J. Mol. Biol. (1975) 97, 593-609

Structure and Replication of Mitochondrial D N A from Paramecium aurelia JuDY M. GODDARD AND DONALD J. CUMMINGS

Department of Microbiology University of Colorado Medical Center Denver, Colo. 80220, U.8.A. (Received 27 February 1975, and in revisedform 20 May 1975) Mitochondrlal DNA from Paramecium aurelia was purified by CsC1 density equilibrium centrifugation and characterized by examination in the electron microscope and sedimentation in sucrose gradients. I t was found that 96% of the molecules were linear; and 65% of these were between 13 pm and 15 pm in length, with an average of 13.8 can. 3% of the molecules were lariats in which the lengths of the circles and tails varied with respect to each other. The proportion of these molecules was increased from 3 to 30% by growth of cells in ethidium bromide. In both cases, the sum of one-half the circle length plus the tail length was equal to 13.8 ~m (0-5o + t ~ 13.8 pro). In mitochondrial DNA preparations from balanced growth cells, approximately 10% of the molecules were linear duplexes of dimer length (25 to 28 pro); this value dropped markedly under conditions of stationary growth. The dimer population could be enriched by growth of cells in chloramphenicol, indicating the need for mitochondrial protein synthesis for processing mitochondrial I)NA. A pathway is presented illustrating the types of molecules seen and a possible sequence by which they might arise. 1. I n t r o d u c t i o n

Characterization of DNA from mitochondna was not reported until the early 1960s (Nass & Nass, 1963a, b; Luck & Reich, 1964). Since t h a t time, however, mitoehondrial DNAs from m a n y different organisms have been studied and, in the case of the higher animal systems, shown to be strikingly similar in their size, topology and mode of replication. These molecules are a p p r o ~ m a t e l y 5 p m in length; t h e y are circular, and they replicate according to a modified Cairns displacement mechanism (Borst & Flavell, 1972; Kasamatsu & Vinograd, 1974). These typical characteristics, however, appear to occur only among the higher animal systems and do not extend to the plants and protists. For example, mitochondrial DNA can range from 0.3/~m in the klnetoplast of flagellate protozoa (Wesley & Simpson, 1973) to 25 /~m in yeast (Hollenberg et aL, 1970); and the m t I ) N k of the ciliate protozoan, Tdrahymena ~yriformis, exists as a 15/~m linear duplex rather t h a n as a circular form (Suyama & Miura, 1968; Flavell & Follett, 1970). iKitochondrial I)NA replication in protists has not been extensively studied, but information does exist for Tdrahymena (Upholt & Borst, 1974; Arnberg d a/., 1974; Clegg d al., 1974). In this case, however, replication is more similar to the basic Cairns mechanism than to the displacement mode of higher animals. More information is obviously needed in order to determine whether 4O

593

594

J . M. G O D D A R D A N D D. J . C U M M I N G S

t h e l i n e a r i t y a n d r e p l i c a t i o n m o d e o f m t D N A i n o r g a n i s m s like TeJrahyme~ a r e s i m p l y u n i q u e e x c e p t i o n s o r w h e t h e r some n e w b a s i c p a t t e r n s will emerge. Paramecium aurdia is a ciliate p r o t o z o a n f r o m w h i c h m t D N A w a s first i s o l a t e d b y S u y a m a & P r e e r (1965). A l t h o u g h a l~inetic c o m p l e x i t y o f 35 × l 0 s d a l t o n s w a s l a t e r r e p o r t e d b y F l a v e l l & J o n e s (1971), n e i t h e r t h e l e n g t h o f t h e g e n o m e n o r i t s m o d e o f r e p l i c a t i o n h a s y e t b e e n d e t e r m i n e d . I n t h i s p a p e r we p r e s e n t evidence t h a t t h e m i t o c h o n d r i a l g e n o m e i n Paramecium is a 13.8 F m l i n e a r d u p l e x a n d t h a t i t s r e p l i c a t i o n results in t h e f o r m a t i o n o f l a r i a t molecules a n d linear dimers. T h e evidence indicates, however, t h a t t h e l a r i a t s do n o t arise b y a rolling-circle t y p e o f m e c h a n i s m . A s c h e m a t i c d i a g r a m is p r e s e n t e d w h i c h i l l u s t r a t e s t h e t y p e s o f molecules seen a n d offers a possible sequence b y w h i c h t h e y m i g h t arise.

2. Materials and M e t h o d s (a) Cells and culture condiOiona P. aurelia syngen 1, stock 513 obtained from the l a b o r a t o r y of G. H. Beale was used in all experiments. The mitochondria wore isolated from exautogamous clones which h a d undergone a p p r o x i m a t e l y 30 to 50 divisions. Cells were cultured a t 27°C in Scotch grass infusion inoculated with Klebaiella aerogenes (Sonneborn, 1950; Cummings, 1972). Ckflture volumes of 2 1 were m a i n t a i n e d in a series of 5-1 Diptheria culture flasks incubated in a horizontal position to ensure m a x i m u m aeration. Cell growth was initiated b y the addition of cells to a s t a r t i n g d e n s i t y of 150 to 200 cells/ml. During exponential growth t h e cultures were supplemented twice with additional bacteria obtained b y collecting overnight 1.5-1 b a c t e r i a l - n u t r i e n t b r o t h cultures b y centrifugation a n d then re-suspending the pellets in a small volume of the grass infusion. I n general, pellets from approx. 6 1 of bacterial culture were a d d e d to 20 1 of cell culture. Small samples were removed periodically for counting of cells u n d e r t h e microscope, a n d cells were harvested either in balanced growth (1200 cells/ml), in late log (3 to 4000 cells/ml) or in s t a t i o n a r y phase. The generation time was a p p r o x i m a t e l y 7 h. A representative growth curve is included in Fig. 1. (b) Cell grouch in the preserve qf dr~js E t h i d i n m bromide was a d d e d to exponentially growing cultures (400 to 600 cells/ml) a n d the cells were harvested several generations later. I n some experiments the drug was a d d e d a t 4 gg/ml a n d the cells were harvested 20 h later. I n other cases, t h e cells were exposed to 10 gg/ml for a p p r o x i m a t e l y 10 h, t h e n diluted to a final drug concentration o f 2 gg/ml a n d harvested a p p r o x i m a t e l y 20 h after the dilution. Similar results were obtained in b o t h cases. Fig. 1 shows the effects of different concentrations of E t h B r t on cell growth. A similar procedure was used for growth of cells in chloramphenicol. The d r u g was a d d e d to a concentration of 25 gg/ml during balanced growth, a n d t h e cells were harvested 20 h later. I n some experiments the Cam was a d d e d after an initial incubation in E t h B r . I n this case, 10 gg E t h B r per m l were a d d e d during balanced growth, a n d approx. 10 h later the cells were concentrated in t h e D e L a v a l gyrotester using two 500-ml washes with grass infusion containing 25 gg of Cam p e r ml. The cells were t h e n resuspended in t h e original volume of culture m e d i a b u t this time containing 25 pg o f Cam per ml. After harvesting in the gyrotester, there was a characteristic 5 to 7 h lag, after which the cells again exhibited exponential growth. The cells were t h e n harvested in the usual m a n n e r 10 h later. This sequence is shown in Fig. 2. (c) P u r i f i c a ~ o$ ,nitoehond~ Cells were harvested on a D e L a v a l gyrotester a t approx. 3000 tees/rain a t a flow-rate of a b o u t 800 ml[min, resuspended in a small volume o f D r y l ' s solution (Dryl, 1959), a n d t h e n t Abbreviations used: EthBr, ethidium bromide; Cam, ehlorampheniool.

MITOCHONDRIAL

DNA FROM

PARAMEOIUM

595

6000 4000

2000

"- IO00 800 6OO

400

!Br

200,,

, ;

I

I00

I

I

20

I

I

..................I

I

40 60 Incubation (h)

I

I

80

Fzo. 1. Growth curve of Paramecium u n d e r normal conditions a n d in t h e presence of v a r y i n g concentrations of E t h B r . Resuspended bacterial pellets were a d d e d a t 300 a n d 800 cells]ml during t h e b a l a n c e d growth period; a n d t h e cells were t h e n h a r v e s t e d either in b a l a n c e d growth a t 1200 cellsfml, in late log a t 3000 eells]ml or in s t a t i o n a r y phase after 90 h. E t h B r was a d d e d %o t h e cells mid-way in t h e b a l a n c e d growth period as indicated. Concentrations of 2 t o 10 ~g]ml were used for all subsequent experiments: ( • ) 0 o r 4 ~g/ml; ([[3} 10 ~g]ml; ( O ) 25 ~g/ml; ( A ) 50 ~g/ml.

4000

2000

Horvest -~ I000800 u

arrest

o/~.---'

600-

/

4O0

?

o~j Resuspend E in Cam lhBr

200

I00

1

I I0

I

I 20

I

I

I

I

30 40 Incubation (h)

I

I 50

I

I

60

I

70

Fro. 2. Growth curve of Paramec4um exposed to E t h B r a n d t h e n Cam. 10 ~g of E t h B r per m l were added a t a cell density of 400 cells]ml. A resuspended bacterial pellet was also a d d e d a t this time, a n d t h e cells were h a r v e s t e d 10 h later on a D e L a v a l gyrotester. The concentrated cells were t h e n resuspended in t h e original volume of culture containing 25 ~g Cam per ml. After a characteristic lag of a b o u t 5 h, t h e cells began to grow exponentially a n d were h a r v e s t e d 10 h later. The generation time in Cam was approximately 9 h compared to 7 h for n o r m a l cells. A small a m o u n t of t h e culture was r e t a i n e d so t h a t t h e growth curve could be continued.

596

J.M.

G O D D A R D AND D. J. CUMMINGS

subjected to a final packing b y centrifugation a t 1500 revs/min in pear-shaped centrifuge bottles. I n general, a balanced growth culture yielded a p p r o x i m a t e l y 0.25 ml p a c k e d cells per 1 of culture. F o r p r e p a r a t i o n of the mitochondria, the p a c k e d cells were resuspended in 2 vol. of ice-cold m a n n i t o l buffer (0-44 M-mannitol, 0.001 M-potassium p h o s p h a t e buffer (pH 6-8) a n d 0.25~o bovine sertun albumin (J. Doussier, personal communication)), a n d then r u p t u r e d b y one passage through a stainless steel milk.homogenizer. The homogenate was diluted to approx. 30 ml with m a n n i t o l buffer a n d the mitochondria purified b y differential centrffugation (Suyama & Preer, 1965) as follows. The s u p e r n a t a n t o b t a i n e d from centrffugation a t 600 g for 6 rain a t 0°G, was recentrifuged a t 5000 g for 6 mln a n d the resulting mitochondrial pellet resuspended gently in a small volume (0"5 ml[ml p a c k e d cells) of m~nnltel buffer containing 1 0 n g o f DNAase p e r ml plus 5 mM-MgC12. A f t e r incubation for 20 min in ice, the DNAase was diluted o u t with 30 m l mannitol buffer plus 10 mM-EDTA followed b y centrifugation a t 5000 g for 6 rain. The final mitochondrial pellet was examined in the electron microscope b y negative staining with 1~/o phosphotungstic acid (pH 7.2). Preparations from balanced growth cells contained 75 to 80~/o mitechondria, 10~/o bacteria a n d 5 to 10~o trichocysts, m e m b r a n e fragments a n d other debris; in preparations from s t a t i o n a r y phase, the p u r i t y of the mitochondria was greater t h a n 95%. (d) Extra,ion and p u r i ~ oJ mitochondrial DiVA The final mitochondrial pellet was gently resuspended a t 0°C in 3 ml of a solution containing 0"5 ~ - E D T A , 0.01 ~-Tris (pH 8.5) plus 2 m g of self-digested Pronase/ml and 1% Sarkosyl or digitonin (Kavenoff & Zimm, 1973). E x t r a c t i o n with digitonin yielded less bacterial I ) N A ; otherwise t h e two detergents a p p e a r e d to give identical results. The suspension was incubated a t 0°C for 1 to 2 h, w a r m e d to 21°C for 30 rain a n d t h e n h e a t e d a t 65°0 for 15 m~n (Kavenoff eta/., 1973). The extraction m i x t u r e was dialyzed for a short t i m e a t 21°C a n d then overnight a t 4°G against two 500-ml vol. D!~A buffer (0.25 ~-NaC1, 0-05 ~-Tris, 0.01 M-Na~ E D T A . 2 H 2 0 (pH 8-5)). The dialysate was diluted to 9.0 ml, a n d 11.0 g CsC1 a d d e d to give a final density of 1.68 g/em 3. The D N A was centrifuged to equilibrium in a Beckman 50Ti fixed-angle rotor for 67 h at 36,000 revs/m~n, 65°F. Sixdrop fractions were collected from the b o t t o m of t h e t u b e through a hole m a d e b y a n I8-gauge needle. The fractions were diluted with 0.5 m l I ) N A buffer a n d t h e absorbanee r e a d a t 260 nm.

(e) Sedimentation velocity F o r application to sucrose gradients, the D N A sample was diluted fourfold with halfstrength D N A buffer a n d t h e n layered on t o p of an 80/o to 25% sucrose gradient in 10 m ~ Tris, 1 mM-EDTA, 1 ~-NaC1 (pH 8.1). 5 to 10 ~g D N A were applied a n d t h e gradient was centrifuged in t h e B e c k m a n SW27 rotor a t 21,400 revs/min, 15 h a t 4°C (Upholt & Borst, 1974). Fractions of a p p r o x i m a t e l y 0.8 ml were collected from the b o t t o m of the t u b e through a hole m a d e b y an 18-gauge needle. The absorbance at 260 n m was determined using the 80/o sucrose solution as a blank.

(f) Electron microscopy of the D N A D N A was spread from either t h e CsC1 or sucrose gradients using the formamlde procedure of Davis et al. (1971) a n d was examined in an RCA4 electron microscope. The h y p o p h a s e contained 0.004 M-Tris, 0-0004 M-EDTA (pH 8"5) a n d 17~o formamide which was a d d e d j u s t before spreading. The hyperphase contained 0.25 to 0.5 ~g DNA/ml, 0.02 M-Trls, 0-002 M-EDTA (pH 8"5), 50 ~g cytoehrome c/ml a n d 45% formamide in a spreading volume of 50 ~1. The D N A was picked up on copper grids coated w i t h 1 ~o collodion, stained in u r a n y l acetate and r o t a r y shadow cast with p l a t i n u m - p a l l a d i u m . Negatives were enlarged on a Scherr-Tnm~co optical comparater, traced, and measured with a Keuffel a n d Esser m a p measurer. The double-stranded replicative form of phage ~X174 was included in each spread as a reference size marker. The size of this D N A was t a k e n to be 3.44 × 10 e

M I T O C H O N D R I A L DNA FROM P A I ? A M E C I U M

597

daltons which is an average of values obtained in other laboratories (Davis et al., 1971; Sharp e$ al., 1972). Assuming a linear density of 1.96 X 106 daltons/micron (Thomas, 1966), the length of the mtDNA was then expressed in terms of microns. (g) Measurement of length diat~b~ior~ To ensure random sampling of the DNA molecules in the population, a series of photographs (about 6) was taken across one entire grid opening. The negatives were developed and any DNA molecules that were only partially contained on the negative were relocated on the grid and photographed in their entirety. Thus any DNA molecules that originated on the initial series of photographs were photographed, traced and measured. The process was repeated on other grid openings until the desired number of molecules was obtained. Molecules measuring less than 5 ~m were not included in the distributions. The proportion of these molecules was low, and many could have been originally present in the doublestranded ~X174 DNA preparation added during the spreading procedure. (h) MaZer/a/8 The following chemicals were obtained from Sigma Chemical Co. : bovine serum albumin, DNAase I from beef pancreas, chloramphenicol, ethidium bromide, digitonin, grade-1 sucrose and mannitol. Pronase (B grade) was obtained from Calbiochem; Sarkosyl NL30 was purchased from CIBA-GEIGY; CsC1 (sequanal grade) was obtained from Pierce Chemical Co. ; disodium EDTA was obtained from Baker Chemical Co. ; and formamide was purchased from Matheson, Coleman and Bell. Scotch grass was purchased from Grass Products Ltd, Castleton, Eassie, Scotland. The replicative form of bacteriophage ¢X174 DNA was obtained from the laboratory of R. L. Sinsheimer, and the DI~A from Mu- 1 bacteriophage was a gift from the laboratory of A. L. Taylor.

3. R e s u l t s (a) Purification of mitochondrial D N A The results of the purification of m t D N A b y CsC1 equilibrium centrifugation are shown in Figure 3. I f the purified mitochondria were not treated with DNAase, the major species of D N A was nuclear, and the m t D N A fraction appeared only as a shoulder (Fig. 3(a)). The nuclear DNA, however, could be removed b y t r e a t m e n t of the mitochondria with DNAase before extraction, so t h a t only the m t D N A and a small amount of bacterial D N A contamination remained in the gradient (Fig. 3(b)). Identification of this remaining D N A as mitochondrial was established in two ways. First, m a r k e r D N A was centrifuged to equilibrium in the same gradient as:the D N A preparation from the mitochondria. [SH]DNA extracted from purified maeronuclei banded four to five fractions on the light side of the main D N A peak, while Klebsiella DNA, either labelled or unlabelled, banded five fractions on the h e a v y side of the D N A peak. This is shown in Figure 3(b). I n addition, the peak fractions of the assumed m t D N A were pooled and analyzed in the model E analytical centrifuge using Klebsiella D N A (p = 1.718) and macronuclear D N A (p------1.686) as markers. The patterns obtained indicated t h a t the D N A was indeed mitochondrial with a density of 1-699 g/cm s (Flavell & Jones, 1971) and was essentially free of contamination from either the bacterial or nuclear DNA. The D N A preparations shown in Figure 3 were obtained from cells in late log growth. D N A from balanced growth cells included a higher proportion of bacterial DNA, b u t the basic p a t t e r n was the same. The m t D N A was resistant to ribonuclease and sensitive to deoxyribonuclease. After heat denaturation in the presence of 0 . 3 ~ formaldehyde, it appeared as a single p e a k in a CsC1 density equilibrium gradient and showed an increase in density of 0.019 g/era 8.

598

J.M.

GODDARD

AND D. J. CUMMINGS

i7,=r

o

E

500

0.3C

400 '~

c

3oo

0.20

200

100 O0

t

I

t~

I

t

20

I

1

I

I 0

I ,,, ~ 10

I 20

I

I 30

0

Fraction no.

(a)

(b)

Fro. 3. Purification of m t D N A b y CsC1 equilibrium centrifugation. (a) D N A from a p r e p a r a t i o n of mitochondria which h a d n o t been t r e a t e d w i t h DNAase. A small a m o u n t of bacterial (Baot.) D N A is also present, as indicated. (b) D N A from m i t o e h o n d r i a which h a d b e e n t r e a t e d w i t h DNAase. T h e m t D N A p e a k is indicated. Nuclear a n d bacterial m a r k e r D N A b a n d e d several fractious to t h e light a n d h e a v y side of t h e peak, respeetively, as indicated b y t h e b r o k e n lines. The D N A was extracted from cells in late log growth in b e t h cases. The density increase is from r i g h t to left. Approximately 3 times t h e n u m b e r of eells were used for t h e p r e p a r a t i o n in (b) compared w i t h t h a t in (a). - - O - - O - - , Absorbanee; - - O - - O - - , radioactivity.

(b) dhare~teriration of mitod~dria2 D2qA from normal cdl,s Purified mtDNA isolated from cells in both balanced and stationary growth was examined in t~le electron microscope in order to determine the size and conformation of the genome as well as the types of molecules present in the population. A random population of appro~mately 100 molecules was therefore photographed and measured. The size distributions obtained are shown in Figure 4 and Table 1. Approximately 96% of the molecules were linear, and although the distribution was generally more nn!form for the balanced growth cells, the peak occurred at about 14 V2min both cases. A genome length of 13-8 van was then obtained for both phases of growth by averaging the lengths of the DNA in the peak regions between 13/~m and 15 i%m. The major difference in the distribution of linear molecules in these two preparations was found in the proportion of molecules measuring greater than 15 ~m. In balanced growth, the longest molecules observed were of dimer length, 25 to 28/~m, and these comprised 8% of the total linear population. This percentage dropped dramatically as the cells progressed from balanced to stationary growth. The dimer thus appears to be a predominant replication intermediate in periods of rapid growth. The molecules between 15 vm and 25/,m could have arisen from breakage of either the dimers or the lariats at the fork. The major non-linear species of DNA was a circle with a taft, or lariat, which comprised 3 to 4% of the population in either stage of growth. In some of these molecules, a short single-stranded region was seen in the circle at the fork, but in

PLATE I. E l e c t r o n m i c r o g r a p h s i l l u s t r a t i n g ty~pical l a r i a t s f r o m m t D N A p r e p a r a t i o n s . (a) A l a r i a t w h i c h c o n s i s t s o f a v e r y s m a l l circle a n d a l o n g t a i l ; (b) a l a r i a t in w h i c h t h e circle is s l i g h t l y larger b u t t h e tail is s h o r t e r ; a n d (c) a l a r i a t w h i c h c o n t a i n s a large circle b u t s h o r t tail. All t h r e e m o l e c u l e s c o n f o r m to t h e r e l a t i o n t h a t 0.5c + t = 14 ~ m . T h e f o r k s a r e i n d i c a t e d b y t h e arrows. T h e m o l e c u l e s in (a) a n d (b) a p p e a r to be c o m p l e t e l y d o u b l e - s t r a n d e d while t h a t in (c) c o n t a i n s a s h o r t s i n g l e - s t r a n d e d region a t t h e fork. A p h a g e ¢ X 1 7 4 D N A circle c a n be s e e n in (b). T h e bar represents 1 ~m. [faci~g p. 599

MITOCHONDRIAL

DNA

FROM

599

PAI~AMECIUM

TABLE 1

Distribution of mitovhondriaZ D N A molevules isolated under different conditio~ of cell growth Condition of the cells Normal balanoed growth No. molecules measured

Normal stationary growth

Ethidium bromidetreated

92

113

159

96 65 8

96 62 m

63 31 3

3

3

28