Molec. gen. Genet. 169, 237-243 (1979)

MGG © by Springer-Verlag 1979

Regulation of RNA Synthesis in Yeast III Synthesis During the Cell Cycle Steven G. Elliott and Calvin S. McLaughlin Department of Biological Chemistry College of Medicine University of California, Irvine, Irvine, CA 92717, USA

Summary. Centrifugal elutriation was used to separate cells in different stages of the cell cycle from a culture of Saccharomyces cerevisiae in balanced exponential growth. The rate of DNA and RNA synthesis was determined using a pulse - long-term label technique that is capable of distinguishing between exponential, linear, and periodic variations in the rate of synthesis through the cell cycle. It was found that while the rate of DNA synthesis varies periodically through the cell cycle, the rate of synthesis of mRNA, rRNA, and tRNA increases exponentially through the cell cycle. The implications of these findings for the control of RNA synthesis are discussed.

Introduction The rate of DNA synthesis varies periodically through the cell cycle in Saccharomyces cerevisiae. The rate reaches its maximum in the first third of the cell cycle and thereafter declines (Williamson, 1965; Hartwell, 1974). The impact of the doubling of the gene number on the rate of synthesis of the various RNA species is the subject of conflicting reports. Sogin et al. (1974), using the incorporation of carrier-free 32p into 18S and 25S RNA as a measure of the rate of synthesis, suggested that these species are synthesized at an exponentially increasing rate during the cell cycle. This agrees with a study by Tauro et al. (1969), who followed the incorporation of uracil into RNA in a culture synchronized by feeding and starving. They suggested that in addition to 25S and 18S rRNA, tRNA also is synthesized at an exponentially increasing rate during the cell cycle. Recently however, two other models have been proposed. Fraser For offprints contact: Calvin S. McLaughlin

and Carter (1976), using adenine to measure synthetic rates, suggested that 18S and 25S rRNA and poly A containing mRNA double in rate during the cell cycle due to a doubling in DNA content. However, Hynes and Phillips (1976), who also followed adenine incorporation, suggested that the rate of synthesis of poly A containing RNA does not change at all during the cell cycle. In the present study, the rate of synthesis of total ribosomal, transfer, and poly A containing RNA are followed through the cell cycle using the incorporation of methionine and uracil into macromolecules as a measure of the rate of synthesis. In all cases, we have employed a dual-label technique, using pulse and long-term labels to measure the relative rate of synthesis. The long-term label provides a measure of the amount of material present and the pulse provides a measure of the rate of synthesis. The ratio of the two is then a measure of the relative rate of synthesis. Ratio determinations correct for recovery since the ratio is independent of the amount of material present. Although the ratio is a measure of the rate of synthesis divided by the amount of material present, it is sensitive to changes in rate. Measurement of this ratio can unambiguously distinguish between exponential, linear, and periodic changes in rate of synthesis. Campbell (1957) and Sebastian et al. (1971) have established the importance of balanced growth during cell cycle studies. The analysis of the cell cycle by centrifugal elutriation presented here is nearly ideal from that standpoint since the cells are labeled during exponential growth and subsequently separated into various cell cycle fractions under conditions that preclude macromolecular synthesis. Gordon and Elliott (1977) have shown that separation of yeast cells according to their position in the cell cycle by centrifugal elutriation compares favorably with other separation methods. The fractionation is very reproducible, al-

0026-8925/79/0169/0237/$01.40

238

S.G. Elliott and C.S. McLaughlin: Regulation of RNA Synthesis in Yeast

l o w i n g e v e n t s to be a c c u r a t e l y a s s i g n e d to specific t i m e s in t h e cell cycle.

Methods Strains. A diploid of Saccharomyces cerevisiae with the classification SKQ2n was used in all the experiments. It was obtained from Brian Cox (University of Oxford, England) and has the genotype a/c~, adel/+, +/ade2, +/hisl. Chemicals. Glusulase was from Endo Laboratories. All other chemicals were from standard commercial sources. All radioactive labels were from Schwarz/Mann. Growth and Labeling of Cells. Cells were grown to midlog phase at 23 ° on a rotary shaker in medium containing per liter: 6.7g yeast nitrogen base minus amino acids (Difco), 21 mg each of all amino acids with the exception of methionine and cysteine, 20 g glucose and 10 mg adenine and uracil. The medium was buffered at pH 5.8 with 10 g/liter succinate and 6 g/liter NaOH. The relative rate of synthesis was determined by a dual-label method. Cells either were labeled for 3 hours with 0.25 gCi/ml [2-1~C] uracil, then pulse labeled for 10minutes with 4~,tCi/ml [3HI methionine, or long-term labeled with 0.SgCi/ml [2-14C] uracil and pulse labeled with 5gCi/ml [5-31-I] uracil, depending on the experiment. In all cases, incorporation was stopped by the addition of ice to the medium. The cells were immediately pelleted, washed once and resuspended in ice cold distilled water.

Cell Cycle Fractionation. The procedure which was used has been described previously (Gordon and Elliott, 1977). Cells grown and labeled as described above were sonified and loaded at 2° into a Beckman JE-6 rotor spinning at 3000rpm in a J-21 centrifuge at a.flow rate of 9 ml/min. One hundred fifty mls were collected at 9 ml/min and successive fractions were obtained by increasing the flow rate in 2 ml/min increments. Sodium chloride was added to a final concentration of 0.10 M and the cells collected by centrifugation. Purification of RNA. Cells were chilled on ice throughout the procedure. Cell pellets were suspended in one volume of buffer containing 0.5 M sodium thioglycollate, and 0.1 M Tris-HC1, pH 8.8. After 30 rain on ice, the cells were collected by centrifugation, washed once with one volume 1 M sorbitol and resuspended in 0.6 volumes 1 M sorbitol; after which, 0.01 volumes of glusulase was added and the cells placed on ice for 60 rain. Spheroplasts were washed once with 1 M sorbitol, then lysed by adding 0.6 volumes of TNS buffer (2% sodium tri-isopropylnaphthalene sulfonate, 0.1 M NaC1, 50 mM Tris-HC1, pH 7.5). The cell extract was mixed with an equal volume of water saturated phenol-cresol containing 0.1% 8-hydroxyquinoline. The aqueous phase was separated by centrifugation, brought up to 0.3 M NaC1, and mixed again with phenolcresol. RNA in the aqueous phase was precipitated at - 2 0 ° C by adding 2 volumes of 95% ethanol. After 8 hours, the RNA was collected by centrifugation, dissolved in 0.5% SDS, 0.15 M sodium acetate and precipitated again in ethanol. The obtained RNA was stored frozen at - 2 0 ° C.

Fractionation of RNA Species. Poly A containing RNA was purified by the poly U filter disc method of Stringer et al. (1976). High molecular weight RNA was separated on 2.6% gels 0.6 mm in diameter and 70 mm long. Electrophoresis was at 3.3 mA per gel for seven hours. Low molecular weight RNA was separated on 8% gels 0.5 mm in diameter and 100 mm long. Electrophoresis

was at 5 mA per gel until the bromophenol blue marker reached the bottom. Electrophoresis was performed according to the method of Loening (1967). Both 2.6% and 8% polyacrylamide gels were scanned at 260 nm, then frozen in dry ice and sliced in 1 mm sections. RNA in the gel slices was solnbilized in 0.3 ml protosol at 23 ° for 24 hours. The amount of radioactivity in the gel slices was determined by scintillation counting in 4.5ml of toluene based scintillation fluid. Gel slices selected to determine the ratio of counts for a particular RNA species were chosen according to three criteria: (1) they came from the region of the gel that corresponded with the desired OD2eo RNA peak; (2) they were slices that had minimal precursor overlap; and (3) they were adjacent slices that had the same ratio of counts. For the selected slices, the ratio of counts in each slice was determined and the average of the ratios calculated. The ratio of counts in total RNA and RNA separated on poly U columns was determined by measuring the ratio of counts of aliquots in Aquasol.

Results Separation o f R N A Species on Gels. E x p o n e n t i a l cult u r e s o f Saccharomyces cerevisiae w e r e g r o w n in m i n i m a l m e d i u m a n d l a b e l e d for 1 z/2 g e n e r a t i o n s (3 h) w i t h [~4C] uracil. Cells w e r e t h e n p u l s e l a b e l e d for 1/12 o f a g e n e r a t i o n (10 m i n ) w i t h [3H] m e t h i o n i n e o r [3H] uracil. T h e cells w e r e lysed, t h e R N A e x t r a c t e d a n d t h e R N A species s e p a r a t e d o n acryla m i d e gels. A t y p i c a l 0 D 2 6 0 scan o f a 2 . 6 % gel is s h o w n in Fig. l a a n d o f a n 8 % gel in Fig. l b . I n 2 . 6 % gels, f o u r d i s t i n c t R N A species are o b s e r v e d . T h e s e a r e a 35S r R N A p r e c u r s o r , a s h a r p d o u b l e s t r a n d e d R N A p e a k , a n d 25S a n d 18S r R N A peaks. I n 8 % gels, t h r e e d i s t i n c t p e a k s are o b s e r v e d . T h e s e are a 5.8S r R N A p e a k , a 5S r R N A p e a k , a n d a 4S t R N A p e a k . F i g u r e 2 a a n d b s h o w t y p i c a l results o f l o n g - t e r m [14C] u r a c i l a n d [3H] m e t h i o n i n e p u l s e labeling. T h e l o n g - t e r m ~4C c o u n t s are d i s t r i b u t e d t h r o u g h o u t all t h e R N A species; h o w e v e r , 3H c o u n t s are a b s e n t f r o m t h e d s R N A , 5.8S a n d 5S R N A p e a k s . T h e s e R N A species a r e n o t m e t h y l a t e d by l a b e l e d m e t h i o n i n e . F i g u r e 2 c a n d d s h o w t y p i c a l gels o f R N A , l o n g t e r m l a b e l e d w i t h [~4C] u r a c i l a n d p u l s e d w i t h [3HI uracil. I n this case, all the R N A species h a v e i n c o r p o r a t e d b o t h 14C a n d 3H l a b e l e d p r e c u r s o r s . Synthesis of Total R N A Through the Ceil Cycle. Cells l a b e l e d l o n g t e r m w i t h [~4C] u r a c i l a n d p u l s e l a b e l e d w i t h e i t h e r [3H] m e t h i o n i n e o r [3H] u r a c i l w e r e fract i o n a t e d b y e l u t r i a t i o n a n d t h e R N A e x t r a c t e d as d e s c r i b e d in M e t h o d s . T h e r a t i o o f 3H to ~4C c o u n t s in R N A was t h e n d e t e r m i n e d for e a c h f r a c t i o n . T h e r a t i o o f c o u n t s u s i n g m e t h i o n i n e or u r a c i l as t h e p u l s e l a b e l is d e p i c t e d in Fig. 3 a a n d b, r e s p e c t i v e l y . I n b o t h cases, t h e r a t i o r e m a i n s c o n s t a n t t h r o u g h t h e cell cycle at a r a t i o o f 0.27 for t h e m e t h i o n i n e p u l s e e x p e r i m e n t a n d at 0.70 for the u r a c i l pulse e x p e r i -

S.G. Elliott and C.S. McLaughlin: Regulation of R N A Synthesis in Yeast

uracil were fractionated by centrifugal elutriation, the RNA was extracted and the RNA passed through a poly U binding column. RNA was then separated into binding (poly A +) RNA and non-binding (poly A - ) RNA. The ratio of long and short-term label in the two types of RNA was then determined. Both types of RNA.are synthesized exponentially (Fig. 4). The poly A + R N A has a much higher average ratio of counts, 3.5 vs 0.61 for the poly A- species. This higher value is consistent with the unstable nature of the poly A + RNA in yeast (Petersen et al., 1976).

ment. This indicates that rate of total R N A synthesis increases exponentially through the cell cycle.

Synthesis of poly A + and poly A - RNA During the Cell Cycle. Cells dual-labeled with [3H] and [1¢C]

Synthesis Through the Cell Cycle of RNA Species Separated on Polyacrylamide Gels. The rate of synthesis of the R N A species long-term labeled with [14C] uracil and pulsed' with [3H] methionine is depicted in Fig. 5. The ratio for 25S RNA remains constant at a value of 0.11, as shown in Fig. 5a. The ratio for 18S R N A also remains constant, 0.115, as shown in Fig. 5 b. The ratio for 4S t R N A also remains constant through the cell cycle at a value of 1.08. When [3H] uracil is substituted for [3H] methionine, similar results are obtained. As shown in Fig. 6, 25S, 18S and tRNA have a 3H/14C ratio that remains constant at values of 0.62, 0.56, and 0.67, respectively. Using uracil as a pulse label, it is possible to determine the rate of synthesis of two additonal species of RNA, 5.8S and 5S RNA. As shown in Fig. 7, both of these species have constant ratios through the cell cycle at values of 0.62 and 0.96, respectively. Therefore, these species also are synthesized at an exponentially increasing rate through the cell cycle. In Table 1, the average ratios for the various RNA species are displayed. When either methionine or uracil is used as the pulse, 25S, 18S and 5.8S RNA have approximately the same constant ratios through

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240

S.G. Elliott and C.S. McLaughlin: Regulation of R N A Synthesis in Yeast FLOW RATE (ml/min) 11

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the cell cycle. This is consistent with the fact that these species are all part of the same precursor molecule and are stable under these conditions, t R N A also is stable under these conditions. These four species make up most of the R N A of the cell; therefore, it is not surprising that poly A - R N A has a ratio close to the average ratio of these four species of RNA. Interestingly, both 5S and poly A ÷ R N A have higher ratios. This suggests that these two species have higher relative synthetic rates and a higher rate of turnover. The methyl-labeled 4S R N A has a much higher ratio than 18S and 25S RNA. Since uracil is the long-term label, this is consistent with the higher degree of methylation of t R N A compared with rRNA.

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Discussion

Much effort has been devoted to determining whether or not variations in macromolecular synthesis occur during the cell cycle (Mitchison, 1971). Saccharomyces cerevisiae has a number of advantages as the experimental organism for such a study because its cell cycle has been the subject of extensive biochemical

S.G. Elliott and C.S. McLaughlin: Regulation of R N A Synthesis in Yeast FLOW 11

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Table 1. The ratio of counts incorporated into various R N A species-

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and genetic studies; and events can be precisely located in the cell cycle by simple cytological and biochemical tests (Hartwell, 1974). Furthermore, cell cycle studies are simplified because cell volume increases linearly through the cell cycle (Sebastian et al., 1971). Therefore, size selection can synchronize populations of cells in balanced growth. Centrifugal elutriation takes advantage of this property by separating cells into distinct size classes. Using this technique, it has been demonstrated that cell size increases linearly with fraction number and that cells are indeed separated according to their position in the cell cycle (Gordon and Elliott, 1977; Elliott and McLaughlin, 1978). When methionine is used to pulse label RNA, the rate of synthesis (pulse methionine counts) divided by the amount of RNA present (long-term uracil counts) remains constant during the cell cycle for total RNA, 18S and 25S rRNA, and tRNA. This indicates that the rate of synthesis increases exponentially during the cell cycle for these RNA species. These results are confirmed when uracil is substituted for methionine, since the ratio for these RNA species again remains constant. In addition, the uracil pulse label results demonstrate that 5.8S and 5S rRNA, and poly A containing RNA also are synthesized at an exponentially increasing rate during the cell cycle. The incorporation of labeled precursors into macromolecules is an accurate measure of rate of synthesis if two criteria are met: (1) the label is corrected for precursor pool saturation, and (2) the specific activity of the pool remains constant through the cell cycle. The first criteria is fulfilled for methionine pulse label experiments since the methionine pool is half saturated in 20 seconds. Therefore, pool corrections are negligible and the apparent rate of synthesis accurately reflects the absolute rate of synthesis. For the uracil pulse label experiment, precursor pool saturation to a first approximation may also be overlooked since the pulse time is twice the time necessary to saturate the pool.

242

S.G. Elliott and C.S. McLaughlin: Regulation of RNA Synthesis in Yeast

The second criteria is actually demonstrated by the experiment itself. A change in the pool specific activity would be reflected by a change in the apparent rate of incorporation of precursors. But since the ratio of counts in RNA remains constant through the cell cycle, the specific activity of the pool must also remain constant during the cell cycle for both methionine and uracil. We consider it unlikely that the specific activity of both the methionine and uracil pools varies periodically through the cell cycle with precisely the same periodicity and extent for both pools and in a manner that exactly compensates for the hypothetical change in the rate of synthesis required to convert a pattern of periodic or linear synthesis to the observed pattern of an exponentially increasing rate of synthesis through the cell cycle. Sogin et al. (1974) directly determined the 32p specific activity of the nucleoside triphosphate precursor pool and found no periodic variation through the cell cycle in the specific activity of the precursor pool. The simple interpretation of this data suggests that the specific activity of the precursor pool remains constant through the cell cycle. Our results agree with previous studies which demonstrated that the rate of synthesis of total purified RNA, 18S and 25S rRNA, and tRNA increases exponentially through the cell cycle (Tauro et al., 1969; Sogin et al., 1974). However, Fraser and Carter (1976) reported a doubling in rate near the time of DNA synthesis for both poly A containing RNA and 18S and 25S rRNA. Their results are supported by observations of Fraser and Moreno (1976) on Sehizosaccharomyces pombe which demonstrated that the rate of rRNA and mRNA increase in a step function after DNA synthesis in synchronized cultures. These different results cannot be explained by a difference in growth rate since our cells have the same doubling time as theirs (1.7 h). Our results also are different from those of Hynes and Phillips (1976) who suggested that poly A containing RNA is synthesized at a constant linear rate of synthesis during the cell cycle. At this time, there is no readily apparent explanation for the three different results that have been obtained by ourselves, Fraser and Carter (1976), and Hynes and Phillips (1976). Further studies will be required to determine why these differences have been observed. Nevertheless, we suggest that the pattern of an exponential increase in the rate of RNA synthesis through the cell cycle should be typical of a prototrophic yeast strain growing in a minimal medium and thus represents the basic mechanism of growth. The data indicate that the ratio of pulse to longterm label varies among the different species of RNA. The ratio will vary for different RNA species if (1)

the RNA species are degraded at different rates; (2) different precursors are used for the pulse and longterm label. The data in Table 1 invoNing a uracil pulse divided by a uracil long-term label suggest that the 5S RNA is degraded 40% faster than the other rRNA species while the poly A containing RNA is degraded at least 6-fold faster than rRNA. This is consistent with the half-life for mRNA (Petersen et al., 1976) and suggests that 5S RNA is synthesized in excess and some of the molecules are degraded. DNA doubles during a short period of the cell cycle (Williamson, 1965; Hartwell, 1974; Elliott and McLaughlin, 1978). However, RNA synthesis is not periodic but increases exponentially. This indicates that either the new DNA is not immediately used, or that the regulatory system which sets the rate of synthesis of the different RNA species is easily able to compensate for a two-fold increase in the number of genes for RNA. The data suggests that RNA synthesis is not under simple promotor control. The control system increases the rate of RNA synthesis in a continuous exponential manner through the cell cycle. The molecular nature of this control system is an open question. Our experiments here and elsewhere (Elliott and McLaughlin, 1978) raise another fundamental question. What are the molecular mechanisms that drive the obvious periodic events in the cell cycle? Acknowledgements. This investigation was supported by grant CA 10628 awarded by the National Cancer Institute, DHEW.

References Campbell, A.: Synchronization of cell division. Bacteriol. Rev. 21, 263-272 (1957) Elliott, S.G., McLaughlin, C.S.: The rate of macromolecular synthesis through the cell cycle of the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA, 75, 4384-4388 (1978) Fraser, R.S.S., Carter, B.L.A. : Synthesis of polyadenylated mRNA during the cell cycle of Saccharomyces cerevisiae. J. Mol. Biol. 104, 223 242 (1976) Fraser, R.S.S., Moreno, F. : Rates of synthesis of poly (A) mRNA and rRNA during the cell cycle of Schizosaccharomyces pombe. J. Cell Sci. 21, 497-521 (1976) Gordon, C.N., Elliott, S.G. : Fractionation of Saccharomyces cerevisiae cell populations by centrifugal elutriation. J. Bacteriol. 129, 97-100 (1977) Hartwell, L.H. : The Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38, 168-198 (1974) Hartwell, L.H., Culotti, J., Pringle, J., Reid, B.: Genetic control of the cell division cycle in yeast. Science 183, 46-51 (1974) Hynes, N., Phillips, S. : Rate of synthesis of polyadenylate containing RNA during the yeast cell cycle. J. Bacteriol. 128, 502-505 (1976)

S.G. Elliott and C.S. McLaughlin: Regulation of RNA Synthesis in Yeast Loening, U.E. : The fractionation of high-molecular weight ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102, 251-257 (1967) Mitchison, M. : The biology of the cell cycle. I. Cambridge, United Kingdom: Cambridge University Press 1971 Petersen, N.S., McLaughlin, C.S., Nierlich, D.P. : Half-life of yeast messenger RNA. Nature 260, 70 72 (1976) Sebastian, J., Carter, B.L.A., Halvorson, H.O. : Use of yeast populations fractionated by zonal centrifugation to study the cell cycle. J. Bacteriol. 108, 1045-1050 (1971) Stringer, J.R., Holland, L.E., Swanstrom, R.I., Pivo, K., Wagner, E.K.: Quantitation of herpes simplex virus type 1. RNA in infected HeLa cells. J. Virol. 21,889-901 (1977) Sogin, S.J., Carter, B.L.A., Hal~7orson, H.O. : Change~ in the rate

243 of ribosomal RNA synthesis during the cell cycle of Saccharomyces cerevisiae. Exp. Cell Res. 89, 127-138 (1974)

Tauro, P., Schweizer, E., Epstein, R., Halvorson, H.O. : Synthesis of macromolecules during the cell cycle in yeast. In: The cell cycle. Gene-enzyme interactions. 1. New York: Academic Press 1969 Williamson, D.H. : The timing of deoxyribonucleic acid synthesis in the cell cycle of Saccharomyces cerevisiae. J. Cell Biol. 25, 517 528 (1965)

Communicated by W. Gajewski Received September 26, 1978

Regulation of RNA synthesis in yeast. III. Synthesis during the cell cycle.

Molec. gen. Genet. 169, 237-243 (1979) MGG © by Springer-Verlag 1979 Regulation of RNA Synthesis in Yeast III Synthesis During the Cell Cycle Steven...
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