[CANCER RESEARCH 33,3312-3318, December 1973]
Similarity of Ribosomal and Ribosomal Precursor RNA's from Rat
Liver and the Novikoff Ascites Hepatoma1
Thomas O. Sitz, Ross N. Nazar, William H. Spohn, and Harris Busch
Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77025
SUMMARY
The 32P-labeled ribosomal
RNA's and their precursors
in
rat liver and the Novikoff hepatoma have been examined by
two competitive hybridization techniques and by nucleotide
analyses. Significant differences were not found between the
corresponding
RNA of these tissues by competition
of
labeled RNA from the hepatoma with unlabeled 18 and 28
S ribosomal RNA and 28 and 45 S nucleolar RNA of liver
and hepatoma by sequential or simultaneous competition
hybridization. Differences were found in the specific activity
of 32P-labeled nucleotides, but no significant differences
were found by ultraviolet light absorbancy in the distribu
tion of short oligonucleotides after digestion with pancreatic
ribonuclease. These data provide evidence that the previ
ously reported differences in nucleotide composition and
oligonucleotide
distribution
were probably the result of
nonuniform labeling rather than differences in the primary
sequences. Accordingly, the primary structures of ribo
somal RNA's and their precursors appear to be conserved
in oncogenesis.
INTRODUCTION
A number of reports from this and other laboratories
suggested that there are significant differences, in tumors
and other cells (6, 13, 18, 31, 32), between the primary
structures of rRNA's and their precursors. 32P-Labeled
nucleolar precursor RNA's in several transplantable tumors
were found to contain less adenylic and more cytidylic acid
than does normal or regenerating rat liver (6). This was also
true for a primary hepatoma induced by 3'-methyl-4-dimethylaminoazobenzene
(18) and, although considerably
less pronounced, for 28 S rRNA of Novikoff hepatoma and
normal liver (30). In contrast, similar nucleotide composi
tion studies in which UV absorbance (6) or analysis of
nucleoside derivatives (22) were used have shown no sig
nificant differences.
Further analyses of the distribution of short, 32P-labeled
oligonucleotides
after complete pancreatic or T, RNase
digestion have also indicated sequential differences in these
RNA species. The differences were most pronounced in the
dinucleotides
of nucleolar precursors, where the G-Up
content was about 50% higher in the tumors (32); similar
1These studies were supported in part by USPHS Grants CA-10893 and
CA-5154.
Received July 24, 1973; accepted September 18, 1973.
3312
differences were also found during 2 other studies ( 13, 27) of
28 S rRNA. A 3rd study on fragments released by partial T!
RNase hydrolysis (30) indicated there were also significant
differences in the content of U-Gp and C-Gp dinucleotides.
When simultaneous
hybridization
competition
of 28 S
rRNA from the Novikoff hepatoma and normal liver was
performed, an approximate
10% difference was noted that
was not found for the corresponding
18 S rRNA's (31).
These data suggested that some sequences in hepatoma 28 S
rRNA were not present in normal liver.
In contrast, analyses of polypurines (1) and the longer
polypyrimidines (23) after complete pancreatic or combined
U2 and T! RNase digestions did not reveal differences in the
primary sequences of 28 S rRNA from normal and
malignant tissues. In addition, 28 S rRNA-32P from HeLa
cells was competed equally well by simultaneous competi
tive hybridization with unlabeled HeLa 28 S rRNA and
RNA from various normal human tissues (1).
To resolve these conflicting reports, in this study 32Plabeled rRNA's and ribosomal precursor RNA's were
competed with unlabeled RNA from normal liver and
hepatoma cells. Both simultaneous and sequential competi
tive hybridizations
were used to preclude artifacts that
might result from the use of a single method. The effects of
32P labeling also were examined in structural comparisons
between normal and tumor tissues.
MATERIALS
AND METHODS
Labeling and Preparation of rRNA. Novikoff hepatoma
ascites cells were maintained in male Holtzman rats for 6
days and then labeled in vitro for 18 hr with 32Pi (Union
Carbide, Tuxedo, N. Y.) essentially as described by Mauritzen et al. (19). At the end of the incubation or when unla
beled RNA was prepared, the cells were pelleted by centrifugation at 7000 x g for 10 min at 0°.
Normal rat livers were labeled by a single i.p. injection of
32P, into fasted Holtzman rats 24 hr prior to sacrifice. The
rats were killed by decapitation and bled; the livers were
removed, minced, and washed directly in cold 0.25 M
sucrose (27).
Cells were homogenized and polysomes were isolated at
0-4° essentially as described by Rich (25). Ribosomal
subunits were prepared by homogenization
in 8 mM EDTA:5 mM Tris (pH 7.5) and
10 to 40% linear sucrose gradients in 10
(Spinco SW-27 rotor for 18 hr at 25,000
of the polysomes
centrifugation on
mM Tris (pH 7.5)
rpm). The RNA
CANCER RESEARCH
VOL. 33
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
Ribosomal and Precursor RNA's
was extracted by a SDS2:phenol method at 20°or 65°(28) fore use. The filters were uniformly loaded with DNA (10
to 15fig/0.6-cm disc).
and purified on 5 to 40% sucrose gradients (12, 24).
Labeling and Preparation of Nucleolar RNA. Cells were
DNA Content of the Filters. The DNA content of the
prepared and labeled for 6 hr as described above. Nuclei filters was analyzed by the diphenylamine method (4) as
were prepared by methods described by Chauveau et al. (8), modified for DNA bound to filters by Meijs and Schilperoot
and the nucleoli were isolated after sonic treatment (5). (21). Highly polymerized Novikoff hepatoma nuclear and
RNA was extracted by the SDS: phenol method (28) at 65° calf thymus DNA (Worthington Biochemical Corp., Free
and purified by repeated sucrose density gradient centrifu- hold, N. J.) were used as standards.
gation in a SW-27 rotor at 26,000 rpm for 16 hr (12, 24).
RNA-DNA Hybridization. RNA-DNA hybridization was
The RNA species were precipitated with 2% potassium carried out essentially by the procedure of Gillespie and
acetate in ethanol and desalted with ethanol at -20°.
Spiegelman (9), with the use of formamide to lower the
Isolation of DNA. DNA was isolated by means of a temperature of hybridization (2, 10, 20). Experiments were
modified Marmur (17) procedure, with extensive digestion performed in 30% formamide:70% (2x SSC; 5 mM morwith nuclease-free Pronase (Calbiochem, San Diego, Calif.) pholinopropane sulfonic acid, pH 7.0) in a volume of 0.3 ml
in the initial stage of the isolation. The pellet of nucleoli or at 45°.3RNA samples were adjusted to the appropriate
nuclei was suspended in about 100 volumes of 1 x SSC2 concentrations and percentage formamide, heated at 70°for
(0.15 M NaCl:0.015 M sodium citrate):0.1 M EDTA, pH 5 min, and then rapidly chilled (24). Then the reactants were
8.0, at 4°,and nuclease-free Pronase (predigested at 2 to 5 allowed to reach 45°and the DNA and blank filters were
mg/ml at 37°for 0.5 hr) was added to a concentration of 0.5 added.
mg/ml. If this initial Pronase stage was eliminated, the
Simultaneous competition experiments were run by add
percentage hybrid of the nucleolar DNA with 28 S rRNA ing RNA-32P at saturating amounts with increasing concen
was reduced by one-half the normal value, indicating a trations of unlabeled competing RNA. In the sequential
selective loss of rDNA genes into the interphase upon competition procedure, increasing inputs of unlabeled RNA
deproteinization with chloroform :isoamyl alcohol. This in excess of saturation were hybridized. The filters were then
mixture was kept at room temperature until it reached 15° washed at 45°in 30% formamide:70% (2x SSC); each set
and then was placed in a 37°H2O bath, with the addition of was incubated under hybridization conditions for 1 hr.
SDS to 0.5%, for 1 hr. SDS was added to a concentration of These filters were then transferred to vials containing the
1%, and sodium perchlorate was added to a concentration of RNA-32P and incubated for the same length of time. At
1 M.This mixture was shaken gently for 1 hr at 37°and then the end of the hybridization the filters were incubated for
was combined with an equal volume of chloroform : isoamyl 1 hr at 45°in fresh buffer, then twice in 2x SSC at room
alcohol (24:1) with shaking for another hour. After centrif- temperature for 10 min, and then treated with T! RNase
ugation to separate the phases, the aqueous layer was and pancreatic RNase (previously heated at pH 5.1 for 10
min at 90°)at concentrations of 20 units and 40 ng/ml, re
collected and reextracted twice with chloroform:isoamyl
alcohol. The DNA was spooled out of the aqueous phase spectively, at 37°in 2x SSC for 0.5 hr. The filters were
with the addition of 1 volume of 100% ethyl alcohol. The washed twice with 2x SSC, dried in the vacuum oven at
DNA was dissolved in 1/100 SSCI mM EDTA at a con 80°,and then counted in a toluene fluor (4 g PPO and 100
centration of 0.5 mg/ml and treated with pancreatic and mg POPOP per liter of toluene).
T! RNase (previously heated at pH 5.1 for 10 min at 90°)at
Specific Activities by Phosphodiesterase Digestion. The
a concentration of 100 ¡¿g
and 50 units/ml, respectively, for specific activities of nucleotides in RNA's were determined
1 hr at 37°.Pronase, nuclease-free and predigested, was by complete digestion (2.5 mg of enzyme per ml for 24 hr at
added at a concentration of 50 /^g/rnl for 1 hr. SDS, sodium 37°)of the 32P-labeled RNA's with venom phosphodiesperchlorate, and chloroform:isoamyl alcohol were added as terase (Worthington) and determination of their nucleotide
described above. The DNA was spooled, dissolved in compositions by both 32P and absorbance measurements.
1/100 SSCI mM EDTA, digested again with Pronase, and Routinely, the 32Pcomposition was measured by separating
deproteinized as before. The DNA was spooled from the the nucleotides electrophoretically at pH 3.5 on Whatman
aqueous phase, dissolved in 1/100 SSCI mM EDTA and No. 3MM paper (26). A Picker Nuclear (Varian Aero
frozen at -20° until loaded on filters. The A260:A280ratios graph, Walnut Creek, Calif.) nucleotide analyzer was used
of these preparations were 1.87.
to determine the UV compositions. When the labeled
Loading DNA on Filters. The DNA was denatured in 0.5 nucleotides were also separated on the analyzer, the re
N NaOH for 0.5 hr at 37°to ensure denaturation and as an sults were identical with those obtained by electrophoresis.
additional purification step (15). This solution was mixed The activities were expressed as 32Picontent per nucleotide
with 300 ml of 6x SSC, neutralized with HC1, and then relative to adenylic acid.
filtered through a 142-mm, 0.45-^m Millipore filter. The
discs (0.6 cm in diameter) were punched out, stored in a
3A comparison of hybridization in 2x SSC, 65°,and 30% formadesiccator and baked in a vacuum oven at 80°for 4 hr be
2The abbreviations used are: SDS, sodium dodecyl sulfate: rDNA,
ribosomal DNA; SSC, 0.15 Msodium chloride:0.015 Msodium citrate.
DECEMBER
mide:70% 2x SSC, 45°,was made using 28 S rRNA. Both conditions
gave similar RNase-resistant percentage hybrids: however, the rate of the
reaction in formamide at 45°was about one-half the rate at 65°.The
percentage hybrid was also the same when intact or partially degraded ( l N
NaOH, 90 sec, 0°,8 to 18 S in size) 28 S rRNA was used.
1973
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
3313
T. O. Sitz, R. N. Nazar, W. H. Spohn, and H. Busch
with the other species of RNA (Charts 2 and 3). When 45 S
nucleolar RNA-32P was competed with 18 S plus 28 S
rRNA by the sequential technique, its binding was reduced
by 50% if the temperatures of the reaction and the wash
were maintained at 45°or the filters were treated with
RNase after the 1st stage. However, when the reactions with
unlabeled RNA were allowed to reach room temperature
and then washed at 45°,the 45 S RNA was competed by
95% (Chart 4). Specificity was maintained in the sequential
technique in these experiments by never allowing the
temperature to drop below 45°during the wash.
When 32P-labeled 18 S rRNA from hepatoma cells was
competed with unlabeled RNA from hepatoma and normal
liver, no differences were demonstrated (Chart 2). The lack
of significant competition by 28 S rRNA from both tissues
demonstrated the specificity of hybridization and purity of
the RNA molecules. This lack of difference in liver and
hepatoma 18 S rRNA was shown previously by Wikman et
al. (31). In Chart 3, 28 S rRNA-32P from hepatoma cells
was competed with unlabeled RNA from both hepatoma
Simultaneous
Chart 1. DEAE-Sephadex A-25 column chromatography of pancreatic
R Nase digestion products from 28 S rRNA. Neutral columns (left) were
eluted with 600-ml gradients of 0.05 to 0.2 MNaCl in 7 Murea and 0.05 M
Tris-CI, pH 7.5. Dinucleotide fractions were pooled and further separated
on acidic columns (right) eluted with 400-ml gradients of 0 to 0.3 MNaCl in
0.003 M HC1.
A Liver-rl8S
A Liver-r28S
O Hepatoma-rl8S
•¿
Hepatoma-r28S
NHAC-rl8S-32P
o
1(1
Oligonucleotide Analysis by UV Absorbance. Purified
nucleolar or cytoplasmic RNA (1 to 10 mg) was digested
with pancreatic RNase (26) or with U2 RNase followed by
T! RNase (23) and applied to a DEAE-Sephadex A-25
column (Chart 1). The oligonucleotides were separated first
according to chain length on DEAE-Sephadex A-25 (Phar
macia Fine Chemicals, Piscataway, N. J.) as described by
Tener (29). Columns (0.7 x 10 cm) were eluted with a linear
sodium chloride gradient from 0.05 to 0.2 M in 7 M urea in
0.05 M Tris-CI, pH 7.5 (total volume, 600 ml/gradient);
3-ml fractions were collected every 5 min and their absorbance was measured at 260 nm. The mono- and dinucleotides
were further separated according to base composition on
columns (0.7 x 15 cm) eluted with a linear sodium chloride
gradient from 0 to 0.3 M in 0.003 M HC1 (total volume, 400
ml/gradient). Three-mi fractions were collected every 5
min and their absorbance was measured at 260 nm. For
nucleolar RNA's, the dinucleotides were analyzed by the
Picker Nuclear nucleotide analyzer at 254 nm.
o
o
246
10
;ig UNLABELED-RNA
RESULTS
Competitive Hybridization. To demonstrate specificity of
competitive hydridization by both the simultaneous and
sequential techniques, 32P-Iabeled 18 S and 28 S rRNA was
competed with either unlabeled 28 S or 18 S rRNA. Both
procedures demonstrated lack of significant competition
3314
Chart 2. Competition experiments between 5 fig, of "P-labeled 18 S
rRNA from hepatoma cells and unlabeled 18 S and 28 S rRNA from
hepatoma and normal liver. The competition in the upper graph was
performed by the simultaneous technique with a 100% value of 392 cpm
(rl8S, 18 S RNA; r28S, 28 S RNA; NHAC, Novikoff hepatoma ascites
cells). The competition in the lower graph was performed by the sequential
procedure with a 100%value of 300 cpm.
CANCER RESEARCH
VOL. 33
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
Ribosomal and Precursor R N A's
contrast, similar studies in which UV absorbance (6) or a
nucleoside derivative method (22) was used suggest no
significant differences. To explain the discrepant nucleotide32P compositions, the specific activities of labeled nucleotides were measured in rRNA's and their precursors.
RNA's labeled under standard conditions were completely
digested with venom phosphodiesterase-liberating
5'nucleotides, the natural precursors for these RNA's. The
amount of each nucleotide was determined with the Picker
Nuclear nucleotide analyzer, and the 32P content was
measured by electrophoresis at pH 3.5. Table 1 summarizes
the specific activities of the nucleotides relative to adenylic
acid and indicates there are significant differences in the
degree of labeling for rat liver and hepatoma RNA's. While
Simultaneous
A Liver-r28S
A Liver-rl8S
Hepatoma-r28S
Hepatoma-rl8S
o
o
i
6
¿igUNLABELED-RNA
RNA - 32P
2468
10
¿ig UNLABELED-RNA
Chart Ì.
Competition experiment between 5 /jg 32P-labe!ed28 S rRNA
from hepatoma cells and unlabeled 18 S rRNA and 28 S rRNA from
hepatoma and normal liver. The competition in the upper graph was
performed by the simultaneous procedure with a 100% value of 250 cpm
(28 S RNA, r28S; 18 S RNA, rl8S; Novikoff hepatoma ascites cells
(NHAC). The competition in the lower graph was performed by the
sequential technique with a 100% value of 214 cpm.
the hepatoma rRNA approaches uniform nucleotide label
ing at 18 hr, that of the rat liver RNA was not uniformly
labeled even after 24 hr.
Studies on the distribution of 32P-labeled nucleotides in
short oligonucleotides, particularly dinucleotides, after pan
creatic (6, 13, 27, 31, 32) or Tt RNase (30) digestion have
also indicated sequential differences in rihosomal preribosomal RNA's. In nucleolar RNA's, the GU content was about
50% higher in malignant tissues (32), and a somewhat lower
30% differences has been found in 28 S rRNA (13, 27). To
evaluate the effects of nonuniform labeling on the oligonucleotide distribution, the differences were reexamined by
means of absorbance measurements. Chart I compares the
separation of dinucleotides after complete digestion with
pancreatic RNase. Table 2 summarizes the data for both
ribosomal and preribosomal RNA's. Since no differences in
GU content were observed, these RNA's appear to be the
same from this point of view as well.
and normal liver, and no significant differences were noted.
In 3 other preparations of labeled and unlabeled RNA, no
significant difference was found.
The 32P-labeled 28 S and 45 S nucleolar RNA's from
hepatoma cells were competed with unlabeled RNA from
hepatoma cells and normal liver. Both species were com
peted equally well by the liver and hepatoma RNA's
(Charts 5 and 6). Repetition of this experiment with
different preparations of labeled and unlabeled RNA pro
duced similar results. The absolute level of competition was
low, perhaps due to contamination from nonribosomal
RNA or even due to 32P-labeled DNA. There were no
significant differences between the nucleolar ribosomal
precursor RNA's from hepatoma and liver as determined
by simultaneous and sequential competitive hybridization.
Structural Analyses of 32P-labeled RNA's. Earlier stud
ies on the nucleotide-32P compositions of ribosomal and preribosomal RNA's from rat liver and various tumors (6)
reported large differences in the AMP and CMP content. In
DECEMBER
o
Q£
U
a.
100
90
80
70
60
50
40
30
20
10
45S-nRNA-32P
•¿
-RNase + PRONASE
A-45° MAINTAINED
0-REACTION COOLED
-O024
/jg
6
UNLABLED
18S AND
8
10
28S rRNA
Chart 4. Sequential competition between 10 fig "P-labeled 45 S
nucleolar RNA (nRNA) from hepatoma cells and unlabeled hepatoma 18
S plus 28 S rRNA. After unlabeled 18S plus 28 S rRNA was hybridized on
3 sets of filters, 1 set was maintained carefully at 45°and washed; the other
2 sets were allowed to reach room temperature. Of the filters allowed to
reach room temperature, one set was treated with RNase and Pronase
while the other was washed at 45°.
1973
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
3315
T. O. Sitz, R. N. N azar, W. H. Spohn, and H. Busch
Simultaneous
A Liver-n45S
O Hepatoma- n45S
A Liver-n28S
O Hepatoma-n28S
o
ce
O
o
2
10
4
6
;U9 UNLABELED-RNA
8
10
ug RNA - 32P
¿igRNA - 32P
A Liver - n45S
O Hepatoma- n45S
Sequential
72 Hr : 72 Hr
o
o
-Q-
-ö
8
l
l
10
2468
¿ig UNLABELED
Chart
5. Competition
experiments
¿ugUNLABELED-RNA
between
Chart
RNA
5 pg "P-labeled
28 S
nucleolar RNA (n28S) from Novikoff hepatoma ascites cells (NHAC) and
unlabeled 28 S nucleolar RNA from hepatoma and normal liver. The
competition
in the upper graph was performed by the simultaneous
procedure with a 100% value of 2219 cpm. The competition in the lower
graph was performed by the sequential technique with a 100% value of 1500
cpm.
DISCUSSION
6. Competition
3316
between
10 ¿ig32P-labeled
45 S
Table I
Specific activities ofS'-nucleotides in 3*P-labeledR NA's from normal
livers and the Novikoff hepatoma
Purified
In this study, no significant differences were found by
simultaneous and sequential competitive hybridization be
tween liver and hepatoma rRNA and their nucleolar
precursors. Arnaldi and Attardi ( 1) also found no significant
difference in 28 S rRNA from HeLa cells and various
normal human tissues when compared by simultaneous
competition. All the hybridization experiments were per
formed with tumor DNA and 32P-labeled tumor RNA that
would detect additional sequences in the tumor RNA.
However, the sequence (23) and base composition (6, 22)
data would suggest no large deletions or additions in the
rRNA's from normal and malignant tissues. Recently,
Hashimoto and Muramatsu (II) reported the presence, in
18 S rRNA of normal mouse liver, of a tetranucleotide and
a hexanucleotide that were absent from mouse hepatoma.
Such limited differences would not be detected by the
techniques used in this paper.
experiments
nucleolar RNA (n45S) from hepatoma cells and unlabeled 45 S nucleolar
RNA from hepatoma and normal liver. The competition in the upper
graph was performed by the simultaneous procedure with a 100% value of
3593 cpm. The competition in the lower graph was performed by the
sequential technique with a 100% value of 3885 cpm.
RNA's
were completely
digested
with venom phosphodies-
terase, and the nucleotides were determined by UV absorbance after
liquid chromatography,
or by radioactivity after paper electrophoresis.
Specific activities, which are tabulated relative to adenylic acid, are
averages of 4 to 6 determinations.
Specific activity relative
to adenylic
acidRNA18
rRNA28
S
PA
PCp(ipU
hr)0.64
hr)1.19
±0.07
1.00
0.66 ±0.03
0.67
0.020.68
±
=fc0.07
1.00
0.96 ±0.04
0.93
0.021.07
±
±0.03
±0.06
1.00
1.00
pA
0.94 ±0.03
PCpGLiver(24 0.67 ±0.07
0.59 ±0.03Novikoff(18
0.76 ±0.02
S rRNANucle-otidePU
CANCER RESEARCH
VOL. 33
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
Ribosomal and Precursor RNA's
Table 2
Distributions ofoligonucleotides in nucleolar and rR NA 'sfrom raÃ-liver
and ¡heNovikoff hepaloma after digestion by pancreatic R Nase
Fragments were separated by DEAE-Sephadex A-25 column chromatography (Chart 1) at neutral or acid pH, and the elution profiles were
monitored by the absorbance at 260 nm for the rRNA's or at 254 nm for
the precursors. The values are averages of 2 determinations.
UV distribution
(% total absorbancy)
RNA
Nucleolus
45 S nucleolar
RNA
Oligonucleotide
Liver
Novikoff
hepatoma
A-Cp
G-Cp
A-Up
G-Up
9.4
12.5
8.7
4.7
9.2
12.2
8.9
4.6
these rDNA transcripts. This high degree of fidelity has also
been demonstrated in HeLa cell rRNA and normal human
tissues (1). Perhaps a model such as the one proposed by
Howell (14) for Chlamydomonas, which is a variation on
the master gene concept (7), might explain the lack of
heterogeneity. Brown ei al. (3) found, in 2 different species
of Xenopus, that their 18 S and 28 S rRNA's were identical
but that a difference existed in the 40 S precursor molecules.
In contrast, the nonconserved regions of 45 S nucleolar
RNA in Novikoff hepatoma cells appear to be maintained
in oncogenesis and in subsequent cell growth.
REFERENCES
1. Arnaldi, F., and Attardi, A. Comparison of the Primary Structures of
28 S and 18 S Ribonucleic Acid from HeLa Cells and Different
Human Tissues. Biochemistry, 10: 1478 1483, 1971.
Ribosome
2. Bonner, J., Kung, G., and Bekhor, I. A Method for Hybridization of
28
rRNA18
S
Nucleic Acid Molecules at Low Temperature. Biochemistry, 6:
3650 3653, 1967.
3. Brown, D. D., Wensink, P. C., and Jordan, E. A Comparison of the
Ribosomal DNA's of Xenopus laevis and Xenopus mulleri: The
S rRNAA-CpG-CpA-UpG-UpA-CpG-CpA-UpG-Up5.610.52.65.16.79.83.23.85.510.32.64.86.29.83.33.9
Evolution of Tandem Genes. J. Mol. Biol., 63: 57 73, 1972.
4. Burton, K. Determination of DNA Concentration with Diphenylamine. Methods Enzymol., 12B: 163-166, 1968.
5. Busch, H. Isolation and Purification of Nucleoli. Methods Enzymol.,
12A: 448 464, 1967.
6. Busch, H., and Smetana, K. The Nucleolus. New York: Academic
Press, Inc., 1970, pp. 209-284.
Although previous reports had suggested that additional
sequences were present in hepatoma 28 S rRNA and its 7. Callan, H. G. The Organization of Genetic Units in Chromosomes. J.
Cell Sci., 2: 1-7, 1967.
nucleolar precursors (31, 32), the nucleotide analyses re
8.
Chaveau,
J., Moule, Y., and Rouiller, C. Isolation of Pure and
ported here indicate that at least some of these previously
Unaltered Liver Nuclei Morphology and Biochemical Composition.
reported differences in nucleotide composition and oligonuExptl. Cell Res., //. 317 321, 1956.
cleotide distribution probably were the result of nonuniform
9. Gillespie, D., and Spiegelman, S. A Quantitative Assay for DNAlabeling rather than actual differences in the primary
RNA Hybridization with DNA Immobilized on a Membrane. J. Mol.
sequences. Earlier comparisons of polypyrimidines (23)
Biol., 12: 824 842, 1965.
would support this conclusion.
10. Gillespie, S.. and Gillespie, D. Ribonucleic Acid-Deoxyribonucleic
The phenomenon of "super" competition was observed
Acid Hybridizations in Aqueous Solutions and in Solutions Contain
ing Formamide. Biochem. J., 125: 481-487, 1971.
with the sequential technique when reactants were allowed
to reach room temperature and then were washed at 45°. 11. Hashimoto, S., and Muramatsu, M. Differences in Nucleotide Se
This interference probably results from "tails" of unlabeled
quences of Ribosomal RNA between the Liver and a Hepatoma of
C3H/He Mice. European J. Biochem., 33: 446 458, 1973.
RNA that are sticking off the DNA and that then bind to
adjacent regions when the temperature is lowered. Lucas 12. Higashi, K., and Busch, H. Preservation of Rapidly Synthesized
Ribonucleic Acid of High Molecular Weight in Frozen Nuclei of Rat
and Ginsberg (16) reported that this interference was
Liver. Biochim. Biophys. Acta, 103: 339-341, 1965.
eliminated by RNase or RNA partially degraded by NaOH. 13. Higashi, K., Matsukisa, T., Gotoh, S., Nishinaga, K., and Sakamoto,
The short time required for the interference to occur would
Y. Different Gene Expression for Ribosomal RNA of AH-130 Tumor
indicate that it arose from RNA already bound to the
and Embryonic and Adult Rat Liver. Biochim. Biophys. Acta, 232:
352-358, 1971.
surface. Once this nonspecific binding occurs, reheating to
hybridization temperature does not remove it. In the 14. Howell, S. H. The Differential Synthesis and Degradation of Ribo
somal DNA during the Vegetative Cell Cycle in Chlamydomonas
experiments reported using the sequential method, the
hybridization temperature of 45°was carefully maintained
reinhardi. Nature New Biol., 240: 264-267, 1972.
during the wash, and nonspecific interference or "super" 15. Kennell, D., and Kotoulas, A. Titration of the Gene Sites on DNA by
DNA-RNA Hybridization. J. Mol. Biol., 34: 71 81, 1968.
competition did not arise.
The high degree of similarity between rRNA's from 16. Lucas, J. J., and Ginsberg, H. S. Synthesis of Virus-Specific Ribonu
cleic Acid in KB Cells Infected with Type 2 Adenovirus. J. Virol., 8:
normal rat liver and those from the Novikoff hepatoma cell
203 213, 1971.
line that has been maintained by serial transplants for many 17. Marmur, J. A Procedure for the Isolation of Deoxyribonucleic Acid
generations suggests a high degree of fidelity in replication
from Microorganisms. J. Mol. Biol., 3: 208 218, 1961.
and transcription of the approximately 200 gene copies of 18. Matsuhisa, T., Higashi, S., Gotoh, S., and Sakamoto, Y. Changes in
rDNA and also a high degree of precision in processing
the Nucleotide Compositions of Nucleolar 45 S RNA of Azo
DECEMBER 1973
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
3317
T. O. Sitz, R. N. N azar, W. H. Spohn, and H. Busch
Dye-induced Hepatoma. Cancer Res., 30: 162-166, 1970.
19. Mauritzen, C. M., Choi, Y. C., and Busch, H. Preparation of
Macromolecules of Very High Specific Activity of Tumor Cells in
Vitro. In: H. Busch (ed.), Methods in Cancer Research, Vol. 6, pp.
253-282. New York: Academic Press, Inc., 1970.
20. McConaughy, B. L., Laird, C. D., and McCarthy, B. J. Nucleic Acid
Reassociation in Formamide. Biochemistry, 8: 3289-3295, 1969.
21. Meijs, W. H., and Schilperoot, R. A. Determination of the Amount of
DNA on Nitrocellulose Membrane Filters. Federation European
Biochem. Soc. Letters, 12: 166 168, 1971.
22. Nazar, R. N. Evidence for Nonuniform Labeling in Comparisons of
"P-labeled RNAs from Rat Liver and the Novikoff Hepatoma.
Federation Proc., 32: 853, 1973.
23. Nazar, R. N., and Busch, H. A Comparison of Polypyrimidine
Fragments in 28 S Ribosomal RNA from Rat Liver and the Novikoff
Ascites Hepatoma. Cancer Res., 32: 2322-2331, 1972.
24. Quagliarotti, G., Hidvegi, E., Wikman, J., and Busch, H. Structural
Analysis of Nucleolar Precursors of Ribosomal Ribonucleic Acid.
Comparative Hybridizations of Nucleolar and Ribosomal Ribonucleic
Acid with Nucleolar Deoxyribonucleic Acid. J. Biol. Chem., 245:
1962-1969, 1970.
25. Rich, A. Preparation of Polysomes from Mammalian Reticulocytes,
Lymph Nodes and Cells Grown in Tissue Culture. Methods Enzymol.,
3318
I2A: 481-491, 1967.
26. Sanger, F., and Brownlee, G. G. A Two-Dimensional Fractionation
Method for Radioactive Nucleotides. Methods Enzymol., 12A:
361-381, 1967.
27. Seeber, S., and Busch, H. Comparative Oligonucleotide Sequences of
28 S Ribosomal RNA of Normal Liver and Novikoff Hepatoma
Ascites Cells. Cancer Res., 31: 1888-1894, 1971.
28. 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.
29. Tener, G. M. Ion-Exchange Chromatography in the Presence of Urea.
Methods Enzymol., 12A: pp. 398-404, 1967.
30. Wikman, J., Howard, E., and Busch, H. 32PDistribution in Oligonucleotides of 28 S Ribosomal RNA of the Novikoff Hepatoma and
Normal Rat Liver. Cancer Res., 30: 773-775, 1970.
31. Wikman J., Quagliarotti, G., Howard, E., Choi, Y. C, and Busch, H.
A Comparison of 32P Distribution in Oligonucleotides of Ribosomal
28 S RNA from Normal Liver and Novikoff Hepatoma Ascites Cells.
Cancer Res., 30: 2749-2759, 1969.
32. Yazdi, E., Ro-Choi, T. S., Wikman, J., Choi, Y. C., and Busch, H. "P
Distribution in Oligonucleotides of Rapidly Sedimenting Nucleolar
RNA's of Hepatomas and Normal Rat Liver. Cancer Res., 29:
1755-1762, 1969.
CANCER RESEARCH
VOL. 33
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.
Similarity of Ribosomal and Ribosomal Precursor RNA's from
Rat Liver and the Novikoff Ascites Hepatoma
Thomas O. Sitz, Ross N. Nazar, William H. Spohn, et al.
Cancer Res 1973;33:3312-3318.
Updated version
E-mail alerts
Reprints and
Subscriptions
Permissions
Access the most recent version of this article at:
http://cancerres.aacrjournals.org/content/33/12/3312
Sign up to receive free email-alerts related to this article or journal.
To order reprints of this article or to subscribe to the journal, contact the AACR Publications
Department at [email protected].
To request permission to re-use all or part of this article, contact the AACR Publications
Department at [email protected].
Downloaded from cancerres.aacrjournals.org on June 16, 2017. © 1973 American Association for Cancer Research.