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Synchronous DNA synthesis following heat-synchronized cell division in Tetrahymena
Synchronous
DNA
231
synthesis
(c) A vacuole such as that containing the “inside” EAT cell has been described in other well-documented cases of emperipolesis involving lymphocytes and other “host” cells [4, 51. However, in 14-day-old EAT one frequently observes balloon EAT cells which contain one very large vacuole and a displaced flattened nucleus. Thus these two cases may represent a “normal” EAT cell within a balloon EAT cell. REFERENCES 1. HUMBLE, .I. 2. IOACHIM, H. 3. SHARP, J. A. 4. SHELTON, E. 5. TRO~ELL, 0.
C., JAYNE, W. H. W. and PULVERTAFT, H. J. V., &it. J. Haemat. L., Lab. Znoest. 14, 1784 (1965). and BURWELL, R. G., Nature 188, 474 (1960). and DALTON, A. J., J. Biophys. Rio&m. Cytol. 6, 513 (1959). A., Znt. Reo. CytoI. 7, 235 (1958).
SYNCHRONOUS HEAT-SYNCHRONIZED K. From
the Biological
DNA SYNTHESIS CELL DIVISION K.
Institute
HJELMI
and
qf the Carlsberg Received
June
2, 283 (1956).
FOLLOWING IN TETRAHYMENA
E. ZEUTHEN
Foundation,
Copenhagen,
Denmark
8, 1967
Cu~nmm
of Tetrahymena pyriformis, amicronucleate strain GL, were synchronized for cell division with 7 heat shocks[2]. In the present study zero time is the end of the synchronizing heat treatment (EH). 3H-thymidine incorporation into the macronuclei was followed through synchronous cell division, using autoradiographic procedures [l]. The specificity of the incorporation into DNA was checked with DNase. Fig. 1 showsresults of continuous (curve A) and pulse incorporations (curve B). In A the tritium label was added at 0 min.; in B it was supplied to separate aliquots 5 min. before each sampling. All results represent one synchronous population growing at 28°C in 2 per cent proteose-peptone +0.4 per cent liver extract [a]. Curve A checks with Fig. 3 in [ 11.It showsgradual increasein the percentage of labelled cellsfor the first 50 min. after EH. This demonstrates asynchronous engagement of the cells in DNA synthesis. We have previously observed this asynchrony both for this period and for the period with heat shocks [l]. After EH +50 min. there is a loss of previously incorporated label, and this results in a minimum labeling at 70 min. From this time there is quick new labeling, so that at 130 min. 90 per cent of the macronuclei are labelled. The results obtained with pulse labelling (B) show that some cells are in DNA synthesis after EH. For a short time around 70 min. no or very few cellsare in synthesis, but from 70-110 min. the percentage labelling increasesfrom 0 to 80 per cent. Subsequently, there is a new decreaseto 7 per cent labelling at 160min. The shapeof curve B indicates an average of 45 min. for the S-period in these 80 per cent of the cells. It is to be noted that Gi is short or absent. Curves A and B suggestthat the events described around division 1 repeat themselves at the time of division 2.
1 Present
address:
Department
of Molecular
Biology,
Odense
University,
Odense,
Experimentnl
Denmark.
Cell Research
48
Ii’. K. Hjelm
232
and E. Zeufhen
$00 % .; s 550 ; E P $ $ -0
0
60
120
180
minutes after EH Fig.
1.
Fig. l.-Curue A, Percentage of macronuclei labelled in DNA when the synchronized population is incubated with 3H-thymidine from EH (end of the period with heat shocks). Curve B, Percentage of macronuclei labelled in 5-min. pulses placed at various times after EH. The times indicate the termination of the pulses. Dose in both experiments: 3H-thymidine from Schwarz; 0.36 C/mM; 0.50 mC/ml. 12.5 ~1 added per ml culture. Fig. 2.-Histograms of numbers of silver grains over labelled macronuclei. Analysis of separate points from curve B in Fig. 1. The times indicate when the 5-min. pulses were terminated. Fig. 2.
Some of the autoradiograms from the pulse experiment of Fig. 1 B have been counted for the number of silver grains over the macronuclei (Fig. 2). In view of the wellknown limitations of the autoradiographic method, here used in its crudest form, the results can be considered only semiquantitative. However, they permit the suggestions (i) that at different times the individual macronuclei incorporate exogenous thymidine at rather different intensities, and (ii) that the incorporation rate in the average active macronucleus is low immediately after EH, increases until 35 min. and again decreases to a very low value at 79-84 min. From this time the rate of incorporation again increases (see also [4]). These changes may reflect variations in the rate of DNA synthesis through the synchronized cell cycle, but other interpretations are possible. For example, the results might reflect changes in the relative contribution to the new DNA by thymidine compounds from endogenous and from exogenous sources (cf. 13, 41). REFERENCES 1. 2. 3. 4.
HJELM, K. K. and ZEUTHEN, E., Compt. Rend. Trav. Lab. Carlsberq 36, 127 (1967). (ed.), Synchrony PLESNER, P., RASMUSSEN, L. and ZEUTHEN, E., in E. ZEUTHEN Division and Growth, p. 543. Interscience Publishers, New York, 1964. STONE, G. E., MILLER, 0. L. and PRESCOTT, D. M., J. Cell Biol. 25, 171 (1965). ZEUTHEN, E., Exptl Cell Res. In press (1967).
Experimental
Cell Research
48
in
Cell
DNA
231
synthesis
(c) A vacuole such as that containing the “inside” EAT cell has been described in other well-documented cases of emperipolesis involving lymphocytes and other “host” cells [4, 51. However, in 14-day-old EAT one frequently observes balloon EAT cells which contain one very large vacuole and a displaced flattened nucleus. Thus these two cases may represent a “normal” EAT cell within a balloon EAT cell. REFERENCES 1. HUMBLE, .I. 2. IOACHIM, H. 3. SHARP, J. A. 4. SHELTON, E. 5. TRO~ELL, 0.
C., JAYNE, W. H. W. and PULVERTAFT, H. J. V., &it. J. Haemat. L., Lab. Znoest. 14, 1784 (1965). and BURWELL, R. G., Nature 188, 474 (1960). and DALTON, A. J., J. Biophys. Rio&m. Cytol. 6, 513 (1959). A., Znt. Reo. CytoI. 7, 235 (1958).
SYNCHRONOUS HEAT-SYNCHRONIZED K. From
the Biological
DNA SYNTHESIS CELL DIVISION K.
Institute
HJELMI
and
qf the Carlsberg Received
June
2, 283 (1956).
FOLLOWING IN TETRAHYMENA
E. ZEUTHEN
Foundation,
Copenhagen,
Denmark
8, 1967
Cu~nmm
of Tetrahymena pyriformis, amicronucleate strain GL, were synchronized for cell division with 7 heat shocks[2]. In the present study zero time is the end of the synchronizing heat treatment (EH). 3H-thymidine incorporation into the macronuclei was followed through synchronous cell division, using autoradiographic procedures [l]. The specificity of the incorporation into DNA was checked with DNase. Fig. 1 showsresults of continuous (curve A) and pulse incorporations (curve B). In A the tritium label was added at 0 min.; in B it was supplied to separate aliquots 5 min. before each sampling. All results represent one synchronous population growing at 28°C in 2 per cent proteose-peptone +0.4 per cent liver extract [a]. Curve A checks with Fig. 3 in [ 11.It showsgradual increasein the percentage of labelled cellsfor the first 50 min. after EH. This demonstrates asynchronous engagement of the cells in DNA synthesis. We have previously observed this asynchrony both for this period and for the period with heat shocks [l]. After EH +50 min. there is a loss of previously incorporated label, and this results in a minimum labeling at 70 min. From this time there is quick new labeling, so that at 130 min. 90 per cent of the macronuclei are labelled. The results obtained with pulse labelling (B) show that some cells are in DNA synthesis after EH. For a short time around 70 min. no or very few cellsare in synthesis, but from 70-110 min. the percentage labelling increasesfrom 0 to 80 per cent. Subsequently, there is a new decreaseto 7 per cent labelling at 160min. The shapeof curve B indicates an average of 45 min. for the S-period in these 80 per cent of the cells. It is to be noted that Gi is short or absent. Curves A and B suggestthat the events described around division 1 repeat themselves at the time of division 2.
1 Present
address:
Department
of Molecular
Biology,
Odense
University,
Odense,
Experimentnl
Denmark.
Cell Research
48
Ii’. K. Hjelm
232
and E. Zeufhen
$00 % .; s 550 ; E P $ $ -0
0
60
120
180
minutes after EH Fig.
1.
Fig. l.-Curue A, Percentage of macronuclei labelled in DNA when the synchronized population is incubated with 3H-thymidine from EH (end of the period with heat shocks). Curve B, Percentage of macronuclei labelled in 5-min. pulses placed at various times after EH. The times indicate the termination of the pulses. Dose in both experiments: 3H-thymidine from Schwarz; 0.36 C/mM; 0.50 mC/ml. 12.5 ~1 added per ml culture. Fig. 2.-Histograms of numbers of silver grains over labelled macronuclei. Analysis of separate points from curve B in Fig. 1. The times indicate when the 5-min. pulses were terminated. Fig. 2.
Some of the autoradiograms from the pulse experiment of Fig. 1 B have been counted for the number of silver grains over the macronuclei (Fig. 2). In view of the wellknown limitations of the autoradiographic method, here used in its crudest form, the results can be considered only semiquantitative. However, they permit the suggestions (i) that at different times the individual macronuclei incorporate exogenous thymidine at rather different intensities, and (ii) that the incorporation rate in the average active macronucleus is low immediately after EH, increases until 35 min. and again decreases to a very low value at 79-84 min. From this time the rate of incorporation again increases (see also [4]). These changes may reflect variations in the rate of DNA synthesis through the synchronized cell cycle, but other interpretations are possible. For example, the results might reflect changes in the relative contribution to the new DNA by thymidine compounds from endogenous and from exogenous sources (cf. 13, 41). REFERENCES 1. 2. 3. 4.
HJELM, K. K. and ZEUTHEN, E., Compt. Rend. Trav. Lab. Carlsberq 36, 127 (1967). (ed.), Synchrony PLESNER, P., RASMUSSEN, L. and ZEUTHEN, E., in E. ZEUTHEN Division and Growth, p. 543. Interscience Publishers, New York, 1964. STONE, G. E., MILLER, 0. L. and PRESCOTT, D. M., J. Cell Biol. 25, 171 (1965). ZEUTHEN, E., Exptl Cell Res. In press (1967).
Experimental
Cell Research
48
in
Cell