tCANCER
RESEARCH
27 Part 1, »49-854,May 1967]
Studies on Nucleolar
Novikoff Tumors1
STEVEN J. SMITH, KEN HIGASHI,
RNA Fractions
of the Walker
and
AND HARRIS BUSCH
Department of Pharmacology, Baylor University College of Medicine, Houston, Texas 77026
SUMMARY
Following separation of various sedimentation classes of
nucleolar RNA on linear sucrose gradients, they were analyzed
for UV and 32Pbase composition. The nucleolus of the Walker
tumor contained mainly 6 S, 28 S, 35 S, 45 S, and 55 S RNA's;
occasionally a small shoulder of 18 S RNA was found. After a
15-min pulse of orthophosphate-32P, most of the radioactivity
api>eared in the 45 S and 55 S regions. Analysis of base composi
tion of newly synthesized nucleolar 45 S RNA of the Walker
tumor by distribution of orthophosphate-82P showed that the
content of adenylic acid was very low (13%) ; this result is similar
to the 32Pbase composition of the nucleolar RNA of the Walker
tumor previously reported (10). By UV base analysis, the 6 S
RNA had a higher content of adenylic and uridylic acid than the
more rapidly sedimenting nucleolar RNA fractions. There were
marked differences between the values obtained for base composi
tion of newly synthesized nucleolar RNA of the Walker tumor
and of normal liver. The 6 S nucleolar RNA of the Walker tumor
also had a significantly different UV base composition from that
of the normal liver.
When labeled nucleolar 45 S RNA of the Novikoff hepatoma
was fractionated by partition chromatography on Sephadex
G-25 using a biphasic organic solvent system, 10 radioactive
fractions were obtained. By analysis for 32Pdistribution in the
nucleotides, it was found that those fractions with the highest
content of adenylic acid were eluted first and those fractions
with the lowest content of adenylic acid were eluted last.
INTRODUCTION
Recent studies from this laboratory have indicated that the
nucleoli of liver cells serve as sites of synthesis of 28 S RNA
(12, 13), which is the RNA component of the 50-60 S ribosomal
nucleoprotein (3, 19). The nucleolar 45 S RNA and 55 S RNA are
apparently the initial products of the synthesis of RNA, and
subsequently, these rapidly sedimenting RNA's are converted
to 28 S RNA. Nucleolar 55 S, 45 S, 35 S, and 28 S RNA (17, 18,
20) have very similar base compositions in normal liver, and all
contain high concentrations of guanylic and cytidylic acids (12).
In addition to these sedimentation classes of RNA, nucleoli of
liver cells contain 6 S RNA, which has a higher content of both
1These studies were supported in part by grants from the
American Cancer Society, the Jane Coffin Childs Fund, the Na
tional Science Foundation, and the TJSPHS (CA 08182).
Received September 12, I960; accepted December 19, 196T>.
adenylic and uridylic acids, i.e., the ratio of A + U/G + C2 is
0.96 (12).
Earlier studies from this laboratory had shown that the 32P
base composition of the nucleolar and nuclear RNA of Walker
tumor differed from that of normal liver (10, 15). The content of
adenylic acid was substantially lower in the 45 S and 55 S nuclear
RNA (15) and nucleolar aRNA and iRNA (10) obtained from
the Walker tumor than in corresponding preparations from
normal liver. Additional evidence for differences in the nucleolar
RNA of Walker tumor and normal liver was obtained by studies
on the nearest neighbor frequencies of newly synthesized nucle
olar RNA (16). Scaled-up procedures permit the isolation of
highly purified nucleolar preparations from tumors in sufficient
amounts for separation of the sedimentation classes of RNA on
a scale sufficient to permit analysis of both UV and 32P base
composition of these RNA fractions. The present study was
designed to determine the sedimentation characteristics of the
newly labeled nucleolar RNA of tumors and to determine the
base comjwsitions of the various sedimentation classes of RNA.
The rapidly labeled nucleolar 45 S RNA of the Walker tumor had
a low adenylic acid content, as was found previously for the whole
nucleolar RNA. In addition, the nucleolar 6 S RNA of the Walker
tumor had a lower content of uridylic acid and a higher content
of guanylic acid than the 6 S nucleolar RNA of normal liver.
Partition chromatography of nucleolar 45 S RNA on Sephadex
columns showed the presence of several RNA subfractions of
varying base composition.
MATERIALS AND METHODS
Animals. Male albino rats obtained from the Cheek-Jones
Company (Houston, Texas), weighing 175-225 gm, were fed ad
libitum on Purina laboratory chow. Both the Walker 256 carcinosarcoma and the Novikoff ascites tumor were transplanted
6-7 days prior to the experiment. In ex[x;riments with radioactive
2The following abbreviations are vised: A + U/G + C, ratio
of the sum of percentages of bases in adenylic and uridylic acids
to the sum of the bases in guanylic and cytidylic acids, or the
ratio of the sum of the percentages of the total 32Pin adenylic and
uridylic acids to the sum of the percentages of the total 32P in
guanylic and cytidylic acids; aRNA (aqueous RNA), RNA re
leased into the aqueous phase by treatment with phosphate buffer
and phenol; iRNA (interphase RNA), RNA that remains in the
interphase or phenol layer after treatment with phosphate buffer
and phenol (10); EDTA, disodium ethylenediamine tetraacetate;
S, sedimentation
coefficient.
MAY 1967
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849
Steven J. Smith, Ken Higashi, and Harris Busch
INITIAL GRADIENT
FOR NUCLEOLAR RNA OF WALKERTUMOR
15,000 z
O
- lo.oooy
(T
U.
- 5,000
E
a.
•O
10
15
20
TUBE NUMBER
25
CHART1. Density gradient sedimentation patterns for Walker tumor nurleolar RNA. The approximate sedimentation coefficients
are shown above the peaks and the shoulders. The distribution of radioactivity after a 15-min pulse of orthophosphate-T is shown as
the dashed line. The arrow indicates the direction of sedimentation.
phosphate, 2 me of orthophosphate-'2P (carrier-free orthophosphate, code P-I, Union Carbide Nuclear Company, Oak Ridge,
Tenn.) were injected via the jugular vein into rats bearing the
Walker tumor and ¡ntra]>eritoneallyto rats bearing the Xovikoff
ascites tumor. Fifteen min later the animals were anesthetized
with diothyl ether, and those bearing the Walker tumor were
exsanguinated by aortic transection. The Walker tumors were
excised quickly and placed in ice-cold 0.25 M sucrose. Hemorrhagic and necrotic tissue was removed from the Walker tumors
in the cold room. The animals bearing the Xovikoff hepatomas
were killed by cervical dislocation; the peritoneal fluid was
drained through a small ajxrture into beakers. The ascites fluid
was centrifuged for 10 min at 10,000 rpm in a Servali refrigerated
centrifuge. The supernatant fraction was discarded, and the
sediment was suspended in 2 M sucrose to separate the tumor
cells from erythrocytes.
Isolation of Nuclei and Nucleoli. The methods used for
isolation of nucleoli of Walker tumor were similar to those used
previously (10-14). In expriment* using orthophosphate-32P,
totals of 30-50 gm of Walker tumor and 60-75 gm of Novikoff
hepatoma were obtained from 15 rats. In the other experiments,
100-150 gm of Walker tumor were obtained from 50 to 70 rats.
Freshly excised Walker tumor was minced and placed in a
stainless steel tissue press to remove connective tissue. The
pressed Walker tumor or the Novikoff ascites was then homoge
nized in 2.0 M sucrose containing 3.3 mM CaCl2 (1:15 w/v) with
a Teflon ]>estle in a glass homogenize!- (0.006-inch pestle clear
ance) after filtration through 4 layers of cheesecloth. The
homogenate was centrifuged at 40,000 X g for 1 hr to sediment
the nuclei (15). The nuclear precipitate was resuspended in
0.25 M sucrose (1 ml/gm original wet weight of tissue) and
sonicated (14) for 60 to 80 sec in a Raytheon sonic oscillator
(1.0-1.1 amp.). Twenty ml of the sonicated suspension were
layered over 20 ml of 0.88 Msucrose and centrifuged at 2000 X g
for 10 min. The purified nucleoli contained very few nuclei or
other contami nan is (14).
Isolation of RNA. Purified nucleoli from Walker tumor were
homogenized in a solution containing 0.3%, sodium dodecylsulfate
(21), 0.14 M NaCl, and 0.05 M sodium acetate at pH 5.1 (2 ml
sodium dodecylsulfate solution/gm tissue). The homogenization
was carried out for 1 min (15 strokes) with a loose-fitting Teflon
pestle. An equal volume of 909¿phenol, containing 0.1% hy-
droxyquinoline (saturated with 0.05 M sodium acetate, pH 5.1),
was then added, and the sample was homogenized again for 1
min. The suspension was incubated while shaking in a water
bath at 65°Cfor 10 min and then was shaken in an "Equiix>se"
shaker at room terni>erature (25°C)for 20 min (15, 21). After
the mixture was centrifuged at 17,000 X g for 10 min in a Servali
centrifuge, the aqueous phase was removed and fresh phenol
(I volume) was added. After shaking for 10 min, the layers were
again separated by centrifugation at 17,000 X g for 10 min.
Fresh phenol was added a 2nd time to the separated aqueous
pha^e; after shaking for 5 min and centrifugation as described
above, the aqueous layer was removed and the RNA was pre
cipitated overnight at —20°C
with 2.0-2.5 volumes of ethanol
containing 2% potassium acetate (21). The precipitated RNA
was washed once with 75% ethanol. The RNA was then dissolved
and stored at -20°C in 2.0-4.0 ml of 0.01 M sodium acetate
buffer, pHS.l.
Sucrose Density Gradient Centrifugation.
For sucrose
density gradient centrifugation, a volume of 0.5 ml to 1.0 ml of
sodium acetate buffer containing 1-2 mg of RNA was layered
over 26.5 ml of a 10-40% linear gradient of sucrose solution
containing 0.1 MNaCl, 1.0 mMKDTA, and 0.01 Msodium acetate,
pH 5.1 (17).
For experiments on 32P-labeled nucleolar RNA, it was necessary
to purify the RNA with Sephadex G-25 to remove contaminating
phosphate before the preparation of the gradients (12). The
gradient was centrifuged in a Spinco SW 25.1 rotor at 25,000
rpm for 16 hr at 5°C.Fractionation of the gradient was carried
out with the aid of an ISCO automatic fractionator (obtained
from Instrument Specialties Co., Lincoln, Neb.) (1, 21).
For purification of the individual sedimentation classes of
nucleolar RNA (Chart 1), fractions from 3 to 5 initial gradient
runs were i»oledand fractionated in a linear gradient of 10-40%
sucrose; all conditions for repurification of peaks were the same
as described above for the initial gradient runs. Fractions under
each optical density and radioactivity jjeak (Chart 1) were
pooled separately, and the RNA was precipitated with 2.0 to
2.5 volumes of ethanol containing 2% potassium acetate. As
described earlier, the precipitate of RNA was routinely washed
with 75% ethanol and stored in 0.01 M sodium acetate, pH 5.1,
at -20°C.
Base Analyses. RNA fractions were hydrolyzed in 0.3 N
CANCER RESEARCH VOL. 27
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Nucleolar RNA Tumor Fractions
PURIFICATION
OF 45S
NUCLEOLAR RNA OF WALKER TUMOR
NUCLEOLAR
28 ** «
RNA
2.0
1.0
in
CM
I
phases were separated. The ratio of upj>er (organic) to lower
(aqueous) phase was 1:1. The optimal temjierature range for
use of the Kirby solvent system was 20-23°C.Below 19CC the
> 2.0 List PURIFICATION
phases became miscible in each other, forming 1 phase.
Partition Chromatography on Sephadex. Following the
method of Muench and Berg (9), 200 gm of Sephadex G-25 fine
beads were allowed to swell in 1 liter of saturated aqueous phase
for 48 hr at 19-22°C.The mixture was then carefully ]X)ured
into a jacketed column (180-cm height x 2.5-cm diameter), which
was maintained at 21°Cby a Forma bath (Forma Co., Marietta,
ä "O
O
r-
a.
O
40 volumes of an organic phase (upper phase) and 52 volumes
of an aqueous phase (lower phase). The organic phase contained
28 volumes of an organic mixture (tertiary amyl alcohol, 5 parts;
butyl Cellosolve, 4 parts; methyl Cellosolve, 1 part) and 12
volumes of an amine solution (redistilled tripentylamine, 6.0 ml;
glacial acetic acid, 1.08 ml; the above organic mixture, 100 ml).
The aqueous phase contained 10 volumes of 0.033 M trilithium
citrate and 42 volumes of distilled water.
The phases were mixed and shaken together in a separatory
funnel and allowed to equilibrate at 21°C;after saturation, the
2.0 L 2nd PURIFICATION
1.0
Ohio). The hydrostatic head was kept below 20 cm during pack
ing of the column. Approximately 1 bed volume of the organic
phase (contained in a jacketed reservoir maintained at 21°C)was
2.0 .3rd PURIFICATION
passed through the newly constructed column under hydrostatic
pressure. The flow rate was about 11 ml |>er hr.
Application of Sample. One mg of nucleolar 45 S RNA
containing 160,000 dpm was dissolved in 1 ml of distilled water
and mixed with 5 ml of organic phase and directly applied to the
top of the column bed. The sample was washed into the column
bed with two 10-ml volumes of up])er phase. The elution was
begun with the upper phase flow rate maintained at 10 ml per hr.
Fraction volumes of 10 ml were collected. One-mi aliquote
were taken for counting of radioactivity as described previously.
The fractions were then [looled and each ¡»oled
fraction made
2% with respect to jxjtassium acetate to separate the organic
and aqueous phases. The upper phase containing the organic
solvents was discarded, and the lower aqueous phase containing
the RNA was precipitated overnight with 2 volumes of 95%
ethanol. Purified yeast RNA was used as a coprecipitant, and
the RNA was sedimented at 2000 X g for 20 min in an Inter
national refrigerated centrifuge. It was then dissolved in 1 to 2
ml of 0.02 M sodium acetate, pH 5.1. Aliquots of each pooled
fraction were taken for (a) hydrolysis of the RNA in 1 ml of
0.3 N KOH for 18 hr and subsequent chromatography on Dowex
1-formate for analysis of the nP distribution of nucleotides and
(6) sedimentation analysis of the RNA on a 10-40% linear
sucrose density gradient.
1.0
0
5
10
15
20
25
TUBE NUMBER
CH.UÃŒT
2. Purification of 45 S nucleolar RNA on sucrose density
gradient as described in text. The shadowed portion was precipita
ted with 2 volumes of ethanol containing 2% potassium acetate
and rerun a 2nd and 3rd time on a 10—10%
linear sucrose density
gradient. The other sedimentation classes of nucleolar RNA were
purified in the same manner. The arrow indicates the direction of
sedimentation.
KOH at 37°Cfor 18 hr (4). The hydrolysate was adjusted to
pH 3-4 with 5.0 N perchloric acid in the cold and centrifugea.
The supernatant solution was adjusted to pH 6-7 with 0.5 N
KOH and centrifuged. The su]>ernatant solution was chromatographed on a Dowex 1 formate column for analysis of the nucleotides (6). For 32Panalysis of nucleotides, yeast RNA hydrolysate
was added as a carrier to the purified peaks. After desiccation,
the nucleotides were dissolved in 0.1 N HC1 (11). An aliquot was
taken from each nuoleotide fraction for analysis of optical
density and for the determination of radioactivity.
Determination of Radioactivity. To each 1.0-ml fraction
of sucrose density gradient, 0.05 ml of 5.0 N perchloric acid was
added, and the sample was hydrolyzed at 70°Cfor 15 min. To
these samples and also to each nucleotide solution obtained by
column chromatography, 8.0 ml of fluor-containing solution were
added (2). The radioactivity was determined in an automatic
liquid scintillation spectrometer (Packard Tri-Carb Series 3000).
Partition Chromatography on Sephadex G-25 Using a
Biphasic Solvent System. The solvent system employed in
these studies was developed by Kirby et al. (8). It consisted of
RESULTS
Sedimentation
Profiles. Four major peaks, indicated in
Chart 1 as 6 S, 28 S, 35 S, and 45 S, were found after the initial
centrifugation of nucleolar RNA of either the Walker or the
Novikoff hepatoma ascites tumor in a 10-40% linear sucrose
gradient. A small 18 S peak or shoulder was occasionally but
not always present. A shoulder of 55 S RNA was found adjacent
to the 45 S peak. Planimetrie analysis showed that the 55 S,
45 S, 35 S, 28 S, and 6 S peaks contained 16, 28, 23, 20, and 6%
of the total nucleolar RNA, res]>ectively. As is shown in Chart
1, most of the radioactivity was recovered in the 45 S peak and
MAY 1967
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851
Steven J. Smith, Ken Higashi, and Harris Busch
TABLE l
Base Composition of Subfractions
Adenylic acid (A)
Uridylic acid (U)
Guanylic acid (G)
Cytidylic acid
(C)A
of Nucleolar RNA of Walker Tumor Determined by Ultraviolet
Absorption"
15]19.4
S
[3]16.0S
[2]15.4 S
[2]14.6 S
12]15.3 S
±0.4
22.4 ±0.6
33.8 ±0.1
24.40.40.7228
±
±0.4
19.0 ±0.7
35.3 ±0.8
29.60.20.5435
±
±0.7
19.0 ±0.6
35.0 ±0.0
30.60.20.5245
±
±0.5
20.5 ±0.6
35.1 ±0.2
29.70.00.5455
±
±1.0
19.2 ±0.6
36.7 ±1.3
28.80.90.53
±
+ U/G + C6
" The values for each purine or pyrimidine are averages of the percentage of total purine and pyrimidine bases in the UNA fraction determined by ultraviolet absorption at the specific wave length for
each nucleotide. The number of experiments is presented in brackets for each class of nucleolar RNA.
Where 2 experiments were carried out, the range of variation for the 2 values is indicated. Where 3 or
more experiments were carried out, the standard errors are indicated as calculated from the equation:
S.E. = VZxVnCn - 1)
TABLE 2
Distribution
of "P in Nucleotides of Early-Labeled Nucleolar RNA of the Walker Tumor with
Different Sedimentation Constants"
Adenylic acid (A)
Uridylic acid (U)
Guanylic acid (G)
Cytidylic
(C)A
acid
[3]12.5 S
[3]13.8 S
[4]13.2 S
[4]12.7 S
±0.1
24.7 ±0.7
32.1 ±0.9
30.80.70.5935
±
±0.3
20.1 ±0.3
34.6 ±0.2
31.60.20.5145
±
±0.1
20.6 ±0.6
34.4 ±0.2
31.70.50.5155
±
±0.4
20.5 ±0.4
34.7 ±0.6
31.90.20.50
±
+ U/G + C28
" The values are averages of the percentage of total radioactivity in the RNA fraction that was
present in the individual 2', (3')-mononucleotides. The standard errors were determined as indicated for
Table 1. Each animal received 2 me of 32P-labeled orthophosphate 15 min before it was sacrificed. The
number of experiments is shown in brackets. For the 6 S RNA, a 32Panalysis of nucleotides was not pos
sible since, as shown in Chart 1, insufficient radioactivity was present in this class of RNA for statis
tically valid determination of radioactivity.
the 55 S shoulder after a 15-min pulse of orthophosphate-32P.
of uridylic acid and a lower content of guanylic acid than the
other factions of newly synthesized RNA. Unlike the results
RNA from the 28 S, 35 S, and 45 S [leaks in the initial gradient
was rerun repeatedly on 10-^090 linear sucrose gradients for obtained for liver nucleoli, the nucleotide composition of these
32P-labeled fractions of nucleolar RNA was similar to the UV
purification, as shown in Chart 2 for 45 S RNA.
analyses of these fractions. The A + U/G + C ratio for distribu
Base Composition Determined by Ultraviolet Analysis
of Nucleotides. The base compositions of the RNA in the tion of 32Pin the nucleotides was approximately the same as the
individual sedimentation peaks are shown in Table 1. The RNA ratio of total bases as determined by ultraviolet absorption
in the 28 S, 35 S, 45 S, and 55 S regions had essentially the same studies.
Fractionation of Nucleolar 45 S RNA. Chart 3 shows the
nucleotide composition. Each of these rapidly sedimenting RNA
fractions had a high content of guanylic and cytidylic acids and fractionation pattern of 32P-labeled nucleolar 45 S RNA of the
a very low content of adenylic acid. The RNA in the 6 S [>eak Novikoff hepatoma after partition chromatography on Sephadex
G-25 using the Kirby solvent system.3 The 10 fractions were
had a higher content of adenylic acid than the more rapidly
analyzed for 32Pbase composition (Table 3). The fractions with
sedimenting RNA and the A + U/G + C ratio was significantly
greater than those of other fractions. In view of the very small the highest content of adenylic acid (Table 3) were eluted first
amount of RNA recovered, the nucleotide composition of the and those with the lowest content of adenylic acid were eluted
last in agreement with the findings for mobility in counter18 S region was not determined.
Distribution of 32Pin Nucleotides of Early Labeled RNA. current distribution systems (8). Approximately 50% of the total
Table 2 shows the percentages of 32Precovered in the nucleotides
RNA recovered in the j)eaks was eluted in Fractions 8-10, in
of fractions of nucleolar RNA of the Walker tumor. The nucleo
tide composition of the newly synthesized RNA in the 35 S, 45
3Countercurrent distribution using the Kirby solvent system
S, and 55 S peaks was the same after a 15-min pulse. However,
was also attempted, but poor resolution of peaks was obtained by
newly synthesized RNA in the 28 S region had a higher content
this method.
CANCER RESEARCH VOL. 27
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Nucleolar RNA Tumor Fractions
45S NUCLEOLAR RNA OF THE NOVIKOFF HEPATOMA!
PARTITION CHROMATOGRAPHY ON SEPHADEX G-25
USING THE KIRBY SOLVENT SYSTEM
^2000
§1500
^1000
* 500
150
200
250
300
350
TUBE NUMBER
CHART3. A representative chromatogram following partition chromatography of 32P-labeled nucleolar 45 S RNA of Novikoff tumor
using the Kirby solvent system. UNA containing 100,000dpm was applied directly to the column (180cm in height; 2.5 cm in diameter)
in a volume of 5 ml. The Sephadex G-25 was swelled in lower phase for 48 hr and then the column was washed with a bed volume of
upper phase. After the sample was added, the column was developed with upper phase. A quantitative recovery of radioactivity was
obtained. RNA from the fractions was precipitated with ethanol as described in the text and analyzed for the 32Pbase composition
and sedimentation characteristics (Table 3).
100
which the A -f U/G -f C ratio was approximately 0.48. The
RNA recovered from these fractions was sedimented in sucrose
density gradients, and the radioactivity was found in the 35-45 S
region.4 The radioactivity of the RNA eluted earlier was found
to sediment in the 10-28 S regions. The recovery of radioactivity
was 96-100%.
DISCUSSION
A number of previous studies have shown that the nucleus of
tumor cells contains RNA that sediments rapidly in sucrose
density gradients and is rapidly labeled with radioactive RNA
precursors. This rapidly sedimenting, rapidly labeled RNA has
been found in other mammalian tissues (5, 7, 11, 15, 20), and the
base composition of the RNA has been found to be different in
both normal and regenerating liver from that of the nuclear RNA
of the Walker tumor (11, 15, 22). In nucleoli of neoplastic cells,
the rates of biosynthesis of RNA are rapid by comparison with
normal liver but are only twice those of regenerating liver (7).
However, the biosynthetic activity of the neoplastic cells for
RNA is directed to a greater extent to the synthesis of nucleolar
RNA than in other tissues (10). The question raised by these
earlier studies was whether the nucleoli of tumor cells were
producing different types of RNA from those produced in nucleoli
of other cells or whether the RNA produced differed in quantita
tive composition of a number of subspecies of nucleolar RNA.
The present study shows that newly synthesized nucleolar
RNA sediments with the 45 S to 55 S peaks, as was found for
the liver. In addition, the 32Pbase composition of the nucleolar
45 S RNA of the Walker tumor was virtually identical with that
of the whole nucleolar RNA or the aRNA and iRNA subfractions
(10). As shown in earlier studies (7, 13), this RNA is the pre
cursor of 28 S nucleolar RNA. Unlike the values obtained in
studies on the corresponding RNA of the liver, the KP base
composition and UV base composition of the nucleolar 45 S RNA
of the Walker tumor were very similar and the values for adenylic
acid content were low by either determination.
Studies in progress in this laboratory (T. Nakamura and H.
Busch, unpublished data) on the nucleoli of a variety of tumors
have indicated that the results obtained with the Walker tumor
are general for the transplantable tumors studied. It seems
possible that the RNA fractions richer in adenylic acid that are
synthesized in liver nucleoli are related to the specialized func
tions in the activity of the liver cells, but this possibility remains
to be tested. As pointed out earlier (11), the differences in the
base composition of the newly synthesized nucleolar RNA are
TABLE 3
Å“PBase Composition of RNA Fractions Obtained by
Chromatography of Nucleolar 45 S of the Novikoff
Hepatoma (Chart 3)°
acid
acid
acid
acid
Fraction
No.12345678910Adenylic
(A)18.214.914.913.013.411.814.010.711.19.5Uridylic
(U)22.022.522.520.422.520.820.920.221.621.5Guanylic
(G)31.834.031.633.632.034.432.734.734.334.9Cytidylic
(C)28.028.530.933.032.133.032.433.333.034.1
4 Although the original RNA sedimented in the 45 S region, the
only fractions recovered from partition chromatography on long
columns (Chart 3) that sedimented rapidly were in Fractions 6, 7,
and 8-10. It is not yet clear whether the 45 S RNA is undergoing
degradation on the column or in the collecting system or if it con
sists of a number of tightly aggregated subspecies of RNA. In
recent studies, short columns (30 X 0.9 cm) with rapid flow rates
have been employed. From these columns, 2 large and 1 small
peaks of radioactivity were eluted in positions analogous to those
of Fractions 2-4, 6-7, and 8-10. The RNA in the large peaks cor
responding to 2-4 and 8-10 in Chart 3 both sedimented in the 45 S
region. The RNA in the peak corresponding to Fractions 8-10 in
Chart 3 composed 60-85% of the total recovered.
C0.670.600.600.500.560.48
+
0 The results are average values for 2 experiments.
MAY 1967
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803
Steven J. Smith, Ken Higashi, and Harris Busch
not due to growth alone, since the base composition of the
corresponding RNA's from regenerating liver nucleoli were
essentially the same as those found in normal liver nucleoli. In
studies in this laboratory (T. S. Ro and H. Busch, unpublished
data) on the nearest neighbor frequencies of the newly syn
thesized nucleolar RNA of regenerating liver, no significant
differences have been found from the results obtained with the
normal liver.
It has not been fully established whether 45 S RNA is a single
molecular species or consists of 2 or more species of RNA.
Initial attempts to fractionate nucleolar 45 S RNA were made
with countercurrent distribution and the Kirby solvent system
(8) as well as other systems, but there was poor resolution of
components. In the present studies with partition chromatography (9), in which the Kirby solvent system (8) was used, the
fractionation of the nucleolar 45 S RNA seemed to occur partially
on the basis of A + U/G + C ratios of the 45 S RNA. Other
factors may be involved, such as absorption, charge density, and
distribution of like groups of the 45 S RNA on the surface of the
Sephadex. Although some fractionation of 45 S nucleolar RNA
has been achieved, characterization of the fractions and sequen
tial analysis of nucleotides will be required to determine the
differences between nucleolar 45 S RNA of the tumors and other
tissues.
Thus far, the function of the nucleolar 6 S RNA has not been
defined, although it is clearly not soluble in the medium used
for preparation of the nucleoli. In the normal liver, the 6 S
nucleolar RNA contained more uridylic acid (28.6%) and less
guanylic acid (28.990) than that of the Walker tumor. As a
result, the A + U/G + C ratio was 0.96 by comparison with
0.72 for the Walker tumor. Both of these values are higher than
the value of 0.60 obtained for the transfer RNA's.
ACKNOWLEDGMENTS
The authors wish to acknowledge the excellent technical
assistance of Mrs. Helen Adams and their appreciation to Mrs.
Rose K. Busch for transplantation of the tumors.
REFERENCES
1. Brakke, M. K. Photometric Scanning of Centrifuged Density
Gradient Columns. Anal. Biochem., 5: 271-283, 1963.
2. Bruno, G. A., and Christian, J. E. Determination of carbon14 in Aqueous Bicarbonate Solutions by Liquid Scintillation
Counting Techniques. Application to Biological Fluids. Anal.
Chem., S3: 1216-1218, 1961.
3. Busch, H., Byvoet, P., and Smetana, K. The Nucleolus of
the Cancer Cell: A Review. Cancer Res., 23: 313-339, 1963.
4. Davidson, J. N., and Smellie, R. M. S. Phosphorous Com
pounds in the Cell. II. The Separation by lonophoresis on
Paper of the Constituent
Nucleotides of Ribonucleic Acid.
Biochem. J., 52: 594-599, 1952.
854
5. Hiatt, H. H. A Rapidly Labeled RNA in Rat Liver Nuclei.
J. Mol. Biol., 5: 217-229, 1962.
6. Huribert, R. B., Schmitz, H., Brumm, A. F., and Potter,
V. R. Nucleotide Metabolism. II. Chromatographie
Separa
tion of Acid-Soluble Nucleotides. J. Biol. Chem., 209: 23-39,
1954.
7. Jacob, S. T., Steele, W. J., and Busch, H. Turnover of 45 S
UNA of Regenerating Liver and the Walker Tumor. Cancer
Res., 27: 52-60, 1967.
8. Kirby, K. S., Hastings, J. H. B., and O'Sullivan, M. A. Coun
tercurrent Distribution
of Ribonucleic Acids. IV. Biochim.
Biophys. Acta, 61: 978-979, 1962.
9. Muench, K. H., and Berg, P. Fractionation
of Transfer
Ribonucleic Acid by Gradient Partition Chromatography
on
Sephadex Columns. Biochemistry, S: 970-981, 1966.
10. Muramatsu, M., and Busch, H. Studies on Nucleolar RNA of
the Walker 256 Carcinosarcoma
and the Liver of the Rat.
Cancer Res., 24: 1028-1034, 1964.
11. Muramatsu, M., and Busch, H. Studies on the Nuclear and
Nucleolar Ribonucleic Acid of Regenerating Rat Liver. J.
Biol. Chem., 240: 3960-3966, 1965.
12. Muramatsu, M., Hodnett, J. L., and Busch, II. Base Composi
tion of Fractions of Nuclear and Nucleolar UNA Obtained by
Sedimentation
and Chromatography.
J. Biol. Chem., 241:
1544-1550, 1966.
13. Muramatsu, M., Hodnett, J. L., Steele, W. J., and Busch, H.
Synthesis of 28S RNA in the Nucleolus. Biochim. Biophys.
Acta, 123: 116-125, 1966.
14. Muramatsu, M., Smetana, K., and Busch, H. Quantitative
Aspects of Isolation of Nucleoli of the Walker Carcinosarcoma
and Liver of the Rat. Cancer Res., 23: 510-518, 1963.
15. Okamura, N., and Busch, H. Base Composition of High
Molecular Weight Nuclear RNA of Walker Tumor and Liver
of the Rat. Cancer Res., 25: 693-697, 1965.
16- Ro, T. S., and Busch H. In Vitro Labeling of RNA in Isolated
Nucleoli of the Walker Tumor and Liver. Cancer Res., 24:
1630-1633, 1964.
17. Scherrer, K., and Darnell, J. E. Sedimentation Characteristics
of Rapidly Labeled RNA from IleLa Cells. Biochem. Biophys.
Res. Commun., 7: 486-490, 1962.
18. Scherrer, K., Latham, H., and Darnell, J. E. Demonstration
of an Unstable UNA and of a Precursor to Ribosomal RNA in
HeLa Cells. Proc. Nati. Acad. Sei. U. S., 49: 240-248, 1963.
19- Sirlin, J. L. The Nucleolus. Progr. Biophys. Biophys. Chem.,
12: 25-66, 1962.
20- Sporn, M. B., and Dingman, W. The Fractionation
and
Characterization
of Nuclear Ribonucleic Acid from Rat Liver.
Biochim. Biophys. Acta, 68: 389-400, 1963.
21. Steele, W. J., Okamura, N., and Busch, H. Effects of Thioacetamide on the Composition and Biosynthesis of Nuclear
and Nucleolar RNA. J. Biol. Chem., 240: 1742-1749, 1965.
22. Whitcutt, J. M., and Roth, J. S. Hibonucleic Acid Synthesis
and Turnover in Rat Liver and in a Rapidly Growing Transplantable Hepatoma. Biochim. Biophys. Acta, 87: 380-387,
1964.
CANCER RESEARCH VOL. 27
Downloaded from cancerres.aacrjournals.org on June 14, 2017. © 1967 American Association for Cancer Research.
Studies on Nucleolar RNA Fractions of the Walker and Novikoff
Tumors
Steven J. Smith, Ken Higashi and Harris Busch
Cancer Res 1967;27:849-854.
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