Vol. 129, No. 2

JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 1066-1071 Copyright © 1977 American Society for Microbiology

Printed in U.S.A.

Volume-Related Mitochondrial Deoxyribonucleic Acid Synthesis in Zygotes and Vegetative Cells of Saccharomyces cerevisiael ENG-HONG LEE2 AND BYRON F. JOHNSON* Division of Biological Sciences, National Research Council of Canada, Ottawa, Canada KiA OR6 Received for publication 27 September 1976

The synthesis of mitochondrial deoxyribonucleic acid (DNA) in Saccharomyces cerevisiae cells has been examined during conjugation, in preconjugal conditions, and in control cultures that were not exposed to obverse diffusible sex factors. The ratios of mitochondrial to nuclear DNA varied from about 0.1 in control cells, to about 0.3 in a cells exposed for 180 min to cell-free culture medium from a cells, and to about 0.4 in conjugating cells 150 min after mixing. The enhanced levels of mitochondrial DNA during preconjugal and conjugal conditions seem correlated with enhanced cell volumes. Likewise, amounts of mitochondrial DNA in vegetative cells were found to be correlated with cytoplas-

mic volumes. Mitochondrial deoxyribonucleic acid (mtDNA) synthesis in vegetative cells of Saccharomyces cerevisiae has been shown to be continuous throughout the cell cycle (13, 18), even in circumstances in which nuclear DNA (nDNA) synthesis is inhibited (6, 11, 12). Discontinuous synthesis has also been suggested (2), but indirectly from mutational analysis rather than by direct measurement. On the other hand, recombination would be expected to be a function of synthesis, and recombination between old and newly synthesized strands of mtDNA is more or less continuous (18; and E. P. Sena, Ph.D. thesis, University of Wisconsin, Madison, 1972). Little is known about mtDNA synthesis in zygotes of S. cerevisiae, primarily because samples of zygotes adequate for biochemical analysis were not previously available (7). However, biochemical analysis has shown that recombination of mtDNA is occurring two generations after conjugation (14) and in cells formed many generations later (9), and genetic evidence suggests that the percent suppressiveness in "petite" matings is determined essentially after the first diploid daughter cell is detached (4). We show here that mtDNA synthesis in zygotes through the first diploid cell cycle is essentially similar to that observed in haploid cells treated with sex factors, with the amounts of mtDNA following the increase in volume of the precon' National Research Council of Canada publication no. 15750. 2 Present address: Department of Pathology, University

of Guelph, Guelph, Ontario, Canada NlG 2W1.

jugal cells or zygotes. Further, vegetative cells having different volumes have related amounts of mtDNA. MATERIALS AND METHODS Wild-type Rho' strains of S. cerevisiae were used; XY 223-1A (ATCC 32147), a, and MI-24D (ATCC 32145), a, were both kindly provided by A. P. James. They were cultured and mated as previously described (7), and the resultant zygotes were isolated by centrifugation through a series of step and linear gradients (to be presented elsewhere). For studies on vegetative cells, strain NCYC 239 (originally obtained from D. H. Williamson) was grown on malt extract medium (20), containing [3H]adenine, for 10 days at 25°C with constant shaking (150 rpm). In such cultures, the last cycle of buds mature and absciss from their mother cells, resulting in a mixture of old large and new small ("0" age) cells. These large and small cells were separated by repeated centrifugation of the mixture in 10% Ficoll (wt/vol) solution for 10 min at 2,000 x g. The large cells were collected as a pellet, and the small cells were gathered from the supernatant fluid. The separated large and small cells and the original mixture were all photographed and measurements were made on enlargements. The resultant size distributions (Fig. 1) were deemed adequate to permit comparative analysis. DNA was isolated according to the method of Williamson et al. (19) or by a method modified from Zeman and Lusena (23). The latter includes centrifugation at 18,000 rpm for 10 min in a Spinco SW30 rotor, after which a portion of the vesicular pellet was suspended and lysed in a detergent mixture previously layered upon a sucrose gradient and centrifuged according to Michels et al. (10). The DNAcontaining fractions were then pooled, dialyzed in

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MITOCHONDRIAL DNA SYNTHESIS IN S. CEREVISIAE

standard saline-citrate (23) for at least 4 h, freezedried, redissolved in a small volume of water, and redialyzed against standard saline-citrate. Finally, the DNA solution was brought to a density of 1.680 g/cm3 by addition of CsCl and was centrifuged in a Spinco SW50 rotor at 30,000 rpm for 20 min at allow removal of most of the protein and polysaccharide, which floated. The DNA was then analyzed either

by preparative ultracentrifugation according to Zeman and Lusena (23) or by analytical ultracentrifu-

gation in an ultracentrifuge (Spinco model E), in which case the absorbance was directly measured with the photoelectric scanner (Beckman) equipped with a multiplexer. The density of the DNA in the peak was calculated according to Mandel et al. (8), with DNA from Micrococcus lysodeikticus taken as 1.731 g/cm3 for a standard. In all experiments, the densities of mtDNA and nDNA were found to be closely comparable to values in the literature, 1.683 and 1.699 g/cm3, respectively (12, 18). RESULTS mtDNA in the haploid a strain constituted about 9% of the total (n plus mt)DNA, in terms of radioactivity in preparative gradients (Fig. 2a), and about 10% if measured by optical density in the analytical ultracentrifuge (Fig. 3). Comparable proportions of mtDNA and nDNA

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have been observed by others (5). When cells of the a strain were treated with cell-free culture supernatant from an a strain culture for 3 h as described earlier (7), about a threefold increase in their proportion of mtDNA was observed (Fig. 4b), whereas the proportions remained constant in the untreated control culture (Fig. 4a). In addition, 93 to 98% ofthe treated cells became unbudded. A similar relative increase of mtDNA in the converse experiment, a cells treated with a sex factor, has been observed (12), and unbudded cell formation has been taken as a characteristic consequence of the a factor on a cells (1, 3, 12) and vice versa (16, 22). Zygotes were isolated from mating mixtures (2.5 h after mixing haploids) to approximately

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FIG. 2. Isopycnic preparative CsCl gradients of S. cerevisiae DNA. (a) XY 223-1A, and (b) XY 223-lA x MI-24D zygotes. XY 223-1A was labeled with [3H]adenine (New England Nuclear Corp.) for 18 h in yeast extract-peptone medium with 2% glucose (7). A portion was used for (a), and the remainder, after it had been washed once, was mated with unlabeled MI-24D to obtain (b); [14C]adenine (New England Nuclear Corp.) was added immediately after the two strains were mixed. Eleven grams of DNA/CsCl solution at 1.680 g/cm3 was used, with a Spinco 40 rotor at 33,000 rpm for 60 to 65 h; temperature was 18°C and 200-pl fractions were counted.

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LEE AND JOHNSON

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FIG. 3. Isopycnic analytical gradients of S. cerevisiae DNA in CsCl. (a) Equiualent to Fig. 2a; (b) equivalent to Fig. 2b. Initial density was 1.680 glcm:'. An An-F four-hole rotor was used at 45,000 rpm for 18 to 21 h.

90 to 95% purity (Fig. 5), with zygotes and unmated haploid cells each tabulated as single units. On the average, the wet weights of these mating mixtures increased by 2.5 times during the incubation. The proportion of mtDNA in zygotes increased about fourfold in terms of

radioactivity (Fig. 2b) and about threefold by the optical density comparison (Fig. 3b). It is noteworthy that the mating medium contains glucose at 10% (7); hence, mtDNA metabolism seems not to be subject to glucose repression during conjugation or preparation for conjugation. The ratio of mtDNA to total DNA was 0.20 in the small vegetative cells, 0.28 in the unfractionated mixture, and 0.35 in the large vegetative cells (Fig. 6). These values lie within 1% error when calculated either by weighing areas under the curves or by computed analysis (23); the values can be seen (Fig. 7) to be well correlated with the mean cell volumes.

DISCUSSION It is clear that DNA synthesis during cellular preparation for conjugation ("preconjugal" events) differs in yeast nuclei and mitochondria. Thus, preconjugal treatment of a strain cells with medium from a strain cells inhibits nDNA synthesis (15, 16, 24), but permits continued mtDNA synthesis (Fig. 4b) in a manner wholly comparable with a factor effects on a cells (12). The difference is further amplified in zygotes (Fig. 2, 3). Moreover, the magnitude of the demonstrated differences between mtDNA and nDNA synthesis is likely to be diminished by the nDNA synthesis occurring in those cells that fail to respond (3 to 7%, in the mediumexchange experiment, and 5 to 10% nonmating cells assayed among the zygotes) to preconjugal or conjugal stimuli. It is noteworthy that this is genuinely a conjugation phenomenon, and not merely a haploid/diploid difference, for haploids and diploids have been shown to have identical mtDNA/nDNA ratios (17). The enhanced amounts of mtDNA are roughly proportional with the wet weight increase and with the enhanced cell volumes of preconjugal and conjugating yeast cells described by Yanagishima (21) and by Zuk et al. (24). Furthermore, it is clear that when vegetatively growing cells divide, the mtDNA present at the end of that cell cycle is distributed between mother and daughter approximately according to their respective cytoplasmic volumes. It seems to us that whatever the nature of the cellular controls on mtDNA synthesis (17), a strict stoichiometry with nDNA is not maintained. Because strict stoichiometry is more or less implicit in discreet S-periods, or discontinuous synthesis, our observations are more comparable with observations of continuous mtDNA synthesis (12, 13, 17, 18), and with reports that mtDNA continues to be synthesized in the absence of nDNA synthesis in sta-

MITOCHONDRIAL DNA SYNTHESIS IN S. CEREVISIAE

VOL. 129, 1977

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FIG. 5. Phase-contrast photomicrograph of purified zygotes suspended in 25% Ficoll solution. Note different sizes of diploid buds.

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CELL VOLUME (/Lm3) FIG. 7. Relationship between amounts of mtDNA and cell volume in stationary-phase yeast cells (NCYC 239). tot, Total DNA.

creasing (21, 24) suggests that the mtDNA synthesis mechanism may be receiving feedback signals (not only from nDNA [17]) relating to the volumes of cytoplasm that must be served by general mitochondrial metabolism.

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ACKNOWLEDGMENTS We thank R. Whitehead for photographic work, W. G. Martin and J. Giroux for instruction, K. A. Mittelstadt for assistance with ultracentrifuge problems, and Donna McLeish and Isabelle Boisclair-Sarrazin for general assistance. We also thank J. M. Ridgeway for the computed analysis of the curves. E.-H. Lee was a National Research Council of Canada postdoctoral fellow, 1974-1975. LITERATURE CITED 1. Bucking-Throm, E., W. Duntze, L. H. Hartwell, and T. R. Manney. 1973. Reversible arrest of haploid yeast cells at the initiation of DNA synthesis by a diffusible

sex factor. Exp. Cell Res. 76:99-110.

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22 18 26 30 FRACTIONS FIG. 6. Isopycnic preparative CsCl gradients of S. cerevisiae DNA (strain NCYC 239) labeled wth [H]adenine for 10 days. DNAs from large, unfractionated and small cells were isolated as described. Eleven grams of DNAICsCl solution at 1.680 glcm3 was used with a Spinco 40 rotor at 33,000 rpm for 60 to 65 h; temperature was 18°C, and 200-,u fractions were collected and counted for radioactivity. Smooth ) represent computed analyses of respeccurves ( tive radioactivities ( - ).

tionary-phase cells (6) and in some cell cycle mutants (11). Finally, the comparison of mtDNA synthesis patterns with general cytoplasmic growth in zygotes and in haploid cells treated with sex factors whose volumes are in-

2. Dawes, I. W., and B. L. A. Carter. 1974. Nitrosoguanidine mutagenesis during nuclear and mitochondrial gene replication. Nature (London) 250:709-712. 3. Duntze, W., V. Mackay, and T. R. Manney. 1970. Saccharomyces cerevisiae: a diffusible sex factor. Science 168:1472-1473. 4. Ephrussi, B., H. Jakob, and S. Grandchamp. 1966. Etudes sur la suppressivite des mutants a d6ficience respiratoire de la levure. 11. Etapes de la mutation grande en petite provoqu6e par le facteur suppressif. Genetics 54:1-29. 5. Goldthwaite, C. D., D. R. Cryer, and J. Marmur. 1974. Effect of carbon source on the replication and transmission of yeast mitochondrial genomes. Mol. Gen. Genet. 133:87-104. 6. Grossman, L. I., E. S. Goldring, and J. Marmur. 1969. Preferential synthesis of yeast mitochondrial DNA in the absence of protein synthesis. J. Mol. Biol. 46:367376. 7. Lee, E.-H., C. V. Lusena, and B. F. Johnson. 1975. A new method of obtaining zygotes in Saccharomyces cerevisiae. Can. J. Microbiol. 21:802-806. 8. Mandel, M., C. Schildkraut, and J. Marmur. 1968. Use

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10. 11. 12.

13.

14.

15.

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of CsCl density gradient analysis for determining the guanine plus cytosine content of DNA, p. 184-185. In L. Grossman and K. Moldave (ed.), Methods in enzymology. Nucleic acids, vol. 12, part B. Academic Press Inc., New York. Michaelis, G., E. Petrochilo, and P. P. Slonimski. 1973. Mitochondrial genetics. III. Recombined molecules of mitochondrial DNA obtained from crosses between cytoplasmicpetite mutants of Saccharomyces cerevisiae: physical and genetic characterization. Mol. Gen. Genet. 123:51-65. Michels, C. A., J. Blamire, B. Goldfinger, and J. Marmur. 1974. A genetic and biochemical analysis of petite mutations in yeast. J. Mol. Biol. 90:431-449. Newlon, C. S., and W. L. Fangman. 1975. Mitochondrial DNA synthesis in cell cycle mutants of Saccharomyces cerevisiae. Cell 5:423-428. Petes, T. D., and W. L. Fangman. 1973. Preferential synthesis of yeast mitochondrial DNA in a factorarrested cells. Biochem. Biophys. Res. Commun. 55:603-609. Sena, E. P., J. W. Welch, H. 0. Halvorson, and S. Fogel. 1975. Nuclear and mitochondrial deoxyribonucleic acid replication during mitosis in Saccharomyces cerevisiae. J. Bacteriol. 123:497-504. Shannon, C., A. Rao, S. Douglass, and R. S. Criddle. 1972. Recombination in yeast mitochondrial DNA. J. Supramol. Struct. 1:145-152. Shimoda, C., and N. Yanagishima. 1973. Mating reaction in Saccharomyces cerevisiae. IV. Retardation of deoxyribonucleic acid synthesis. Physiol. Plant. 29:54-59.

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16. Wilkinson, L. E., and J. R. Pringle. 1974. Transient G, arrest of S. cerevisiae cells of mating type a by a factor produced by cells of mating type a. Exp. Cell Res. 89:175-187. 17. Williamson, D. H. 1970. The effects of environmental and genetic factors on the replication of mitochondrial DNA in yeast, p. 247-276. In P. L. Miller (ed.), Control of organelle development. Symposium 24, Society for Experimental Biology. Cambridge University Press. 18. Williamson, D. H., and D. J. Fennell. 1974. Apparent dispersive replication of yeast mitochondrial DNA as revealed by density labelling experiments. Mol. Gen. Genet. 131:193-207. 19. Williamson, D. H., E. Moustacchi, and D. J. Fennell. 1971. A procedure for rapidly extracting and estimating the nuclear and cytoplasmic DNA components of yeast cells. Biochim. Biophys. Acta 238:369-374. 20. Williamson, D. H., and A. W. Scopes. 1962. A rapid method for synchronizing division in the yeast, Saccharomyces cerevisiae. Nature (London) 193:256-257. 21. Yanagishima, N. 1969. Sexual hormones in yeast. Planta 87:110-118. 22. Yanagishima, N. 1973. Physiology of the mating reaction in yeast. Curr. Adv. Plant Sci. 7:55-66. 23. Zeman, L., and C. V. Lusena. 1974. DNA synthesis in isolated yeast mitochondria. Can. J. Biochem. 52:941-949. 24. Zuk, J., D. Zaborowski, J. Litwinska, E. Chlebowicz, and T. Bilinski. 1975. Macromolecular synthesis during conjugation in yeast. Acta Microbiol. Pol. Ser. A 7:67-75.