JOURNAL OF BACTERIOLOGY, Oct. 1976, p. 502-505 Copyright © 1976 American Society for Microbiology
Vol. 128, No. 1 Printed in U.S.A.
Rate of Synthesis of Polyadenylate-Containing Ribonucleic Acid During the Yeast Cell Cycle NANCY E. HYNES'
AND
STEPHEN L. PHILLIPS*
Department of Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received for publication 24 June 197Q
The rate of synthesis of polyadenylate-containing ribonucleic acid is constant throughout the cell cycle of Saccharonyces cerevisiae.
During the life cycle of a cell, diverse biological events such as deoxyribonucleic acid (DNA) replication and nuclear division must be coordinated so that they occur in the proper order. Exactly how these events are coordinated is not known, but there is growing evidence that differential gene expression, manifested by restricted periods for the synthesis of individual proteins, may play a role in this coordination process (10). In mammalian cells, studies of the synthesis of nucleohistones have shown that this class of proteins accumulates in the nucleus only during the S phase of the cell cycle (1, 3). Nonhistone, DNA-binding proteins have also been shown to have periodic synthesis (15). The strongest evidence in favor of periodic protein synthesis has come from measurements of enzyme activity in synchronous cultures of yeast (2, 5). Whether enzymes are synthesized in a periodic manner because they play a role in control of cellular events at a specific period in the life cycle is not yet known. Ribonucleic acid (RNA) of the eukaryote cell is synthesized throughout the cell cycle (10). Tauro et al. (17) have presented evidence suggesting that in Saccharomyces cerevisiae the rate of synthesis of ribosomal RNA is constant throughout the cell cycle. However, messenger RNA synthesis has never been subjected to this type of an analysis. Yeast messenger RNA contains polyadenylic acid [poly(A)] (4, 8, 14). Since it is possible to readily isolate poly(A)containing RNA from bulk cellular RNA by virture of its ability to hybridize to oligodeoxyribothymidylic acid [oligo(dT)]-cellulose, a study of the relative rate of synthesis of poly(A)-containing RNA throughout the cell cycle was carried out using this methodology. A variation of a procedure first described by Sebastian et al. (16) was employed to obtain yeast cells at various stages of their cell cycle. I Present address: Max-Planck Institute Genetik, 1 Berlin 33, West Germany.
crease in size throughout the cycle and thus can It is based upon the fact that yeast cells inbe separated into the various stages of the cycle by zonal sedimentation through a sucrose gradient. To document the efficacy of the procedure the appearance of several known cell cycle markers was analyzed. These markers have been described as "landmarks" by Hartwell (6) because they are discontinuous events that occur during a defined interval in the cycle. The DNA of S. cerevisiae replicates at a discrete interval early in the life cycle of the cell (12, 18, 19, 20). An asynchronous yeast population was labeled overnight with ['4C]adenine (at a concentration sufficient to ensure linear uptake of radioactivity) and fractionated by zonal sedimentation. DNA content per cell was measured by determining the cell number and the alkali-resistant, trichloroacetic acid-precipitable radioactivity in each fraction of the gradient (19). The distribution of cells throughout the gradient and the 14C counts per minute in DNA per cell are shown in Fig. 1. The entire cell cycle is displayed in 16 fractions of the gradient. DNA content doubles in four to five fractions, an interval that corresponds quite closely to that fraction of the cell cycle occupied by DNA replication (19). Buds emerge from the cell at the end of the Gl phase and grow in size throughout the cell cycle (9). This is the most convenient landmark to assess since it can be monitored by direct visual examination. Cells were harvested and separated by zonal sedimentation (Fig. 2). Fractions of the gradient were collected, and the total number of cells in each fraction, along with the distribution between budded and unbudded cells, was determined. The percentage of cells with buds for each fraction is shown in Fig. 2. Bud emergence, a discrete event in the cell cycle, occurs as a step in one to two fracfur Molekular tions of the sucrose gradient. Thus, the data presented above confirm that the zonal sedi502
503
NOTES
VOL. 128, 1976
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FRACTION NUMBER FIG. 1. DNA content of cells separated by zonal sedimentation into a sucrose gradient. A 10-ml culture of S288C was grown for 16 h in minimal medium (7 g ofyeast nitrogen base without amino acids and 20 g of glucose per liter of distilled water) containing ["4C]adenine (0.067 iLCi/pg) at a concentration of 15 pg/ml. At a concentration of 2 x 107 cells/ ml, the culture was harvested by pouring over a 0.5culture volume of crushed ice. The cells were collected by centrifugation (6 min at 4,500 x g), washed once, and suspended in 2 ml of cold water. The cell suspension was layered on a 40-ml, 15 to 35% (wtl vol) linear sucrose gradient prepared in water and gently stirred with a thin glass rod to mix it with the top 10 mm of the sucrose gradient. This last step has been reported to prevent cell streaming by producing an inverse gradient of cells at the top of the sucrose gradient (11). The gradient was then centrifuged at 4°C in an SW27 rotor (Beckman) at 2,500 rpm for 2 min (from the moment power was applied to the rotor until the power was switched off). The gradient was collected by pumping its contents into 2-ml fractions. The cells were recovered from each fraction by centrifugation (15 min, 1,000 x g) at 4°C and were su,,Aended in cold water. Cell concentration in a small sample from each fraction was determined using a model b Coulter counter. Radioactivity in DNA was determined as alkali-resistant, trichloroacetic acid-precipitable counts per minute (19). Each fraction was brought to 0.3 N KOH and incubated at 37°C for 18 h. Hydrolysis was terminated, and macromolecules were precipitated by bringing each sample to 10% trichloroacetic acid. Carrier RNA was added, and samples were held on ice for 30 min. Precipitates were collected on glass fiber filters, washed three times with 3 ml of cold 5% trichloroacetic acid and once with 3 ml of cold 95% ethanol, and dried. Radioactivity retained on filters was monitored by scintillation spectrometry in 4.4 ml of scintillation solution [0.01 g of 1,4-bis-2-(4-methyl-5phenyloxazole)benzene and 6 g of 2,5-diphenyl-orazole per liter oftoluene]. Symbols: 0, cells/ml in each fraction (sedimentation is from left to right); x, radioactivity in DNA per cell in each fraction.
w
2 4 6 s FRACTION NUMBER FIG. 2. Bud appearance in cells separated by zonal sedimentation into a sucrose gradient. A culture of S288C (4 x 108 cells) was prepared and fractionated by zonal sedimentation as described in the legend to Fig. 1. The total number of cells, budded and unbudded, was determined visually using light microscopy and a hematocytometer. Symbols: 0, number of cells per fraction; x, percentage of cells in each fraction with buds.
mentation procedure separates an asynchronous population of yeast by position in the cell cycle. A sample of cells was labeled for 3' with [3H]adenine, harvested, and separated by zonal sedimentation according to position in the cell cycle. Fractions were collected, and the number of cells in each fraction was determined (Fig. 3). The RNA was extracted from each fraction and chromatographed on small columns of oligo(dT)-cellulose, and the radioactivity in poly(A)-containing RNA and RNA that does not bind to oligo(dT)-cellulose was determined. Ribosomal RNA does not bind to oligo(dT)-cellulose using our conditions of chromatography (4). Thus, the majority of the pulse-labeled RNA that does not bind to the columns is precursor ribosomal RNA (4). The data presented in Table 1, columns 2 and 3, show that the distribution of pulsed label between poly(A)-containing RNA and RNA that does not bind to oligo(dT)-cellulose does not vary significantly from fraction to fraction. Thus, if there is only one nuclear pool for the nucleoside triphosphates, we conclude that the relative rates of synthesis of poly(A)-containing RNA and ribosomal RNA do not vary significantly during the cell cycle. Based on the observation that the rate of synthesis of ribosomal RNA is constant throughout the cell cycle (17), we conclude that the rate of synthesis of poly(A)-containing RNA is also constant. This conclusion is supported by the data presented in column 4 of Table 1, which demonstrate that
504
NOTES
J. BACTERIOL.
5
4 (I)
3
-J w
2 0
4
6
8
10
12
FRACTION NUMBER FIG. 3. Zonal sedimentation of cells labeled for 3 min with [3H]adenine. See footnote a, Table 1, for details of the experiment.
An41ysis of the rate of RNA synthesis throughout the yeast cell cyclea
TA.:LE 1,
Total radioactivity (%) in: Fractionb
4 5 6 7 8 9 10 11
cpm/cell x 102
Nonbinding RNA 66 65 65 68 67 67 69 71
in
Poly(A)- poly(A)-concontain- taining RNA ing RNA 34 1.0 35 1.2 35 1.0 32 0.8 32 1.3 33 1.2 31 0.9 29
1.3
67 ± 0.7 33 ± 0.7 1.1 + 0.07 Average + SEM a A 10 ml-culture of S288C was labeled for 3 min with 15 ,uCi of [3H]adenine per ml (0.15 Ci/mg). The cells were rapidly chilled, harvested, and fractioned by zonal sedimentation as described in the legend to Fig. 1. The cells from each fraction were suspended in 0.4 ml of sodium dodecyl sulfate (SDS) buffer [0.1 M NaCl-20 mM ethylenediaminetetraacetate-20 mM tris(hydroxymethyl)aminomethanehydrochloride pH 7.5-0.5% sodium dodecyl sulfate). Carrier cells (1.4 x 108) in 0.3 ml of SDS buffer were added to each fraction. Nucleic acids were obtained from each fraction in the following way. The cell suspension was transferred into the chamber of an Eaton press (a kind gift of Walter Vincent, University of Delaware) that was maintained at dry ice temperature. The quick-frozen cell suspension was forced through the 1-mm orifice of the chamber by application of 10,000-lb./in2 pressure. Then the extract was quickly brought to room temperature, and cell debris was removed by centrifugation for 10 min at 4,500 x g. The extract was brought to 2 ml with SDS buffer, and the nucleic acids were isolated using the SDS-hot phenol-chloroform method described by Penman (13). The RNA in each fraction was passed over a column of oligo(dT)-cellulose and fractionated into nonbinding RNA and poly(A)-containing RNA as previously described (8). b Refer to Fig. 3. SEM, Standard error of the mean.
the actual amount of isotope incorporated during a 3-min pulse into poly(A)-containing RNA per cell is essentially uniform during the cell
cycle. Thus, taken together, the data suggests that the synthesis of ribosomal RNA and polyadenylated messenger RNA is constant throughout the cell cycle. Continued metabolism of RNA during the synchronization procedure may have changed the fraction of labeled RNA that binds to oligo(dT)-cellulose. However, this is unlikely to be a major problem because the fraction of RNA that binds to oligo(dT)-cellulose reported here and its size distribution are very similar to these parameters obtained for RNA extracted directly from an asynchronous population of cells labeled for 3 min with [3H]adenine (data not shown). Certainly, this study does not rule out the possibility that the synthesis of some species of mRNA are restricted to a particular time in the cell cycle. Messengers that constitute a small fraction of the poly(A)-containing RNA or which are devoid of polyWA) would have gone undetected in this analysis. Thus, a rigorous test of the periodic synthesis of specific messengers must await their isolation. The investigation was supported by grant GB-26315X from the National Science Foundation to S.L. P.N.E. H. was supported by Public Health Service Predoctroal training grant GM000149 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Bomn, T. W., M. D. Scharff, and E. Robbins. 1967. Rapidly labeled polyribosome associated RNA having the properties of histone messenger. Proc. Natl. Acad. Sci. U.S.A. 58:1977-1983. 2. Donachie, W. D., and M. Masters. 1969. Enzyme interactions, p. 37-76. In G. M. Padilla, G. L. Whitson, and I. L. Cameron (ed.), The cell cycle. Academic Press Inc., New York. 3. Gallwitz, D., and G. C. Mueller. 1969. Histone synthesis in vitro on HeLa cell microsomes: the nature of the coupling to DNA synthesis. J. Biol. Chem. 244:59475952. 4. Groner, B., N. Hynes, and S. Phillips. 1974. Length heterogeneity in the poly(adenylic) acid region of yeast messenger ribonucleic acid. Biochemistry 13:5378-5383. 5. Halvorson, H. O., B. L. A. Carter, and P. Tauro. 1971. Synthesis of enzymes during the cell cycle. Adv. Microbiol. Physiol. 6:47-106. 6. Hartwell, L. H. 1974. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38:164-198. 7. Hynes, N. E., and S. L. Phillips. 1976. Turnover of polyadenylate-containing RNA in Saccharomyces cerevisiae. J. Bacteriol. 125:595-600. 8. McLaughlin, C. S., J. R. Warner, M. Edmonds, H. Nakazato, and M. Vaughan. 1973. Polyadenylic acid sequences in yeast messenger ribonucleic acid. J. Biol. Chem. 248:1466-1471. 9. Mitchison, J. M. 1958. The growth of single cells. II. Saccharomyces cerevisiae. Exp. Cell Res. 15:214-221. 10. Mitchison, J. M. 1973. Differentiation in the cell cycle, p. 1-9. In M. Balls and F. S. Billett (ed.), The cell cycle in development and differentiation. Cambridge
University Press, Cambridge. 11. Mitchison, J. M. and W. S. Vincent. 1965. Preparation
of synchronous cell cultures by sedimentation. Nature (London) 205:987-989.
VOL. 128, 1976 12. Ogur, M., S. Minckler, and D. McClary. 1953. Deoxyribonucleic acid and the budding cycle in the yeasts. J. Bacteriol. 66:642-645. 13. Penman, S. 1969. Preparation of purified nuclei and nucleoli from mammalian cells, p. 35-48. In K. Habel and N. P. Salzman (ed.), Fundamental techniques in virology. Academic Press Inc., New York. 14. Reed, J., and E. Winteraberger. 1973. Adenylic acid rich sequences in messenger RNA from yeast polysomes. FEBS Lett. 32:213-217. 15. Salas, J., and H. Green. 1971. Proteins binding to DNA and their relation to growth in cultured mammalian cells. Nature (London) New Biol. 229:165-169. 16. Sebastian, J., B. L. A. Carter, and H. 0. Halvorson. 1971. Use of yeast populations fractionated by zonal centrifugation to study the cell cycle. J. Bacteriol.
NOTES
505
108:1045-1050. 17. Tauro, P., E. Schweizer, R. Epstein, and H. 0. Halvorson. 1969. Synthesis of macromolecules during the cell cycle in yeast, p. 101-108. In G. M. Padilla, G. L. Whitson, and I. L. Cameron (ed.), The cell cycle. Academic Press Inc., New York. 18. Williamson, D. H. 1964. The timing of deoxyribonucleic acid synthesis in the cell cycle of Saccharomyces cerevisiae. Biochem. J. 90:25-26. 19. Williamson, D. H. 1965. The timing of deoxyribonucleic acid synthesis in the cell cycle of Saccharomyces cerevisiae. J. Cell Biol. 25:517-528. 20. Williamson, D. H., and A. W. Scopes. 1960. The behavior of nucleic acids in synchronously dividing cultures of Saccharomyces cerevisiae. Exp. Cell Res. 20:338349.
Vol. 128, No. 1 Printed in U.S.A.
Rate of Synthesis of Polyadenylate-Containing Ribonucleic Acid During the Yeast Cell Cycle NANCY E. HYNES'
AND
STEPHEN L. PHILLIPS*
Department of Biochemistry, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 Received for publication 24 June 197Q
The rate of synthesis of polyadenylate-containing ribonucleic acid is constant throughout the cell cycle of Saccharonyces cerevisiae.
During the life cycle of a cell, diverse biological events such as deoxyribonucleic acid (DNA) replication and nuclear division must be coordinated so that they occur in the proper order. Exactly how these events are coordinated is not known, but there is growing evidence that differential gene expression, manifested by restricted periods for the synthesis of individual proteins, may play a role in this coordination process (10). In mammalian cells, studies of the synthesis of nucleohistones have shown that this class of proteins accumulates in the nucleus only during the S phase of the cell cycle (1, 3). Nonhistone, DNA-binding proteins have also been shown to have periodic synthesis (15). The strongest evidence in favor of periodic protein synthesis has come from measurements of enzyme activity in synchronous cultures of yeast (2, 5). Whether enzymes are synthesized in a periodic manner because they play a role in control of cellular events at a specific period in the life cycle is not yet known. Ribonucleic acid (RNA) of the eukaryote cell is synthesized throughout the cell cycle (10). Tauro et al. (17) have presented evidence suggesting that in Saccharomyces cerevisiae the rate of synthesis of ribosomal RNA is constant throughout the cell cycle. However, messenger RNA synthesis has never been subjected to this type of an analysis. Yeast messenger RNA contains polyadenylic acid [poly(A)] (4, 8, 14). Since it is possible to readily isolate poly(A)containing RNA from bulk cellular RNA by virture of its ability to hybridize to oligodeoxyribothymidylic acid [oligo(dT)]-cellulose, a study of the relative rate of synthesis of poly(A)-containing RNA throughout the cell cycle was carried out using this methodology. A variation of a procedure first described by Sebastian et al. (16) was employed to obtain yeast cells at various stages of their cell cycle. I Present address: Max-Planck Institute Genetik, 1 Berlin 33, West Germany.
crease in size throughout the cycle and thus can It is based upon the fact that yeast cells inbe separated into the various stages of the cycle by zonal sedimentation through a sucrose gradient. To document the efficacy of the procedure the appearance of several known cell cycle markers was analyzed. These markers have been described as "landmarks" by Hartwell (6) because they are discontinuous events that occur during a defined interval in the cycle. The DNA of S. cerevisiae replicates at a discrete interval early in the life cycle of the cell (12, 18, 19, 20). An asynchronous yeast population was labeled overnight with ['4C]adenine (at a concentration sufficient to ensure linear uptake of radioactivity) and fractionated by zonal sedimentation. DNA content per cell was measured by determining the cell number and the alkali-resistant, trichloroacetic acid-precipitable radioactivity in each fraction of the gradient (19). The distribution of cells throughout the gradient and the 14C counts per minute in DNA per cell are shown in Fig. 1. The entire cell cycle is displayed in 16 fractions of the gradient. DNA content doubles in four to five fractions, an interval that corresponds quite closely to that fraction of the cell cycle occupied by DNA replication (19). Buds emerge from the cell at the end of the Gl phase and grow in size throughout the cell cycle (9). This is the most convenient landmark to assess since it can be monitored by direct visual examination. Cells were harvested and separated by zonal sedimentation (Fig. 2). Fractions of the gradient were collected, and the total number of cells in each fraction, along with the distribution between budded and unbudded cells, was determined. The percentage of cells with buds for each fraction is shown in Fig. 2. Bud emergence, a discrete event in the cell cycle, occurs as a step in one to two fracfur Molekular tions of the sucrose gradient. Thus, the data presented above confirm that the zonal sedi502
503
NOTES
VOL. 128, 1976
U) 0
24 0
0
1
0
l
x
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0
1
D
I
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FRACTION NUMBER FIG. 1. DNA content of cells separated by zonal sedimentation into a sucrose gradient. A 10-ml culture of S288C was grown for 16 h in minimal medium (7 g ofyeast nitrogen base without amino acids and 20 g of glucose per liter of distilled water) containing ["4C]adenine (0.067 iLCi/pg) at a concentration of 15 pg/ml. At a concentration of 2 x 107 cells/ ml, the culture was harvested by pouring over a 0.5culture volume of crushed ice. The cells were collected by centrifugation (6 min at 4,500 x g), washed once, and suspended in 2 ml of cold water. The cell suspension was layered on a 40-ml, 15 to 35% (wtl vol) linear sucrose gradient prepared in water and gently stirred with a thin glass rod to mix it with the top 10 mm of the sucrose gradient. This last step has been reported to prevent cell streaming by producing an inverse gradient of cells at the top of the sucrose gradient (11). The gradient was then centrifuged at 4°C in an SW27 rotor (Beckman) at 2,500 rpm for 2 min (from the moment power was applied to the rotor until the power was switched off). The gradient was collected by pumping its contents into 2-ml fractions. The cells were recovered from each fraction by centrifugation (15 min, 1,000 x g) at 4°C and were su,,Aended in cold water. Cell concentration in a small sample from each fraction was determined using a model b Coulter counter. Radioactivity in DNA was determined as alkali-resistant, trichloroacetic acid-precipitable counts per minute (19). Each fraction was brought to 0.3 N KOH and incubated at 37°C for 18 h. Hydrolysis was terminated, and macromolecules were precipitated by bringing each sample to 10% trichloroacetic acid. Carrier RNA was added, and samples were held on ice for 30 min. Precipitates were collected on glass fiber filters, washed three times with 3 ml of cold 5% trichloroacetic acid and once with 3 ml of cold 95% ethanol, and dried. Radioactivity retained on filters was monitored by scintillation spectrometry in 4.4 ml of scintillation solution [0.01 g of 1,4-bis-2-(4-methyl-5phenyloxazole)benzene and 6 g of 2,5-diphenyl-orazole per liter oftoluene]. Symbols: 0, cells/ml in each fraction (sedimentation is from left to right); x, radioactivity in DNA per cell in each fraction.
w
2 4 6 s FRACTION NUMBER FIG. 2. Bud appearance in cells separated by zonal sedimentation into a sucrose gradient. A culture of S288C (4 x 108 cells) was prepared and fractionated by zonal sedimentation as described in the legend to Fig. 1. The total number of cells, budded and unbudded, was determined visually using light microscopy and a hematocytometer. Symbols: 0, number of cells per fraction; x, percentage of cells in each fraction with buds.
mentation procedure separates an asynchronous population of yeast by position in the cell cycle. A sample of cells was labeled for 3' with [3H]adenine, harvested, and separated by zonal sedimentation according to position in the cell cycle. Fractions were collected, and the number of cells in each fraction was determined (Fig. 3). The RNA was extracted from each fraction and chromatographed on small columns of oligo(dT)-cellulose, and the radioactivity in poly(A)-containing RNA and RNA that does not bind to oligo(dT)-cellulose was determined. Ribosomal RNA does not bind to oligo(dT)-cellulose using our conditions of chromatography (4). Thus, the majority of the pulse-labeled RNA that does not bind to the columns is precursor ribosomal RNA (4). The data presented in Table 1, columns 2 and 3, show that the distribution of pulsed label between poly(A)-containing RNA and RNA that does not bind to oligo(dT)-cellulose does not vary significantly from fraction to fraction. Thus, if there is only one nuclear pool for the nucleoside triphosphates, we conclude that the relative rates of synthesis of poly(A)-containing RNA and ribosomal RNA do not vary significantly during the cell cycle. Based on the observation that the rate of synthesis of ribosomal RNA is constant throughout the cell cycle (17), we conclude that the rate of synthesis of poly(A)-containing RNA is also constant. This conclusion is supported by the data presented in column 4 of Table 1, which demonstrate that
504
NOTES
J. BACTERIOL.
5
4 (I)
3
-J w
2 0
4
6
8
10
12
FRACTION NUMBER FIG. 3. Zonal sedimentation of cells labeled for 3 min with [3H]adenine. See footnote a, Table 1, for details of the experiment.
An41ysis of the rate of RNA synthesis throughout the yeast cell cyclea
TA.:LE 1,
Total radioactivity (%) in: Fractionb
4 5 6 7 8 9 10 11
cpm/cell x 102
Nonbinding RNA 66 65 65 68 67 67 69 71
in
Poly(A)- poly(A)-concontain- taining RNA ing RNA 34 1.0 35 1.2 35 1.0 32 0.8 32 1.3 33 1.2 31 0.9 29
1.3
67 ± 0.7 33 ± 0.7 1.1 + 0.07 Average + SEM a A 10 ml-culture of S288C was labeled for 3 min with 15 ,uCi of [3H]adenine per ml (0.15 Ci/mg). The cells were rapidly chilled, harvested, and fractioned by zonal sedimentation as described in the legend to Fig. 1. The cells from each fraction were suspended in 0.4 ml of sodium dodecyl sulfate (SDS) buffer [0.1 M NaCl-20 mM ethylenediaminetetraacetate-20 mM tris(hydroxymethyl)aminomethanehydrochloride pH 7.5-0.5% sodium dodecyl sulfate). Carrier cells (1.4 x 108) in 0.3 ml of SDS buffer were added to each fraction. Nucleic acids were obtained from each fraction in the following way. The cell suspension was transferred into the chamber of an Eaton press (a kind gift of Walter Vincent, University of Delaware) that was maintained at dry ice temperature. The quick-frozen cell suspension was forced through the 1-mm orifice of the chamber by application of 10,000-lb./in2 pressure. Then the extract was quickly brought to room temperature, and cell debris was removed by centrifugation for 10 min at 4,500 x g. The extract was brought to 2 ml with SDS buffer, and the nucleic acids were isolated using the SDS-hot phenol-chloroform method described by Penman (13). The RNA in each fraction was passed over a column of oligo(dT)-cellulose and fractionated into nonbinding RNA and poly(A)-containing RNA as previously described (8). b Refer to Fig. 3. SEM, Standard error of the mean.
the actual amount of isotope incorporated during a 3-min pulse into poly(A)-containing RNA per cell is essentially uniform during the cell
cycle. Thus, taken together, the data suggests that the synthesis of ribosomal RNA and polyadenylated messenger RNA is constant throughout the cell cycle. Continued metabolism of RNA during the synchronization procedure may have changed the fraction of labeled RNA that binds to oligo(dT)-cellulose. However, this is unlikely to be a major problem because the fraction of RNA that binds to oligo(dT)-cellulose reported here and its size distribution are very similar to these parameters obtained for RNA extracted directly from an asynchronous population of cells labeled for 3 min with [3H]adenine (data not shown). Certainly, this study does not rule out the possibility that the synthesis of some species of mRNA are restricted to a particular time in the cell cycle. Messengers that constitute a small fraction of the poly(A)-containing RNA or which are devoid of polyWA) would have gone undetected in this analysis. Thus, a rigorous test of the periodic synthesis of specific messengers must await their isolation. The investigation was supported by grant GB-26315X from the National Science Foundation to S.L. P.N.E. H. was supported by Public Health Service Predoctroal training grant GM000149 from the National Institute of General Medical Sciences.
LITERATURE CITED 1. Bomn, T. W., M. D. Scharff, and E. Robbins. 1967. Rapidly labeled polyribosome associated RNA having the properties of histone messenger. Proc. Natl. Acad. Sci. U.S.A. 58:1977-1983. 2. Donachie, W. D., and M. Masters. 1969. Enzyme interactions, p. 37-76. In G. M. Padilla, G. L. Whitson, and I. L. Cameron (ed.), The cell cycle. Academic Press Inc., New York. 3. Gallwitz, D., and G. C. Mueller. 1969. Histone synthesis in vitro on HeLa cell microsomes: the nature of the coupling to DNA synthesis. J. Biol. Chem. 244:59475952. 4. Groner, B., N. Hynes, and S. Phillips. 1974. Length heterogeneity in the poly(adenylic) acid region of yeast messenger ribonucleic acid. Biochemistry 13:5378-5383. 5. Halvorson, H. O., B. L. A. Carter, and P. Tauro. 1971. Synthesis of enzymes during the cell cycle. Adv. Microbiol. Physiol. 6:47-106. 6. Hartwell, L. H. 1974. Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38:164-198. 7. Hynes, N. E., and S. L. Phillips. 1976. Turnover of polyadenylate-containing RNA in Saccharomyces cerevisiae. J. Bacteriol. 125:595-600. 8. McLaughlin, C. S., J. R. Warner, M. Edmonds, H. Nakazato, and M. Vaughan. 1973. Polyadenylic acid sequences in yeast messenger ribonucleic acid. J. Biol. Chem. 248:1466-1471. 9. Mitchison, J. M. 1958. The growth of single cells. II. Saccharomyces cerevisiae. Exp. Cell Res. 15:214-221. 10. Mitchison, J. M. 1973. Differentiation in the cell cycle, p. 1-9. In M. Balls and F. S. Billett (ed.), The cell cycle in development and differentiation. Cambridge
University Press, Cambridge. 11. Mitchison, J. M. and W. S. Vincent. 1965. Preparation
of synchronous cell cultures by sedimentation. Nature (London) 205:987-989.
VOL. 128, 1976 12. Ogur, M., S. Minckler, and D. McClary. 1953. Deoxyribonucleic acid and the budding cycle in the yeasts. J. Bacteriol. 66:642-645. 13. Penman, S. 1969. Preparation of purified nuclei and nucleoli from mammalian cells, p. 35-48. In K. Habel and N. P. Salzman (ed.), Fundamental techniques in virology. Academic Press Inc., New York. 14. Reed, J., and E. Winteraberger. 1973. Adenylic acid rich sequences in messenger RNA from yeast polysomes. FEBS Lett. 32:213-217. 15. Salas, J., and H. Green. 1971. Proteins binding to DNA and their relation to growth in cultured mammalian cells. Nature (London) New Biol. 229:165-169. 16. Sebastian, J., B. L. A. Carter, and H. 0. Halvorson. 1971. Use of yeast populations fractionated by zonal centrifugation to study the cell cycle. J. Bacteriol.
NOTES
505
108:1045-1050. 17. Tauro, P., E. Schweizer, R. Epstein, and H. 0. Halvorson. 1969. Synthesis of macromolecules during the cell cycle in yeast, p. 101-108. In G. M. Padilla, G. L. Whitson, and I. L. Cameron (ed.), The cell cycle. Academic Press Inc., New York. 18. Williamson, D. H. 1964. The timing of deoxyribonucleic acid synthesis in the cell cycle of Saccharomyces cerevisiae. Biochem. J. 90:25-26. 19. Williamson, D. H. 1965. The timing of deoxyribonucleic acid synthesis in the cell cycle of Saccharomyces cerevisiae. J. Cell Biol. 25:517-528. 20. Williamson, D. H., and A. W. Scopes. 1960. The behavior of nucleic acids in synchronously dividing cultures of Saccharomyces cerevisiae. Exp. Cell Res. 20:338349.
