THE MECHANISM
SYNCHRONY
IN VI CIA
OF 5 - A M I N O U R A C I L - I N D U C E D
OF C E L L D I V I S I O N
FABA
ROOT M E R I S T E M S
WOLF PRENSKY,
Ph.D., and H A R O L D
H. S M I T H , Ph.D.
From the Biology Department, Brookhaven National Laboratory, Upton, New York. Dr. Prensky's
present address is Department of Physiology, Tufts University, School of Medicine, Boston
ABSTRACT
Cessation of mitosis was brought about in Vicia faba roots incubated for 24 hours in the
thymine analogue, 5-aminouracih Recovery of mitotic activity began 8 hours after removal
from 5-aminouracil and reached a peak at 15 hours. If colchicine was added 4 hours before
the peak of mitoses, up to 80 per cent of all cells accumulated in mitotic division stages. By
use of single and double labeling techniques, it was shown that synchrony of cell divisions
resulted from depression in the rate of DNA synthesis by 5-aminouracil, which brought
about an accumulation of cells in the S phase of the cell cycle. Treatment with 5-aminouracil may have also caused a delay in the rate of exit of cells from the G~ period. It appeared to have no effect on the duration of the G~ period. When roots were removed from
5-aminouracil, DNA synthesis resumed in all ceils in the S phase. Although thymidine
antagonized the effects of 5-aminouracil, an exogenous supply of it was not necessary for
the resumption of DNA synthesis, as shown by incorporation studies with tritiated deoxycytidine.
INTRODUCTION
Smith, Fussell, and Kugelman (10) have reported
partial synchronization of cell division in bean root
meristems during recovery of mitotic activity following incubation in 5-aminouracil (5-AU).
Synchronization of cell division in higher plants
normally occurs only in processes associated with
gametogenesis. This phenomenon has been utilized
extensively by Hotta and Stern (3) to describe
biochemical events associated with gametogenesis,
and by Sparrow (9) to correlate radiosensitivity
with various stages of the meiotic division cycle in
Trillium. The partial synchronization of mitoses
obtained by pretreatment with 5-AU has been
utilized by Prensky and Smith (8) to study chromosomal proteins through successive cell division
cycles in Vicia faba. Both the intrinsic value in
understanding the effects of a chemical synchronizing agent and the practical value of
utilizing such information for other studies of
cellular metabolism have prompted us to investigate further the effects of 5-AU on cellular activity
in bean root meristems.
Woods and Duncan (16) and Duncan and
Woods (1) have shown that 5-AU depresses
mitotic activity in root meristems of Allium cepa.
They also observed a decline in the number of
nuclei with a 4C content of DNA after a 24-hour
treatment with 5-AU. From their data they implied that 5-AU interfered with normal DNA
metabolism. The present study was designed to
extend previous studies on the effects of 5-AU on
processes associated with cell division in Vicia faba
401
a n d to c o r r e l a t e t h e effects of 5 - A U o n D N A synthesis w i t h t h e d e c l i n e a n d r e c o v e r y of m i t o t i c
activity.
MATERIALS
AND
METHODS
Vicia faba Cultures
Seeds were w a s h e d for 15 m i n u t e s in 5 per cent
Clorox a n d detergent, soaked for 24 hours in r u n n i n g
t a p water ( a p p r o x i m a t e l y 16°C), a g a i n disinfected
in Clorox, a n d finally rinsed in t a p water, G e r m i n a tion was t h e n c o n t i n u e d in vermiculite at 24 to 25¢C,
a relatively h i g h t e m p e r a t u r e w h i c h gave smaller
losses of seedlings d u e to fungal infection. After 6
days, seedlings with p r i m a r y roots 5 to 8 c m long were
transferred to 1/~ strength H o a g l a n d ' s m i n e r a l n u trient solution. T h e s e cultures consisted of a b o u t l0
seedlings per liter of m e d i u m , a n d were placed in a
d a r k c h a m b e r at 19°C. If s e c o n d a r y roots were desired, the tip o n t h e p r i m a r y root was broken off
u p o n transfer to liquid culture. O n the other h a n d ,
secondary roots were r e m o v e d daily if meristems of
the p r i m a r y roots were to be e x a m i n e d .
5-Aminouracil Treatments
Studies were first m a d e on the concentration of
5 - A U w h i c h w o u l d result in complete suppression of
mitosis after a 24-hour i n c u b a t i o n a n d s u b s e q u e n t
o p t i m u m s y n c h r o n y of cell division w h e n the seedlings were r e i n c u b a t e d in n u t r i e n t solution. C o n c e n trations of 5 - A U f r o m 100 t h r o u g h 800 p p m were
tested, a n d all concentrations resulted in a mitotic
i n d e x of no m o r e t h a n 0.5 per cent, c o m p a r e d with
control values of f r o m 5 to 12 per cent. A concentration of 500 p p m was the lowest to yield satisfactory
s y n c h r o n i z a t i o n of cell divisions in b o t h p r i m a r y a n d
s e c o n d a r y root meristems recovering from the effects
of 5-AU. M a x i m u m recovery at 19°C was obtained
15 h o u r s after r e m o v a l from t h e 24 h o u r i n c u b a t i o n
period in 5-AU.
Stocks of 5 - A U were obtained f r o m C a l b i o c h e m
Co. (Los Angeles, California) or f r o m E a s t m a n
K o d a k Co. (Rochester, New York). Solutions of t h e
c o m p o u n d varied in color a m o n g lots from colorless
to a deep yellow. T h e 5 - A U was therefore purified
b y recrystallization from cold water after dissolving
to n e a r s a t u r a t i o n levels at 85 to 90°C. Successive
recrystallizations resulted in progressively less colored
5 - A U preparations. C r u d e a n d recrystallized 5 - A U
samples were c o m p a r e d for the effect on mitotic
index depression a n d on mitotic peak at + 1 5 hours
(in t h e text a n d tables, "+" preceding " h o u r s "
signifies time after a 24-hour i n c u b a t i o n period in
5 - A U ; " h o u r s " w i t h o u t a plus sign refers to time
d u r i n g the 5 - A U i n c u b a t i o n period). N o difference
b e t w e e n t h e c r u d e m a t e r i a l a n d samples recrystal-
402
lized u p to 3 times could be observed o n t h e basis
of t h e mitotic index at 24 or + 1 5 hours. For all
labeling e x p e r i m e n t s 5 - A U , obtained from E a s t m a n
K o d a k a n d purified once by recrystallization, was
dissolved in distilled-deionized water to yield a concentration of 500 p p m ( ~ 4 )< 10 -3 M).
Isotope Incorporation Studies
T h y m i d i n e - H ~ ( T D R - H ~) a n d t h y m i d i n e - C 14
( T D R - C 14) were obtained from N e w E n g l a n d
N u c l e a r Corp., Boston, at specific activities of 3,200
a n d 25 me/raM, respectively. T D R - C 14 was used at
a concentration of 1 ~ c / m l . T h e c o n c e n t r a t i o n of
T D R - H ~ varied according to the purpose of the experiment. If root tips were to be fixed i m m e d i a t e l y
after labeling, solution concentrations u p to 1 0 / ~ c / m l
were used to o b t a i n short exposure autoradiographs.
W h e n root tips were to be fixed a considerable
length of time after the labeling period, £e. sufficient
for the incorporated t r i t i u m to affect the cell cycle
(6, 11), 0.5 to 3 ~ c / m l was used. D o u b l e labeling with
T D R - H 3 a n d T D R - C 14, to study the rate of exit of
ceils f r o m t h e D N A synthetic (S) phase, was done on
the basis of premises outlined by W i m b e r a n d
Quastler (15).
Since 5 - A U is a n analogue of t h y m i n e , it was considered advisable to try a labeled precursor other
t h a n t h y m i d i n e for the d e t e r m i n a t i o n of the rates of
D N A synthesis in 5 - A U treated material. Roots of
V. faba seedlings were i n c u b a t e d in concentrations
r a n g i n g from 2 to 5 /~c/ml of deoxycytidine-H 3
( C D R - H ~) p u r c h a s e d from Schwarz BioResearch,
Inc., O r a n g e b u r g , N e w York tspecific activity 1,100
mC/mM).
Roots were g r o w n in dilutions of the labeled precursor in distilled-deionized water or in 500 p p m of
5-AU. T h e y were fixed in alcohol-acetic acid (3:1),
a n d a u t o r a d i o g r a p h s were p r e p a r e d f r o m Feulgenstained squashes of the t e r m i n a l 1.5 m m of the roots.
Slides were dipped in K o d a k N T B liquid emulsion,
a n d processed in the u s u a l m a n n e r (D19 developer,
5 minutes, at 18°C). W h e n a thick emulsion was
desired, to differentiate b e t w e e n C 14 a n d H 3 labeled
cells, one coating of N T B was f o u n d a d e q u a t e w h e n
the slides were dried in a horizontal position.
T h e a u t o r a d i o g r a p h s were m o u n t e d in Euparol,
a n d finished preparations of each root tip were exa m i n e d for nuclear labeling with a Zeiss 100 X oil
i m m e r s i o n lens at a total magnification of 1,280.
RESULTS
Partial Synchronization of Cell Division
I n agreement w i t h p r e v i o u s w o r k ( l , 10), 24
h o u r s i n c u b a t i o n of V. faba seedlings in 700 p p m
a q u e o u s s o l u t i o n of 5 - A U d e c r e a s e d t h e m i t o t i c
THE JOURNAL OF CELL BIOLOGY • VOLUME ~4~, 1965
I~OVRE 1
Viciafaba: squash of root meristem grown in normal growth medium. )< 880.
index in root meristem cells from a control value
of a b o u t l0 per cent to a n index of 0.0 to 0.5 per
cent (Figs. 1 a n d 2). This result was obtained
with Tradescantia paludosa a n d Allium cepa root
meristems as w e l l However, in Tradescantia
(tested at 50, 100, 250, a n d 700 p p m of 5-AU),
recovery of mitotic activity was highly erratic,
a n d work was discontinued with this species.
O n i o n root meristems, treated with 250 p p m of
5-AU, recovered their mitotic activity at a rate
analogous to t h a t of V. faba; the mitotic index at
+ 1 4 hours was a b o u t 48 per cent.
Observations of p r i m a r y root meristems treated
with 500 p p m confirmed the d a t a on secondary
root meristems presented by S m i t h et al. (10).
R e d u c t i o n in the n u m b e r of mitoses after a 24
h o u r 5-AU t r e a t m e n t was not d e p e n d e n t on concentration in the range of 100 to 700 p p m of 5AU. Partial cell division synchrony at -t-15 hours
was not d e p e n d e n t on concentration from 400 to
700 p p m of 5-AU, w h e n i n c u b a t e d at 19°C.
T h e rate of recovery from the effects of 5-AU
is highly d e p e n d e n t on temperature. Limited
observations indicate t h a t following t r e a t m e n t
with 5-AU at 19°C, with a recovery period temperature of 12°C, the peak of mitotic activity is
reached at a b o u t q-30 hours. A t this t e m p e r a t u r e
a large n u m b e r of prophase figures was often
observed at + 2 8 hours (Fig. 3).
Fig. 4 shows the a p p e a r a n c e of a partially
synchronized root tip at + 1 5 hours, recovered at
a t e m p e r a t u r e of 19°C. A t this t e m p e r a t u r e all
division stages are present, a n d a n a c c u m u l a t i o n of
a particular stage has not been observed.
Since cell divisions c o m m e n c e at + 8 hours
a n d reach a peak at q-15 hours, we tried to accumulate mitotic figures by the colchicine m e t h o d
with results as shown in Fig. 5. A b o u t 70 to 80
WOLF PRENSKY AND HAROLD •.
SMITH 5-Aminouracil-Induced Synchrony
403
FI(IURN ~ Squash of root meristem after ~4 hours incubation in 500 ppm of 5-aminouraeil. Cell divisions
absent. X 5~0.
per cent of the cells are in mitosis, which appears
to be the maximal frequency of mitosis one can
obtain with this experimental system.
Reversal of 5-Aminouracil Effects
5-AU is structurally related to thymine through
the replacement of the 5-methyl group of thymine
w i t h a 5-amino group. It is therefore of interest to
see whether the effects of 5-AU can be reversed by
uracil or other pyrimidine-containing compounds.
Seedlings of V. faba were incubated in 200 ppm
5-AU which completely suppressed mitotic activity after 24 hours. Uracil, thymidine, thymidylic
acid, and cytidylic acid were added in equimolar
concentrations to the 5-AU. Meristems from four
primary roots were examined for frequency of cell
divisions after 24 hours of growth in each of the
treatments. Only general observations on the material were made, as shown in Table I. Both thymidine and thymidylic acid completely antagonized 5-AU effects; in fact, the frequency of cell
404
division was about 1.5 X the control value. Cytidylic acid evidently killed most of the root tips,
which became leathery and unsatisfactory for
cytological observation. Uracil had no apparent
effect on the response to 5-AU.
D N A Synthesis in 5-AU Treated Root
M eristems
Since thymidine antagonized the effect of 5-AU,
labeling with T D R - H a might in itself affect D N A
synthesis and obscure results with 5-AU. We
therefore investigated the incorporation of C D R H ~ into root meristem nuclei. Secondary root
meristems were pulse-labeled for 30 minutes (2
/zc/ml) during the middle or end of the 5-AU
treatment period, and root tips were fixed immediately and + 15 hours for each labeling time.
A control lot consisted of pulse-labeled roots which
had not been grown in 5-AU, and which were
fixed immediately after labeling. The results of
this experiment are presented in Table II; the
Tn~. JOURNAL OF CELL BIOLOGY • VOLUME ~4, 1965
appearance of labeled cells at + 15 hours is shown
in Fig. 6.
The labeling index found immediately after
either pulse label did not account for the high
labeling index of dividing and interphase cells in
roots fixed at + 15 hours. Furthermore, there was
a large difference between the labeling index of
roots grown in 5-AU and the controls. The latter
had a labeling index consistent with our experience
with T D R - H a labeling. The comparison between
the 5-AU treated roots and the controls suggests
an inhibition of C D R - H 3 incorporation by 5-AU.
A comparison between the labeling indices in
roots fixed immediately after labeling and at + 15
hours suggests that C D R - H ~ enters a precursor
pool from which it continues to label D N A for
extended periods of time. The following experiment was designed to test whether the continuous
labeling characteristics of C D R - H a are restricted
to 5-AU treated material.
A longitudinal cut, 3 to 4 rnm in length, was
made in each of four primary root tips, which were
then labeled with 5 /~c/ml of C D R - H 3 for 30
minutes. One half of each root tip was fixed immediately after labeling, the other half was fixed
after growing for 8 hours in the absence of exogenous C D R - H 3. The labeling intensity of the two
lots is presented in Table III. Cell division occurring during the 8 hour interval would be expected
to increase the labeling index because of the proliferation of labeled cells. However, if C D R - H 3
were available for a short period of time, i.e. if it
behaved as a pulse label, an increase in the labeling index would be accompanied by a reduction
in the average number of grains per labeled nucleus. O n the other hand, if the label is available
for a considerable amount of time, and in sufficient quantity, then new cells will be labeled as
they come into the S phase. The product of the
labeling index and the grain count would not be
expected to increase with time unless there was
either further incorporation into already labeled
ceils, incorporation into new cells, or both. The
results shown in the last column of Table I I I are
interpreted to mean that C D R - H 3 continued to
label nuclei after the initial treatment period.
l~¢vR~, 3 Synchronization of cell division after 5-aminouracil treatment. Root meristem fixed at q-~8
hours, recovery temperature 12°C. Large numbers of prophases are evident. )< 740.
WOLF PRENSKY 3.NI) HAROr~D H, SMITH 5-Aminouracil-~lnd~tced 8ynchrony
4,05
FmURE 4 Synchronization of cell division after 5-aminouraeil treatment. Root meristem fixed at +15
hours, recovery temperature 19°C. All stages of mitosis are present. )< 4~0.
Fig. 7 compares the pulse labeling characteristics of T D R - H 3 a n d C D R - H 3 after varying periods
of i n c u b a t i o n in 500 p p m of 5-AU. T h e n u m b e r of
cells incorporating T D R - H 3 almost doubles after
a 20 h o u r i n c u b a t i o n period in 5-AU. I n agreem e n t w i t h the experiments discussed above, the
n u m b e r of cells incorporating C D R - H 3 decreases
with time of i n c u b a t i o n in 5-AU. Since T D R - H 3
appears to be incorporated b y all cells in the S
phase, the effect of t h y m i d i n e in antagonizing
5 - A U is postulated as due to its supplying a precursor for D N A synthesis, the supply of w h i c h is
inhibited by 5-AU. C D R - H 3 a p p a r e n t l y does not
satisfy a limiting precursor requirement, a n d the
reduced labeling indices a n d grain n u m b e r s per
labeled nucleus reflect a slower rate of D N A synthesis in 5-AU treated roots. Consequently,
labeling with C D R - H 3 is a better way of measur-
406
ing rate of D N A synthesis in the presence of 5-AU
t h a n labeling with T D R - H ~. Both incorporation
per cell a n d fraction of cells incorporating measurable a m o u n t s of C D R - H 3 decrease with time
of 5-AU incubation. O n the other h a n d , the
labeling index obtained with T D R - H 3 indicates
the proportion of cells t h a t reach the S phase of
the cycle d u r i n g the 5-AU incubation period. It
m a y be concluded t h a t t r e a t m e n t with 5-AU accumulates cells in the S phase because of a n inhibition of D N A synthesis due to 5-AU preventing
cells from completing D N A replication. Since the
pool of cells w h i c h can enter the S phase must
diminish with time, the relatively largest a c c u m u lation occurs in the first 8 hours of the 5-AU treatm e n t period. Experiments with T D R - C ~4, which
were c o m p a r a b l e in protocol with the experiments
THE Jot~aN.~L OF C~LL BmLOGr • VOLVME ~4, 1965
FtGU]~E 5 Chromosome preparation after arrest of mitosis with eolehicine. Root transferred to colchieine
at +11 and fixed at -4-16 hours. X 340.
TABLE
I
Reversal of the Antimitotic Activity of
5-Aminouracil by the Addition of
Other Compounds
Mitotic index
after 24 hrs:~
Treatments*
(percent)
Control (I-/20)
5-AU
5-AU -]- Uracil
5-AU -t- Cytidylic acid
5-AU q- Thymidine
5-AU -+- Thymidylic acid
7-10
0
0
killed
10-15
10-15
* All concentrations at 1.6 X 10-3 M (= 200 ppm of
5-AU) in distilled-deionized water.
Five primary root meristems examined per treatmeat.
above confirmed the data obtained from tritium
labeling (Fig. 7).
The next question is w h e t h e r cells leave the S
phase during the 5-AU incubation period.
W i m b e r and Quastler (15) have shown that w h e n
cells are labeled with H 3 and C 14, with an appropriate interval between labels, two classes of
cells can be distinguished microscopically, those
labeled with H a alone, and those labeled with C 14
with or without H 3. W h e n thymidine is the isotope
carrier, the n u m b e r of cells labeled only with H 3
is a direct measure of the n u m b e r of cells which
were not in the S phase when the T D R - C 14 was
m a d e available. W e therefore labeled separate
lots of root meristems with T D R - H 3 for 30 minutes
before and during the 5-AU treatment period.
After a 24 hour incubation period in 5-AU, all
roots were labeled with T D R - C 14. T h e results of
this study are presented in Table IV. After the
WOLF PRENSKY AND HAROLD H. SMITH 5-Aminouracil-Induced Synchrony
407
T A B L E II
Labeling Index of 5-Aminouracil Treated Root Meristems when Pulse Labeled with C D R - H 3
Labeling index (cellslabeled)
Time of Labeling*
Interphase cells
Before 5-AU (Control)
10.5 w.o. (middle of 5-AU period)
Fixed at
11 hours
Fixed at +15 hours
23.5 w.o. (end of 5-AU period)
Fixed at 24 hours
Fixed at +15 hours
36.4 ~ 2.9
Dividing cells
per cent
18.8 :l: 4.7
74.3 :i: 2.3
97.9 -4- 0.3
7.7 4- 5.2
81.7 4- 2.4
94.2 -4- 2.4
* Time with respect to the start of a 24 hour incubation period in 5-AU ; 2/zc/ml of C D R - H 3 for 30 minutes,
4 secondary root t i p s / t r e a t m e n t ; approximately 250 cells/root were examined.
FIGURE 6 Nuclear labeling with CDR-H 3. Isotope applied during 5-aminouracil treatment period and
fixed at -4-15 hours. )< 840.
entire 24-hour period in 5-AU only 7.31 per cent
of the cells do not incorporate C 14. Half of these
cells probably completed D N A synthesis within
the H 3 trcatment period, and, in fact, most of the
cells labeled with tritium only were rather weakly
408
labeled. T h e values obtained at 16 and 20 hours
probably reflect the n u m b e r of cells which m a y
have completed D N A synthesis because of the
thymidine t r e a t m c n t itself. All cells labeled only
with H 3 were weakly labeled, which indicates that
T~E JOURNAL OF CELL BIOLOGY • VOLUME ~4, 1965
they may have been in the terminal stages of the S
period. We may assume, therefore, that few or no
cells leave the S phase during the 5-AU incubation
period. In our studies of the normal cell cycle in
V. faba, by use of the double-labeling technique,
we found that a 4-hour interval between H 3 and
C 14 resulted in an H3:C 14 ratio of 0.412 (leading to
an estimate of 9.7 hours for the average duration of
S. In the 5-AU treated material the 4.5 hour interval (line 3, Table IV) yielded an H3:C ~4 ratio
of 0.030. At this rate of exit from the S phase,
one cycle of D N A replicaton would presumably
last about 150 hours ( l
TABLE
Labeling Intensity in Root Tip Halves
After a Pulse Label with CDR-H 3.
Half root tips
Fixed immediately
Fixed 8 h o u r s
later
Difference
sE of difference
A
B
Labeling
index
Grain count
per labeled
nucleus
1"
D N A Synthesis Following Incubation in 5-A U
AXB
Total
incorporation
29.3
13.96
411.82
48.8
17.03
831.06
19.3
5.31~
3.07
0.2619~
* L a b e l e d w i t h 5 luc/ml ; d a t a f r o m 4 p r i m a r y r o o t
m e r i s t e m s split l o n g i t u d i n a l l y before labeling.
Averages based on g r a i n c o u n t s of 150 cells per r o o t
tip half.
Significant at the 0.01 level.
x
•
o
•
_
X 4.5~
III
HLCDR
H3-CDR
H3-TDR
C14 TOR
In the previous section we have shown that
incubation of roots in 5-AU causes an inhibition
and eventual suppression of D N A synthesis. T h e
observed synchrony of cell division must therefore
rest on the ability of the cells to resume synthesis
of D N A when the 5-AU titer is reduced by washing
and incubation in normal growth medium. With
about 80 per cent of the cells labeled in all treatments, the C 14 labeling index in Table IV is an
indication of the resumption of D N A synthesis and
we have, in fact, obtained similar values with a
pulse label of T D R - H 3 following a 24-hour incubation period in 5-AU.
Since Littlefield (7) showed that exogenous
T D R was necessary to overcome the inhibitory
effects of 5-fluorodeoxyuridine ( F U D R ) , we tested
the incorporation of C D R - H 3 following an 8 hour
grain count
L.I.
L.I.
L,I. not connected B
IO0
'100
-
--
o
8"8
=8
E
,,8
C
• --
8O
7~Y
I--
%%%
.c -r
--
60
6O
O
~g 40
40
o
T
~e
7 °
~
---
O
U
3"
.--
,~
20
2O
J
0
I
0
I
4
I
I
8
12
Labeling time
( H o u r s after start of
5-AU
I
f6
~ 0
20
¢¢
c
a ®
L) u
T
treatment)
FIGURE 7 Changes of the labeling index (L.I.) during 5-aminouraeil treatment. Roots labeled for 30
minutes and fixed immediately after labeling. Labeling index of control root meristems taken as 100 per
cent
WOLF PRENSKY AND HAROLD H. SMITH 5-Aminouracil-IndUced Synchrony
409
TABLE
IV
Results of Double-labeling to Test Completion of S During 5-Aminouracil Treatment*
Treatment
No.
1
2
3
T i m e of labeling with tt~
Interval between
H 3 and Ca4
hours
hours
--14
16
20
Labeling index
25.0
8.5
4.5
H~ only
C!~
7.31 5= 1.13
2.38 5= 0.47
2.45 5= 1.02
76.67 ::t= 2.51
80.37 5= 1.32
80.69 5= 4.27
* One half hour with ~IDR-H 3, 5/zc/ml in water for t r e a t m e n t No. 1, 10/ac/ml in 500 p p m 5-AU for treatments 2 and 3. T D R - C ~4, 1.5 /~c/ml administered in three 15 minute pulses at + 1 , + 2 , and + 3 hours
after a 24-hour growth period in 5-AU.
i n c u b a t i o n period in F U D R a n d 5-AU, without
the addition of T D R . T h e F U D R - t r e a t e d root
tips did not incorporate any C D R - H * (Table V).
T h e 5-AU treated root tips h a d a higher labeling
index t h a n the controls, reflecting the accumulation of cells in the S phase. T h e y also incorporated
more tritium per labeled cell, reflecting perhaps
a n a c c u m u l a t i o n of a precursor pool which is
utilized w h e n the block induced by 5-AU is released. W e c a n conclude, therefore, t h a t a n
exogenous supply of T D R is not necessary for
cells to resume D N A synthesis after removal of
the seedlings from the 5-AU solution.
A n o t h e r p r o b l e m is the frequency with which
cells t h a t will undergo mitosis at + 1 5 hours c a n
be labeled beforehand in the period of recently
resumed D N A synthesis. T o investigate this we
labeled 5 - A U treated root tips for 6 hours following removal from a 24-hour i n c u b a t i o n in 5AU. T h e root tips were fixed 9 hours after labeling ceased, w h i c h corresponds to + 1 5 hours, a
time w h e n the mitotic index is maximal. W i t h
this protocol, almost all the cell divisions in the
control lot (no 5-AU treatment) should be labeled,
assuming a 4.7 h o u r G~ a n d a n 8 h o u r S period
(2, ~, 5). T h e results are presented in T a b l e V I .
T h e mitotic labeling index of the 5-AU treated root
tips was lower t h a n in the control, which could
come a b o u t if the d u r a t i o n of the G~ period for
some cells is longer in the 5-AU treated root tips.
Effect of 5-aminouracil on the G2 Period
As noted in the preceding section, it a p p e a r e d
t h a t d u r i n g recovery, the G2 period is of somewhat
longer d u r a t i o n t h a n normal. T h e following exp e r i m e n t was designed to test for possible direct
effects of 5-AU on the progress of cells t h r o u g h
the G2 phase of the cell cycle.
410
TABLE
V
Recovery of DNA Synthesis Without Exogenous
Thymidine in 5-Aminouracil Treated Root
Tips*
Treatment before
labeling*
Control
5-AU, 500 ppm,
8 hours
F U D R , 10.7 M,
8 hours
Labeling index
Grains/labeled
nucleus $
25.8 -4- 2 5
62.0 5= 8 7
14.4 5= 2.3
22.2 5= 4.1
0.0
0.0
* Labeled with 4 ~ c / m l of C D R - H 3, 4 root tips
in each t r e a t m e n t 1 hour.
:~ Examined 150 nuclei in each root tip.
TABLE VI
Labeling Index o.f Dividing Cells when
Labeled after 5-Aminouracil Incubation
Period
Treatment before labeling*
Labeled mitoses
per cent
Control
5-AU, 500 ppm, 24 hours
95.9 4- 1.36
81.8 -4- 2.13
* Continuously labeled in 0.5 uc/ml, T D R - H 3, for
6 hours, 7 root tips in each treatment, and fixed 9
hours after labeling (+15 hours).
Seedlings with m a n y secondary roots were
labeled for 20 minutes in 3 ~ c / m l of T D R - H 3,
after which they were rinsed for 10 minutes in
distilled-deionized water, a n d r e t u r n e d to the
n o r m a l growth medium. After postlabeling intervals of 0.5, 1, 2, a n d 4 hours, seedlings were
transferred to 5-AU. T w o sets of controls were
employed, of which one was a n u n l a b e l e d set
THE JOURNAL OF CELL BIOLOGY • VOLCM~ ~4, 1965
entered mitosis after transfer to 5-AU. After
another hour in 5-AU, less than 1 per cent of the
dividing cells were labeled. The rapid decline in
the mitotic index in this treatment precluded
later observations. In the treatment involving
a 2 hour interval (D), the appearance of labeled
mitoses is delayed. However, the high labeling
index at 10 hours is based on very few cells since
the mitotic index at that time is very low (Table
VIII).
The mitotic index of the above material is presented in Table V I I I . The columns were shifted
from the a r r a n g e m e n t in Table V I I to show the
mitotic index after given lengths of time in 5-AU.
The data for unlabeled controls (treatment F)
correspond to those found by D u n c a n and Woods
(1) in Allium cepa. Because of the rather slow decline in the mitotic index u n d e r such conditions,
we had at first discounted the possibility that 5A U affects the G~ stage. With respect to the mitotic index, there appeared to be an interaction between the effects of 5-AU and the time of T D R H ~ labeling. The mitotic index of the treatments
labeled with T D R - H 3 0.05 a n d I hour before the
5-AU incubation period (treatments B and C) was
more variable than that of the control series. T h e
2-hour interval between T D R - H 3 and 5-AU
placed in 5-AU, and the other was labeled but
remained in the normal growth medium. Root
tips were fixed at hourly intervals up to l0 hours
after labeling, as shown in Table V I I .
A recently published estimate for the average
duration of the G2 period is about 4 to 5 hours
(2). It was expected, therefore, that in treatments with 2 and 4 hour intervals between labeling and incubation in 5-AU, an appreciable
n u m b e r of G2 cells would be labeled at the start
of the 5-AU incubation period (about 9 and 18
per cent of all cells, respectively, based on a 36
per cent labeling index and an average duration
of S of 8 hours). If passage through the G~ period,
or its terminal portion, is not delayed by 5-AU,
dividing cells should be labeled with a frequency
comparable with those controls which were not
placed in 5-AU. If the T D R - H 3 labeling interfered with the 5-AU treatment, the non-labeled
controls should have a mitotic index different
from the experimental group.
Autoradiographs of all T D R - H 3 labeled root
tips were examined for frequency of labeled
mitoses. These observations are recorded in Table
V I I . In all 5-AU treated root tips the frequency
of labeled mitoses is greatly reduced. This is apparent even in t r e a t m e n t E, in which at the start
of the 5-AU incubation period about 32 per cent
showed a decline in the rate of cell division similar
of the dividing cells must have been labeled. This
frequency declined to 19.2 per cent 1 hour later,
to the one found in the control, where no T D R - H 3
was used, whereas the 4-hour interval resulted in
indicating that few, if any, of the labeled cells
a decline which was faster than in the control.
TABLE VII
Passage of Labeled Cells Through the G2 Phase of the Cell Cycle." Per Cent Labeled Mitoses at
Various Times after Labeling*
Interval betweenTDR-H~
labeling and start of
Treatment
5-AU incubation
Labeled mitoses,hours after cessationof labeling
2
4
5
6
75
85
95
105
-0
0
.8
1.0
58.0
-.7
5.1
¶
90.3
-1.0
16.7
¶
81.6
-3.3
49.4
¶
per cent
A
B
C
D
E
Control: no 5-AU
1/~ hour§
1 hour§
2 hours
4 hours
0
--0[I
0H
32.1
7.9
--32.1 [[
33.8
1.3
.2
0
19.2
69.3
1.2
0
0
.5
* Secondary roots labeled with 3 # c / m l of T D R - H 8 for 20 minutes, rinsed, and returned to 1/~ Hoagland's.
After stated interval, treatments B-E were incubated in 5-AU, 8 root tips fixed at each period.
Low mitotic indices in treatments C, D, and E.
§ Labeled mitoses had few grains, especially in treatment B.
[I Same as control.
¶ Few mitoses found, all unlabeled, except in one out of 8 root tips fixed at 9 hours, where 31 out of I00
mitoses were labeled.
WOLF PRENSKY AND HAROLD H. SMITH 5-Aminourac~l-Induoed
Sgnehrony
411
TABLE
VIII
Mitotic Index o] Root Meristerns Incubated in 5-Aminouracil alter Labeling with T D R - H ~*
Interval
between
TDR_H3
Treatment
labeling and
start of 5-AU
incubation
Mitoses, hours in 5-AU~
1
2
3
4
5
10.7
--
--
9.3
7.0
---6.7
--
---6.3
10.6
--10.9
3.4
--
5.2
3.6
8.6
1.1
8.8
6.5
8.4
9.9
.9
--
6
7
8
9
10
7.9
--
9.7
9.2
--
7.2
9.5
7.6
.5
4.4
6.5
6.0
3.6
.
3.5
.
7.1
1.6
.
5.4
-.
1.0
.
--.
--
16
per cent
A
B
C
D
E
F
Control :
no 5-AU
1/~ hour§
1 hour
2 hours
4 hours
Control :
no T D R - H ~
.
.
.
1.2
--
--< .1
* Treatments identical with those in Table V I I , except for the addition of an unlabeled control series.
D a t a for t r e a t m e n t A are for hours after T D R - H 3 labeling.
§ Data for treatment B were taken 1/~ hour earlier than indicated in column heading.
T h e large differences in mitotic indices were
not anticipated, and therefore the experiment,
as it stands, is not suitable for elucidating the
cause for the range of effects actually observed.
It is possible that the mitotic index of treatments
B and C was influenced by the small amount of
thymidine available to the root tips shortly before
being transferred to 5-AU. This, however, does
not explain the rapid decline of the mitotic index
in t r e a t m e n t E. T h e failure of large numbers of
labeled cells to enter mitosis could imply a superimposition of a 5-AU induced radiosensitivity to
the beta radiation from the incorporated tritium.
T h e mitotic index of the controls, which were
labeled but not grown in 5-AU (treatment A),
does not appear to have been greatly affected by
the T D R - H 3 treatment. It appears, therefore,
that the structure of the data is markedly affected
by unresolved factors, though it is highly probable
that 5-AU affects the rate of progress of cells
through the G2 period.
Other Observations
It is clear that 5-AU, in depressing D N A synthesis, interferes somehow with thymidine m e t a b olism in V. faba. W e wanted to find out whether
related 5-amino compounds had similar effects.
Therefore, we tested the effect of a 24 hour incubation of V. faba roots in several concentrations of
both 5-aminodeoxyuridine and 5-amino-orotic
acid, but found no decrease in the mitotic index.
412
W i t h our techniques it has therefore been impossible to infer at w h a t step in the biosynthesis
of thymidilic acid 5-AU exerts its inhibitory
effects on D N A synthesis.
DISCUSSION
In this study we were able to correlate the induced
partial synchrony of cell division with a depression
in D N A synthesis during the 5-AU incubation
period, resulting in an accumulation of cells in the
S phase. W i t h a double labeling procedure involving an H a label followed by a C 14 label, it was possible to show that few, if any, cells leave the S
phase. Since a m a x i m u m of about 80 per cent of
the cells accumulates in the D N A synthetic phase
during 24 hours of 5-AU treatment, one m a y be
reasonably certain that the 5-AU treatment did
not materially affect the rate at which cells left
the G1 phase and entered S. H o w a r d and Pclc
(5) obtained up to 75 pcr cent labeling u n d e r
conditions of continuous labeling with pa2, for 24
hours, in V. faba. W i m b e r (12), with repeated
labeling during a 24 hour period with T D R - H 3,
found about 80 per cent of the cells labeled in
Tradescantia root meristem cells. Consequently,
this proportion apparently represents a value
which is close to the m a x i m u m fraction of root
meristem cells that it is possible to accumulate at
any stage of the cycle.
A direct test of whether the rate of entry into
D N A synthesis is affected by 5-AU could pre-
T H ~ JOURNAb OF CELL BIOLOGY - VOLUME ~4, 1965
sumably be m a d e with a C 14 label followed by a n
H 3 label (i.e., reversing the order of labeling used
in the experiment in T a b l e IV). W e did not report
the d a t a from this experiment because the values
obtained for the H3:C 14 ratios could not be interpreted in terms of the effect of 5-AU on the cell
cycle. This is reasonable because of the large difference in specific activities between C 14 a n d t-I3
labeled thymidine. At the specific activities available to us the concentration of thymidine is 128
times lower for c o m p a r a b l e activity per milliliter
w h e n T D R - H 3 is used t h a n w h e n it is substituted
by T D R - C 14. However, since the rate of accumulation of cells in S shown in our experiments appears
to be consistent with the d a t a of H o w a r d a n d
Pelc, a n d of W i m b e r , the failure of the reverse
double-labeling experiment is not a crucial
point. C u r r e n t work in our laboratory on the
labeling kinetics of H 3 after C ~4 labeling in untreated V. faba bears this out; it appears that more
a t t e n t i o n will need to be paid to absolute concentrations of thymidine in double-labeling experiments, especially where the C 14 label precedes
or accompanies the H 3 label.
A l t h o u g h we are reasonably certain t h a t 5-AU
does not affect the G1 period, there is some evidence of a delaying effect in the G: period. Aside
from the data in Tables V I I a n d V I I I , it is difficult to account for the incomplete labeling of the
cells recorded in T a b l e V I except by postulating
some sort of effect in the G2. D u r i n g a n d after the
5 - A U t r e a t m e n t there was a m u c h greater variability in q-DR-H 3 incorporation per cell in those cells
which were scored as " l a b e l e d , " c o m p a r e d with
controls. Part of the deficiency in labeled mitoses
m a y therefore be due to less t h a n detectable in-
corporation of T D R - H 3 in some cells. However,
G2 cells are, by definition (5), those which do not
incorporate detectable amounts of D N A precursor after the detectable D N A synthetic period has
been completed. T h e d a t a of W i m b e r a n d Davies
(13) on the occurrence of F U D R - i n d u c e d breaks
in the G2 phase, a n d observations on the a p p a r e n t
incorporation of T D R - H 3 into chromosomes during p a c h y n e m a of meiosis in male Triturus (14)
m a y be indicative of D N A synthesis in periods
following the m a i n D N A synthetic period. If
t h a t is the case, then the effect of 5-AU on the
G2 phase could occur by the same m e c h a n i s m by
which the S phase is affected, namely t h a t of
suppression of D N A synthesis by interference
with thymidine metabolism. Nor would this be
inconsistent with the d a t a we obtained with C D R H 3 labeling, since the continuous labeling characteristic of this precursor could be responsible for
the registration of cells as labeled w h e n a pulse
label would have placed t h e m in the unlabeled
category.
The authors wish to acknowledge the helpful discussions held in the course of this study with Doctors
Donald E. Wimber and Philip S. Woods. They are
also grateful to Mr. Keith H. Thompson for the
statistical analysis and processing of the data. The
photographs in this paper were taken by Mr. Robert
F. Smith of the Photography Department, Brookhaven National Laboratory. The senior author held
a United States Public Health Service Postdoctoral
Fellowship (GM-12,602) during part of the time
this research was being carried out. Work performed
at Brookhaven National Laboratory was under the
auspices of the United States Atomic Energy Commission.
Received for publication, March 20, 1964.
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WOLF PRENSKY AND HAROLD H. SMITH 5-Aminouracil-lnduced Synchrony
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THE JOURNAL OF CELL BIOLOGY • VOLUME ~4, 1965