EVIDENCE FOR SEQUENTIAL GENE ACTION WITHIN
THE CELL CYCLE OF PARAMECIUM'
EAR:L D. HANSON
AND
MASAO KANEDA2
Shanklin Laboratory, Wesleyan University, Middletown, Connecticut 06457
Received May 10, 1968
0 study the action of genetic material during the interfission cycle, single
Tcells of Paramecium aurelia were exposed for short periods to actinomycin D
under a variety of conditions. The result of such exposures was always a delay
in fission equivalent to an added fission period. Our interpretation of this phenomenon is consistent with a model of sequential gene action occurring within
a cell cycle.
MATERIALS A N D METHODS
The cell system studied here is Paramecium aureZia, syngen 4, stock 5.1, sensitive (i.e., SUCcumbs to killer paramecima (SONNEBORN
1950)). I t is cultured in a bacterised medium (HANSON
1963) in test tubes for stock icultures and in glass, 3-spot depression slides for experimental work.
Single cells are manipulated by micropipettes and are observed and counted for fission rate
determinations under low powers of a stereomicroscope (SONNEBORN
1950).
Except for the preliminary runs (see RESULTS) where the actinomycin was first mixed with
distilled water, uninoculated culture fluid was used to dissolve the antibiotic to provide a stock
solution of 10,OOO pg/ml. This was diluted further with uninoculated medium to provide the
concentration needed experimentally. Apart ifrom the periods of exposure to this medium o r to
its control equivalent, the cells were kept in fresh, inoculated culture medium. The actinomycin
was supplied through the courtesy of Merck, Sharp and Dohme, Rahway, N. J.
Pulsed exposure to actinomycin D at known times in the interfission period was achieved as
follows. A dividing cell was placed i n fresh culture fluid in the center depression of a 3-spot slide.
When it divided, the division time was recorded and then, 'as close to the desired time as possible,
one of the two cells was transferred to a depression which contained the desired concentration of
actinomycin D and the other fission product was transferred to a depression which contained
culture medium identical to that i n the experimental depression except that it lacked the
inhibitor. At the end of the (desiredtime, usually 5 to 6 min but varying between 3 and 30 min,
the cell being exposed to actinomycin was transferred again by a micropipette to a depression of
fresh culture fluid and then again to a second fresh depression. The amount of medium carried
over with the cell at each transfer was less than one hundredth of the volume of the medium in
a depression (ca. 0.5 ml) thus ensuring effective washing of the cell and dilution of the inhibitor
to an insignificant level. The control was similarly transferred.
The developmental or in1,erfissionage of the cells at initiation of the exposure was determined
from the interfission period of the unexposed sister cells. There is a good correlation between the
interfission periods of sister cells (KINBALL et al. 1959) which can give a correlation coefficient
unpublished). We
as high as 0.93 if cells are used within a few fissions of autogamy (KANEDA
!sed such cells in this work. The interfission period, T , of the experimental cell is given by
T = rT f (1-r) T, where T is the interfission period of the sister cell, T is the mean interfission period of all the sister cell controls and r is the correlation coefficient obtained separately
by comparing the interhiom periods of untreated sister cells. The d u r a t i y of the period from
the previous fission to the time at which exposure started is divided by T and gives the interThis work was supported by a research grant from the National Science Foundation (GB 3937).
' Biological Institute, Faculty of General Education, Hiroshima University, Hiroshima, Japan.
Genetics 60: 793-805 December 1968.
794
EARL D. HANSON AND MASAO KANEDA
fission age expressed as a decimal part of the estimated interfission period. I t is critical to the
design of the experiments used here that the interfission age be accurately determined since the
response of the cells to the inhibitor is distinctly different if exposure starts before interfission
age 0.86 as compared to results of exposures coming after that time.
Working with single cells has the advantage of providing careful control of each experimental
organism, but it also demands rigorous attention to the experimental protocol to ensure equivalent
treatment of all cells. In this regard two points were especially important: a) Only those dividing
cells were used which, upon isolation into fresh medium, completed fission normally-they did
not separate as a consequence of expulsion from the micropipette, there was no untoward delay
in separation, there was no evidence of ruptures in the fission plane, normal swimming behavior
was apparent, etc. b) For any given experimental run the same medium was used for all cells
since they appear to be very sensitive to changes in their environment. Gentle manipulations by
means of the micropipette, as for purposes of isolation, had no significant effect on their fission
rate but transfer to a new medium did. On the average, the first interfission period in fresh
culture medium took about 5 hrs at 29°C. The second period in the same medium took about
4.25 hrs provided the cells were minimally disturbed, which included avoidance of even slight
drying out of the culture depressions.
RESULTS
Preliminary pulse exposures to actinomycin revealed unexpectedly that cells
treated before the stabilization point (see below), which is at interfission age 0.86
(GILLand HANSON
1968), would not divide for one whole interfission period in
addition to the normal period they were already in. That is to say, citing a specific
example, a cell exposed at age 0.5 will not divide until another period and a half
have elapsed.
In this preliminary work the concentration of the actinomycin D was thought
to be 125 pg/ml of culture fluid. However, because the inhibitor was not completely dissolved, the actual concentration was far less than that and subsequent
experiments (see below) used other concentrations. The exposures in this preliminary work varied from 5 to 20 min and there was some direct correlation of
duration of exposure with duration of fission delay, i.e., certain of the longest
exposures showed delays somewhat in excess of one added fission period and the
shortest exposures had delays of approximately one extra period or slightly less.
However, there was nothing to suggest direct proportionality, that is, a 20 min
exposure did not give four times the delay of a 5 min exposure. The results of this
work are briefly given as follows: mean interfission period of the control sister
cells was 287.4 min and for the exposed cells 586.5 min (based on a total of 37
pairs of sister cells). The interfission period of the experimental cells is, then,
2.09 times longer than that of the controls. Furthermore, this relationship holds
for all cells regardless of when the exposure occurred, provided that it preceded
and ZEUTHEN1962; FRANKEL
1965 and
the stabilization point (RASMUSSEN
earlier papers; RASMUSSEN
1967; GILLand HANSON1968). This point is defined
as the time in the interfission period after which exposure to various antimetabolites, for example, will not suppress the morphogenetic events which culminate
in successful cell division. This point is interpreted as being the time, especially
with regard to actinomycin exposure, when all transcription necessary for the
next fission has been completed. Hence, exposures after this time do not affect
1965; GILLand HANSON1968).
the next fission (FRANKEL
S E Q U E N T I A L G E N E ACTION
795
Our preliminary results also included 19 exposures after the stabilization point
and in all cases the cells divided at their normal time (mean 260.2 minutes). I n
six cases, the experimeritals were followed through a second fission and in all
cases this fission was delayed by a whole additional fission period (mean 481.0
min, which is 1.95 of the control value).
Since the preliminary results gave no clear indication that the fission delay
was a direct consequencle of the specific experimental conditions used, this possibility needed to be tested further. We, therefore, examined the following four
factors: a ) concentration of the inhibitor, b) length of exposure to the inhibitor,
c) time of exposure in the cell cycle, and d) general culture conditions. The
rationale was simply to vary each of these four factors and look f o r correlated
changes in the fission delay. The results are given after describing an improvement in the experimental design first reported.
The experimental celll receiving a single pulsed exposure to actinomycin prior
to the stabilization point is best compared to the first two fissions of the sister cell.
This is because of the transfer effect, mentioned in the METHODS section, which is
apparent in the first division after transfer. This fission takes 4.5 to 5.5 hrs at
29°C in our material, whereas the second fission takes from 4 to 4.5 hrs. The same
timing would reasonab1.y be found in the experimental sister cells, and hence, it
is more accurate to compare the delayed fission of the experimental to the first
two fissions of the control cell. This ratio, E1/C1+2,where E , is the interfission
period of the first division of the experimental cell and C1+2is the sum of the first
and second interfission periods of the control cells, is expected to be unity, as seen
from the preliminary work.
The results of using three different concentrations of the inhibitor are given
in Table 1. In each case it can be seen that the average E/C value is not significantly different from 1.O. Furthermore, other concentrations lower than those
given in the table were employed. However, two different batches of actinomycin
were used and they slhowed somewhat different minimal levels for causing
delayed fissions. In one batch-used for the 90 and 40 pg/ml concentrations of
Table 1-30 pg/ml hald no effect, the controls and experimentals dividing at
essentially the same time. I n the other batch-used for the 36 p g / d results of
Table 1-25 pg/ml of the inhibitor had no effect. In effect then 36 pg of actinomycin from the one batch is equivalent to 40 pg of actinomycin/ml of culture
from the second batch. Actually, in later experiments (Table 3) this latter concentration was raised slightly to 45 pg/ml of fluid medium.
Before leaving Table 1 it is necessary to comment briefly on the somewhat
different mean interfission periods. We found that within any experimental run
we could get quite consistent interfission periods but from one day to another
there were differences that we can only attribute to slight differences in the
culture conditions, though every effort was made for consistency. For example,
on some days the paramecia had completed four fissions since their last autogamy and some times they would have completed only three. The extra fission
of course doubled the rmmber of cells present and this would have affected the
medium accordingly, so that transfer to fresh medium would probably be a
796
EARL D. H A N S O N A N D MASAO KANEDA
TABLE 1
The effectof pulsed exposure to actinomycin D concentrations of 90,45, and 36 pg/l.O ml
of culture fluid
a) 90 m/m!
Interfission penod
Experiment Control
El
Cl+,
EI/Cl+,
El
Cl,,
435
429
43 1
428
425
448
478
456
469
476
499
48 1
537
542
543
532
487
487
465
451
45 1
471
446
445
448
464
430
476
45 9
441
0.82
0.88
0.88
0.92
0.94
0.99
1.Ol
1.02
1.05
1.06
1.os
1.12
1.13
1.18
1.23
537
546
632
659
494
536
490
532
613
589
668
691
517
564
495
465
...
...
471.8
+. 10.8
463.5
k 6.5
--
4 36.cdd.
Interfission period
Experiment Control
b) y.cg/ml
Interfisslon period
Experiment Control
-
1.02
k 0.03
El/Cl+%
0.88
0.93
0.95
0.95
0.96
0.97
0.99
1.14
...
...
...
-
...
...
-
553.1 574.0
k21.5 k21.7
...
0.97
t0.03
El
Cl,,
EI/Cl+Z
475
678
658
568
591
653
5 93
699
656
696
729
529
685
654
552
543
593
528
612
560
529
532
0.90
0.99
1.01
1.02
1.09
1.10
1.12
1.14
1.17
1.32
1.36
...
...
-
635.4
k22.2
...
574.3
+. 16.5
...
-
1.11
f0.04
The interfission periods are expressed in minutes. The C , value is the average for the two cells
produced from C,. Standard errors are given with each average in this and the other tables.
greater shock for these cells than for those which had affected their medium to a
lesser extent due to their smaller numbers. Further comment on culture conditions will be made below. From these efforts to develop consistent responses on
the part of the paramecia, we adopted, as our standard conditions, exposures of
5 to 6 min in duration, usually applied between 0.6 to 0.7 of the interfission
period and using 36
or 45 pg/ml of actinomycin depending on the batch of
actinomycin used.
I n Table 2 are summarized the results of exposing the cells to other than the
standard conditions. First, the results of three repetitions of the standard conditions (see line I a ) of Table 3) gave an E&+* value of 1.07 +. 0.03 (n=35).
Compared to this we observed no significant differences when cells were a )
exposed to the inhibitor for 20 min, rather than 5-6 min, b) exposed at interfission ages ranging from 0.40 to 0.69, rather than 0.6-0.7, and c) treated at 25°C
for seven min between interfission ages 0.45-0.73, rather than using the standard
29°C treatment.
Some further details can be added for each of these sets of data. Regarding
duration of exposures, in the preliminary experiments various lengths of exposures were used, varying between 5 and 20 min. All of them-5, 8, I O , 15, or
20 min-gave results showing a clear tendency to a delay equal to one added
fission. Also, when we tried even shorter exposures, such as for 3 minutes’ dura-
797
SEQUENTIAL GENE ACTION
TABLE 2
Summary of the results of three different types of pulsed exposures to actinomycin D.
a) Duration
of exposure
20min
(n=12)
E,/C,+,
1.09*0.04
b) T i e of exposure:
interfission age
0.40-0.49
(n=7)
0.05-0.59
(n=8)
0.06-0.69
(n=22)
c ) Growth
E,/C,,,
conditions
0.93t0.05
25°C
(n=14)
E,/C,+,
0.92+0.01
1.02+0.05
1.03t0.04
The number of pairs of sister cells used for each experimental run is given by n.
tion, there was no significant delay; the first division of the controls and experimentals was completed in very much the same time. This same situation was
observed for 5 min exposures at 25°C. Thinking that a 7 min exposure at 25°C
was proportional to a 5 :min exposure at 29"C, because of the shorter interfission
period at the higher temperature, we then used the 7 min exposure and found the
results reported in Table 2. It appears, then, that there is an all or none effect
of the duration of exposure and that exposures of 5 min at 29°C and of 7 min at
25°C are very close to the minimum times needed to get an effect.
Relative to interfission age at time of exposure, the preliminary results always
showed a one-fission delay regardless of the time in the cell cycle when the
exposures were made, confirming the data in Table 2. Also important here is to
try exposures very early in the series of events that determine a given fission.
The earliest possible time would be just after the stabilization point of the preceding interfission period. 'This was done (Table 3, line I b) and again gave us a
delay of one fission period. This experiment will be discussed again later. The
TABLE 3
E / C values for different experimental runs using the experimental design indicated
by In, Ib, etc. (Refer to Figure I )
Experimental design
1
E/C values
2.
3.
0.868+ 0.01
(n=lO)
Mean
Ia
El/C1+2
1.11t0.04
(n=11)
1.18t 0.03
(n=14)
Ib
EZ/Cz+a
1.08t 0.05
(n=3)
0.97 t 0.07
(n=10)
IIa
E&,+,
1.08t 0.03
(n=12)
0.92 t 01.01
(n=13)
0.94+ 0.08
(n=8)
0.98 & 0.02
(n=33)
1.38rt 0.03
(n=14)
1 .I 6 t 0.03
(n=ll)
1.48-C 0.03
(n=39)
1.W?c0.02
(rr=f%)
IIb E1/C1+,
The number of pairs of cells used for each run is given by n.
1.07t0.03
(n=35)
1.00k0.06
(n=13)
798
EARL D. H A N S O N A N D MASAO KANEDA
essential point now is that there is no correlation of delay with age of the cell
when it is exposed to actinomycin.
Finally, changes in growth conditions, in addition to those reported in Table 2,
were consistent in giving the one fission delay. In some cases, when the standard
techniques were still being worked out, slides and culture fluid for the experiment
were inadvertently not prewarmed at 29°C before being used for the cells which
were being grown at that temperature. The cells were, therefore, suddenly immersed in a medium at 25°C (it warmed up subsequently, of course) and they
showed very inconsistent interfission periods from one pair of sister cells to another. We assume this was due to a mild temperature shock. The important point
is, though, that if the interfission period of the control was lengthened-sometimes up to 8 hrs-there was a corresponding delay of the interfission period of
the experimental cell. This also occurred when we used post-autogamous cells
that were poorly fed and not in log phase. The lag in their first fission, as seen
in the control cells was mirrored in the behavior of the experimental cell, i.e.
value was close to 1.O.
again the
In sum, there is no demonstrated correlation between the observed delay in
fission due to brief exposure to actinomycin and any of the four possible causal
factors of concentration of the inhibitor, duration of exposure, time of exposure
in the cell cycle, or growth conditions, as studied here.
The only exceptions to the one fission delay were found when certain combinations of two short exposures were administered to the paramecia. The experiments are diagrammatically summarized in Figure 1. In the first lines of the
figure (I a and I b) are single exposure experiments already discussed. All other
lines, except the one labelled “Control”, are double exposure experiments. The
results of these experiments are given in Table 3. When two exposures are given
in the same interfission period and both come before the stabilization point (IIa) ,
the delay is for one fission period. If, however, one exposure comes before the
stabilization point and the next after that point, then the delay is greater than
one added fission period (IIb). An interpretation of this finding will be presented
in the next section. However, to look further into this phenomenon attempts were
made to expose the cells at equivalent times in two successive interfission periods
(I1 b, and I1 b,, the former representing early-early exposures and the latter
late-late exposures) or at different times (I1 b,, late-early exposures). The results
of these exposures are as follows: early-early exposures (I1b,) gave an E/C value
of 1.43 f 0.03 (n=18); late-late exposures (I1b,) gave E/C = 1.60 2 0.04
(n=9);and late-early exposures (I1b,) gave E/C = 1.45 2 0.06 (n=12). (The
three preceding E/C values are not significantly different. The overall value for
these 39 cases is given in column 3, I1 b, of Table 3. In the two preceding columns
there was no specific attention given to exact timing of the two exposures which
occurred at various times before and after a given stabilization point; they are
like I1 b, in that in most cases the two exposures are not equivalent times in two
successive interfission periods.) In the data from the carefully timed experiments
it is clear that the delay is almost equal to two added fissions. In such a case E
would be the equivalent of three fissions and when compared to the first two
SEQUENTIAL GENE ACTION
799
FIGURE
1.-Diagrammat tc outline of experimental designs used in obtaining the data shown
in Table 3. Single pulse experiments are designated by Ia and Ib, double pulse experiments by
IIa, IIb, etc. Time is indicated by the horizontal lines, proceeding from left to right. Vertical
straight lines represent fission; vertical wavy lines represent the stabilization points. Hatched
areas are exposures to the illhibitor. E and C refer to experimental and control cell interfission
periods, respectively. The subscripts designate the interfission periods following initiation of the
experiment.
fissions of the control it would give E/C = 1.5, which is very close to the observed
value of 1.48 -+ 0.03.
Finally, in reporting our results, we would point out that after the delayed
fission the treated cells ]-evertedto a normal interfission period. In these cases the
E/C value would be 1.0, where E is the first interfission period after the delayed
division and C is the interfission period of the undisturbed control cells. In a
sample of 25 experimental cells compared to the appropriate controls the E/C
value was 0.98 * 0.02.
800
EARL D. HANSON A N D MASAO KANEDA
DISCUSSION
Before turning to a discussion of the one-fission delay reported above, it is to
be noted that other work using a quite similar treatment of P. aurelia did not
find the delay we have reported. RASMUSSEN(1967) using actinomycin D concentrations of 0.04 mM (50 ,pg/ml) and 0.08 miv (100 ,pg/ml) and exposure
times of 30 min, reports delays of about 1.3 to 4.3 hrs in a sample of 42 cells,
where the normal interfission period is around 5.2 hrs. Such delays disappear
when exposures are made after the “transition point” (equivalent, it appears, to
our stabilization point). RASMUSSEN
provides no discussion of his data on the
exposures to ‘actinomycin(summarized in his Figure 5 , p. 135). It is hard to see
where certain differences in experimental techniques-Aerobacter aerogenes
rather than A . cloacae as the food organism, experimental cells held in tubular
“microchambers” rather than depression slides, and cells 8-1 5 fissions, rather
3-5 fissions post autogamy-could cause the differences between RASMUSSEN’S
and the present work. Further investigation of these differences is needed.
Actinomycin D has two known biological effects and both must be considered
in interpreting the fission delays which we have found in P. aurelia. The antimetabolite blocks formation of RNA from a DNA template (HURWITZ,
et al.
1962) and it can block DNA synthesis (REICHand GOLDBERG
1964; and others).
The former effect occurs at lower concentrations of actinomycin than the latter.
It has been suggested (REICH and GOLDBERG
1964) that the block of RNA transcription is due to a steric effect, the actinomycin actually interfering by its
physical presence with the action of the RNA polymerase, and the inhibition of
DNA synthesis is thought to be due to a disruption of the physical properties of
the DNA due to binding of large quantities of actinomycin. Two types of binding,
in terms of their strength, are known between DNA and actinomycin (GELLERT,
et al. 1965) with one a thousand times stronger than the other. The stronger one
is thought to be the cause of inhibition of transcription. More recently, the precise
site of the actinomycin binding has been attributed to the 2-amino groups of
purines in the minor groove of DNA (CERAMIet al. 1967). On present knowledge, then, the effect of actinomycin is attributable to its localization at certain
sites in the smaller groove of the DNA double helix. At lower concentrations of
the antimetabolite the binding is sufficient to block DNA-dependent RNA synthesis, and at higher concentrations synthesis of DNA itself can be disrupted.
Turning now to our results with P. aurelia we are not in a position to decide
unequivocally what effect-on DNA or RNA synthesis, or both-the actinomycin
is having. But whatever the effect, the special fact of the one fission delay requires
explanation and it appears at present that an effect on transcriptional events,
where these events are occurring sequentially in the cell cycle, best accounts for
our observations. Our discussion will first look at possible effects on DNA and
then on RNA formation.
The concentrations of actinomycin used by us can be considered high relative
to doses of 5 Fg/ml or less that are effective with bacterial systems. However,
Paramecium is a large cell (ca. 150 p long) and its very high macronuclear ploidy
SEQUENTIAL GENE ACTION
801
(estimated at 860 n by WOODARD,
GELBER,and SWIFT1961) raises the question
as to how much actinomycin actually was present in the macronucleus. Recall
also that the exposures were short-usually 5-7 min long-and that we were
working close to the lovver limit of the effect we were studying. Nonetheless, it
cannot as yet be ruled out that there was sufficient actinomycin for an effect on
DNA replication. However, if so, we could have expected its effect to be reflected
in changes in concentration of the actinomycin or in length of exposure. The fact
that this did not occur suggests that the DNA effect, if there is one, might be
indirect rather than direct. One might postulate that it took the cell machinery
a whole fission to undo the binding of the actinomycin to the DNA. This however
is unsatisfactory in that the delay is not one fission from time of exposure but one
fission added on to the fission in which the exposure occurred. The delay is somehow tied to a timing device related to the cell cycle. One might, therefore, think
of an indirect effect on DNA as coming from transcription of an RNA necessary
for a DNA polymerase (or other enzymes related to DNA synthesis) which is
part of the cooperative effort to make DNA. Its absence in one cell cycle needs
another cycle to form it and then division can proceed. Speculations of this type
are, of course, postulating the key effect of the actinomycin to be on RNA formation. Therefore, let us tiurn to that discussion after saying, in conclusion, that no
simple hypothesis of direct or indirect action of actinomycin on DNA formation
seems to account for the fission delay.
An effect on transcriptional events in paramecia can be discussed from the
point of view of three models of gene action. One can suppose, first, that all genes
act at the outset of an interfission period and that translational and assembly
events are what determme formation of the materials for orderly morphogenesis.
However, the finding of a stabilization point at interfission 0.86 (GILL and
HANSON
1968) and the same situation also occurring in Tetrahymena (FRANKEL
1965 and earlier papers) and other evidence (WOODARD,
GELBER,and SWIFT
1961; KIMBALLand PE:RDUE
1962) that RNA synthesis occurs throughout the
cell cycle, all argue against transcription being a massive all-at-once formation
of gene products. A second possibility is to think of all genes acting all the time
and to assume that the accumulation of gene products, presumably at different
rates, would provide the necessary proteins for morphogenesis and fission at the
right time. Apart from an a priori arguable inefficiency of the scheme (NANNEY
1960) it also fails to account for the fission delay unless one adds various complicating postulates. Furthermore, in its simplest form, without added postulates,
this hypothesis would suggest that a five-minute cut-off of gene products would
result in a certain delay in their subsequent accumulation which would probably
be reflected in some minutes of fission delay. A five-hour fission delay is hardly
to be expected and if a five-minute exposure resulted in a certain length of delay
one would expect a ten-minute exposure to give a proportionally longer delay.
But this is not observed. This possibility of continuous transcription of all loci
seems unlikely at least in its simplest formulation.
The final hypothesis is one of sequential gene action, where one can suppose
that different genes are turned off and on in a sequence that is appropriate to the
802
EARL D. H A N S O N A N D MASAO K A N E D A
needs of cellular development in the cell cycle. A more detailed formulation of
the model would say that not only is there sequential activation of genes but that
the sequence is obligate, i.e., a gene is activated only at a given time in the cell
cycle and if its product is not formed then, it will not be formed until the cycle
has once again run its course. Such a point of view accords nicely with the results
of the pulsed exposures of paramecia to actinomycin. We can assume that exposure to actinomycin cut off the transcription of certain genes and the absence
of these gene products therefore denied the cell something that would normally
be present for use by the cell. These products would only become available when
those affected genes could again be activated, that is, in the next cell cycle. Hence,
fission-the result of cumulative action of genes in a given cell cycle-would be
delayed until a second, added cell cycle or, better, added cycle of gene activation
could be completed in the absence of the inhibitor.
This model assumes that specific gene products must be formed anew in each
cell cycle to support fission. Such a view is consistent with conclusions drawn
from Tetrahymena (FRANKEL
1965; and work on the “division protein”, RASMUSSEN and ZEUTHEN1962; WATANABE
and IKEDA1965) and with certain
other cases of proteins being formed as needed by cells (FLICKINGER
1963).
Further, this model agrees with other observations made here on the effect of
actinomycin. For example, it predicts that two exposures in the same cell cycle
and before the same stabilization point, would be expected to have the same effect
as one exposure. Two such exposures would deny the cell the products of two
different sets of genes and hence block fission, but one added, undisturbed transcriptional read-out would allow the cell to recover. This is precisely what we
observe. Moreover, two exposures in successive cycles of transcription would
mean each cycle would be incomplete and yet a third cycle would be needed.
This is seen to have occurred in certain of our observations (Table 3, line I1 b,
esp. column 3 ) . However, it did not occur in all cases and it may be that there
can be some complementation of products from one cycle with those for a succeeding one. That is, products that are formed but not used, since fission did not
occur, may persist and be available in the second cycle and if the two cycles are
missing different gene products, the products they have can cooperatively promote fission-somewhat later than normal in some cases (Table 3, line I1 b,
column 2) but nonetheless not quite as late as if there were no complementation.
This pattern of recovery after two exposures in different cell cycles deserves
further study but for the moment the data presented are not inconsistent with
the idea of sequential gene action.
And, finally, on this model of sequential genetic activity, we would expect,
after the delayed fission has occurred, that the normal timing of events would
reassert itself, for the cell has now been able to complete one undisturbed transcriptional cycle and the next should be the same. C h r data on the fission after
the delay show that the expected normal interfission period does occur.
We conclude, on present evidence that the model of obligate sequential gene
action within a cell cycle best explains the observed effects of pulsed exposures
of paramecia to actinomycin D.
The qualification that the sequence of activity is obligate or fixed is the key to
SEQUENTIAL GENE ACTION
803
accounting for the delay of precisely one whole interfission period. This idea has
a further important ccinsequence regarding the control of the transcriptional
read-out. From bacterial systems and the concept of the inducer we are accustomed to looking for control of sequential activity as being the result of gene-cytoplasm interaction with the intermediates being m-RNA (to determine formation
of enzymes which control the formation of various substances) and inducers
(arising from enzymatic activity, perhaps, and controlling the action of operator
loci). In the present case of Paramecium it is postulated that transcription is
blocked but that sequential cistronic activation goes on, which implies that this
activation is not cytoplasmically controlled. Indeed, we are forced to the view
that there is some intranuclear control determining a specific sequence of activation and that this goes on independently of the cytoplasmic events. At present
we have no specific insights as to what this control might be. One possibility
might be, of course, intranuclear histones. This implies that nuclear events have
evolved a pattern of activity, under control of intranuclear suppressors and activators of the histone type, that results, under normal conditions, in cell division.
Blocking the products of this activity suppresses cell division but not the control
mechanism which follows its normal pattern of activity. Whether this activity
might be polarized reading of chromosomes as mediated through histones, is
simply another conjecture at present. What we have shown is the validity of
proposing obligate sequential gene action in the cell cycle of paramecia and what
remains of prime importance is to analyze the basis of this precise control.
Within multicellu1a:r systems the differential activity of genes is well-established (BEERMANN
1963; CLEVER
1966) and also in clones of unicellular systems
(BEALE1954; SIEGELand COHEN1963). In these instances the differences in
genic activity are intercellular, i.e., different cells of the clone show evidence of
different genes being active in them. The further question is whether o r not
differential gene action is also intracellular, that is, whether it occurs within a
single cell to determine, at least in part, developmental events within the cell.
There are various lines of experimental work which provide examples of intracellular differential gene activity. Apparently the phage genome undergoes
sequential transcription following infection of the bacterial host ( COHENet al.
1963). I n bacteria themselves a non-random reading of the genome has been
and SPIEGELMAN
1962) and during the process
thought to occur (KANO-SUEOKA
of germination of spores there is evidence for sequential transcription of at least
parts of the genome (S'rEINBERGand HALVORSON
1968a). This latter work argues
that control of the read-out is not primarily the result of repressors o r inducers
but depends somehow Ion the state of the DNA itself, but not on DNA replication
1968b). In eukaryotic cells, synchronized yeast cells
(STEINBERG
and HALVORSON
have shown sequential appearance of specific enzymes within the first cell cycle
and this has been interpreted as evidence that transcription is ordered (HALVORSON et a2. 1964). Our evidence in this paper argues for ordered transcription in
the normal cell cycle of eukaryotic cells.
The authors thank Miis. CONCETTINA
GILLIE~for her consistently thoughtful comments
throughout the course of this work and for her invaluable technical help. W e also thank MESSRS.
WILLIAM
FISHER
and DAVID
BELLER
for their technical help.
804
EARL D. H A N S O N A N D MASAO KANEDA
SUMMARY
Paramecium aurelia has been exposed to actinomycin D for periods only
minutes in duration and invariably shows a 4 to 6 hour delay in fission equivalent
to an additional interfission perid.-This delay cannot be attributed solely to the
concentration of the inhibitor, to the duration of the exposure, or to the time in
the cell cycle when the exposure was made, nor, finally, to the general culture
conditions.-The delay can be explained by a model of gene action in which
cistrons are transcribed sequentially in the cell cycle and in an invariable order.
Certain predictions arising from this hypothesis have been experimentally fulfilled.
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