This work has been digitalized and published in 2013 by Verlag Zeitschrift für Naturforschung in cooperation with the Max Planck Society for the Advancement of Science under a Creative Commons Attribution4.0 International License.

Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschungin Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung derWissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht:Creative Commons Namensnennung 4.0 Lizenz.

8 2 2 R. L. BRAHMACHARY, D. GHOSAL, AND P. K. TAPASWI

Rhythmic Incorporation of P32 and C14-uracil in Early Mitotic Cycles of Limnaea (Mollusc) Eggs 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

Unit of Embryology, Indian Statistical Institute, Calcutta-35

(Z. Naturforsch. 26 b, 822—824 [1971]; received March 25, 1971)

Incorporation of P3 2 and C14-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 RNA synthesis. 40—50% less RNA 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 review1. "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 incorpora-tion 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 homo-genates 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. CUM-MINS and R U S C H 4 also determined cycles of RNA synthesis in slime mold cells.

Rhythms in the incorporation of P32 and C14-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-incor-poration 5.

Materials and Methods

Uncleaved, freshly laid eggs and early cleaving eggs of Limnaea were collected from vessels at the labora-tory 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.

Reprints request to Dr. R. L. BRAHMACHARY, Unit of Em-bryology, Indian Statistical Institute, Calcutta-35, Indien.

1 D. MAZIA, in: The Cell, III, Academic Press, New York 1961.

2 Y. MANO, B. B. R. C. 33, 877 [1968]. 3 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 in-corporation into acid-insoluble fractions during dif-ferent stages of the uncleaved and cleaving eggs. Simi-larly, the incorporation of C14-uracil was studied. At-tempts 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 homo-genized 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 P32 defi-nitely prove a marked decrease of incorporation into acid-insoluble fraction in the latter part of the uncleaved stage (Table I) .

4 J. E. CUMMINS and H. P. RUSCH, Endeavour, X X V I I , 124 [1968].

5 R. L. BRAHMACHARY, D. GHOSAL, P. K. TAPASWI, and T. K. BASU, Exp. Cell Res. 65, 325 [1971].

6 R. L. BRAHMACHARY, K. P. BANERJEE, and T. K. BASU, Exp. Cell Res. 51 ,177 [1968].

I N C O R P O R A T I O N O F P 3 2 A N D C 1 4 - U R A C I L I N E A R L Y M I T O T I C C Y C L E S

Experi- Cpm in the first Cpm in the fol- Mean de-ment 50' (100' before lowing 50' (50' crease in No. cleavage to 50' before cleavage second

before cleavage) to cleavage) phase

1 2612 1344 4 1 % 2 9519 6200

4 1 %

Table I. Incorporation of P3 2 into successive stages of un-cleaved 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 micro-scope 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 in-corporation 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 incor-poration 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 P32 can be incorporated into RNA, DNA, phosphoprotiens, phospholipids etc., further investigation is necessary for determining the nature of the TCA-insoluble fraction. As shown earlier6

the major part of P32 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 incuba-tion 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

Experi- Cpm during Cpm during Mean de-ment first half second half crease in No. (35') (35') second half

1 6961 3674 4 5 % 2 346 160 3 21153 13879 4 859 504

Table II a. Incorporation of P3 2 during the first and second half of the second cell division cycle (2 — 4 cell).

8 2 3

Experi- Cpm during Cpm during Mean de-ment first half second half crease m No. (30') (30') second half

1 1832 1085 4 4 % 2 2342 1249

4 4 %

Table II b. Incorporation of P3 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 C14-uracil is incorporated into RNA, Table III sums

Cell divi- Cpm during first Cpm during second sion cycles half (30') half (30')

2 - 4 Cell 733 386

Cpm during first Cpm during second half (35') half (35')

4 - 8 Cell 970 470

8 - 1 6 Cell 1170 659

Table III. Incorporation of C14-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 in-creasing basal rate6 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 P32 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-cyto-plasmic and/or other relationships of the integrated cell. On the other hand it is well known that trans-lation 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 pos-sible to detect any well defined rhythm within tho

Homogenate incubated for Homogenate incubated for the first half (30') the second half (30')

216 246

Table IV. Incorporation of P3 2 into homogenates during 2 — 4 cell. Numbers indicate Cpm.

8 2 4 I N C O R P O R A T I O N O F P 3 2 A N D C 1 4 - U R A C I L I N E A R L Y M I T O T I C 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 deter-mined with sucrose density gradient and gel-electro-phoresis. It was at first determined that ion-agar electrophoresis gives essentially the same results as with sucrose density gradient7. In this manner, the percentage of different RNA fractions has now been established in the cleaving Limnaea eggs 8

The rhythmic incorporation of P32 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 P32 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 inter-vals) with non-radioactive phosphate at a low tem-perature liberates the contaminant P32 (at the sur-face etc.) and minimizes exchange reactions with the pool. Concentrations of non-radio active phos-phate (washing solution) were tested for deletereous actions on normal development and a permissible value was selected. However, unlike S35 5, P32

contaminant cannot be so quickly washed away while prolonged washings permit exchange reaction. The results of S35 (inorganic sulphate) intracellu-lar pools show that altered permeability barrier during the two halves of the division cycle is not the cause of differential incorporation5. Though this does not exclude the possibility of differential permeability to P32, it is rendered less likely.

As C14-uracil can be quickly washed away it is possible to measure the intracellular pool after washing the eggs and removing the surface con-taminants. Different volumes of uracil solution and

7 R .L . BRAHMACHARY and P . K . TAPASWI, Current Sei. 3 8 , 4 9 6 [ 1 9 6 9 1 .

8 R . L . BRAHMACHARY, P . K . TAPASWI, and D . GHOSAL, Z . Naturforsch. 26 b, 271 [1971].

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).

First half of Second half of 2 — 4 cell cycle 2 — 4 cell cycle

(35') (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 rate-limiting 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 ap-pearance of non-radioactive uracil from the cellular or capsular material. This cannot be detected by pre-cipitating 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.

T w o o f u s ( R . L . B R A H M A C H A R Y a n d D . G H O S A L ) thank the Indian Council of Medical Research for sup-porting this study.

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 syn-thesis in Limnaea.

9 Y. MANO, Dev. Biol. 22, 433 [1970]. 10 B. J. FRY and P. R. GROSS, Dev. Biol. 21, 105 [1970]. 1 1 B. JOCKUSCH, Z . Naturforsch. 2 3 B, 1 5 1 2 [ 1 9 6 8 ] .