RNA of the Walker tumor in view of the uncertainties of the
contribution of the cytoplasmic RNA.
Recently, the citric acid procedure (5) was restudied for isola
tion of nuclei from the Walker tumor on a large scale (9). These
nuclei were found to be satisfactory from both the morphologic
and chemical point of view for isolation of nucleic acids, although
the losses of protein render them of lesser value for studies on
nuclear proteins. Moreover, it was possible to separate the RNA
into 2 fractions of which 1, the interphase or iRNA fraction,
contained considerably more of the rapidly sedimenting RNA
than the other, or aRNA fraction.
The present studies have reaffirmed the previous experiments
that showed the Walker tumor nucleus rapidly synthesizes
RNA which is rich in guanylic and cytidylic acids and unusually
low in adenylic acid by comparison to the normal liver (19).
Along with the results of studies on the nucleoli of tumor cells
(16), these data support the concept that marked differences
exist in the gene readout in the nuclei of Walker tumor cells by
comparison with liver (16) and regenerating liver (17).
Materials
and Methods
ANIMALS.The male rats used in these experiments were ob
tained from the Cheek-Jones Company, Tomball, Texas; they
weighed 180-220 gm and were fed ad libitum on Purina laboratory
chow. The Walker 256 carcinosarcoma was implanted 7-8 days
prior to the experiment. For studies on base composition by
optical density (UV), 50-100 tumor-bearing rats were used, and
10-15 tumor-bearing rats were used for the analysis of radioac
tivity. From 8 to 10 rats were employed for each study on rat
liver. Orthophosphate-32P (carrier-free), 2 me, was injected i.v.,
20 min prior to sacrificing the rats in the tumor experiments and
15 min before sacrificing the rats in the experiments on liver.
The tumors and livers were treated as previously described (9).
PREPARATION
OF NUCLEI
FROM
WALKER
TUMOR
AND LIVER.
Tumor tissues were fragmented with a tissue press (2), weighed,
and suspended in cold 2.5% citric acid (1:9, w/v). Liver tissue
was suspended in cold 5% citric acid (1:9, w/v). The following
procedures were the same for both tumor and liver.
The tissue suspensions were homogenized in a continuous
tissue homogenize!- (1:9, w/v) in the mass scale experiments
CANCER RESEARCH VOL. 26
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RNA
Fractions of Walker Tumor Nuclei
(9) or in tightly fitting Teflon pestle glass homogenizers (2-3 X
10~3inch clearance) in the small scale experiments. The homogenates were centrifugea at 000 X g for 10 min. The sediments were
resuspended in 0.25 M sucrose (5 ml/gm of tissue) containing
1.5% citric acid with a Teflon pestle and glass homogenizer.
The suspensions were layered over 2 volumes of 0.88 M sucrose
containing 1.5% citric acid and centrifuged at 900 X g for 10
min. The supernatant solution was decanted and discarded.
Cytoplasmic fragments and citric acid adhering to the tubes were
carefully removed.
EXTRACTION
OFNUCLEAR
RNA. For extraction of whole nuclear
RXA, isolated nuclei were homogenized in 0.3 7t sodium clodeoyl
sulfate containing 0.14 M NaCl and 0.05 Msodium acetate buffer
(pH 5.0-5.1) and an equal volume of phenol containing 0.1%
8-hydroxyquinoline. This suspension was treated according to
the method previously described (9).
For extraction of aRXA and iRNA,3 isolated nuclei were ho
mogenized in 5.02 M sodium phosphate buffer (pH 6.8) contain
ing 0.18 M NaCl, 3-4 ml/gm of tissue, plus an equal volume of
saturated phenol (12). The RNA extracted at room temperature
was designated as aRXA. The same buffer was added to the
interphase and phenol phase, and the mixture was shaken at
65°C.The RNA extracted with hot phenol was designated as
iRNA (9).
PURIFICATIONOF RNA. The RNA was precipitated with 2
volumes of ethanol containing 2% potassium acetate, collected
by centrifugation, resuspended in 0.05 M sodium acetate buffer
(pH 5.0) (about 2 mg/ml), and passed through a Sephadex
G-25 column (2.2 x 30 cm) previously buffered at pH 5 at 4°C.
Under these conditions, non-nucleotide phosphate-32P deriva
tives (4) were separated from the RNA. The RNA peak was
recovered from the Sephadex column fractions by reprecipitation with 2 volumes of ethanol containing 27¿potassium acetate.
SUCROSE DENSITY GRADIENT SEDIMENTATION STUDIES. For the
mass isolation of the different sedimenting RNA fractions, a
large scale (1750 ml) linear sucrose density gradient (15-40%)
was made at 5000 rpm in a zonal ultracentrifuge rotor (Beckman)
prior to the addition of the sample. The sucrose gradient solu
tions contained 0.1 M NaCl, 0.02 M sodium acetate buffer (pH
5.0-5.1), and 0.1 HIMEOT A. Nuclear RNA (about 50 mg in 30
ml) was pumped in through the center core and the rotor speed
was accelerated to 40,000 rpm (61,000 X g). The speed was
maintained for 15 hr and then decreased to 5000 rpm; the sample
was then pumped out with 50% sucrose. The optical density at
254 m/* was determined with the aid of an ISCO automatic
recorder (Chart 1). Individual peaks (6 S, 18 S, 28 S, 35 S, 45 S,
and 55 S RNA) were pooled separately and purified by repeated
sucrose density gradients until 1 symmetric peak was obtained
(Chart 2). The SW 25.1 rotor was used for these purifications
and for fractionation of nuclear RNA in the small scale experi
ments.
DETERMINATION
OF BASECOMPOSITION.
The purified RNA of
the fractions was hydrolyzed with 0.3 N KOH for 18 hr at 37°C.
3The conditions for extraction of aRNA and iRNA in this
paper were slightly different from those for aRNA and iRNA of
earlier papers (16-18) from this laboratory (9). The pH of the
mixture of phosphate buffer (pH 6.8), phenol, and nuclei isolated
with citric acid was adjusted to 5.0-5.1 (9).
2000
- 1000
T 2000
_l
- 1000
CL
0
10,000
5000
0
5
10
15
TUBE NUMBER
CHAUT1. Sedimentation profiles of nuclear RNA fractions of
tumor and liver. Orthophosphate-32? was injected i.v. 20 min
prior to killing the tumor-bearing rats and 15 min prior to killing
the normal rats. The arrow indicates the direction of the sedi
mentation. The numbers above the peaks indicate the approximate
sedimentation coefficients. Radioactivity was determined as de
scribed previously (9).
The hydrolysate was adjusted to pH 2-3 with 0.5 N PCA at
0°-2°C
temperature and centrifuged. The precipitate was washed
with 0.5 N PCA. The combined supernatant solution was ad
justed to pH 6-7 with 0.5 N KOH and centrifuged. The super
natant fraction was then applied to a Dowex 1-formate column
and the nucleotides were eluted with a linear formic acid gradient
(H2O to 4.5 N formic acid) (10). The fractions were pooled,
desiccated, and dissolved in 0.1 N HC1. Individual optical densi
ties were determined at the wave length of maximum absorbancy
for each nucleotide.
Yeast RNA was added to some 32P-labeled samples as a carrier
and 0.5-1.0 mg of RNA mixture was chromâtographed on a
Dowex 1 column. The distribution of 32P in the nucleotides was
calculated as a percentage. Radioactivity was determined as
previously described (9).
Results
SUCROSE DENSITY GRADIENT SEDIMENTATION.
The
profiles
OÕ
sucrose density gradient sedimentation of nuclear aRNA and
iRNA of tumor are shown in Chart 1.4, B. The iRNA contained
most of the radioactivity, although the aRNA had some radio
activity in the 6 S, 35 S, 45 S, and 55 S regions. Although a small
peak of radioactivity was present in the 6 S region and somewhat
more in the 12-18 S regions, the main peak of radioactivity of
the iRNA was present in the rapidly sedimenting RNA in the
45 S and 55 S regions.
Chart 1C shows the gradient sedimentation profile of nuclear
OCTOBER 1966
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1966 American Association for Cancer Research.
2197
Ken Higashi, Helen R. Adams, and Harris Busch
PURIFICATION
OF RNA FRACTIONS
45SURNA)
2.0 "1st purification
35S (iRNA)
1.0
2nd purification
IO
15
20
25
TUBE
05
IO
15
20
25
NUMBER
CHART2. Sedimentation profiles of RNA fractions during purification stages. The shaded areas were excluded from the subsequent
gradients. The fractions used for analyses were those obtained in the unshaded areas of the second purification. The arrow indicates
the direction of the sedimentation.
TABLE 1
BASE COMPOSITION
OF aRNA ANDiRNA OF TUMORNUCLEI(ULTRAVIOLET
DETERMINATION)
The values for each purine or pyrimidine are averages of percentage of total purine and pyrimidine
bases in the RNA fraction determined by ultraviolet absorption. The standard errors and the number
of experiments are also presented. A, adenylic acid; U, uridylic acid; G, guanylic acid; C, cytidylic
acid. The numbers of the peaks indicate the approximate sedimentation coefficients. The percentage
of aRNA was 31.1% and that of iRNA was 68.9% of total extractadle RNA, respectively (7).
of
experi
ments33322344333334A19.3
+ U)/
(GC)0.770.750.540.000.540.590.080.870.800.500
+
aRNA6S18
S28
S35S45
S55STotal
aRNAiRNAOS18
S28S35
S45
S55
STotal
iRNANo.
0.821.1
±
0.910.8
±
0.118.1
±
010.0±
0.715.7
±
1.018.7
±
0.322.2
±
0.521.9
±
0.718.2
±
0.519.4
±
1.118.0
±
1.221.3
±
0.821.7
±
0.024.3
±
RNA
OF TUMOR
NUCLEI (ULTRAVIOLET
DETERMINATION).
Although the overall
base compositions of the aRNA and iRNA fractions were similar
(Table 1), the base compositions were quite different for the
RNA fractionated by sucrose density gradients. For example,
the 6 S RNA of the iRNA had a higher content of adenylic
acid than the 6 S RNA of the aRNA; the content of guanylic
acid was correspondingly less than that of aRNA. Thus, the
2198
0.923.5
±
1.528.5
±
0.029.1
±
0.827.4
±
0.223.0
±
1.327.4
±
0.723.9
±
0.420.±
1.123.7
±
1.332.9
±
0.522.8
±
±1.117.9
6
0.717.9
±
1.034.8
±
0.329.4
db
0.417.2
±
0.020.2
±
0.735.8
±
0.920.8
±
0.519.7
±
0.510.9
±
0.237.0
±
1.020.4
±
0.818.1
±
0.120.9
±
0.035.0
±
0.325.3
±
0.218.8
±
1.522.1
±
1.330.8
±
0.928.3
±
±0.5G33.8
±0.4U24.2
±0.5c22.7
±0.7(A
RNA of rat liver as extracted with the SDS-phenol procedure.
As found previously for liver nuclei obtained with the sucrosecalcium procedure (19), the radioactivity
peak was located
mainly in the RNA of 45 S and 55 S regions.
BASE COMPOSITION OF 8UBFRACTIONATED
1.133.5
±
2.030.5
±
0.333.4
±
0.337.4
±
0.439.4
±
0.932.2
±
0.429.0
±
ratio (A + U:G + C) of the 6 S RNA of the iRNA fraction was
higher than that of aRNA. A similar difference was found for
the 55 S RNA. For this fraction, the ratios of (A + U) : (G + C)
were slightly higher than those of corresponding 45 S RNA
in both the aRNA and iRNA fractions. However, the 55 S RNA
of the iRNA fraction contained more adenylic acid and less
guanylic acid than the corresponding fraction of the aRNA.
In both aRNA and iRNA, the base compositions of 18 S and
28 S RNA were not significantly different. The 18 S RNA had a
higher content of adenylic and uridylic acids than the 28 S RNA
and correspondingly less guanylic and cytidylic acids. Accord
ingly, the ratios of (A + U):(G + C) for the 18 S fractions of
CANCER
RESEARCH
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VOL. 26
RNA Fractions of Walker Tumor Xuclei
TABLE 2
BASE COMPOSITIONOF aRNA AND iRNA OP TUMOR NUCLEI (32P DETERMINATION)
The values are averages of the percentage of total radioactivity in the RNA fraction that was pres
ent in the individual 2'or 3'mononucleotides.
The values of 2 experiments are also presented in the
parentheses. Each animal received 2 me of orthophosphate-32P i.v. 20 min before it was killed.
+
U)/(G
C
+ )0.740.520.490.900.970.860.690.610.650.
aRNA6S35
S45
SiRNA6S18
S28S35
S45
S55
STotal
16.9)14.2
(15.4,
14.0)13.9
(14.3,
13.9)22.2
(13.8,
25.3)19.0
(25.1,
18.5)19.1
(19.6,
18.8)25.2
(19.4,
30.7)32.4
(34.5,
37.0)34.5
(34.3,
34.0)31.4
(35.0,
20.9)31.2
(25.0,
30.6)32.6
(31.7,
33.3)21.4
(31.8,
23.1)21.9
(21.2,
23.0)20.6
(20.7,
21.2)17.6
(20.0,
17.1)15.3
(18.0,
15.5)16.3
(15.0,
15.3)15.3
(17.3,
25.3)27.5
(25.0,
27.5)25.8
(27.4,
25.8)23.1
(25.7,
23.2)22.8
(23.0,
23.2)23.0
(22.4,
22.5)22.1
(23.5,
30.7)25.5
(32.0,
25.6)28.6
(25.3,
27.8)30.8
(29.3,
30.2)31.9
(31.4,
31.4)32.4
(32.4,
33.7)32.4
(31.0,
20.9)25.2
(21.8,
23.9)25.1
(26.5,
25.1)28.6
(25.1,
29.5)30.1
(27.6,
29.8)28.4
(30.4,
28.6)30.3
(28.2,
SDS-ex-tractableRNA45
S55
SA16.2
22.2)24.0
(22.0,
33.1)30.6
(31.6,
29.7)28.0
(30.8,
14.9)17.5
(15.6,
(31.0, 30.1)C23.0
(23.4, 24.5)G32.6
(28.4, 27.6)(A
(17.2, 17.8)U25.2
TABLE 3
BASE COMPOSITIONOF RAT LIVER NUCLEI (32P DETERMINATION)
The values are averages of the percentage of total radioactivity in the RNA fraction that was present
in the individual 2' or 3' mononucleotides. The values of 2 experiments are also presented in the paren
theses. Each animal received 2 me of orthophosphate-32P i.v. 15 min before it was killed. Liver nuclei
were also isolated with the citric acid procedure.
+
U)/(G
C
+ )1.000.990.980.96
Total
SDS-ex-tractableRNA28
S35
S45
S55
SA26.3
25.7)24.9
(24.6,
25.2)25.2
(24.7,
26.5)25.8
(26.0,
22.6)23.8
(24.7,
26.0)25.7
(23.7,
25.8)25.7
(25.8,
21.8)24.0
(25.8,
25.6)24.8
(24.7,
24.8)26.5
(26.5,
25.0)25.3
(24.5,
26.8)26.0
(24.5,
23.0)22.8
(24.5,
(27.0, 26.0)C24.9
(25.2, 25.3)(A
(24.8, 27.2)U23.7
(23.0, 22.5)G25.2
the a- and iRNA were 0.75 and 0.80, respectively, and the corre
sponding ratios for thé28 S fractions of the a- and iRNA were
0.54 and 0.56, respectively.
32P BASE COMPOSITION OF SUBFRACTIONATED RNA
OF TUMOR
NUCLEI.The newly synthesized RNA was found predominantly
in the iRNA, and because of the lower uptake of isotope in the
aRNA it was not possible to determine the 32Pbase composition
for each peak in the aRNA fraction (Table 2). The newly syn
thesized RNA of the 6 S peak of the iRNA fraction had a higher
adenylic acid content than the 6 S fraction of aRNA. The ratio
of (A + U) : (G + C) in both 6 S and 18 S in iRNA were higher
than those of other fractions. The lowest values for adenylic and
uridylic acids were found in the 45 S peak of the aRNA.
BASE
COMPOSITION
OCTOBER
OF
WHOLE
NUCLEAR
RNA
EXTRACTABLE
WITH SDS-PHENOL.The 45 S and 55 S fractions of the SDSextractable RNA of the citric acid nuclei had base compositions
very similar to those of the iRNA which contains most of the
labeled RNA. The (A + U) : (G + C) ratio was 0.60 for 45 S
RNA and 0.71 for 55 S RNA, respectively. These results agreed
with those of the previous studies on nuclear preparations ob
tained with the sucrose-calcium procedure (19).
BASE COMPOSITION OF RAT LIVER
NUCLEI
ISOLATED
WITH
CITRICACIDPROCEDURE
(32PDETERMINATION).
Since it has been
shown previously (9) that some RNA was lost from nuclei
isolated with the citric acid procedure, it was necessary to de
termine whether AU-rich RNA was lost preferentially. As shown
in Table 3, nuclei of rat liver isolated with the citric acid pro
cedure contain 55 S RNA with a base composition identical with
1966
Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1966 American Association for Cancer Research.
2199
Ken Higashi, Helen R. Adams, and Harris Busch
that found when the nuclei were isolated with the sucrose-cal
cium procedure (19).
Discussion
The differences found in the base composition of 45 S and 55 S
RNA in rat liver and Walker 256 Carcinosarcoma (19) have been
confirmed in these experiments in which improved procedures
were utilized for isolation of nuclei and purification of the
RNA fractions. Isolation of nuclei from Walker tumor with the
citric acid procedure made it possible to remove the cytoplasmic
matrix from tumor nuclei. Improved methods for sucrose density
gradients and larger scale isolations of RXA made it possible to
obtain sufficient RNA for base analysis, after purification of
RNA of each peak by repeated sucrose density gradients.
Sibatani et al. (22) and Kimura (12) showed that 2 metabolically distinct classes of RXA exist in cells by phenol fractionation
of RNA. Their procedure was modified with the use of thermal
fractionation of RNA (16-18) based on the evidence of Georgiev
et al. (6, 7) that an optimal temperature for extraction of rapidly
synthesized RNA was 55°-65°C.
It was found that the overall
UV-base composition was similar for the total aRNA and iRNA
fractions, but the individual sedimentation classes of these
fractions had different UV and 32P base compositions. These
data provide support for the concept that a multiplicity of types
of RNA is present in the whole nuclear RNA and its various
fractions. Clearly, a number of technical improvements are
required for separation of the various molecular species in the
fractions. A number of workers (1, 13-15, 20) have determined
the base composition of ribosomal RNA. In general, those results
have shown that 18 S and 28 S RNA have different base composi
tion and the ratios of (A + U) : (G + C) for 18 S RNA were
higher than those of 28 S RNA. The present report is compatible
with all these results and is in agreement with the hypothesis
that the ribosomal RNA is synthesized on at least 2 distinct
DNA templates in mammalian cells. The similarities of the UV
base compositions of 45 S, 35 S and 28 S RNA support the
hypothesis that the more rapidly sedimenting RNA fractions are
precursors of 28 S RNA (21), but the origin of 18 S RNA is not
yet defined.
The lower content of adenylic and uridylic acids in the rapidly
sedimenting RNA of Walker tumor nuclei may be a relatively
common occurrence in nuclei of neoplastic cells (6, 8, 16, 21, 23).
Since newly synthesized 45 S RNA in nucleoli has a low content
of adenylic acid in both Walker tumor and other neoplastic
cells,4 the low content of adenylic and uridylic acids in nuclear
RNA may reflect the rapid synthesis of the nucleolar RNA in
the tumors. In the nontumor tissues such as liver (19) and kidney5
as well as rapidly growing nontumor tissues such as regenerating
liver (17), the rapidly sedimenting nuclear RNA has a much
higher content of adenylic and uridylic acids than the tumors
4The nucleolar 45 S RNA of Ehrlich ascites tumor also has a
low content of adenylic and uridylic acids and approximates that
of the Walker tumor (T. Nakamura and H. Busch, unpublished).
6 Recently, it was found that the 45 S RNA of nuclei of the kid
ney has a high content of adenylic and uridylic acids by compari
son with that of Walker tumor (S. Schwartz and H. Busch, un
published).
2200
studied. These results may reflect the synthesis of different RNA
species or different percentages of similar types of molecules.
Studies on fractionation of molecular species of RNA by various
types of Chromatographie procedures (11) are in progress in an
effort to differentiate between these alternatives.
Acknowledgments
The authors wish to express their appreciation to L>r.William
J. Steele for his helpful suggestions and to Mr. Charles Taylor
for supplying the transplanted tumors.
References
1. Brown, F., and Martin, J. S. Base Composition of Ribosomal
Ribonucleic Acid Fractions From Five Mammalian TissueCulture Strains. Biochem. J., 97: 20c-22c, 1905.
2. Busch, H., and Desjardins, R. A Continuous Tissue Homogenizer. Exptl. Cell Res., 40: 353-59, 1965.
3. Chauveau, J., Moule, Y., and Rouiller, C. Isolation of Pure
and Unaltered Liver Nuclei. Morphology and Biochemical
Composition. Ibid., 11: 317-21, 1950.
4. Davidson, J. N., and Smellie, R. M. S. Phosphorus Compounds
in the Cell. 3. The Incorporation of Radioactive Phosphorus
into the Ribonucleotide Fraction of Liver Tissue. Biochem.
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5. Dounce, A. L., Witler, R. F., Monty, K. J., Pate, S., and
Cottine, M. A. A Method for Isolating Intact Mitochondria
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Mitochondrial Destruction on the Properties of Cell Nuclei.
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7. Georgiev, G. P., Samarina, O. P., Lerman, M. I., Smirnov,
M. N., and Severtzov, A. N. Biosynthesis of Messenger and
Ribosomal Ribonucleic Acids in the Nucleolochromosomal
Apparatus of Animal Cells. Nature, 200: 1291-94, 1903.
8. Harel, J., Harel, L., Lacour, F., Boer, A., and Imbenotte, J.
Fractions with Differing Base Composition in RNA from
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9. Higashi, K., Shankar, Narayanan K., Adams, H. R., and
Busch, H. Utilization of the Citric Acid Procedure and Zonal
Ultracentrifugation for Mass Isolation of Nuclear RNA from
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Nucleotide Metabolism. II. Chromatographie Separation of
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11. Kidson, C., and Kirby, K. S. Recognition of Altered Patterns
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12. Kimura, K. Fractionation of RNA with Phenol (III). Biochim.
Biophys. Acta, 55: 22-30, 1902.
13. Kirby, K. S. Isolation and Characterization of Ribosomal
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OCTOBER 1966
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2201
Base Composition of RNA Fractions of Nuclei of Walker Tumor
Isolated with the Citric Acid Procedure
Ken Higashi, Helen R. Adams and Harris Busch
Cancer Res 1966;26:2196-2201.
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