OF BACTERIOLOGY, Feb. 1979, 0021-9193/79/02-1048/03$02.00/0
JOURNAL
p.
Vol. 137, No. 2
1048-1050
Synthesis of Ribosomal Proteins During the Cell Cycle of the Yeast Saccharomyces cerevisiae STEVEN G.
ELLIOTT,'
JONATHAN R. WARNER,2 AND CALVIN S. McLAUGHLIN1*
Department of Molecular Biology and Biochemistry, University of California-Irvine, Irvine, California 927171 and Departments of Biochemistry and Cell Biology, Albert Einstein College of Medicine, Bronx, New York 104612 Received for publication 5 October 1978
Centrifugal elutriation was used to separate yeast cells by their cell cycle position. The rate of synthesis of ribosomal proteins showed a constant exponential increase through the cell cycle. FLOW PATE The cell cycle of yeast has been studied exteil27 25 21 23 17 19 15 sively. In Saccharomyces cerevisiae, it has been 4 demonstrated that total protein, rRNA, mRNA, and tRNA are synthesized at a continuous rate during the cell cycle (5, 8, 9). In contrast, in 3 Schizosaccharomyces pombe, ribosomal proteins and rRNA show step changes in synthesis 2 (11) consistent with a linear doubling model which predicts a doubling in the rate of synthesis a * of a gene product when the gene dosage doubles ... during S phase. In both organisms, the activity of many enzymes has been shown to increase in a periodic manner during the cell cycle (5). Fur.4 b thermore, the synthesis of one defined class of proteins, the histones, has been shown to have .3 a variable rate of synthesis with a peak during S phase in S. cerevisiae. However, other studies in S. pombe suggested that cellular proteins, re.2 solved by sodium dodecyl sulfate gels, are synthesized in a nonperiodic manner (10). Shulman * 0 et al. (7) examined the synthesis of ribosomal proteins separated on one-dimensional gels in S. 1 cerevisiae from different periods of the cell cycle lI .1 .2 .3 .4 .5 .6 .7 .8 .9 1.0 and also could see no discontinuous synthesis. nm c ds cs ids CELL CYCLE POSITION In the latter two cases, the gel system did not FIG. 1. Cells were labeled for 3 h (1.5 generations) resolve individual proteins. Therefore, it is conceivable that peaks in synthesis of individual with 10 ,iCi of 3H-labeled yeast protein hydrolysate (Schwartz/Mann) per ml, then pulse-labeled for 10 proteins are dispersed throughout the cell cycle, min with 0.75 1Ci of r5S]methionine per ml (4). Inproviding a total constant rate of synthesis. In corporation was stopped by the addition of 0.5 volume the light of these problems, we decided to reex- crushed ice. Cells were separated by centrifugal eluamine the question of periodic synthesis by triation as previously described (2). Electrophoresis studying the synthesis of ribosomal proteins by ofpurified ribosomal proteins and total proteins was means of three improvements: two-dimensional performed as previously described (3). Spots were cut gels to resolve individual ribosomal proteins (3); out, and the radioactivity in them was determined by scintillation counting. The ratio of 35S/3H obtained a dual-label technique to label protein; and a selective technique, centrifugal elutriation (2), was corrected by dividing by the '5S/3H ratio observed to separate cells into different size classes rep- for total protein from the corresponding fraction. The corrected ratio then becomes independent of the resenting different stages of the cell cycle. amount of counts added. Its absolute value reflects The dual-label technique involves labeling the abundance of methionine in that protein. (a) protein continuously for more than a generation, Nonribosomal protein A. (b) Ribosomal protein 8. ids, followed by a second short pulse. The long-term Initiation of DNA synthesis; cds, cessation of DNA counts are a measure of the amount of protein, synthe.sis; nm, nuclear migration; cs, cell separation. -
V)
.1
0
--
O
1048
VOL. 137, 1979
NTS NOTES
14 1049
TABLE 1. Ratio of counts in each spot in each fraction Flow rate
Protein 15
1 2 5 6 8 9
0.409 0.207
17
0.686 0.598 0.339 0.266 0.272 0.328 0.303 0.270 0.374 0.303 0.133 0.166 0.155 0.307 0.334 0.307 9P 0.347 0.475 0.418 11 0.223 0.192 0.171 12 0.193 0.238 0.203 13 0.686 0.954 0.736 14 0.485 0.483 0.486 19 0.126 0.146 0.131 21 0.379 0.365 0.288 22 0.219 0.237 0.197 23 0.216 0.199 0.184 24 0.582 0.737 0.610 26 0.188 0.198 0.151 27 0.145 0.123 0.106 28 0.242 0.253 0.236 29 0.154 0.188 0.158 33 0.486 0.719 0.479 37 0.254 0.198 0.173 38 0.520 0.543 0.448 39 0.602 0.668 0.598 40 0.266 0.222 0.244 41 0.320 0.366 0.352 42 0.456 0.379 0.368 45 0.667 0.609 46 0.336 0.346 0.310 48 0.101 0.079 0.092 50 0.156 0.094 0.116 51 0.298 0.305 0.093 52 0.156 0.185 0.140 56 0.321 0.373 0.399 57 0.882 0.880 0.923 58 0.598 0.705 0.587 59 0.659 0.484 0.428 60 0.255 0.263 0.264 62 0.282 0.350 0.313 63 1.237 1.586 1.408 64 0.586 0.703 0.681 A 1.275 0.937 0.943 B 0.756 0.605 0.968 C 1.013 0.729 0.839 D 1.383 1.537 1.290 E 0.865 0.696 0.561 F 0.806 0.998 0.890 G 0.920 1.203 1.251 I 0.246 0.261 0.259 " SD, Standard deviation. See the text.
% SD
21
23
25
27
0.521 0.233 0.272 0.288 0.142 0.278 0.376 0.149 0.195 0.709 0.451 0.127 0.263 0.205 0.175 0.621 0.144 0.146 0.230 0.175 0.618 0.181 0.500 0.598 0.276 0.373 0.401 0.542 0.326 0.067 0.090 0.274 0.130 0.423 0.811 0.553 0.534 0.208 0.321 1.064 0.809 0.875 0.602 0.745 1.216 0.893 0.859 1.219 0.264
0.487 0.254 0.268 0.278 0.131 0.271 0.413 0.182 0.189 0.668 0.402 0.123 0.304 0.197 0.180 0.580 0.162 0.138 0.224 0.149 0.555 0.198 0.473 0.639 0.257 0.337 0.360 0.636 0.365 0.101 0.126 0.298 0.168 0.314 0.784 0.576 0.451 0.239 0.269 1.307 0.582 1.093 0.613 0.683 1.209 0.598 0.873 1.102 0.271
0.564 0.316 0.302 0.334 0.158 0.312 0.425 0.186 0.197 0.787 0.444 0.140 0.322 0.238 0.211 0.570 0.138 0.123 0.236 0.146 0.517 0.169 0.492 0.615 0.217 0.359 0.305 0.557 0.302 0.059 0.116 0.260 0.135 0.331 0.792 0.518 0.353 0.229 0.248 1.379 0.584 0.962 1.082 0.757 1.187 0.637 0.864 1.082 0.253
0.514 0.283 0.288 0.302 0.139 0.263 0.340 0.144 0.200 0.678 0.394 0.115 0.325 0.233 0.203 0.528 0.146 0.170 0.207 0.121 0.473 0.188 0.437 0.573 0.231 0.359 0.354 0.568 0.317 0.080 0.098 0.224 0.204 0.359 0.724 0.508 0.383 0.198 0.304 1.193 0.585 1.058 1.013 0.703 1.175 0.564 0.834 1.040 0.241
19
and the pulse counts are a measure of the rate of synthesis. The ratio of the two provides a number that is a measure of the rate of protein synthesis divided by the total amount of protein present. This ratio is independent of the actual recovery. Furthermore, it can distinguish unambiguously between step, periodic, and expo-
a
16.2 4.5 7.6 11.8 9.2 8.7 12.0 15.1 8.1 13.4 8.7 8.0 12.8 8.5 8.1 10.9 14.4 15.2 6.2 13.8 16.5 14.7 7.8 5.1 9.2 5.2 12.3 8.2 6.7 19.7 20.1 29.9 17.3 11.4 8.4 11.3 21.9 11.1 11.5 15.5 13.5 13.2 26.1 14.5 10.3 20.2 6.9 10.5 4.1
nential modes of synthesis. Step synthesis patterns are predicted by the linear doubling model in which the rate of synthesis of a gene product is proportional to the gene dosage, which doubles in S phase. Periodic synthesis patterns are predicted when genes are periodically activated or inactivated during the cell cycle. The expo-
1050
J. BACTERIOL.
NOTES
nential pattern occurs when the level of gene activity increases with the mass of the cell. The experiment is to label for 3 h with 3H-labeled yeast protein hydrolysate, followed by a 10-min pulse with [35S]methionine. The ribosomal proteins are extracted and run on two-dimensional gels as described previously (3). Sixty-five ribosomal proteins and several other cellular proteins are resolved by this system. The numbering system is as described previously (3). Dual-labeled cells prepared as described in Fig. 1 were fractionated into discrete cell types by centrifugal elutriation, and the proteins were extracted and separated on two-dimensional gels. In this procedure, ribosomal proteins are obtained from the total cellular protein rather than purified ribosomes. Individual spots were cut out, and the ratio of counts in them was determined. Figure lb is a typical plot of the ratio of counts versus cell cycle position for a ribosomal protein. The ratio remains constant, indicating that the synthesis of that protein is increasing in an exponential manner. In Fig. la, a nonribosomal protein also shows an exponentially increasing rate during the cell cycle. Table 1 depicts the ratio of counts of each spot in each fraction. Virtually all the proteins showed a constant ratio with points randomly distributed around the exponential accumulation line, indicating that all the ribosomal proteins and cellular proteins examined are synthesized at an exponentially increasing rate during the cell cycle. The percent standard deviation (standard deviation x 100/average) through the cell cycle was calculated for each protein. The average percent standard deviation was 11.9%. In contrast, DNA synthesis, which is clearly an example of periodic synthesis, showed a 75% standard deviation (1). The linear doubling model predicts a 17.1% standard deviation. Of the 49 proteins examined, only 8 had percent standard deviations greater than 17.1%. In addition, none of the proteins exhibited a pattern of change in ratio similar to that predicted by either the linear doubling model or the periodic synthesis model. If a protein exhibited periodic synthesis, a variation in ratio of greater than 10-fold would be expected. The linear doubling mode of accumulation would result in a twofold change. Therefore, these models can be ruled out. The only viable alternatives to the simple exponential model are more involved ones, such as periodic degradation and periodic synthesis, both of which occur at the same time; or models including posttranscriptional regulation in conjunction with one or both of the former. However, these models appear unlikely. The observation that ribosomal proteins are synthesized at a constant rate is not surprising. All the rRNA's are synthesized at a constant
rate (2; submitted for publication), and rRNA and ribosomal proteins react coordinately in response to nutritional changes (12). Therefore, the synthesis of ribosomal proteins and rRNA should increase in amount in the same exponential manner during the cell cycle. Although all the ribosomal proteins and some nonribosomal proteins are synthesized in an exponentially increasing manner, it is still possible that some proteins are synthesized periodically, e.g., histones (6). However, since total protein (1, 5), mRNA (submitted for publication), and the proteins reported on here show no periodic changes in rate, it is unlikely that doubling in gene number during S phase causes a generalized doubling in rate of synthesis of the majority of proteins, or that many proteins exhibit periodic synthesis at all. It would appear, therefore, that the numerous observations of periodic activity during the cell cycle may be due to periodic changes in regulatory molecules affecting enzyme activity and not enzyme synthesis. This research was supported by grant PCM75-03938 from the National Science Foundation to J.W. and by Public Health Service grant CA-10628 from the National Cancer Institute to C.M.
LITERATURE CITED 1. Elliott, S. G., and C. S. McLaughlin. 1978. Rate of
macromolecular synthesis through the cell cycle of the yeast Saccharormyces cereclisiae. Proc. Natl. Acad. Sci. U.S.A. 75:4384-4388. 2. Gordon, C. N., and S. G. Elliott. 1977. Fractionation of Saccharomyces cereuisiae cell populations by centrifugal elutriation. J. Bacteriol. 129:97-100. 3. Gorenstein, C., and J. R. Warner. 1976. Coordinate regulation of the synthesis of eukaryotic ribosomal proteins. Proc. Natl. Acad. Sci. U.S.A. 73:1547-1551. 4. Graham, R., and W. M. Stanley. 1972. An economical procedure for the preparation of 1- 'S-methionine of high specific activity. Anal. Biochem. 47:505-51:3. 5. Mitchison, J. M. 1971. The biology of the cell cycle. Cambridge University Press, Cambridge, Great Britain. 6. Moll, R., and E. Wintersberger. 1976. The synthesis of yeast histones in the cell cycle. Proc. Natl. Acad. Sci. U.S.A. 73:1863-1867. 7. Shulman, R. W., L. H. Hartwell, and J. R. Warner. 1973. Synthesis of ribosomal proteins during the yeast cell cycle. J. Mol. Biol. 73:513-525. 8. Sogin, S. J., B. L. A. Carter, and H. 0. Halvorson. 1974. Changes in the rate of ribosomal RNA synthesis during the cell cycle of Saccharomyces cereuisiae. Exp. Cell Res. 89:127-138. 9. Tauro, P., E. Schweizer, R. Epstein, and H. 0. Halvorson. 1969. Synthesis of macromolecules during cell cycle arrest in yeast, p. 101-118. In G. M. Padilla, G. L. Whitson, and I. L. Camieron (ed.), The cell cycle gene enzyme interactions. Academic Press, New York. 10. Wain, W. H. 1971. Synthesis of soluble protein during the cell cycle of the fission yeast Schizosaccharomvces pombe. Exp. Cell Res. 69:49-56. 1 1. Wain, W. H., and W. D. Staatz. 1973. Rate of synthesis of ribosomal protein and total RNA through the cell cycle of the fission yeast Schizosacchatoomces pornbe. Exp. Cell Res. 81:269-278. 12. Warner, J. R., and C. Gorenstein. 1978. Yeast has a true stringent response. Nature (Lontion) 275:338-3:39.
JOURNAL
p.
Vol. 137, No. 2
1048-1050
Synthesis of Ribosomal Proteins During the Cell Cycle of the Yeast Saccharomyces cerevisiae STEVEN G.
ELLIOTT,'
JONATHAN R. WARNER,2 AND CALVIN S. McLAUGHLIN1*
Department of Molecular Biology and Biochemistry, University of California-Irvine, Irvine, California 927171 and Departments of Biochemistry and Cell Biology, Albert Einstein College of Medicine, Bronx, New York 104612 Received for publication 5 October 1978
Centrifugal elutriation was used to separate yeast cells by their cell cycle position. The rate of synthesis of ribosomal proteins showed a constant exponential increase through the cell cycle. FLOW PATE The cell cycle of yeast has been studied exteil27 25 21 23 17 19 15 sively. In Saccharomyces cerevisiae, it has been 4 demonstrated that total protein, rRNA, mRNA, and tRNA are synthesized at a continuous rate during the cell cycle (5, 8, 9). In contrast, in 3 Schizosaccharomyces pombe, ribosomal proteins and rRNA show step changes in synthesis 2 (11) consistent with a linear doubling model which predicts a doubling in the rate of synthesis a * of a gene product when the gene dosage doubles ... during S phase. In both organisms, the activity of many enzymes has been shown to increase in a periodic manner during the cell cycle (5). Fur.4 b thermore, the synthesis of one defined class of proteins, the histones, has been shown to have .3 a variable rate of synthesis with a peak during S phase in S. cerevisiae. However, other studies in S. pombe suggested that cellular proteins, re.2 solved by sodium dodecyl sulfate gels, are synthesized in a nonperiodic manner (10). Shulman * 0 et al. (7) examined the synthesis of ribosomal proteins separated on one-dimensional gels in S. 1 cerevisiae from different periods of the cell cycle lI .1 .2 .3 .4 .5 .6 .7 .8 .9 1.0 and also could see no discontinuous synthesis. nm c ds cs ids CELL CYCLE POSITION In the latter two cases, the gel system did not FIG. 1. Cells were labeled for 3 h (1.5 generations) resolve individual proteins. Therefore, it is conceivable that peaks in synthesis of individual with 10 ,iCi of 3H-labeled yeast protein hydrolysate (Schwartz/Mann) per ml, then pulse-labeled for 10 proteins are dispersed throughout the cell cycle, min with 0.75 1Ci of r5S]methionine per ml (4). Inproviding a total constant rate of synthesis. In corporation was stopped by the addition of 0.5 volume the light of these problems, we decided to reex- crushed ice. Cells were separated by centrifugal eluamine the question of periodic synthesis by triation as previously described (2). Electrophoresis studying the synthesis of ribosomal proteins by ofpurified ribosomal proteins and total proteins was means of three improvements: two-dimensional performed as previously described (3). Spots were cut gels to resolve individual ribosomal proteins (3); out, and the radioactivity in them was determined by scintillation counting. The ratio of 35S/3H obtained a dual-label technique to label protein; and a selective technique, centrifugal elutriation (2), was corrected by dividing by the '5S/3H ratio observed to separate cells into different size classes rep- for total protein from the corresponding fraction. The corrected ratio then becomes independent of the resenting different stages of the cell cycle. amount of counts added. Its absolute value reflects The dual-label technique involves labeling the abundance of methionine in that protein. (a) protein continuously for more than a generation, Nonribosomal protein A. (b) Ribosomal protein 8. ids, followed by a second short pulse. The long-term Initiation of DNA synthesis; cds, cessation of DNA counts are a measure of the amount of protein, synthe.sis; nm, nuclear migration; cs, cell separation. -
V)
.1
0
--
O
1048
VOL. 137, 1979
NTS NOTES
14 1049
TABLE 1. Ratio of counts in each spot in each fraction Flow rate
Protein 15
1 2 5 6 8 9
0.409 0.207
17
0.686 0.598 0.339 0.266 0.272 0.328 0.303 0.270 0.374 0.303 0.133 0.166 0.155 0.307 0.334 0.307 9P 0.347 0.475 0.418 11 0.223 0.192 0.171 12 0.193 0.238 0.203 13 0.686 0.954 0.736 14 0.485 0.483 0.486 19 0.126 0.146 0.131 21 0.379 0.365 0.288 22 0.219 0.237 0.197 23 0.216 0.199 0.184 24 0.582 0.737 0.610 26 0.188 0.198 0.151 27 0.145 0.123 0.106 28 0.242 0.253 0.236 29 0.154 0.188 0.158 33 0.486 0.719 0.479 37 0.254 0.198 0.173 38 0.520 0.543 0.448 39 0.602 0.668 0.598 40 0.266 0.222 0.244 41 0.320 0.366 0.352 42 0.456 0.379 0.368 45 0.667 0.609 46 0.336 0.346 0.310 48 0.101 0.079 0.092 50 0.156 0.094 0.116 51 0.298 0.305 0.093 52 0.156 0.185 0.140 56 0.321 0.373 0.399 57 0.882 0.880 0.923 58 0.598 0.705 0.587 59 0.659 0.484 0.428 60 0.255 0.263 0.264 62 0.282 0.350 0.313 63 1.237 1.586 1.408 64 0.586 0.703 0.681 A 1.275 0.937 0.943 B 0.756 0.605 0.968 C 1.013 0.729 0.839 D 1.383 1.537 1.290 E 0.865 0.696 0.561 F 0.806 0.998 0.890 G 0.920 1.203 1.251 I 0.246 0.261 0.259 " SD, Standard deviation. See the text.
% SD
21
23
25
27
0.521 0.233 0.272 0.288 0.142 0.278 0.376 0.149 0.195 0.709 0.451 0.127 0.263 0.205 0.175 0.621 0.144 0.146 0.230 0.175 0.618 0.181 0.500 0.598 0.276 0.373 0.401 0.542 0.326 0.067 0.090 0.274 0.130 0.423 0.811 0.553 0.534 0.208 0.321 1.064 0.809 0.875 0.602 0.745 1.216 0.893 0.859 1.219 0.264
0.487 0.254 0.268 0.278 0.131 0.271 0.413 0.182 0.189 0.668 0.402 0.123 0.304 0.197 0.180 0.580 0.162 0.138 0.224 0.149 0.555 0.198 0.473 0.639 0.257 0.337 0.360 0.636 0.365 0.101 0.126 0.298 0.168 0.314 0.784 0.576 0.451 0.239 0.269 1.307 0.582 1.093 0.613 0.683 1.209 0.598 0.873 1.102 0.271
0.564 0.316 0.302 0.334 0.158 0.312 0.425 0.186 0.197 0.787 0.444 0.140 0.322 0.238 0.211 0.570 0.138 0.123 0.236 0.146 0.517 0.169 0.492 0.615 0.217 0.359 0.305 0.557 0.302 0.059 0.116 0.260 0.135 0.331 0.792 0.518 0.353 0.229 0.248 1.379 0.584 0.962 1.082 0.757 1.187 0.637 0.864 1.082 0.253
0.514 0.283 0.288 0.302 0.139 0.263 0.340 0.144 0.200 0.678 0.394 0.115 0.325 0.233 0.203 0.528 0.146 0.170 0.207 0.121 0.473 0.188 0.437 0.573 0.231 0.359 0.354 0.568 0.317 0.080 0.098 0.224 0.204 0.359 0.724 0.508 0.383 0.198 0.304 1.193 0.585 1.058 1.013 0.703 1.175 0.564 0.834 1.040 0.241
19
and the pulse counts are a measure of the rate of synthesis. The ratio of the two provides a number that is a measure of the rate of protein synthesis divided by the total amount of protein present. This ratio is independent of the actual recovery. Furthermore, it can distinguish unambiguously between step, periodic, and expo-
a
16.2 4.5 7.6 11.8 9.2 8.7 12.0 15.1 8.1 13.4 8.7 8.0 12.8 8.5 8.1 10.9 14.4 15.2 6.2 13.8 16.5 14.7 7.8 5.1 9.2 5.2 12.3 8.2 6.7 19.7 20.1 29.9 17.3 11.4 8.4 11.3 21.9 11.1 11.5 15.5 13.5 13.2 26.1 14.5 10.3 20.2 6.9 10.5 4.1
nential modes of synthesis. Step synthesis patterns are predicted by the linear doubling model in which the rate of synthesis of a gene product is proportional to the gene dosage, which doubles in S phase. Periodic synthesis patterns are predicted when genes are periodically activated or inactivated during the cell cycle. The expo-
1050
J. BACTERIOL.
NOTES
nential pattern occurs when the level of gene activity increases with the mass of the cell. The experiment is to label for 3 h with 3H-labeled yeast protein hydrolysate, followed by a 10-min pulse with [35S]methionine. The ribosomal proteins are extracted and run on two-dimensional gels as described previously (3). Sixty-five ribosomal proteins and several other cellular proteins are resolved by this system. The numbering system is as described previously (3). Dual-labeled cells prepared as described in Fig. 1 were fractionated into discrete cell types by centrifugal elutriation, and the proteins were extracted and separated on two-dimensional gels. In this procedure, ribosomal proteins are obtained from the total cellular protein rather than purified ribosomes. Individual spots were cut out, and the ratio of counts in them was determined. Figure lb is a typical plot of the ratio of counts versus cell cycle position for a ribosomal protein. The ratio remains constant, indicating that the synthesis of that protein is increasing in an exponential manner. In Fig. la, a nonribosomal protein also shows an exponentially increasing rate during the cell cycle. Table 1 depicts the ratio of counts of each spot in each fraction. Virtually all the proteins showed a constant ratio with points randomly distributed around the exponential accumulation line, indicating that all the ribosomal proteins and cellular proteins examined are synthesized at an exponentially increasing rate during the cell cycle. The percent standard deviation (standard deviation x 100/average) through the cell cycle was calculated for each protein. The average percent standard deviation was 11.9%. In contrast, DNA synthesis, which is clearly an example of periodic synthesis, showed a 75% standard deviation (1). The linear doubling model predicts a 17.1% standard deviation. Of the 49 proteins examined, only 8 had percent standard deviations greater than 17.1%. In addition, none of the proteins exhibited a pattern of change in ratio similar to that predicted by either the linear doubling model or the periodic synthesis model. If a protein exhibited periodic synthesis, a variation in ratio of greater than 10-fold would be expected. The linear doubling mode of accumulation would result in a twofold change. Therefore, these models can be ruled out. The only viable alternatives to the simple exponential model are more involved ones, such as periodic degradation and periodic synthesis, both of which occur at the same time; or models including posttranscriptional regulation in conjunction with one or both of the former. However, these models appear unlikely. The observation that ribosomal proteins are synthesized at a constant rate is not surprising. All the rRNA's are synthesized at a constant
rate (2; submitted for publication), and rRNA and ribosomal proteins react coordinately in response to nutritional changes (12). Therefore, the synthesis of ribosomal proteins and rRNA should increase in amount in the same exponential manner during the cell cycle. Although all the ribosomal proteins and some nonribosomal proteins are synthesized in an exponentially increasing manner, it is still possible that some proteins are synthesized periodically, e.g., histones (6). However, since total protein (1, 5), mRNA (submitted for publication), and the proteins reported on here show no periodic changes in rate, it is unlikely that doubling in gene number during S phase causes a generalized doubling in rate of synthesis of the majority of proteins, or that many proteins exhibit periodic synthesis at all. It would appear, therefore, that the numerous observations of periodic activity during the cell cycle may be due to periodic changes in regulatory molecules affecting enzyme activity and not enzyme synthesis. This research was supported by grant PCM75-03938 from the National Science Foundation to J.W. and by Public Health Service grant CA-10628 from the National Cancer Institute to C.M.
LITERATURE CITED 1. Elliott, S. G., and C. S. McLaughlin. 1978. Rate of
macromolecular synthesis through the cell cycle of the yeast Saccharormyces cereclisiae. Proc. Natl. Acad. Sci. U.S.A. 75:4384-4388. 2. Gordon, C. N., and S. G. Elliott. 1977. Fractionation of Saccharomyces cereuisiae cell populations by centrifugal elutriation. J. Bacteriol. 129:97-100. 3. Gorenstein, C., and J. R. Warner. 1976. Coordinate regulation of the synthesis of eukaryotic ribosomal proteins. Proc. Natl. Acad. Sci. U.S.A. 73:1547-1551. 4. Graham, R., and W. M. Stanley. 1972. An economical procedure for the preparation of 1- 'S-methionine of high specific activity. Anal. Biochem. 47:505-51:3. 5. Mitchison, J. M. 1971. The biology of the cell cycle. Cambridge University Press, Cambridge, Great Britain. 6. Moll, R., and E. Wintersberger. 1976. The synthesis of yeast histones in the cell cycle. Proc. Natl. Acad. Sci. U.S.A. 73:1863-1867. 7. Shulman, R. W., L. H. Hartwell, and J. R. Warner. 1973. Synthesis of ribosomal proteins during the yeast cell cycle. J. Mol. Biol. 73:513-525. 8. Sogin, S. J., B. L. A. Carter, and H. 0. Halvorson. 1974. Changes in the rate of ribosomal RNA synthesis during the cell cycle of Saccharomyces cereuisiae. Exp. Cell Res. 89:127-138. 9. Tauro, P., E. Schweizer, R. Epstein, and H. 0. Halvorson. 1969. Synthesis of macromolecules during cell cycle arrest in yeast, p. 101-118. In G. M. Padilla, G. L. Whitson, and I. L. Camieron (ed.), The cell cycle gene enzyme interactions. Academic Press, New York. 10. Wain, W. H. 1971. Synthesis of soluble protein during the cell cycle of the fission yeast Schizosaccharomvces pombe. Exp. Cell Res. 69:49-56. 1 1. Wain, W. H., and W. D. Staatz. 1973. Rate of synthesis of ribosomal protein and total RNA through the cell cycle of the fission yeast Schizosacchatoomces pornbe. Exp. Cell Res. 81:269-278. 12. Warner, J. R., and C. Gorenstein. 1978. Yeast has a true stringent response. Nature (Lontion) 275:338-3:39.
