Download Transcription of the Repetitive DNA Sequences

[CANCER RESEARCH 35, 1559-1562,June 1975]
Transcription of the Repetitive DNA Sequences in
Polyoma-transformed and Nontransformed Mouse Cells in Culture'
Leo J. Grady and Wayne P. Campbell
Division of Laboratories and Research, New York State Department of Health. Albany, New York 1220/
SUMMARY
regular medium and in medium containing 3 @zgof the
thymidine analog BUdR2 per ml have been described (6).
RNA-DNA saturation hybridization experiments were
Radioactive DNA. Secondary cultures of mouse embryo
cells were grown in half-gallon roller bottles in the same
used to estimate the extent of transcription
of repetitive
DNA sequences in polyoma-transformed and nontrans medium as above. While still subconfluent, the cultures
15 jzCi/ml (specific
formed mouse cells in culture. Measurements
were made were exposed to [methy/-3H]thymidine,
activity, 50.7 Ci/mmole; New England Nuclear, Boston,
with RNA from nontransformed cells in both the subconflu
ent and confluent stages of growth, transformed cells in Mass.), for 24 hr. The cells were then harvested and the
normal growth medium, and transformed cells grown in DNA was extracted and purified according to published
procedures (4). The final DNA preparation had a specific
medium containing 5-bromodeoxyuridine,
3 sg/ml, a treat
activity of 2.5 x 10@cpm4ig.
ment which reduces their tumorigenic potential. No differ
ences were observed in the amount of repetitive DNA
RNA. The methods for cell culture and for the extraction
transcribed or in the families of sequences expressed, except and purification of RNA have been described elsewhere (4).
in transformed cells grown in the presence of 5-bromodeox
For BUdR, cells were grown in the presence of the analog
yuridine, in which case the extent of transcription
was for 10 passages before their RNA was extracted.
reduced.
DNA Fractionation. Tritium-labeled
DNA was sus
pended in 0.0015 M NaCl-0.000IS M sodium citrate at 300
@zg/mland sheared by passage through a French pressure
INTRODUCTION
cell at 25,000 psi. Based on sedimentation
in alkaline
sucrose gradients, DNA sheared in this manner is indistin
One of the interests of our laboratory has been to guishable from DNA sheared at 50,000 psi in an American
Instrument Co. No. 46-13715 pump. The DNA concentra
determine whether virus-induced transformation
is accom
panied by measurable changes in the proportion of the tion was adjusted to 50 @g/ml,and the buffer was adjusted
to 0.12 M PB. The DNA was denatured by immersion in
cellular genome that is transcribed. Previously, we reported
that, at least in culture, differences in the transcription of boiling water for 5 mm and then incubated at 60°to C0t
[nucleotide concentration
x time (moles x sec/liter)J = 3
the nonrepetitive DNA sequences do exist between trans
x lO_2 (1). At this point, the sample was passed over a
formed and nontransformed
mouse cells (4). The present
hydroxylapatite
column (DNA grade; Bio-Rad, Richmond,
communication
describes experiments designed to measure
the degree to which repetitive DNA is transcribed in the Calif.) at 60°,and the column was washed with 5 column
same cell lines. Taken together with the earlier studies on volumes of 0.12 M PB + 0.4% SLS to remove single
the expression of nonrepetitive DNA, these measurements stranded DNA. Finally, double-stranded
DNA was eluted
provide a reasonably complete picture of the extent of by washing with 5 column volumes of 0.4 M PB + 0.4%
transcription
of cellular DNA in cultures of transformed
SLS. This procedure served to remove the 10% ofthe mouse
and nontransformed
cells.
genome associated with the very rapidly reassociating
satellite DNA. The removal ofthis DNA component should
result in lower backgrounds due to DNA-DNA self-reaction
MATERIALS AND METHODS
in subsequent RNA-DNA
hybridization experiments.
At
the same time, since satellite sequences are not transcribed
Cells and Cell Culture. The nontransformed AL/N line of (3), their absence should not alter the results of such
mouse cells (1 1) and a polyoma-transformed
derivative, PY experiments.
AL/N clone 3, were used. The cells were grown in a
RNA-DNA Hybridization. Hybridization mixtures con
modified Eagle's medium (No. 71035; Grand Island Biolog
tamed denatured, fractionated
DNA, 0.1 jzg/ml, labeled
ical Co., Grand Island, N. Y.) supplemented with 10% fetal with tritiated thymidine and various concentrations
of
bovine serum (Grand Island Biological Co.). The growth unlabeled RNA in 1.0 ml of 0.12 M PB. Each experiment
properties and the tumorigenicity of these cell lines in both also included 2 control samples that contained only 0. 1 jsg of
1 This
work
was
supported
in
part
by
USPHS
Grant
CA
the National Cancer Institute.
ReceivedJanuary 10, 1975;acceptedMarch 5, 1975.
JUNE
13401-02
from
2 The
abbreviations
used
are:
BUdR,
phate buffer composed of equimolar
5-bromodeoxyuridine;
concentrations
PB,
of NaH,P04
phos
and
Na,HPO4; SLS, sodium lauryl sulfate.
1975
Downloaded from cancerres.aacrjournals.org on August 3, 2017. © 1975 American Association for Cancer Research.
1559
L. J. Grady and W. P. Campbell
labeled DNA per ml. Reactions were run in 45- x 15-mm
glass vials (No. 3-33EA; Fisher Scientific Co., Rochester,
N. Y.), and the samples were layered with paraffin oil to
prevent evaporation.
Incubations were at 60° for 16 hr.
Reactions were terminated by diluting samples into 5.0 ml
of 0. 12 M PB + 0.4% SLS at
@O
followed by immediate
0
Ui
4
U
0
12
4
Ui
4
z
0
passage over a column of hydroxylapatite, also at 60@.
Single-stranded DNA was washed from the column with
0.12
M PB
+
0.4%
SLS
as above.
DNA-DNA
and
RNA-DNA hybrids were then collected by washing the
column with 0.4 MPB + 0.4% SLS. The nucleic acids in the
0.12 M PB and 0.4 M PB fractions were precipitated with
trichloroacetic
acid (final
precipitates were collected
5'
concentration,
5%), and the
on HAWP filters (Millipore,
Bedford, Mass.).
Absorption within the precipitate of the low-energy beta
particle released by tritium decay can produce serious errors
if direct counting of precipitates
on filter supports is
attempted. To circumvent this problem, filters were placed
in glass counting vials and incubated overnight at 68°with
cot (moles x sec/liter)
Chart
1. Reassociation
kinetics
of fractionated
and unfractionated
mouse DNA. Labeled DNA was fractionated as describedunder “Mate
rials and Methods.― The reassociation kinetics of this DNA was then
compared to that of similarly sheared, but unfractionated, DNA. In order
to follow the reactions over a wide range of C0t values, 0. 1 @gof each
labeledDNA wasmixed with enoughsheared,unlabeledDNA to give: C0:
= l0@ to lO_2,0.9 @g/ml;C0: = lO@ to 6.0, 15 @g/ml;C0: = 6.0 to
10', 600
@g/ml.Samples were denatured and then incubated at 6O@in
0.12 M PB. At various intervals, portions of each samplewere removed
and the amount of DNA reassociatedwas determined with columns of
hydroxylapatite.
•,fractionated DNA; 0, unfractionated
DNA.
0.6 ml of 1.0 N HCI (7). After cooling, 15 ml of Aquasol
@
(New England Nuclear, Boston, Mass.) were added to each
vial, and the samples were counted in a Beckman LS250
scintillation spectrometer.
In reconstruction
experiments,
the counts per sample recovered with this procedure were
independent of the amount of unlabeled carrier DNA added
to the sample. The amount of DNA in hybrid form
(DNA-DNA and RNA-DNA) was taken as the proportion
of the total counts recovered that were found in the 0.4 MPB
fraction. The quantity of DNA in RNA-DNA hybrids was
determined
by subtracting
from this value the average
extent of DNA-DNA self-reaction (2 to 3%) that took place
in the controls that contained DNA alone.
The hybridization technique for these experiments used
unlabeled RNA to drive reactions containing a small
amount of labeled DNA. Since the reactions were carried
out in solution, DNA-DNA as well as RNA-DNA hybrids
were able to form. To keep the background due to the
DNA-DNA hybrids to a minimum, the labeled DNA was
fractionated prior to use in hybridization experiments so as
to remove the very rapidly reassociating
satellite DNA
sequences. A comparision of the reassociation kinetics of
mouse DNA before and after such fractionation is given in
Chart I . These data establish that the procedure used to
remove the satellite sequences did not alter the reassociation
properties of the remainder of the DNA. Inasmuch as
Flamm et a!. (3) have shown that satellite DNA is not
transcribed, the absence of this fraction should not change
the outcome of the hybridization experiments.
A summary
of RNA-DNA
saturation
hybridization
using RNA from subconfluent and confluent
AL/N cells is presented in Chart 2.4. Also
the results from an experiment that contained a
RNA from the 2 growth states. In every
saturation value obtained was about 15%. The
range for 2 experiments was 3.3 to 3.9% using RNA from
I560
RNA C0N@ENTRATI0N
IN ,ug/mI
Chart 2. RNA-DNA saturation hybridization experiments.A, results
with RNA from:
.,
a mixture
of
@,
confluent AL/N
confluent
and
cells; V , subconfluent AL/N
subconfluent
AL/N
cells.
B,
cells;
reactions
containing RNA from: 0, PY Al/N cells; A, a mixture of PY AL/N and
subconfluent AL/N cells; 0, PY AL/N cells grown in medium containing
BUdR, 3 @ig/ml.
subconfluent cells, 3.3 to 3.8% with RNA from confluent
cells, and 3.4 to 3.8% when RNA mixtures were used. These
results strongly suggest that there are neither quantitative
RESULTS
experiments
cultures of
included are
mixture of
instance, the
EL@0@:@:t
nor qualitative differences in the families of repetitive DNA
transcribed
experiment
in the 2 conditions of growth. When an identical
was performed
using purified nonrepetitive
DNA, prepared as described elsewhere (4), no reaction was
observed. Thus, it can be concluded that only repetitive
DNA formed hybrids under the reaction conditions used.
The labeled DNA utilized in all of the experiments
described thus far had been fractionated
to remove the
satellite DNA. Consequently,
the 3.5% saturation values
obtained are actually equivalent to 3. 1% of the whole mouse
genome. In an experiment carried out with subconfluent
AL/N RNA and unfractionated
mouse DNA, the satura
tion value was 2.8% (DNA-DNA self-reaction was 10% in
this case). This number is in good agreement with the 3.1%
calculated above and supports the contention that removal
ofthe satellite DNA does not alter the experimental results.
The results of similar experiments using RNA from PY
AL/N cells grown either in regular medium or in medium
supplemented with BUdR, 3 jsg/ml, are illustrated in Chart
2B. The saturation value obtained with RNA from trans
formed cells grown in regular medium is similar to that for
nontransformed
cells, ranging from 3.2 to 3.7% for 3
experiments. In contrast, experiments with RNA from cells
CANCER
RESEARCH
VOL. 35
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Repetitive
grown in the presence of BUdR reveal a decrease in the
extent of repetitive DNA transcribed, the saturation values
being 2.0 and 2.4% for 2 experiments.
Chart 2B also
includes the data from a reaction that contained a mixture
of RNA from subconfluent AL/N cells and PY AL/N cells.
No differences were observed in the families of repetitive
DNA transcribed in transformed and nontransformed cells.
Since repetitive DNA consists of families of closely
related, but not identical, base sequences, it is highly
improbable that a RNA molecule will hybridize with a
region of DNA precisely like that from which it was
originally transcribed.
The actual extent to which mis
matching of base pairs occurs depends largely on the salt
concentration and temperature used in the reactions and can
be estimated from the thermal stability of the hybrids. An
example of the thermal stability of the hybrids formed in
this study is shown in Chart 3. The Tm for repetitive
DNA-DNA
hybrids was 72°, whereas that for repetitive
DNA-RNA hybrids was 67°.Even in cases where there are
few, if any, mismatched base pairs, there is a 4°difference
between the Tm'5 Of DNA-DNA and RNA-DNA hybrids.
Therefore, the above values suggest that the extent of
mismatching of base pairs in the RNA-DNA
hybrids is
essentially the same as that in the DNA-DNA hybrids. On
the other hand, the Tm'5 for both types of hybrid are
approximately
12°lower than those for nonrepetitive DNA
hybrids formed under similar conditions (4). Ifthe relation
ship determined by Laird et al. (9) that a °decrease in Tm
is equivalent to 1.5% mismatching ofbase pairs is applied to
our data, it can be estimated that the repetitive DNA
hybrids contain
18 to 20% mismatched
base pairs.
Ui
DNA
Transcription
in Mouse Cells
DISCUSSION
The 3.5% of the DNA that formed hybrids in these
experiments can be viewed in several ways. If this number is
corrected for the 10% of the mouse genome removed by the
initial fractionation of the DNA, then it represents 3. 1% of
the whole mouse genome. However, since only repetitive
DNA was able to form hybrids under the experimental
conditions used, and since repetitive sequences comprised
about 20% of the DNA used, the 3.5% of the DNA that did
hybridize is equivalent to 17.5% of the repetitive sequences.
These data provide information only with regard to the
transcription
of families of repetitive DNA; they do not
permit conclusions to be drawn regarding the expression of
specific sequences. Also, the saturation values obtained are
for a given salt concentration and temperature,
and reac
tions involving repetitive DNA are quite sensitive to
changes in these parameters (2).
No differences in the families of repetitive DNA ex
pressed were found between confluent and subconfluent
nontransformed
cells in culture. Likewise, there were no
differences detected between nontransformed cells and those
transformed by polyoma virus. This latter result agrees with
the findings of Levine et a!. (10), who worked with
SV4O-transformed
and nontransformed
hamster cells, and
supports their conclusion that for established cell lines in
culture viral transformation
does not lead to changes in
transcription
of the repetitive
DNA
sequences
of the cell.
In the case of transformed cells grown in the presence of
the thymidine analog BUdR, we observed a decrease in the
transcription of repetitive DNA. Similar results were also
found by Kotzin and Baker (8) who studied the effect of
BUdR on the transcription of repetitive DNA in the sea
urchin. It is also consistent with our previous report of a
decrease in the expression of nonrepetitive DNA in PY
AL/N cells grown in medium containing BUdR (5).
The results obtained thus far in our studies of the
transcription
of the cell genome in nontransformed
and
virus-transformed
mouse cells in culture can be summarized
as follows: (a) no difference is observed in the families of
repetitive DNA expressed in the 2 types of cells; (b) there
are differences in the transcription of nonrepetitive DNA
sequences between transformed and nontransformed
cells;
(c) growth of transformed
cells in medium containing
BUdR, 3 j.tg/ml, a treatment that reduces but does not
60
@2
TO
TEMPERATURE
80
eliminate tumorigenicity (6), results in a general suppres
sion of the transcription of both repetitive and nonrepetitive
90
DNA sequences.
(°C)
Chart 3. Thermal stability of the RNA-DNA
hybrids. After 16 hr
incubation at f,@0 the hybridization mixture was diluted into 5.0 ml of 0.12
M PB
+
0.4%
SLS
and
passed
over
a hydroxylapatite
column
at
Single-strandedDNA waswashedfrom the column with 0.12MPB + 0.4%
SLS, and the temperatureof the column wasthen raisedin 5°increments.
After eachincreasein temperature,the column waswashedwith 3 column
volumes of 0. 12 M PB + 0.4% 515 at the new temperature. The dissocia
tion of the RNA-DNA hybrids was followed by measuring the percentage
of the total trichloroacetic acid precipitable counts, which bound to the
column at 600 that eluted at each temperature.•,hybrid of PY AL/N
RNA and repetitiveDNA; 0, DNA-DNA hybrid after incubationto
C0: = 10.
REFERENCES
6O@.
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/61:529-540,1968.
2. Church, R. B., and McCarthy, B. J. Related Base Sequences in the
DNA of Simpleand ComplexOrganisms.II. The Interpretationof
DNA/RNA
Hybridization Studies with Mammalian Nucleic Acids.
Biochem. Genet., 2: 55-73, 1968.
3. Flamm, W. G., Walker, P. M. B., and McCallum, M. Some
Propertiesof the Single Strands from the DNA of the Nuclear Satel
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I561
L. J. Grady and W. P. Campbell
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CANCER
RESEARCH
VOL.35
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Transcription of the Repetitive DNA Sequences in
Polyoma-transformed and Nontransformed Mouse Cells in
Culture
Leo J. Grady and Wayne P. Campbell
Cancer Res 1975;35:1559-1562.
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