822
R. L. B R A H M A C H A R Y , D. GHOSAL, A N D P. K. T A P A S W I
Rhythmic Incorporation of P32 and C14-uracil in Early
Mitotic Cycles of Limnaea (Mollusc) Eggs
R . L . BRAHMACHARY, D . GHOSAL, a n d P . K . TAPASWI
Unit of Embryology, Indian Statistical Institute, Calcutta-35
(Z. Naturforsch. 26 b, 822—824 [1971]; received March 25, 1971)
Incorporation of P 3 2 and C 14 -uracil into RNase-sensitive, acid-insoluble fraction takes place in
well defined cycles in course of the early cleavage in Limnaea embryos. This suggests a cyclic
R N A synthesis. 4 0 — 5 0 % less R N A is transcribed during the latter half of each cell division cycle.
The rhythm is absent in the homogenate. The relevance of the findings has been discussed.
The biosynthesis of various molecules are known
to slow down during mitosis as pointed out by
M A Z I A in his very comprehensive review 1 . "Hard
data" on such rhythmic biosynthesis especially as
regards the metabolism of RNA and protein are very
few. Recently M A N O 2 reported an interesting rhythm
of protein biosynthesis (as evident from incorporation of C14-amino acids) superimposed on a basal
rate of increase in early mitotic cycles of sea-urchin
embryos. The rhythm is evident also in the homogenates and as such seems to be independent of
normal nuclear-cytoplasmic relationship. Mitotic
rhythm in the early embryo can be considered to
be a genetically well-defined process for a particular
species 3 and M A N O ' S finding is another interesting
aspect of this rhythm at the molecular level. CUMMINS and R U S C H 4 also determined cycles of RNA
synthesis in slime mold cells.
Rhythms in the incorporation of P 32 and C 14 uracil in early cleaving embryos of Limnaea are
being reported in the present paper. The results
suggest a well-defined cycle in the synthesis of RNA
during the first two cell cycles. A more marked
cycle exists in case of inorganic sulphate S35-incorporation 5 .
Materials and Methods
Uncleaved, freshly laid eggs and early cleaving eggs
of Limnaea were collected from vessels at the laboratory where Limnaea were reared on dry lettuce. Eggs
were treated with P32 and the counts measured in the
TCA-insoluble part following an earlier investigation 6 .
1
2
3
Reprints request to Dr. R. L. BRAHMACHARY, Unit of Embryology, Indian Statistical Institute, Calcutta-35, Indien.
D. MAZIA, in: The Cell, III, Academic Press, New York
1961.
Y. MANO, B. B. R. C. 33, 877 [1968].
R. L. BRAHMACHARY, in: Intern. Rev. of Cytology 21, 65,
Academic Press, New York 1967.
Definite numbers of eggs from the same egg mass
were left in the isotope for successive periods and then
treated with "carrier" and TCA and incorporation into
acid-insoluble part was then measured. Incorporation
into RNase-sensitive acid-insoluble fraction was shown
with the help of 20jug RNase/ml (final concentration).
Thus it was now possible to compare the rates of incorporation into acid-insoluble fractions during different stages of the uncleaved and cleaving eggs. Similarly, the incorporation of C14-uracil was studied. Attempts were made to measure the intracellular pool
as explained in the discussion. Normal development of
eggs in the P32 and C14-solution was observed before
carrying out the actual experiments. P32 counts were
taken with a Geiger Counter and C14 counts with a
Panax windowless phosphor scintillation counter.
Egg cells were homogenized in tris buffer of same
alkalinity (pH 7.2) in which part of the egg mass
developed as control. Homogenization was effected
in an all-glass homogenizer and it was ensured by
microscopic observation that no intact egg was present.
The homogenate was incubated with measured amounts
of isotope solution for 30' or so i. e. up to the time
when the intact eggs from the same egg mass had
atttained the mid point of cleavage cycle (see results).
At that stage the same number of eggs was again homogenized and incubated till the onset of next cleavage in
controls. Bacterial population of the egg mass was
shown to be negligible by plating the egg homogenate
and counting colonies.
Results and Discussions
Freshly laid (fertilized) Limnaea eggs cleave
after about 2 — 3 hours. The results with P 32 definitely prove a marked decrease of incorporation
into acid-insoluble fraction in the latter part of the
uncleaved stage (Table I ) .
4
5
6
J. E. CUMMINS and H. P. RUSCH, Endeavour, X X V I I , 124
[1968].
R. L. BRAHMACHARY, D. GHOSAL, P. K. TAPASWI, and T. K.
BASU, Exp. Cell Res. 65, 325 [ 1 9 7 1 ] .
R. L. BRAHMACHARY, K. P. BANERJEE, and T. K. BASU,
Exp. Cell Res. 5 1 , 1 7 7 [1968].
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INCORPORATION
OF P32 A N D
C14-URACIL IN
Experiment
No.
Cpm in the first
5 0 ' (100' before
cleavage to 5 0 '
before cleavage)
Cpm in the following 5 0 ' (50'
before cleavage
to cleavage)
Mean decrease in
second
phase
1
2
2612
9519
1344
6200
41%
Table I.
Incorporation of P 3 2 into successive stages of uncleaved egg.
The second mitotic cycle i. e. 2 — 4 cell last about
1 hour. The first set of eggs were treated from the
initiation of the first cleavage (i. e., the formation
of two cells) to the mid point of the cycle i. e. about
30 min after cleavage. This stage is recognizable
in Limnaea on visual examination under the microscope because the two blastomeres appear in a
strongly compressed phase. At this moment the
radio-active eggs were treated with carrier and TCA
and the second set of eggs (from the same egg
mass) were put in the isotope and incorporation
allowed to proceed for next 30 min or so i. e.,
up to the initiation of a clear 4-cell stage.
Table II furnishes representative examples of incorporation during the first and second half of this
cell cycle. Thus, for both the first (uncleaved to
2-cell) and second (2 to 4-cell) cell division incorporation during the latter half of the cell cycle is
„ about 40 — 45% less. (The question of permeability
barrier and intracellular pool has been discussed
below). As P 32 can be incorporated into RNA,
DNA, phosphoprotiens, phospholipids etc., further
investigation is necessary for determining the nature
of the TCA-insoluble fraction. As shown earlier 6
the major part of P 32 incorporation is due to RNA
synthesis. In the present experiment it was now
shown that at least about 85% of this TCA-insoluble
fraction is RNase sensitive after 1 hour of incubation with the enzyme. This would suggest at least
2 6 - 3 0 % less RNA synthesis in the second half
of the cell-cycle. In order to further investigate the
Experiment
No.
Cpm during
first half
(35')
Cpm during
second half
(35')
Mean decrease in
second half
1
2
3
4
6961
346
21153
859
3674
160
13879
504
45%
Table II a. Incorporation of P 3 2 during the first and second
half of the second cell division cycle (2 — 4 cell).
EARLY
MITOTIC
CYCLES
823
Experiment
No.
Cpm during
first half
(30')
Cpm during
second half
(30')
Mean decrease m
second half
1
2
1832
2342
1085
1249
44%
Table II b. Incorporation of P 3 2 during the first and second
half of the third cell division cycle (4 — 8 cell).
rhythmic synthesis (if any) of RNA, incorporation
of C14-uracil was noted because, as is known from
a wide field of experiments, 90% or more of C 14 uracil is incorporated into RNA, Table III sums
Cell division cycles
2 - 4 Cell
Cpm during first
half (30')
Cpm during second
half (30')
733
386
4 - 8 Cell
Cpm during first
half (35')
970
Cpm during second
half (35')
470
8 - 1 6 Cell
1170
659
Table III.
Incorporation of C 14 -uracil during the first and
second half of cell division cycles.
up the data which shows clearly that in each cleavage
cycle ( 1 — 2 — 4 — 8 — 16 cell) incorporation is
about 50% less during the latter half of the cell
division. This rhythm is superimposed on the increasing basal rate 6 and the general picture is
similar to that of M A N O 2 . Results obtained with cell
homogenates do not show however the rhythmic
incorporation of P 32 as evident in intact cells
(Table IV). This perhaps indicates that normal
transcription i. e. RNA synthesis at this stage of
development depends on an intricate nuclear-cytoplasmic and/or other relationships of the integrated
cell. On the other hand it is well known that translation i. e. protein synthesis in early sea-urchin
embryos is governed by cytoplasmic fractors only
which is in agreement with M A N O ' S results. In
Limnaea the rate of protein synthesis as measured
by incorporation of C14-amino-acids is very slight
in the early stages and so far it has not been possible to detect any well defined rhythm within tho
Homogenate incubated for
the first half (30')
Homogenate incubated for
the second half (30')
216
246
Table IV.
Incorporation of P 3 2 into homogenates
2 — 4 cell. Numbers indicate Cpm.
during
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824
INCORPORATION
OF
P32 A N D
C14-URACIL
IN
EARLY
MITOTIC
C Y C L E S 824
very small rates of incorporation in the early cell
cycles.
On the other hand, the nature of RNA synthesis
during the early cell cycles of Limnaea can be determined with sucrose density gradient and gel-electrophoresis. It was at first determined that ion-agar
electrophoresis gives essentially the same results as
with sucrose density gradient 7 . In this manner, the
percentage of different RNA fractions has now been
established in the cleaving Limnaea eggs 8
The rhythmic incorporation of P 32 or C14-uracil
could be due to a rhythmic change in permeability
of Limnaea eggs. It is therefore necessary to
measure the intracellular pools of P 32 during the
two phases of cell cleavage. This was attempted as
in the earlier work 5 with the assumption that rapid
and successive washings (one min or half-min intervals) with non-radioactive phosphate at a low temperature liberates the contaminant P 32 (at the surface etc.) and minimizes exchange reactions with
the pool. Concentrations of non-radio active phosphate (washing solution) were tested for deletereous
actions on normal development and a permissible
value was selected. However, unlike S 35 5 , P 32
contaminant cannot be so quickly washed away
while prolonged washings permit exchange reaction.
The results of S 35 (inorganic sulphate) intracellular pools show that altered permeability barrier
during the two halves of the division cycle is not
the cause of differential incorporation 5 . Though
this does not exclude the possibility of differential
permeability to P 32 , it is rendered less likely.
water were tested as washing medium. It was seen
after successive washings in 250 ml water (each
washing for 105 sec) that 4 washings remove the
contamination. Thus after 7 min of washing it is
possible to measure the TCA-soluble intracellular
pool in terms of cpm (see table).
As C14-uracil can be quickly washed away it is
possible to measure the intracellular pool after
washing the eggs and removing the surface contaminants. Different volumes of uracil solution and
Note added to Proof: (1) MANO has further confirmed the
findings on sea-urchin 9 but GROSS and FRY did not notice the
same rhythmic pattern 10.
(2) JOCKUSCH 11 reported a cyclic pattern of protein synthesis in Limnaea.
7
R . L . BRAHMACHARY a n d P . K . TAPASWI, C u r r e n t S e i . 3 8 ,
496
8
R . L . BRAHMACHARY, P . K . TAPASWI, a n d D . GHOSAL,
Z.
Second half of
2 — 4 cell cycle
(35')
577
715
Thus, the intracellular pool of C14-uracil during
the second half of the cell division is certainly not
less than in the first half. Therefore absence of pool
due to decreased permeability cannot be the ratelimiting factor of RNA synthesis i. e. incorporation
of C14-uracil into TCA-insoluble fraction. There is
however the possibility that the specific activity of
the pool may decrease in the second half due to appearance of non-radioactive uracil from the cellular
or capsular material. This cannot be detected by precipitating the total phosphorous or uracil because,
unlike sea-urchin Limnaea eggs are not available in
huge quantities. However, such a regular appearance
of phosphorous or uracil in the second half of
mitosis would be an interesting rhythm in itself.
Two
of
us
(R.
L.
BRAHMACHARY
and
D.
GHOSAL)
thank the Indian Council of Medical Research for supporting this study.
9
10
[19691.
First half of
2 — 4 cell cycle
(35')
11
Y. MANO, Dev. Biol. 22, 433 [1970].
B. J. FRY and P. R. GROSS, Dev. Biol. 21, 105 [1970].
B . JOCKUSCH, Z . Naturforsch. 2 3 B, 1 5 1 2
[1968].
Naturforsch. 2 6 b, 271 [1971].
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