JOURNAL OF BACTERIOLOGY, Sept. 1977, p. 735-740 Copyright C) 1977 American Society for Microbiology

Vol. 131, No. 3

Printed in U.S.A.

Killer Double-Stranded Ribonucleic Acid Synthesis in Cell Division Cycle Mutants of Saccharomyces cerevisiael CHANNA SHALITIN* AND IRITH WEISER Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel Received for publication 7 March 1977

The synthesis of killer double-stranded ribonucleic acid (dsRNA) in Saccharomyces cerevisiae was examined in seven different cell division cycle mutants (cdc) that are defective in nuclear deoxyribonucleic acid replication and contain the "killer character." In cdc28, cdc4, and cdc7, which are defective in the initiation of nuclear deoxyribonucleic acid synthesis, and in cdc23 or in cdcl4, defective in medial or late nuclear division, an overproduction of dsRNA at the restrictive temperature was observed. In contrast to the above mutants, the synthesis of killer dsRNA is not enhanced at the restrictive temperature in either cdc8 or cdc2M, which are defective in deoxyribonucleic acid chain elongation. Examination of killer sensitive strains (cdc7 K- and cdc4 K-) has shown that the complete killer dsRNA genome is essential for the overproduction of dsRNA at the restrictive temperature. Many strains of the yeast Saccharomyces cerevisiae produce an extracellular substance that kills sensitive cells (K+ denotes the presence of the killer substance, and K- denotes the nonkiller sensitive phenotype). This subject has been recently reviewed by Wickner (22). Cells secreting the killer toxin also carry two doublestranded ribonucleic acid (dsRNA) species (2.5 x 106 and 1.4 x 106 daltons). These dsRNA species are encapsulated in intracellular viruslike particles. The maintenance or replication ofthe killer dsRNA requires at least 10 chromosomal genes in addition to 3 other chromosomal genes that determine the expression of killing and resistance to it, whereas the killer phenotype is determined by a non-Mendelian genetic element (22). In a recent communication (7), the relative synthesis of killer dsRNA was studied in a cell division cycle mutant, cdc4, that is known to be defective in the initiation of deoxyribonucleic acid (DNA) synthesis and to contain the "killer character." Evidence was presented that this mutant synthesizes 2.5-fold more killer dsRNA species at the restrictive temperature. Killer dsRNA refers to synthesis of both the large (2.5 x 106 daltons) and small (1.4 x 106 daltons) species of dsRNA, although the presence of only the smaller species is essential for killer toxin production (21). The present study was undertaken to inquire whether a rare sensitive variant, cdc4 K-, carrying only the larger dsRNA species (molec' This work is dedicated to the memory of Lea Bi-

chowsky-Slomnizky, a valued colleague and friend.

ular weight, 2.5 x 106) and no dsRNA component with a molecular weight of 1.4 x 106 (21), would also show a higher level of dsRNA species at the restrictive temperature. Our results show that in this mutant there is no augmented dsRNA synthesis under nonpermissive growth conditions. This communication also reports a more extensive analysis of cell division cycle mutants carrying the killer character in an attempt to find additional mutants that show an increased killer dsRNA synthesis under restrictive growth conditions. Evidence will be presented that four additional mutants-cdc28 and cdc7, defective in the initiation of DNA synthesis, and cdc23 or cdc14, defective in medial or late nuclear division-show an augmented dsRNA level formed at 37°C. Mutants cdc8 and cdc2l showed no such overproduction at 370C. MATERIALS AND METHODS Strains. The temperature-sensitive cdc mutants of S. cerevisiae and their wild-type parent (A364A) were obtained from L. H. Hartwell. All have been described previously (12, 13). Strains used (genotype, allele number, and phenotype are in parentheses) are as follows: A364A (wild type, K+), 185.3.4 (cdc28-1 K+), 314-D5 (cdc4-1 K+), 4008 (cdc7-4 K-), E247 (cdc7-7 K+), 198-Dl (cdc8-1 K+), 146.2.3 (cdc21-1 K+), 9013 (cdc23-1 K+), and 214 (cdc14-3 K+). ST1 (cdc4-1 K-) was a spontaneous mutant selected from 314-D5 by replica plating onto methylene blue agar (6) previously spread with a background lawn of X2180-lAa sensitive cells. After 48 h at room temperature, killer colonies were identified as those with a surrounding clear zone in which the 735

736

SHALMN AND WEISER

background cells had been killed. Nonkiller colonies were identified as those colonies lacking such a zone. Suspected nonkiller colonies were purified by three successive single-cell isolations and retested for phenotype as described above. The resulting strains were checked for sensitivity to the killer toxin, and a sensitive nonkiller strain, ST1, was chosen for further study. Medium. YEPD medium contains 1% yeast extract, 1% peptone, 2% dextrose, 2% agar, and 0.002% adenine. Chemicals. [5,6-3H]uracil (specific activity, 53 Ci/ mmol) and [5-3H]uracil (specific activity, 25 Ci/ mmol) were purchased from the Radiochemical Centre, Amersham, England. DNA synthesis. DNA synthesis was measured by the incorporation of [5-3H]uracil. Determination of radioactivity incorporated into DNA was performed as described by Hatzfeld (14). Preparation of 3H-labeled RNA. Cells were grown overnight to a concentration of 5 x 106 cells/ml in a synthetic medium (10) containing 10 ,uCi of [5,63H]uracil and supplemented with 20 to 40 i.g of nonradioactive adenine and uracil per ml. When indicated, part of the culture was shifted for 6 h to 37°C. The cells were collected by centrifugation, washed in cold water, and suspended at a concentration of 108/ml in a buffer containing 0.1 M sodium acetate (pH 5.2), 2% sodium dodecyl sulfate, 1% sodium triisopropyl naphthalenesulphonate (Eastman), and 1% diethyl pyrocarbonate (Sigma). Subsequently, the cells were frozen in an Eaton pressure cell and passed through the cell under pressure of 16,000 lb/in2. The RNA was extracted at 68°C by water-saturated phenol. The RNA was ethanol precipitated in the presence of 0.24 M ammonium acetate. The RNA pellet was suspended in a solution containing 0.30 M sodium chloride and 0.03 M sodium citrate. Then 2 M LiCl was added to precipitate ribosomal RNA (2). Finally, the RNA was reprecipitated by ethanol. Preparation of nonlabeled RNA followed a similar procedure. Gel electrophoresis. Samples of RNA to be analyzed by gel electrophoresis were in electrophoresis buffer [40 mM tris(hydroxymethyl)aminomethaneacetate buffer, pH 7.6, containing 2 mM ethylenediaminetetraacetic acid and 20 mM sodium acetate]. Gel electrophoresis was done in 2.7% acrylamide0.14% methylene bisacrylamide-0.5% agarose composite gels (16), as previously described (18). CF11-cellulose chromatography. Isolation of dsRNA by CF11-cellulose chromatography was done as previously described (7).

RESULTS Characterization of ST1 (cdc4 K-). To characterize a spontaneous cdc4 mutant defective in the initiation of nuclear DNA synthesis and lacking both the toxin and immunity functions of the killer character, the following experiments were performed. (i) The morphology of the mutant cells grown at the permissive temperature (25°C) was compared to that of cells shifted to the restrictive temperature (370C). As

J. BACTERIOL.

shown in a previous publication, using the parent 314-D5 (cdc4 K+) cells (11), cells of strain ST1 or 314-D5 continued to undergo bud initiation at the restrictive temperature so that most cells terminated with three to five elongated buds attached to the parent cell. (ii) DNA synthesis was monitored at the permissive and restrictive temperatures. The results from an asynchronous culture of ST1 cells are presented in Fig. 1. Examination of the labeling pattern of a culture at various times after a temperature shift up indicates that incorporation of radioactivity into DNA stops after about 45 min. Since the DNA-synthetic interval constitutes about 25% of the cell division cycle, the cessation of incorporation of isotope into DNA after 45 min is consistent with this mutant having a temperature-sensitive block in DNA initiation, as reported by Hartwell (11). These results are similar to those obtained with 314D5 cells (data not shown). A slow rate of DNA synthesis is apparent at the restrictive temperature. This residual synthesis might be accounted for by mitochondrial DNA replication (4, 5, 15). (iii) The number of viable cells was monitored at the permissive and restrictive temperatures. The generation time of 314-D5 2

2

0

0C 0

30

60

90 120 150 TIME (mh)

FIG. 1. DNA synthesis in a culture of strain STI (cdc4 K-) at the permissive (25C) and restrictive (37°C) temperatures. At time zero of the epxeriment, 1 ,uCi of[5-3H]uracil per ml was added to the culture (final specific activity, 0.1 uCi/mmol). At the time indicated by the arrow, the culture was divided into two portions, one remaining at 25°C (0) and the second raised to 370C (a). Ten-milliliter samples were removed at various times, and DNA synthesis was monitored by the incorporation of radioactivity into DNA, as described in the text.

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KILLER dsRNA SYNTHESIS IN S. CEREVISIAE

was 120 min at 250C, as compared with 140 min for ST1 cells at the same temperature (Fig. 2). The longer generation time of strain ST1 is not yet understood. Furthermore, loss of viability occurred at 37°C after 120 min in 314-D5 cells (Fig. 2A), as compared with ST1 cells, which remained viable at 37°C although growth was arrested (Fig. 2B). In both strains cells are blocked in nuclear division and there is no detachment of buds (11). The loss of viability in 314-D5 cells has been previously described (5, 11). Since ST1 cells showed no such loss, it is clear that this drop in viability depends on the presence of the killer character. Since killer cells are themselves immune to the action of their toxin (3), the cell death in such strains remains unexplained. However, cell death of the mutant cells at 370C could be prevented by treatment of the culture with cycloheximide (2 ,tg/ml), an inhibitor of protein synthesis (Gold and Shalitin, unpublished results). Gel electrophoresis of killer RNA. Since the ST1 cells are defective for both the toxin and immunity functions of the killer character, it was of interest to identify the genomic constituents of the killer dsRNA. The LiCl-soluble fraction was subjected to CF11-cellulose chromatography to select dsRNA molecules. The dsRNA was then analyzed by gel electrophoresis. The results are shown in Fig. 3. Molecular weight estimates are based on comparisons of the migration of killer RNA with that of reovirus dsRNA (19). Whereas two species of dsRNA were observed in the K+ strain (2.5 x 106 and 1.4 x 106 daltons), in K- strains used, the smaller species of dsRNA (1.4 x 106 daltons) was missing. This finding is in accord with previous observations (21). Killer dsRNA formed at restrictive, compared with permissive, growth conditions. In view of the results reported here and our previous findings concerning the overproduction of dsRNA in 314-D5 cells at the restrictive temperature (7), it was of interest to determine whether ST1 cells carrying only the larger dsRNA species (2.5 x 106 daltons) would also show a higher level of dsRNA species at the restrictive temperature. Furthermore, it was of interest to test whether the augmented synthesis of dsRNA occurs at the nonpermissive temperature in all cell division cycle mutants carrying the killer factor, or whether this effect is specific to certain cell division cycle mutants. To this end, cells of the appropriate strain were grown for at least five generations at the permissive temperature (250C) in the presence of [3H]uracil to uniformly label the RNA and were then shifted to 37°C. After 6 h at 370C, estimates of the dsRNA content of the RNA were

737

TIME (min)

FIG. 2. Increase in cell number of a culture of strain 314-D5 (A), compared with strain STI (B), at 25 and 37°C. Cells were grown in synthetic medium supplemented with 40 pg each of adenine and uracil per ml at 25°C. At time zero of the experiment, the culture was divided into two portions, one of which remained at 25°C (-), while the other was placed at 37°C (0). Samples were removed from the cultures, diluted, and plated onto YEPD plates at 250C to determine the number of viable cells.

made using CFli-cellulose chromatography (8). Our previous results have shown that CF11cellulose chromatography renders a reliable relative estimate of killer dsRNA (7). Furthermore, as shown in Fig. 3, the RNA eluted from the CF11 column with 0% ethanol is, in fact, killer dsRNA. Analyses of the RNA preparations from cells grown at the permissive and restrictive temperatures were performed for the wild type (A364A K+) and for mutant strains defective in the initiation of nuclear DNA synthesis, in DNA chain elongation, or in medial or late nuclear division. The results are summarized in Table 1. The percentage of dsRNA among the total RNA in the wild type (A364A K+) was about 1.1 at 250C and remained close to this value at 370C. Among the mutants, only those defective in the initiation of nuclear DNA synthesis and carrying the killer factor (cdc28 K+, cdc4 K+, and cdc7 K+), and two defective in medial or late nuclear division (cdc23 K+, cdc14 K+), showed an increased synthesis of dsRNA at the restrictive temperature. The ratio of dsRNA to total RNA was also tested by electrophoresis of

738

J. BACTRIUOL.

SHALITIN AND WEISER

ml) for at least six generations. The cultures then washed and suspended in fresh medium containing 100 ,ug of nonlabeled adenine per ml and shifted to the restrictive temperature for 6 h. The amount of radioactivity found in RNA after 6 h at the restrictive temperature was approximately the same as that at the permissive temperature. Therefore, for all strains tested there is little or no degradation of bulk RNA at the restrictive temperature, and the increased ratio shown in Table 1 must be the result of increased synthesis of dsRNA.

were

TArnZ 1. Proportion of dsRNA under various growth conditions 7

Gcondi-

dsRNA/total DNA (%)b

370C

1.10 ± 0.05 0.94 + 0.03

0.85

25°C

370C

1.78 ± 0.07 3.84 0.30

215

250C

1.02 ± 0.15 2.13 ± 0.20

210

250C

370C

1.59 ±± 0.08 1.32 0.10

0.83

250C 37°C

0.39 ± 0.04 1.24 ± 0.06

3.1 3.5

cdc7 K-

250C

370C

0.95 ± 0.07 1.03 ± 0.11

108

cdc8 K+

250C

370C

0.68 ± 0.05 0.70 ± 0.07

103

370C

250C

1.13 ± 0.10 1.22 ± 0.15

1.08

cdc23 K+

250C

0.40 ± 0.04 0.72 ± 0.07

1.80

cdcI4 K+

250C

Growth

Strain

A364 K+ FIG. 3. Comparison of dsRNA extracted from K+ and K- ceUs. Nonlabeled RNA samples were prepared and subjected to CF11-cellulose chromatography. cdc28 K+ The fraction eluted with 0% ethanol was ethanol precipitated with 250 pg of carrier calfliver transfer RNA (Sigma) per ml and suspended in electrophore- cdc4 K+ sis buffer. Approximately 10- to 13-pg portions of dsRNA were analyzed by electrophoresis on 2.7% polyacrylamide gels, stained in 01% toluidine blue 0 cdc4 Kand destained in distilled water. Reovirus dsRNA (10 pg) was analyzed as a control. (a) Reovirus RNA; (b) cdc4 K+; (c) cdc4 K-, (d) cdc7 K-. Migra- cdc7 K+ tion is from top to bottom. Running time, 3.5 h.

the [3H]dsRNA fractions separated by CF11 column chromatography. The increase in the ratio of dsRNA to total RNA formed at the restrictive temperature was also observed by using gel electrophoresis. The overproduction of dsRNA concerns both the large and small dsRNA species. In contrast, there was no such increase in the ratio of dsRNA to total RNA after a shift to 370C in the two mutants defective in nuclear DNA chain elongation, cdc8 and cdc2l (Table 1), or in mutants defective in the initiation of nuclear DNA synthesis but missing one killer dsRNA species (cdc4 K- and cdc7 K-). We conclude that the presence of the complete killer dsRNA genome is essential for the overproduction of dsRNA at the restrictive temperature in mutants defective in the initiation of nuclear DNA synthesis. To insure that the greater 37°C/250C ratios that we observed for several mutants were the results of dsRNA synthesis rather than differential degradation of ribosomal RNA, the following experiment was performed. Cultures of cdc4 K+, cdc28 K+, cdc7 K+, cdc23 K+, and cdc14 K+ and their isogenic wild-type strain, A364A K+, were grown at the permissive temperature in the presence of [3Hladenine (1 IACi/

cdc2l K+c

tion" 250C

37°C

370C

30/~

MOT

0.92 ± 0.15 1. 63 1.50 ± 0.10 a Cultures were grown ovemight at 25°C in the presence of 10 ,uCi of [5,6-3H]uracil per ml. At zero time part ofthe culture was shifted to 370C. After 6 h the cells were collected, 3H-labeled RNA was extracted, and dsRNA was isolated by CF11-cellulose chromatography as described in the text. b The percentage of total radioactivity found in dsRNA was estimated from the radioactivity found in the dsRNA peak (0% ethanol) of the CF11-cellulose column. Total radioactivity was that present before precipitation with LiCl. Values are based on 2 to 3 experiments. c The cdc:21 mutant has been shown by Game (9) to be a temperature-sensitive thymidylate auxotroph. The cdc21 gene is identical to the tmpl gene.

370C

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KILLER dsRNA SYNTHESIS IN S. CEREVISIAE

DISCUSSION Here, we have shown that there is a two- to threefold increase at the restrictive temperature in the relative amount of killer dsRNA found in strains carrying the killer character and mutations in genes cdc28, cdc4, and cdc7, which are required for the initiation of nuclear DNA synthesis, and a 1.6- to 1.8-fold increase in cdc14 and cdc23, which are required for the completion of nuclear division (Table 1). In contrast, other mutant strains (cdc8 and cdc2l) that affect nuclear DNA chain elongation show an inhibition ofboth nuclear and mitochondrial DNA synthesis at 360C (4, 5, 23). In these strains we found no increase in the relative amount of dsRNA at the restrictive temperature (Table 1). Although there may be a correlation between increased relative synthesis of mitochondrial DNA and killer dsRNA, this need not be the case. Since killer dsRNA is also present in cytoplasmic petite mutants having no detectable mitochondrial DNA (1), it is possible that nonmitochondrial circular DNA may account for our findings. In this connection, it is noteworthy that Cottrell et al. (4) observed a low-level synthesis of DNA of nuclear density in 314-D5 K+ (cdc4) mutant cells grown at the restrictive temperature. They suggested that a nonmitochondrial cytoplasmic circular DNA having the buoyant density of yeast nuclear DNA might account for this low-level DNA synthesis (4). Since the homology between dsRNA and nuclear DNA has been demonstrated (7, 20) and since we have shown the ribonucleotide sequence homology between some abundant polyadenylic acid RNA species (killer messenger RNA) and dsRNA (7), it seems possible that the killer messenger RNA is transcribed from a circular DNA template (7, 18, 20). If this is the case, then it is expected that its replication would be dissociated from nuclear DNA replication, similar to mitochondrial DNA replication. In fact, our recent experiments have shown preferential synthesis of 2-,um circles at the nonpennissive temperature in cdc4 K+ and cdc7 K+ cells (Shalitin and Gold, unpublished results). Moreover, the replication of 2-,um DNA circles has been previously shown (17) to require the function of a nuclear gene (cdc8) necessary for the elongation of DNA chains. As demonstrated in Table 1, the overproduction of dsRNA at the restrictive temperature is only expressed when genes required for the initiation of nuclear DNA synthesis are defective and when the small dsRNA piece (1.4 x 106 daltons) is also present. The requirement for the small dsRNA is not yet understood but should be clarified upon isola-

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tion and characterization of circular DNA templates. Attempts to isolate these circular DNA templates for killer messenger RNA are underway. ACKNOWLEDGMENTS Thanks are due to Rivka Pusin for capable technical assistance and to I. Fischer for useful discussions. Some of L. Hartwell's strains were generously provided by G. Simchen. We also thank Aaron J. Shatkin for his generous gift of a reovirus RNA sample. The authors acknowledge with thanks the support of the Technion Research Fund. LITERATURE CITED 1. Al-Aidroos, K., J. M. Somers, and H. Bussey. 1973. Retention of cytoplasmic killer determinants in yeast cells after removal of mitochondrial DNA by ethidium bromide. Mol. Gen. Genet. 122:323-330. 2. Barlow, J. J., A. P. Mathias, R. Williamson, and D. B. Gammack. 1963. A simple method for the quantitative isolation of undegraded high molecular weight ribonucleic acid. Biochem. Biophys. Res. Commun.

13:61-66. 3. Bussey, H., D. Sherman, and J. M. Somers. 1973. Action of yeast killer factor: a resistant mutant with sensitive spheroplasts. J. Bacteriol. 113:1193-1197. 4. Cottrell, S. C., M. Rabinowitz, and G. S. Getz. 1973. Mitochondrial deoxyribonucleic acid synthesis in a temperature-sensitive mutant of deoxyribonucleic acid replication of Saccharomyces cerevisiae. Biochemistry 12:4374-4378. 5. Cryer, D. R., C. D. Goldthwaite, S. Zinker, K. B. Lam, E. Storm, R. Hirschberg, J. Blamire, D. B. Finkelstein, and J. Marmur. 1973. Studies on nuclear and mitochondrial DNA of Saccharomyces cerevisiae. Cold Spring Harbor Symp. Quant. Biol. 38:17-29. 6. Fink, G. R., and C. A. Styles. 1972. Curing of a killer factor in Saccharomyces cerevisiae. Proc. Natl. Acad.

Sci. U.S.A. 69:2846-2849. 7. Fischer, I., and C. Shalitin. 1977. Increased synthesis of abundant poly(A) RNA in a DNA defective mutant of Saccharomyces cerevisiae containing the "killer character." Biochim. Biophys. Acta 475:64-73. 8. Franklin, R. M. 1966. Purification and properties of the 9.

10. 11. 12.

13.

replicative intermediate of the RNA bacteriophage R17. Proc. Natl. Acad. Sci. U.S.A. 55:1504-1511. Game, J. C. 1976. Yeast cell cycle mutant cdc21 is a temperature-sensitive thymidylate auxotroph. Mol. Gen. Genet. 146:313-315. Hartwell, L. H. 1970. Periodic density fluctuation during the yeast cell cycle and the selection of synchronous cultures. J. Bacteriol. 104:1280-1285. Hartwell, L. H. 1971. Genetic control of the cell division cycle in yeast. fl. Genes controlling DNA replication and its initiation. J. Mol. Biol. 59:183-194. Hartwell, L. H. 1973. Three additional genes required for deoxyribonucleic acid synthesis in Saccharomyces cerevisiae. J. Bacteriol. 115:966-974. Hartwell, L. H., R. K. Mortimer, J. Culotti, and M. Culotti. 1973. Genetic control ofthe cell division cycle in yeast. V. Genetic analysis of cd mutants. Genetics

74:267-286. 14. Hatzfeld, J. 1973. DNA labeling and its assay in yeast. Biochim. Biophys. Acta 299:34-42. 15. Newlon, C. S., and W. L. Fangman. 1975. Mitochondrial DNA synthesis in cell cycle mutants of Saccharomyces cerevisiae. Cell 5:423-428. 16. Peacock, A. C., and C. W. Dingman. 1968. Molecular weight estimation and separation of ribonucleic acid by electrophoresis in agarose-acrylamide composite gels. Biochemistry 7:668-674.

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17. Petes, T. D., and D. H. Williamson. 1975. Replicating circular DNA molecules in yeast. Cell 4:249-253. 18. Shalitin, C., and I. Fischer. 1975. Abundant species of poly(A)-containing RNA from Saccharomyces cerevisiae. Biochim. Biophys. Acta 414:263-272. 19. Shatkin, A. J., J. D. Sipe, and P. Loh. 1968. Separation of ten reovirus genome segments by polyacrylamide

gel electrophoresis. J. Virol. 2:986-991. 20. Vodkin, M. 1977. Homology between double-stranded RNA and nuclear DNA of yeast. J. Virol. 21:516-521.

J. BACTERIOL. 21. Vodkin, M., F. Katterman, and G. R. Fink. 1974. Yeast killer mutants with altered double-stranded ribonucleic acid. J. Bacteriol. 117:681-686. 22. Wickner, R. B. 1976. Killer of Saccharomyces cerevisiae: a double-stranded ribonucleic acid plasmid. Bacteriol. Rev. 40:757-773. 23. Wintersberger, U., J. Hirsch, and A. M. Fink. 1974. Studies on nuclear and mitochondrial DNA-replication in a temperature-sensitive mutant of Saccharomyces cerevisiae. Mol. Gen. Genet. 131:291-299.