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Transcript
Molecular
Events in the Reproduction
of Animal Cells
I . The Effect of Puromycin on the
Duplication of DNA*
GERALD
C.
MUELLER,
KAZUTO
AND
K@&jiwu@,
ROLAND
R.
ELTON
STUBBLEFIELD,
RUECKERT
(McArdZe Memorial Laboratory, University of Wisconsin Medical *SCFiOO4
Madison, Wiaxnwin)
SUMMARY
The initiation of DNA synthesis was studied in cultures of HeLa cells which were
synchronized by the induction and timely reversal of a thymidineless
state. It was
.
@
observed
that
the
synthesis
of
DNA
was
initiated
whereas
its effects
on protein
synthesis
were
a
immediate.
that the synthesis of DNA in the mammalian
localized changes involving protein synthesis
process.
slow
rate
but
was
more
than
(1@) it was
demonstrated
that
DNA
replication
occurs only after a specific triggering event and
that the formation of new DNA is a nonlinear
process within a single nucleus. Under the condi
tions of these experiments
levels of puromycin
which inhibit protein synthesis prevented the ac
celeration of DNA synthesis without inhibiting
the established rate of DNA synthesis.
On the basis of the results of these studies and
the studies on chromosome labeling in the second
paper
of this series,
it is proposed
that
the DNA
of the triggered IleLa cell exists in two physiologi
cal states : a fraction which is competent for repli
cation, and another which becomes competent for
a This work was supported
United States Public Health
by grant CY-1897-C7 from the
Service and by funds from the
Alexander and Margaret Stewart Trust.
The
data
support
the
concept
cell is a focalized phenomenon and that
are required to initiate the replication
replication through a puromycin-sensitive
presumed to be protein synthesis.
The replication of mammalian cells can be visu
alized as a cyclic process in which the cell prog
resses through a sequence of molecular events in
volving the timely synthesis or activation of spe
cific macromolecules.
The present study concerns
the mechanisms underlying the initiation of DNA
synthesis.
With use of synchronized
cultures of HeLa
@
at
doubled after
hours. This acceleration of DNA synthesis could be prevented with
amounts of puromycin which simultaneously
inhibited protein synthesis. Puromycin,
however, did not inhibit DNA synthesis which was in progress at the time of addition,
process
MATERIALS
AND METHODS
The origin and maintenance
of the HeLa cell
strain used in these experiments has been described
in detail previously
(1@). Stock cultures were
grown in spinner flasks using Eagle's HeLa medi
um (7) from which the calcium and magnesium
were omitted. This medium was supplemented
further
with
0.1
m@& glycine,
0.1
m@ serine,
0.01
mM inositol, 0.1 per cent pluronic F-681 (14), and
10 per
cent
bovine
serum
(medium
is referred
to
as BEHM) . Stock culture suspensions were di
luted daily with fresh medium to an initial cell con
centration of 1.5 X 1O@cells per milliliter. Cells
were thus maintained in logarithmic growth with
the growth potential of the medium approximate
ly half expended at the time of refeeding. The gen
eration time under these conditions
averaged
around
hours.
For the experimental
studies replicate mono
layer cultures were prepared by inoculating 3—5X
10@ cells
in
scription
Received for publication May 11, 1962.
1 Wyandotte
10 milliliters
bottles.
Chemical
After
Co.,
BEHM
@4hours'
Wyandotte,
into
3-oz.
incubation
pre
at
Michigan.
1084
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
MUELLER ci @2l.—Effectof Puromycin
370
C.
in
an
atmosphere
of
5
per
cent
CO2
in
air
to permit attachment
of the cells to the glass, a
thymidine deficiency was produced by the addition
of 1.1 ml. of BEHM containing 10@ M amethop
term and 5 X 10@ M adenosine. At the indicated
time, usually 16 hours after the addition of ame
thopterin, the cultures were rescued by the addi
tion of thymidine in the amounts described.
Allhandling of the cells was carried out in a 37°C.
room, and all media were prewarmed to 37°C. in a
thermostated
water bath prior to use in order to
avoid any cold shock synchronization
of the cul
tures.
Experimental
cultures
were
harvested
dicated
times by rinsing cell sheets
0.9
cent
per
saline
followed
by
at the
in
successive
@.0ml. of 88 per cent
cell residue was dissolved
formic
acid,
RESULTS
and
suitable
in
au
quots taken for analysis. Isotopic determinations
were carried out on aliquots plated on clean aiumi
num planchets and counted for radioactivity
in a
gas-flow proportional
counter. DNA was deter
mined by the modified fluorometric procedure of
Kissane and Robins (8), with salmon-sperm DNA
used as the reference standard (California Corp.
for Biochemical Research). Protein determinations
were made with the Oyama and Eagle modification
of the Lowry procedure (11).
For the autoradiographic
studies with tritium
labeled thymidine the cells were planted on cover
slips (10 X 50 mm.)
The culture
1.0 ml.;
inserted
into Leighton
size was reduced
all other
conditions
tubes.
proportionately
were
maintained
to
iden
tical to those of the 10-mi. cultures in prescription
bottles. At the indicated times the cells were la
beled with tritiated thymidine (@curies per milli
mole). At the conclusion of the labeling period the
cover slip with its adherent cells was removed from
the Leighton tube, rinsed with water, and fixed in
methanol—acetic
acid
(3 : 1) at
room
temperature
for 30 minutes. The cover slip was then rinsed in a
second change of fixative and was rapidly air
dried. This procedure flattened the nuclei and fa
cilitated grain counting subsequently.
The cover
slip was mounted to a microscope slide and coy
ered with stripping film according to the procedure
of Pelc (5). After the proper exposure the auto
graphs
were developed
in Kodak
of the emulsion with dilute HC1. The preparations
were then air-dried, and a second cover slip was
mounted with Permount. Localization and count
ing of the silver granules were carried out with
phase-contrast
microscope.
Thymidine
@-C'@and leucine-1-C'4 were oh
tamed from New England Nuclear Corporation;
tritium-labeled
thymidine was obtained from the
Schwartz Biochemical Company. The puromycin
was a generous gift of Dr. Nestor Bohonas and
the Lederie Company. These materials were dis
solved in double-distilled water and added as a @5or 50-microliter aliquot to the culture medium as
indicated.
washes
to dry.
The dry, defatted
1085
@2times with
with two 1O-ml. volumes of 4 per cent perchioric
acid, 80 per cent ethanol, and absolute ethanol.
The cell sheets were then rinsed with ether and
permitted
on DNA
D-19
developer
for 7 minutes, rinsed in distilled water, and fixed
in Kodak acid fixer. The nuclei were stained
through the stripping film with Harris' hematoxy
un for 30 minutes, followed by a brief de-staining
Synchronization
of the culturea.—The induc
tion of a thymidineless state with amethopterin in
HeLa cultures was described previously (1@). This
state, which is analogous to that obtained in bac
terial cultures with a thymidine deficiency (3, 4),
is characterized
by the immediate cessation of
DNA synthesis and cell divisions while RNA and
protein continue to accumulate. After an optimal
exposure
to amethopterin
(16 hours)
the
addition
of exogenous thymidine results in a synchronized
wave of DNA synthesis which is followed by a
similar wave of cell divisions. The wave of cell
divisions, however, takes place only after the cell's
complete complement of DNA has been synthe
sized. The typical response of the basic thymidine
rescue experiment is depicted in Charts 1 and %.
In all experiments it was observed that DNA syn
thesis was resumed on addition of exogenous thy
midine at a rate one-third to one-half the maxi
mum rate; the maximal rate was attained in the
cultures
only after
an initial
s-hour
period
during
which the acceleration took place.
In order to elucidate further the molecular
events taking place in this system it was first nec
essary to establish the manner in which the popu
lation of cells was responding. For this purpose
preliminary kinetic experiments were carried out
on the ability of cultures to incorporate thymidine
@-C'4 after
various
periods
of pretreatment
with
amethopterin.
Table 1 illustrates that a maximal
rate of DNA synthesis was obtained only after 16
hours of pretreatment;
this is in accord with earlier
studies which showed that 16 hours was the opti
mum time for reversal of the thymidineless state
so as to obtain a maximal synchronization
of cell
divisions with a minimum of thymidineless death
(1@).
Susceptibility
of the incorporated thymidine-2-C'4
to the action of DNa@e.—Inorder to assure that
the incorporated
thymidine-@-C'4
resided in DNA,
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
Cancer Research
1086
experiments
@
were
carried
out to correlate
the
loss of
label from the nucleoprotein residue with the hy
drolysis of DNA by DNase. In Table
it is dem
onstrated that the release of label parallels com
pletely the solubilization of the DNA by the spe
cific enzyme.
The triggering of individual ce& for DNA syn
thesis.—While the kinetic studies in whole cultures
suggested strongly that individual cells were ac
0
Ui
M
(1)
Ui
1.9
DNA
by autoradiography.
C)
that
1
MENT ON ABILITY OF HELA CUL
TURES To INCORPORATE THYMI.
DINE.2.C―
U)
>-
U)
0)
Pretreatment
with
amethopterin
1.3
0
8 illustrates
EmcT OFAMETHOPTERIN
PRETREAT.
I.,
0
Chart
TABLE
l-7I@
z
196@
cumulated by the thymidine deficiency in a state
of readiness for DNA synthesis, it was necessary
to verify this picture at the level of individual
cells. For this purpose cultures were permitted to
incorporate
tritium-labeled
thymidine
for a 10minute period at indicated times after the start
of the amethopterin treatment; the individual cells
were then examined for the amount of isotope in
P1
z
@,October
no more than 30 per cent of the cells at any one
time in a logarithmically
growing HeLa culture
are in the process of synthesizing their DNA; the
rest are in the interphase or post-DNA synthesis
12.1
II
Vol.
incorporation
(counts/minXlO')4
(hr.)Thymidine.5-C1'
I. I
6.37
7.65
7.79
7.42
8
12
16
184.58
HOURS AFTER
THYMIDINE ADDITION
Cultures
of
HeLa
pared and pretreated
Ciwrr 1.—Theaccumulation of DNA and cells in cultures
cells
were
pre
with amethopterin
10-' M. At the indicated times 10 pg.
following thymidine rescue. Cultures were prepared as under
“Methods―and treated with amethopterin
for 16 hours prior
to the addition of 10 pig. of thymidine-2-04
per culture. DNA
synthesis was measured from the amount of thymidine-2-C'4
incorporated
into DNA.
thymidine.2-C― were added to the cal
tures and the incorporation
into DNA
was measured over a 4-hour period.
TABLE
2
PARALLEL REMOVAL BY DNASE OF DNA AND
II
2.32j@/HR,
§ 0
8
RADIOACTIVITY
FROM
THYMIDINE.
2-C'4LABELEDCELLS
N.J
•@Gi•
10
Houss
£Pm
7.
TUTMIDDII
5-C―
aaa&zanioR.4DI0AcTITITT
aa@[email protected]
ia
DNA5EDNA
centCounts/
sascusHocus
cent6
z@
/
0@3-
2
4
80
@°Ij
I
I
mm/flaskPer
I
Ill
I 2 3 4 5 6 7 8 9 10 II 12
HOURS AFTER
THYMID1NE-2-@d4' ADDITION
0
2
414.2
6.4
2200
45
48
2.4
17
800
16
100
6140
100
15.6
3.8
1600
26
25
1.6100 10.55180 600100 10.8
Replicate monolayer cultures were sacrificed 6 or 8 hours
after
rescue
with
thymidine-2.C'4.
The cell sheets
were washed
8 times with saline, precipitated on the glass with 4 per cent
perchioric acid for 2 minutes at room temperature,
washed
with distilled water, and incubated
at 87°C. with DNaae
(Nutritional Biochemicals Corporation 1 X recrystallized) 250
CHART
rescue
2.—The
acceleration
of thymidine-deficient
of DNA
cultures
synthesis
following
as measured
accumulation of thymidine-2-C―in DNA.
the
by the
pg/mi in 0.1 M tris buffer containing 10' M MgSo4. Monolay.
ers were prepared and analyzed for DNA as described under
“Materials and Methods.―
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
MUELLER ci al.—Effect of Puromycin
@
state. Since the generation time of the cells was
about
hours in monolayers, these data corre
spond to an over-all DNA synthesis time of 6.6
hours; this agrees with observations on other ani
mal cells in culture.
While the addition of amethopterin
blocks the
corporation
10 ag/mi
permitted
synthesis
attached
to the glass in a morphologically
of thymidine
deficiency
process
state
and
trated
@
hours'
90
per
cent
and
nearly
all the
cells
to remain
intact
synthesis,
With this concentration
of puromycin the in
hibition of protein synthesis could be reversed
readily by removing the puromycin
medium,
washing the cell sheet once with 10 ml. of fresh
chart,
this
the
synthesis
continues
results
of ribo
(1@). As illus
in a progressively
greater number of cells becoming triggered for or
achieving the basic competency to initiate the syn
thesis of their DNA: by 16 hours after the addition
of amethopterin,
over 90 per cent of the cells are
able to synthesize DNA if provided with a source
of exogenous thymidine. Accordingly, the acceler
ation of DNA synthesis observed during the first
hours of the thymidine rescue must take place in
cells already in the process of DNA synthesis.
Counting the number of silver grains in the au
toradiographs permitted a comparison of the rela
tive abilities of the individual cells to incorporate
tritiated thymidine into DNA during a short ex
posure. In the case of the logarithmically
growing
cultures, 30 per cent of those cells which were in
the process of DNA synthesis were labeled ap
proximately twice as heavily as the rest of the cells
which were making DNA. A similar examination
of cells from cultures which were synchronized by
16
@
a thymidine
by
all incorporation
(Chart 4).
concentrations
of puromycin
proportion of the cells to de
a puromycin concentration of
form over a period
of DNA
in the
produces
of leucine-1-C'4
@5j@g/ml blocked
Whereas the higher
caused a significant
tach from the glass,
in those cells which are in the
nucleic acid and protein
@
1087
on DNA
pretreatment
with
amethopterin
8I0
I4
HOURS AFTER
AMETHOPTERIN ADDITION
re
vealed that the actual number of heavily labeled
cells remained the same while the percentage
dropped to 8 per cent. This indicates that the
newly triggered cells entered into a state of light
labeling when first rescued from the thymidine de
ficiency. Two hours later and after the acceleration
had taken place, almost all cells could be classified
as labeled heavily. This proved further that the
observed acceleration of DNA synthesis was tak
ing place in a large majority of the cell population.
The influence of puromycin on the accelera2ion of
DNA synthesis.—A number of investigators have
demonstrated
that puromycin is a highly effective
inhibitor of protein synthesis in both intact ani
mals and cell-free systems (@, 9, 10, 15, 16). This
inhibition results from the blocking of the trans
fer of activated amino acids from soluble RNA into
the formation of peptide bonds in ribosomes (16).
Since the observed acceleration of DNA synthesis
in thymidine rescued cultures might well depend
on protein synthesis, experiments with puromycin
were done. In the initial studies it was demon
strated that puromycin was indeed highly effective
in blocking protein synthesis in HeLa cultures and
acted immediately;
10 pg/mi inhibited the in
of 8—1@hours.
CHART 8.—The accumulation
synthesis following the induction
of nudei capable of DNA
of a thymidine deficiency.
Populations of HeLa cells grown for the indicated times in
amethopterin
medium were permitted
thymidine
for a 10-minute period,
to incorporate tritiated
and the percentage
of
labeled nuclei was determined (upper curve). The lower curve
depicts the expected cell-number
increase from control cultures
supplemented with thymidine throughout
triggering
of nuclei
are plotted
The data for the
as N plus 100 per cent
to show
the relationship of the triggering event to the expected cell
number increase.
BEHM,
and
replacing
this
wash
with
10 ml.
of
puromycin-free medium. Although the resumption
of protein synthesis was almost immediate, the
rate of leucine incorporation
was slightly slower
than the controls after
hours of puromycin treat
ment (Chart 5). This effect was exaggerated some
what by an additional
hours of puromycin treat
ment.
The viability of the puromycin-treated
cells was
further
tested
by the ability
of individual
cells to
give rise to clones. Such studies demonstrated that
cloning efficiency declined only 15 per cent after
3 hours
in puromycin
dicate, however,
medium.
Other
that puromycin-treated
studies
in
cells re
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
@
Cancer Research
1088
tamthecapacity
tosynthesize
poliovirus2
onre
versal of the inhibition. Accordingly the puromy
cm effects are largely and readily reversible within
the exposure periods used.
In Chart 6 the effect of puromycin treatment on
the acceleration of DNA synthesis in cultures res
cued with thymidine is illustrated. It is evident
that
puromycin
does
not
inhibit
DNA
synthesis
which is in process, but acts to prevent the accelera
Vol.
October
196@
demonstrated
in Chart 7, where the removal of
puromycin resulted in an immediate acceleration
of DNA synthesis in the cultures. It was thus
demonstrated
that
the
cultures
were
still
compe
tent to respond. It is of interest that the kinetics
of the acceleration of DNA synthesis were similar
whether the puromycin was removed after
@,4,
or 8 hours.
Of additional interest in these studies was the
observation that cells blocked with puromycm 4
hours after the reversal of the thymidineless state
failed to divide until the puromycin was removed
from the cultures. The possibility that the syn
thesis of a particular protein necessary for mitosis
occurs at this time is being investigated.
However,
in accord with the findings of DNA it is also pos
sibie the puromycin sensitive reaction was affect
ing the duplication of the last few DNA molecules
30
LEUCINE-C14
LNCORPORATLO@4
/Jc3
PUROMYCIN
PER
ML
z
[email protected] 4.—Suppression by puromycin of leucine-1-C―incor
poration into HeLa protein. Replicate monolayer cultures in
the logarithmic phase of growth, prepared as described under
“Methods,―were overlaid with S ml. prewarmed BERM con
taming puromycin at the indicated concentrations
and 10 pg.
DL-leuclne-1-C― (sp. act. 8.75 mc/m.mole). After an incorpora
tion
period
described
of 4 hours
under
at 87°C., monolayers
“Methods―and counted
were sacrificed
as
for radioactivity
in
0
20
0.
@1
0
0
the acid-insolubledefatted monolayer. Data are expressedas
per cent of incorporation
occurring
mycin (6,820 counth/min/culture).
in controls
without
10
pure
0.
0
@
tion of DNA synthesis. Thus the addition of puro
mycin 4 hours after the addition of exogenous
thymidine had little influence on the established
maximal rate of DNA synthesis. However, when
added
hours after thymidine rescue, labeling
proceeded at almost control rates for the first few
hours, then decelerated as the amount of DNA
synthesized
@
in the
culture
tended
to plateau.
When
added at zero time, however, puromycin permitted
a steady linear rate of labeling but completely oh
literated the acceleration of labeling noted in the
controls. A similar result was obtained when puro
mycin was added
hours prior to thymidine
rescue.
Of particular
interest
in these
experiments
was the observation that the amount of DNA syn
thesized under the influence of puromycin tended
to plateau at different levels depending at which
point in the wave of DNA synthesis the puromycin
was added.
The reversibility
of this puromycin
effect is
2 J@ Olson
and
G. C. Mueller
(unpublished
data).
2
4
6
8
10
“OURS
CHART 5.—Reversal
synthesis.
Cultures
of the puromycin
inhibition
of protein
of HeLa cells were exposed to puromycin
10 pg/mi for 2 or 4 hours, at which time the monolayers
were washed with BEHM and the medium was replaced with
puromycin-free medium. Leucine-1-C― was present throughout
the experiment.
Data are expressed as counts/mm
in total
protein of the culture.
of the cell's complement of genetic material. In
any case puromycin appears also to offer a tool
for studying the specific events associated with
mitosis.
DISCUSSION
The data on the molecular events in the repro
duction of mammalian cells in this and the subse
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
MUELLER
et al.—Effect
of Puromycin
quent papers of this series can best be integrated
in terms of a fundamental
“cell
cycle―such as de
picted in Chart 8. As illustrated, the daughter cells
formed by a previous mitosis enter the interphase
state; in this condition they synthesize the macro
molecular and micromolecular
products
which
characterize them as specific cell types (i.e., skin,
liver, kidney, etc.). Cells may remain in interphase
for weeks, months, or even years depending on ex
ternal stimulating factors and the reactive poten
tial of the particular cell type to its environment.
on DNA
1089
mulates the rest of the cells in the triggered state
in which they are ready to initiate DNA synthesis,
but are unable to do so because of the deficiency
of thymidine. From these data it can be concluded
that the triggering phenomenon in itself is inde
pendent
of the
I0
presence
of thymidine
derivatives
THVMIDINE-C14
-INTODNA
Ui
CONTROL
a:
PURO-2HRS
Lii
PURO
4HRS
a:
PURO
8HRS
U
a:
Lii
0@
MEDIUM
0
(3
CHART
synthesis.
CHANGED
I
I
2
8HOURS
4
7.—Reversal
of
the
Thymidine-rescued
6
puromycin
cultures
effect
of HeLa
on
DNA
cells were
treated with puromycin 10 pg/mI for 2, 4, or 8 hours and
then changed to puromycin-free
medium. Thymidine-2-C'4
was present throughout the experiment. Data are expressed as
HOURS AFTER
THYMIDINE RESCUE
CHART
corporation
6.—Effect
of
puromycin
on
into DNA of synchronized
the accumulated counts/zulu of thymidine-2-C―
incorporated
into the DNA of the culture.
thymidine-2-C―
in
HeLa cells. Replicate
monolayer cultures of HeLa were previously grown for 16
hours in amethopterin
medium to induce the critical thymidine
deficiency state as described under “Methods.―At the time
L.:..@;'
(DAUGHTER
CEL.t.S)
designated “zero,―
cultures wererescuedby adding thymidine
2-C'4 (sp. act. 1.15 X 10 counts/min/pmole) to a final con
centration
of 10'
is. Puromycin
(10 pg/mi)
was added
at —2,
0, 2, and 4 hours with respect to the addition of thymidine.
.
@
=controls
(no
puromycin);
J =puromycin
added
2
hours prior to thymidine;
®=puromycin
added at time of
thymidine rescue; 0 =puromycin
added 2 hours after thymi
dine rescue; 0 =puromycin
added 4 hours after thymidine
rescue.
However, through processes which require both
protein and RNA synthesis (1@) they may over
come the proposed barriers (A-E) and progress to
the “triggered―state. In this state they are en
dowed with a basic competency to initiate the
duplication of their genetic material, DNA.
In the logarithmically
growing HeLa cultures
used in these experiments only 80 per cent of the
cells of the random population
are engaged in
DNA synthesis at any one time. However, growth
in the amethopterin
medium for 16 hours accu
I
A
@.INTERPHASE
DNA
SYNTHESIS
FUNCTIONS
@t
C
NUCLEAR
AND
CELL.
GROWTH
TRIGGER
CELL
CHART
8.—A
hypothetical
CYCLE
reproductive
cycle
of
a mam
malian cell. A—'E constitute
stages or barriers within the
interphase period which have to be overcome in the progression
of a cell toward the initiation of a nuclear replication.
Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
@
Cancer Research
1090
and that this state can be maintained for appre
ciable periods of time in the absence of DNA syn
thesis (i.e., at least 8—1@
hours).
The reversal of the thymidineless state was at
tended by the initiation of DNA synthesis in all
cells simultaneously;
however the newly synthe
sized DNA was accumulated at a slow rate in the
first hour of the reversal and only after the second
hour was the maximal rate of accumulation
at
tamed. The observation
that this acceleration
could be prevented with puromycin under condi
tions which blocked protein synthesis suggests
that the synthesis of some specific proteins may
be required for the acceleration.
As shown by the autoradiographic
studies of
the subsequent paper (13), the DNA which is syn
thesized during the first hour after reversal of the
thymidineless
state is distributed
nonrandomly
among the various chromosomes of the HeLa cell
and is also distributed
nonrandomly
along the
length of a specific chromosome. These studies
also have demonstrated
that the acceleration of
DNA synthesis observed in the present study is
correlated
with
the
activation
of many
more
sites
of DNA synthesis along the chromosomes.
Thus
the synthesis of DNA in this mammalian cell nu
Vol.
October
196@
ACKNOWLEDGMENTS
The authors are very grateful for the excellent and cc
operative assistance of Mrs. Eleanor Erikson and Mrs. Kath
leen Deighton.
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Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1962 American Association for Cancer Research.
Molecular Events in the Reproduction of Animal Cells: I. The
Effect of Puromycin on the Duplication of DNA
Gerald C. Mueller, Kazuto Kajiwara, Elton Stubblefield, et al.
Cancer Res 1962;22:1084-1090.
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