Current Genetics

Current Genetics (1982)6:147-152

© Springer-Verlag 1982

The Regulation of Mitochondrial DNA Levels in Saccharomyces cerevisiae Michael N. Conrad and Carol S. Newlon Department of Zoology, Universityof Iowa, Iowa City, Iowa 52242, USA

Summary. Mitochondrial DNA (mtDNA) synthesis can continue under conditions which block cell division and nuclear DNA (nDNA) synthesis, producing cells with several times the normal level of mtDNA. We have examined mtDNA synthesis in cultures recovering from such cell cycle blocks. Our results show that the rate ofmtDNA synthesis is not affected either during a block of the cell cycle with e~-factor or during recovery from a perturbation in the amount of mtDNA/cell induced by blocking the cell cycle with or-factor or cdc4. The normal mtDNA content was restored a period of several generations when permissive conditions were restored. These results suggest that mtDNA synthesis is coupled to cell growth. Key words: Mitochondrial DNA cerevisiae

Cell cycle - S.

Introduction

The mitochondrial genome of Saccharomyces cerevisiae contains genetic information vital for the assembly of an organelle capable of respiration. Specifically, yeast mitochondfial DNA (mtDNA) has been shown to contain the genes for three subunits of cytochrome oxidase, four subunits of the F 1 ATPase, and cytochrome b. In addition, genes for the 21S and 15S mitochondrial ribosomal RNA's and tRNA's are located in the mitochondrion (reviewed by Dujon 1981). The replication and transmission of mtDNA is essential for the production of respiration competent progeny. To ensure that this is accomplished, mtDNA replication must be coordinated with the cell division cycle. Offprint requests to: C. S. Newlon

The mitochondrial genome of S. cerevisiae is a 75 kb double-stranded circular molecule (Hollenberg e t al. 1970). Yeast mtDNA has a lower buoyant density in CsC1 (1.685 g/cc) than nuclear DNA (nDNA) (1.699 g/cc) (Tewari et al. 1965; Corneo et al. 1966; Sinclair et al. 1967), allowing the two DNA species to be easily separated. In a given genetic background mtDNA appears to be present in constant ratio to nuclear DNA, with diploids containing twice as much mtDNA per cell as haploids and tetraploids four times as much (Fukuhara 1969; Williamson 1970; Grimes et al. 1974). mtDNA level is probably not controlled by the mitochondrial genome, since many cytoplasmic petite mutants, deleted for large portions of the mitochondrial genome, contain about as much mtDNA per cell as grande strains (Nagley and Linnane 1972). From a genetic analysis of strains exhibiting high and low levels of mtDNA, Hall et al. (1976) concluded that cellular mtDNA content is under the control of at least two nuclear genes. The level of mtDNA can vary with the conditions of growth. Cells grown on low concentrations of glucose or on non-fermentable carbon sources such as ethanol, lactate or glycerol can contain up to twice as much mtDNA as cells grown on high concentrations of glucose, although this can vary widely from strain to strain (Fukuhara 1969; Williamson 1970; Mian et al. 1973; Goldthwaite et al. 1974). This suggests that amplification ofmtDNA may be under the same control mechanisms which regulate the derepression of mitochondrial enzyme activities associated with the switch from glycolytic to oxidative metabolism (reviewed by Perlman and Mahler 1974). However, mtDNA amplification in derepressed cells is not a prerequisite for heightened expression of mitochondrial enzymes, since derepression can occur when mtDNA synthesis is inhibited by the cdc8 or cdc21 mutations (Mahler et al. 1975). 0172-8083/82/0006/0147/S 01.20

148 Although the correlation of ploidy and mtDNA content suggests a coupling of nuclear and mtDNA synthesis, the two DNA species exhibit different temporal patterns of replication. Nuclear DNA synthesis is confined to 1 / 4 1/2 of the cell cycle (Williamson 1965; Barford and Hall 1976; Slater et al. 1977; Rivin and Fangman 1980), while mtDNA synthesis takes place throughout the cell cycle (Williamson and Moustacchi 1971 ; Sena et al. 1975). In addition, mtDNA synthesis continues under a variety of conditions in which nDNA synthesis is inhibited. These include entry into stationary phase (Goldthwaite et al. 1974) and interruption of the cell cycle with cycloheximide or amino acid starvation (Grossman et al. 1969), with the mating pheromones o~-factor and a-factor (Petes and Fangman 1973; Lee and Johnson 1977), or with a variety of G1- or G2-arrest cell cycle mutations (Cottrell et al. 1973; Cryer et al. 1973; Newlon and Fangman 1975). Since the amount of mtDNA increases by a factor of greater than two when nDNA synthesis is inhibited, mtDNA molecules appear capable of initiating new rounds of replication in the absence of nDNA synthesis. Newlon and Fangman (1975) found that the size ofmtDNA made during cell cycle arrest was the same as that from dividing cells~ indicating that the mitochondfial DNA synthesized was not the result of producing fragmented molecules or of rolling circle replication. Both mtDNA and nDNA synthesis are interrupted by the mutations cdc8 and cdc21, each of which interrupts nucleotide chain elongation (Hartwell 1971, 1973; Cryer et al. 1973; Wintersberger et al. 1974; Newlon and Fangman 1975). Thus, nDNA and mtDNA synthesis share some elements of the fork propagation machinery, but control of initiation of replication of the two DNA's is independent. The ability of mtDNA synthesis to be uncoupled from nDNA synthesis raises questions as to how mtDNA synthesis is integrated into the cell cycle. Three models can be formulated to explain the control of mtDNA levels. These models predict different responses to the presence of a higher than normal level of mtDNA induced by cell cycle arrest. In the first model mtDNA content is measured relative to nuclear DNA, and the correct level of mtDNA is some fraction of the mass of nuclear DNA. Interruption of progress through the cell division cycle is presumed to prevent the functioning of the mechanism which normally regulates mtDNA synthesis, since mtDNA content continues to increase under these conditions. Upon return to a state permissive for cell division, the postulated regulatory mechanism would be expected to bring about a downward adjustment in the level of mtDNA, either by shutting off mtDNA synthesis or by degrading excess mtDNA. In the second model mtDNA content is set by cell size (or mass), and thus mtDNA synthesis is coupled to cell growth. Since cells arrested with mating factor and cdc

M.N. Conrad and C. S. Newlon: Mitochondriat DNA Levelsin Yeast mutants incubated at the restrictive temperature continue to increase in size (Johnston et al. 1977), mtDNA synthesis would be expected to continue as long as the cell continued to grow, despite the inhibition of nDNA synthesis and cell division. When the cell is permitted to divide again, mtDNA would continue to be synthesized in proportion to the mass of the cell. However, the average cell size would be expected to decline. Johnston et al. (1977) have demonstrated that large cells resulting from c~-factor treatment produce buds which are larger than normal cells but much smaller in proportion to their mothers. This pattern of division would return the size distribution to near-normal in a few generations. If the mtDNA level were determined by cell size, the amount of mtDNA per cell would also decline to normal at the same rate as the cell size since smaller buds would contain proportionally smaller amounts of mtDNA. One would expect neither degradation of mtDNA nor a shut-off of mtDNA replication. As in the first model, the normal ratio of mtDNA/nDNA would be re-established, but the stability of mtDNA and its rate of synthesis relative to cell growth would not be affected. In the third model mtDNA is synthesized at a rate proportional to the amount of mtDNA present, doubling the amount of mtDNA each generation time. According to this model, the perturbation of mtDNA level would be permanent. Cells which started with an elevated level of mtDNA would produce progeny that would also possess a high mtDNA/nDNA ratio. To differentiate among these models, we have used arrest in the G1 phase of the cell cycle induced with either c~-factor or the temperature sensitive cell cycle mutation cdc4 to increase the ratio of mtDNA/nDNA by two- to four-fold. We then measured the rate of synthesis and stability of mtDNA following release from G1 arrest.

Materials and Methods Strains and Culture Conditions. The strains used were A364A (MATa adel ade2 ural his7 lys2 tyrl gall, Hartwell 1967) and 135.1.1 (MATa adel ade2 ural lys2 tyrl gall (?J cdc4-4, Hart-

well 1973). 135.1.1 was grown in Y minimal medium, pH 5.8 (Newlon et al. 1974), supplemented with (per liter) 20 g glucose, 0.5 g yeast extract, 50 mg each histidine, lysine, tyrosine and methionine, 10 mg adenine and 3 mg uracil; the permissivetemperature was 23 °C and the restrictive temperature was 37 °C. A364A was grown at 30 °C in the same medium but at pH 3.5. This allows the more economical use of a-factor. Chemicals. a-factor was prepared by the method of Duntze et al.

(1973) and added at a concentration sufficient to prevent budding for the desired period. [6-3H]uracil and [2-14C]uracil were obtained from Amersham (Arlington Hts., ILL). Monitoring Cell Number and DNA Synthesis. Cell number was

determined by sonicating a portion of the culture to break apart clumps of cells and counting cells in a hemacytometer. Incorpo-

M. N. Conrad and C. S. Newlon: Mitochondrial DNA Levels in Yeast r

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Fig. 2. Stability of mtDNA synthesized prior to release from an a-factor block. The proportion of mtDNA was estimated in the label-chase experiment described in the legend to Fig. 1. The percentage of mtDNA in a CsC1gradient was determined by analyzing a plot of the 3H-DNA profile with a Hewlett-Packard curve resolver, or by summing the cpm's in each peak. The arrow (~) indicates the time at which a-factor and 3H-uracil were removed.

ration of radioactive precursors into DNA was monitored as alkali stable, TCA precipitable radioactivity (Hartwell 1970).

Analysis of mtDNA. At intervals, a 5-10 ml sample of the culture (2-8 x 106 cells/ml) was harvested and spheroplasts prepared with Glusulase (Endo) by the method of Forte and Fangman (1976). The spheroplasts were resuspended in 0.5-1 ml SCE (1 M sorbitol, 0.1 M Na citrate, 0.06 M EDTA, pH 5.8) and lysed by the addition of 5% sarkosyl to a final concentration of 1%. Proteinase K was added to N50 ~g/ml and the lysate incubated at 37 °C for 1 h. CsC1 gradients contained 0.5-0.6 ml of lysate, 1 mM Tris-C1, pH 8, 1 mM EDTA and CsC1 to give a refractive index of 1.3990-1.4000. Experiments with cdc4 employed gradients containing ethidium bromide (Newlon and Fangman 1975) to produce a sharper separation of nuclear and mitochon-

The first experiment examined the accumulation o f excess m t D N A during treatment with a-factor and the stability o f that DNA when the a-factor was removed. Cultures were grown in 3H-uracil to uniformly label DNA, treated with a-factor for the equivalent o f three generations, then washed free o f a-factor and resuspended in non-radioactive medium containing an excess of cold uracil. At intervals, aliquots o f the culture were removed, and lysates were prepared for centrifugation in CsC1 gradients to separate mitochondrial and nuclear DNA. Figure 1 shows the distribution o f nuclear and mitochondrial DNA at three points in the experiment. The percentage of 3H.DNA as mitochondrial DNA is plotted against time in Fig. 2. At t = 0, m t D N A was about 8% of total DNA. After 270 rain in a-factor, m t D N A had increased to 26% o f total DNA. During the chase period, in which the cell number increased four-fold, the proportion o f 3H-DNA in the mitochondrial fraction remained nearly constant, representing 23% o f the 3H-DNA at t = 570 mln. Thus under these conditions mitochondrial DNA is as stable as nuclear DNA. To detect changes in the rate o f synthesis o f m t D N A and the ratio o f mitochondrial to nuclear DNA, a second experiment was conducted with a continuously labeled culture so that the mass o f DNA could be monitored. Strain A364A was grown in medium containing 3H-uracil for 5 - 6 generations to uniformly label DNA. a-factor was added to the culture and, after 240 min, the cells were washed and resuspended in fresh medium containing 3H-uracil at the same specific activity. Cell density and total DNA synthesis were monitored in the a-factor treated culture and in a parallel asynchronous culture (Fig. 3). Cell density remained constant during the period of treatment with a-factor, and total DNA synthesis proceeded at a much slower rate than in the asynchronous culture. The rate o f DNA synthesis increased and cell division resumed when the a-factor was removed. The proportion of m t D N A was determined from CsC1 gradient profiles and is presented in Fig. 4A. mtDNA represented approximately 5% o f total DNA in asynchronous culture and increased to 17% after 225 rain in afactor. After the a-factor was removed, the proportion of m t D N A dropped slowly to a level of 6 - 9 % o f total DNA 475 rain. The m t D N A content of the culture can be estimated b y multiplying the cpm in DNA in a sample o f the culture (the data in Fig. 3) b y the percentage o f DNA which is m t D N A at that time. The results are plotted in Fig. 4B. m t D N A increased exponentially for the course

M. N. Conrad and C. S. Newlon: Mitoehondrial DNA Levels in Yeast

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Fig. 3A and B. Cell division and DNA synthesis in asynchronous and a-factor-treated cultures. Strain A364A was grown 5 - 6 generations in medium containing 5 vCi/ml 3H-uracil. At t = 0 part of the culture was removed and maintained as an asynchronous culture; a-factor was added to the remainder. At t = 240 min (*) the a-factor-treated culture was centrifuged and resuspended in fresh medium containing 3H-uracil at the same specific activity but in a volume which represented a 2.5-fold dilution o f the cell density. This dilution has been taken into account in the presentation of the results. At intervals aliquots of the culture were removed to determine cell number and the incorporation of 3H into DNA; A Cell density; B DNA synthesis, e - o , asynchronous culture; o-Q, a-synchronized culture

Fig. 4A and B. mtDNA levels and rates of synthesis during and after a-factor induced arrest; A The % mtDNA was determined from CsC1 gradient analysis of aliquots of the cultures in Fig. 3 (not shown). Culture treated with a-factor ( o - o ) ; culture after removal of the a-factor ( e - o ) ; B The amount of mtDNA per ml of culture was estimated by multiplying the DNA content o f l ml of the culture (Fig. 3B) by the proportion of mtDNA at that time (Fig. 4A). The slope of the line represents the rate of mtDNA synthesis.

of the experiment, exhibiting a doubling time of about 90 min, the same as the cell doubling time in an asynchronous culture (see Fig. 3). Thus the rate of mtDNA synthesis is not affected during cell cycle arrest or during recovery from such a block. In summary, the mtDNA synthesized during a prolonged period of a-factor-induced G1 arrest was not degraded when division resumed. The ratio of mtDNA/ nDNA declined gradually over the course of several cell generations following release from the a-factor block, even though the rate of mtDNA synthesis did not change. Since the level of mtDNA relative to nDNA did decline, a doubling of mtDNA content must not be required for cell division. These results are most consistent with a model in which mtDNA synthesis is coupled to growth rather than to nuclear DNA levels. To confirm these results, the control of mtDNA level was examined in the cell cycle mutant cdc4. At 37 °C this mutant arrests in G1 and continues to produce buds at intervals of roughly one generation, even though nDNA synthesis and nuclear division are blocked (Hartwell 1971). The cdc4 mutant was chosen for this study because

strain 135.1.1 contains a relatively high level of mtDNA (10-14% of total DNA, Newlon and Fangman 1975, this paper) and retains a high degree of viability (70-80%) after the equivalent of one generation at 37 °C. Sinceloss of viability does occur with extended incubations at the restrictive temperature, the cdc4 cultures could not be arrested for as long a period as with a-factor. The following regime of radioactive labelling allowed us to monitor simultaneously both the stability of mtDNA made at 37 °C and any change in the proportion of mtDNA in total DNA. Strain 135.1" 1 was grown at 23 °C for 5 - 6 generations in medium containing both 3H- and 14C-uracil to label DNA uniformly. At t = 0 the culture was shifted to 37 °C for 2.75 h. The cells were then filtered and resuspended at 23 °C in fresh medium containing 3H-uracil at the same specific activity as before, but free of 14C-uracil. 3H-DNA then reflects the mass of DNA in the culture. If the mtDNA made prior to the shift-down is stable, the mtDNA/nDNA ratio in 14C.DN A should not change. Cell density and DNA synthesis over the course of the experiment are plotted in Fig. 5, illustrating the interrup-

M. N. Conrad and C. S. Newlon:~Mitochondrial DNA Levelsin Yeast

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Fig. 5. Cell division and DNA synthesis in cdc4 before, during and after arrest at 37 °C. Strain 135-1-1 was grown 5-6 generations in medium containing 2 ~Ci/ml 3H-uracil and 1 ~Ci/ml 14C-uracil. At t = 0, the culture was shifted to 37 °C. After 2.75 h (40, the culture was filtered and resuspended at 23 °C in fresh medium containing only 3H-uracil at the same specific activity. Cell number (panel A) and DNA content (panel B) were monitored as in Materials and Methods. e-e, 3H-DNA(cpm x 10--1); 0-% 14C-DNA (cpm x 10-2)

151 tion of cell division at 37 °C and recovery of the culture when shifted to 23 °C. The incorporation of 14C into DNA ceased when cells were shifted to medium containing only 3H-uracll, indicating that uracil pools are small (Fig. 5B). The percentage of mtDNA in 14C- and 3H-DNA is plotted in Fig. 6A. The proportion of mtDNA rose from 12% in asynchronous cells to approximately 30% in a culture held at 37 °C for one generation. Following return of the culture to 23 °C, the proportion ofmtDNA present in DNA synthesized before the shift-down (14C-DNA) remained essentially constant (Fig. 6B). The percentage of mtDNA in total DNA (3H-DNA) declined to approximately 14% in the 10 h after return to 23 °C. As before, mtDNA synthesis continued at a constant rate after the shift to 23 °C with a doubling time of about 2.5 h (Fig. 6C). Yeast cells containing an elevated level of mtDNA accumulated during arrest induced by the cdc4 mutation behave in the same manner as cells recovering from an a-factor block. There is no degradation of mtDNA and the rate ofmtDNA synthesis is unaltered when the culture is returned to the permissive temperature. The mtDNA/ nDNA ratio declines over a period of several generations, gradually restoring the normal distribution.

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Fig. 6A-C, mtDNA levels and rates of synthesis in cdc4 during growth at 37 °C and after return to 23 °C. A At various times during the course of the experiment shown in Fig. 5 lysates were prepared from aliquots of the culture and centrifuged in CsC1 gradients. The % mtDNA was determined for 3H- and 14C-DNA profiles (not shown). The % mtDNA at t = 0 (X) was determined from a separate, asynchronous culture; B The amount of 14CmtDNA per ml of culture was determined from the % mtDNA and the total 14C-DNA per mt as in Fig. 4B; C The amount of 3H-mtDNA per ml of culture was determined as in 4B. The lines on the figure after the shift from 37 °C to 23 °C were calculated from a least squares regression, omitting what appears to be an anomalously low value in the last 14C point (see Fig. 6B). e - e , 3H-DNA; o - % 14C-DNA

The response of yeast cells to a perturbation of mtDNA level eliminates two of the three models proposed to explain regulation of mtDNA levels. Since mtDNA made during the period of cell division arrest is stable and since the rate of mtDNA synthesis is unaffected by the perturbations in the cell cycle, it is unlikely that mtDNA synthesis is tightly coupled to nDNA levels. The amount of mtDNA per cell does decline, however, when cell division resumes, ruling out the model which requires a doubling of mtDNA content each cell cycle regardless of the amount of mtDNA present. The only model supported by the data is one which postulates that mtDNA synthesis is not coupled to nDNA replication and the cell division cycle, but is linked instead to the growth of the cell. The view that mtDNA synthesis is dependent on cell growth is supported by a strong correlation between cell volume and mtDNA content in stationary phase cells. Furthermore, the ratio of mtDNA/nDNA can increase three- to six-fold when nuclear DNA synthesis is blocked under conditions where growth continues (Petes and Fangman 1973; Newlon and Fangman 1975; Lee and Johnson 1977, this paper). In contrast, when yeast cells are treated with cychoheximide, only a two-fold increase in the relative amount of mtDNA occurs. Radioactive adenine continues to be incorporated into mtDNA in the presence of cycloheximide even after the increase in mtDNA mass has ceased (Grossman et al. 1969), pre-

152 sumably reflecting repair or turnover synthesis. Thus, while inhibition o f protein synthesis can dissociate nuclear and mitochondrial DNA synthesis, continued cell growth appears necessary for the synthesis o f large amounts o f mtDNA. The results o f an experiment b y Johnston et al. (1977) suggest that restoration o f a normal cell size and, consequently, a normal m t D N A / n D N A ratio will occur after a period o f cell division arrest. They observed that the first buds produced b y cells which had been arrested with afactor for two generations were smaller in proportion their mothers than buds in an asynchronous culture. A continuation of this trend would restore the normal cell size in a few generations. Although we made no attempt to measure ceil size in our experiments, the average ceil size three generations after release from the a-factor block was clearly smaller than the average cell size in a culture treated with a-factor for three generations. The stability o f mtDNA made during cell cycle arrest indicates that the m t D N A is probably intact, normal DNA, which is not subject to degradation. This interpretation is consistent with the observations o f Newlon and Fangman (1975) who found that mtDNA synthesized at 37 °C b y strains carrying the cdc7 m u t a t i o n sedimented at the same rate as m t D N A from an asynchronous culture. While our results are consistent with a coupling o f m t D N A synthesis to cell growth, they do n o t explain the higher m t D N A content o f cells grown on non-fermentable carbon sources. Cells grown in glycerol or very low concentrations o f glucose have more m t D N A per cell despite being smaller in volume than cells grown on high concentrations o f glucose (Adams 1977; Stevens 1977). One plausible explanation o f this paradox is that m t D N A synthesis is coupled not to cytoplasmic growth in general, b u t to a specific component o f cytoplasmic growth, namely mitochondrial growth. This interpretation is supported b y the results o f a serial section study o f mitochondria which found that the mitochondria o f glucose-grown cells occupy 3.1% o f the cell volume, and that the mitochondria of glycerol-grown cells occupy 12.6% of the cell volume (Stevens 1977). The increase in m t D N A level during arrest o f cell division would be explained b y continued mitochondrial growth.

Acknowledgements. We thank Michael C. Newlon and Lolya Lipehitz for helpful comments on the manuscript. This work was supported by NIH research grant GM21510 and RCDAGM00265 to CSN and a predoctoral traineeship from the Cell and Molecular Biology Training Grant GM07228 to MNC. References Adams J (1977) Exp Cell Res 106:267-275 Barford JP, Hall RJ (1976) Exp Cell Res 102:276-284 Corneo G, Moore C, Sanadi DR, Grossman LI, Marmur J (1966) Science 151:687 -689

M.N. Conrad and C. S. Newlon: Mitochondrial DNA Levels in Yeast Cortrell S, Rabinowitz M, Getz GS (1973) Biochemistry 12: 4374-4378 Cryer DR, Goldthwaite CD, Zinker S, I_am K-B, Storm E, Hirsehberg R, Blamire J, Finkelstein DB, Marmur J (1974) Studies on nuclear and mitochondrial DNA of Saceharomyces cerevisiae. Cold Spring Harbor Symp Quant Biol 38:17-29 Dujon B (1981 ) Mito chondrial genetics and functions. In: Strathern JN, Jones EW, Broach JR (eds) The Molecular Biology of the Yeast Saccharomyces, Life Cycle and Inheritance. Cold Spring Harbor, pp 505-635 Duntze W, Stotzler D, Biicking-Throm E, Kalbitzer S (1973) Eur J Biochem 35:357-365 Forte MA, Fangman WL (1976) Cell 8:425-431 Fukuhara H (1969) Eur J Biochem 11:135-139 Goldthwaite CD, Cryer DR, Marmur J (1974) Mol Gen Genet 133:87-104 Grimes GW, Mahler HR, Perlman PS (1974) J Cell Biol 61:565574 Grossman LI, Goldring ES, Marmur J (1969) J Mol Bio146:367376 Hall RM, Nagley P, Linnane AW (1976) Mol Gen Genet 145: 169-175 Hartwell LH (1967) J Bacterio193:1662-1670 Hartwell LH (1970) J Bacteriol 104:1280-1285 Hartwell LH (1971) J Mol Biol 59:183-194 Hartwell LH (1973) J Bacteriol 115:966-974 Hereford LM, Hartwell LH (1974) J Mol Biol 84:445-461 Hollenberg CP, Borst P, Van Bruggen EFJ (1970) Bioehim Biophys Acta 209:1-15 Johnston GC, Pringle JR, Hartwell LH (1977) Exp Cell Res 105: 79-98 Lee E-H, Johnson BF (1977) J Bacterio1129:1066-1071 Mahler MR, Assimos K, Lin CC (1975) J Bacterio1123:637-641 Mian FA, Kuenzi MT, Halvorson HO (1973) J Bacteriol 115:876881 Nagley P, Linnane AW (1972) J Mol Biol 66:181-193 Newlon CS, Fangman WL (1975) Cell 5:423-428 Newlon CS, Petes TD, Hereford LM, Fangman WL (1974) Nature 247:32-35 Perlman PS, Mahler HR (1974) Arch Biochem Biophys 162:248271 Petes TD, Fangman WL (1973) Biochem Biophys Res Commun 55:603-609 Rivin CJ, Fangman WL (1980) J Cell Biol 85:96-107 Sena EP, Welch JW, Halvorson HO, Fogel S (1975) J Bacteriol 123:497-504 Sinclair JH, Stevens BJ, Sanghavi P, Rabinowitz M (1967) Science 156:1234-1237 Slater ML, Sharrow SO, Gaff JJ (1977) Proc Natl Acad Sci USA 74:3850-3854 Stevens BJ (1977) Biol Cell 28:37-56 Tewaff KK, Jayaraman J, Mahler HR (1965) Biochem Biophys Res Commun 21:141-148 Williamson DH (1965) J Cell Biol 25:517-528 Williamson DH (1970) The effect of environmental and genetic factors on the replication of rnitochondrial DNA in yeast. In: Miller PL (ed) Control of organelle development. Cambridge University Press, pp 247-276 Williamson DH, Moustacchi E (1971) Biochem Biophys Res Comm 42:195-201 Wintersberger U, Hirsch J, Fink AM (t975) Mol Gen Genet 131 : 291-299 Communicated b y C. W. Birky, Jr. Received July 6/July 21, 1982