The fate of ultraviolet-induced pyrimidine dimers in the mitochondrial DNA of Saccharomyces cerevisiae following various post-irradiation cell treatments

241

Biochimica et Biophysica A cta, 366 (1974) 241--250 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 98107

THE FATE OF ULTRAVIOLET-INDUCED PYRIMIDINE DIMERS IN THE MITOCHONDRIAL DNA OF SA CCHA R 0114YCES CERE VI$IAE FOLLOWING VARIOUS POST-IRRADIATION CELL TREATMENTS

R. WATERS and E. MOUSTACCHI

Fondation Curie, Institut du Radium, Section de Biologie, BStiment 110, 91405 Orsay (France) (Received April 17th, 1974)

Summary The photoreactivability of ultraviolet-induced pyrimidine dimers (p~'py) in the mitochondrial genome of stationary phase Saccharomyces cerevisiae has been investigated in conjunction with the fate of these photoproducts following post-irradiation dark incubation in saline and nutrient media. In all instances "petite" induction and survival were measured. Such py~y are directly photoreactivable, and although they do not appear to be removed from mitochondrial DNA on dark liquid holding, an extensive degradation of mitochondrial DNA was observed.

Introduction

The induction of cytoplasmic respiratory deficient "petite" mutants (p-) in Saccharomyces cerevisiae by an intercalating dye such as ethidium bromide and by radiations has been extensively studied [1--4], and the ability to repair the lesions leading to such p- production has been questioned. The removal of ultraviolet-induced pyrimidine dimers (py~py) from bacterial DNA by repair systems has received much attention and at least three major pathways, those of photoreactivation [5] excision--resynthesis [6,7] and recombination [8] have been discovered. A study of the response of the ultraviolet-induced p- production following various post-ultraviolet irradiation treatments under conditions which would allow the operation of these mechanisms has been undertaken by various workers. As for lethality [9], the ultraviolet induction of p- cells is partially reversible when visible light is applied

Abbrevia~ons: pypy, pyrimidine dimers; TT, t h y m i n e - - t h y m i n e dimers; UT, ttracil--thymine dimers, UU, uracil---uracil dimers; p-, respiratory deficient "petite" mutants.

242 immediately after ultraviolet treatment [10]. However, as photoprotection [11,12] had not y e t been well defined, these experiments could not differentiate between this phenomenon and that of direct photoreactivation. The influence of a fractionated ultraviolet dose treatment [13] and of various post~ ultraviolet irradiation treatments such as dark liquid holding in saline and photoreactivation on the observed p- frequency has also been investigated in both wild type and radiation sensitive mutants of this organism [4,14--16]. Similar studies have been carried out in logarithmic and stationary phase cells [17] and in synchronous cultures [18]. These experiments indicate that modifications of the ultraviolet-induced frequencies of p- occur and that these are dependent on the genetic ability to repair radiation damage and the physiological state of the cells. Hence it was decided to determine whether photoprotection occurs in S. cerevisiae and whether the application of visible light after ultraviolet irraA diation results in the direct splitting of ultraviolet-induced p y p y in mitochondrial DNA. We have also examined the fate of ultraviolet-induced p~'py in the mitochondrial DNA of S. cerevisiae following post-irradiation dark liquid holding and attempted to relate these findings to the phenomena observed for p- induction. Materials and Methods Strain and media. The strain used was N123 which gives a R A D response to ultraviolet irradiation [4]. All precultures were grown to a stationary phase (5 • 10 s cells/ml, less than 5% budding cells) in liquid complete medium (1% yeast extract, 2% peptone, 2% glucose). Survivals following irradiation were estimated by plating on solid medium of the same composition plus agar, colonies being counted after 5 days incubation in the dark at 30 ° C. "Petite" colonies were differentiated as previously described [14], both sectored and complete " p e t i t e " colonies were included as p-. On taking the latter only, the overall effect remains unchanged. Labelling and incubation. As previously described [19]. Ultraviolet irradiation and p o s t treatments. As previously described [19] unless otherwise stated. D N A extraction. The extraction and subsequent CsC1 density gradient centrifugations were carried out as previously described [19]. Partial separation of the nuclear and mitochondrial DNAs was achieved by the first centrifugation. A further purification of the mitochondrial DNA peak was afforded by re-running the pooled fractions from a mitochondrial peak in a second CsC1 gradient similar to the first. Hence any contaminating nuclear DNA that had banded near to the mitochondrial peak in the first gradient was removed, and mitochondrial DNA of a considerable purity was obtainable. This purity is verifiable by calculating the C/T ratio of the sample following the subsequent hydrolysis and chromatography. This is 0.25 for mitochondrial DNA and 0.66 for nuclear DNA [20,21]. Chromatography and hydrolysis and liquid scintillation counting were carried out as previously described [ 19].

243 Ultraviolet dose

lOd I

1000

2000 |

3000 I

4000 I

)

10~

Ultraviolet dose

400 I

800 I

1200 I

1

I

!

10

"~ .C

=~.ooI oII

.000

I

I

I

I

I

8C

3ol

"tO

2oi

5C o

.;

3<

=, ,,= 101

1C

Fig. 1. The i n f l u e n c e o f visible light t r e a t m e n t o n ultraviolet survival and p - i n d u c t i o n w h e n applied prior to, o r after ultraviolet irradiation. Ultraviolet irradiation a l o n e ( o ) , visible light t r e a t m e n t prior to u l t r a v i o l e t irradiation (A) and ultraviolet irradiation f o l l o w e d b y v i a b l e light t r e a t m e n t ( o ) . a, for high d o s e s as used for the b i o c h e m i c a l m e a s u r e m e n t of p y p y ; b, for l o w d o s e s as used in m o s t p h y s i o l o g i c a l experiments.

Results

(A) Visible light treatment The effect of visible light treatment prior to ultraviolet irradiation (photoprotection) on cell survival and p- induction is shown in Fig. 1. It can be seen that this treatment does not increase the survival or reduce the percentage of pcells observed on ultraviolet irradiation alone. In fact both lethality and pinduction were slightly greater following this pretreatment. We confirmed that the application of visible light treatment after ultraviolet irradiation considerably increased cell survival and decreased p- induction in the conditions utilized for biochemical estimations of py~py content (Fig. la). Hence such recoveries may be attributed to the direct photoreactivation of ultraviolet-induced py~py and not to the indirect effects of photoprotection. This conclusion also applies to a similar study at low doses (Fig. lb). Following these results it was decided to determine whether the photoreactivation of ultraviolet-induced p~py was a p h e n o m e n o n c o m m o n to both the nuclear and mitochondrial D N A of S. cerevisiae. Fig. 2 shows the effect of photoreactivating light on the percentage of ultraviolet-induced p:~py in nuclear D N A following a dose of 4 0 0 0 ergs/mm 2 . Fig. 3 shows the effect of photoreactivation on the percentage of similar photoproducts induced in mitochondrial D N A by the same ultraviolet dose.

244

SO

A

2C

C

-u u

I

0.8

0.?

u ^

/

10

0.6

I

I

0.5

0.4

0.3

0.2

o.1

0,I

0

Fig. 2. T h e e f f e c t of visible light t r e a t m e n t a f t e r u l t r a v i o l e t i r r a d i a t i o n o n t h e p e r c e n t of r a d i o a c t i v i t y as a f u n c t i o n of R F f o u n d o n c h r o m a t o g r a m s of h y d r o l y s e d n u c l e a r D N A e x t r a c t e d f r o m cells i r r a d i a t e d w i t h 4 0 0 0 e r g s / m m 2. N o n - i r r a d i a t e d cells (o); n o n - i r r a d i a t e d cells g i v e n visible light t r e a t m e n t (G); u l t r a v i o l e t i r r a d i a t e d cells ( , ) a n d u l t r a v i o l e t - i r r a d i a t e d cells f o l l o w e d b y visible light t r e a t m e n t (A).

The C/T ratios in both instances are in accord with the expected 0.66 and 0.25 for the respective nuclear and mitochondrial DNAs of S. cerevisiae. Correspondingly more TT and less CT were induced in mitochondrial DNA as compared to those in nuclear DNA for a given dose. The exact changes in the

3(

- - 0.3

0.2 o I

C 10

0.1

0,8

0.7

0.6

0.5

R!

0.4

O:3

0.2

0.I

Fig. 3. T h e e f f e c t o f visible light t r e a t m e n t a f t e r u l t r a v i o l e t i r r a d i a t i o n o n t h e p e r c e n t of r a d i o a c t i v i t y as a f u n c t i o n o f R F f o u n d o n c h r o m a t o g r a m s o f h y d r o l y s e d m i t o c h o n d r i a l D N A e x t r a c t e d f r o m cells irrad i a t e d w i t h 4 0 0 0 e r g s / m m 2 . N o n - i r r a d i a t e d cells (o), n o n - i r r a d i a t e d cells given visible light t r e a t m e n t (o), u l t r a v i o l e t - i r r a d i a t e d cells ( i ) a n d u l t r a v i o l e t - i r r a d i a t e d cells f o l l o w e d b y visible light t r e a t m e n t (A).

4000 ergs/mm2 of ultraviolet irradiation 4 0 0 0 e r g s / m m 2 of u l t r a v i o l e t i r r a d i a t i o n + visible light t r e a t m e n t 5 0 0 0 e r g s / m m 2 of u l t r a v i o l e t irradiation 5000 e r g s / m m 2 of ultraviolet i r r a d i a t i o n + visible light t r e a t m e n t

Treatment

.

~

.

.

.

0 . 2 6 (0) 0.11 (58) 0 . 3 3 (0) 0.19 (42)

0.70 (0)

0.28 (60)

0 . 9 2 (0)

0.42 (46)

0.69 (37)

1 . 1 0 (0)

0.39 (54)

0 . 8 4 (0)

T~

T~T

C~T

Mitochondrial DNA

Nuclear DNA

0.13 (30)

0 . 1 8 (0)

0.07 (50)

0 . 1 4 (0)

C~

% o f c p m e x p r e s s e d as u l t r a ~ n o l e t - m d u c e d p y p y / t o t a l r a d x o a c h v l t y

•

2.1

1

2

1

DMF for survival

1.5

1

2

1

% p- a f t e r u l t r a v i o l e t + visible light

% p- a f t e r u l t r a v i o l e t

^ T H E E F F E C T OF VISIBLE L I G H T T R E A T M E N T ON T H E P E R C E N T OF U L T R A V I O L E T - I N D U C E D PYPY IN T H E N U C L E A R A N D M I T O C H O N D R I A L DNAs O F S. C E R E V I S I A E A N D ITS RELATIONSHIP TO SURVIVAL AND p-INDUCTION D M F is t h e dose m o d i f y i n g f a c t o r i.e. t h e r a t i o o f t h e d o s e s r e q u i r e d f o r a g i v e n s u r v i v a l s e e n f o l l o w i n g u l t r a v i o l e t plus visible light t r e a t m e n t a n d u l t r a v i o l e t a l o n e ; p e r c e n t o f p- is e x p r e s s e d as p e r c e n t o f p- s e e n p e r s u r v i v o r s . T h e d a t a f o r p- i n d u c t i o n a r e e x p r e s s e d as s u c h since t h e f r e q u e n c y o f p- i n d u c t i o n r e a c h e s a p l a t e a u for high d o s e s (Figs l a a n d l b ) . N u m b e r s in p a r e n t h e s e s r e p r e s e n t p e r c e n t a g e o f r e m o v a l o f p ~ p y .

TABLE I

b~ ~n

246

percentages of TT and CT dimers for both DNAs brought about by this photoreactivation as well as survivals and percentages of p-'s are given in Table I, for two doses. It can be seen that photoreactivation has diminshed to a similar extent the percent of pyApy in both nuclear and mitochondrial DNAs. (B) The effects on mitochondrial DNA of the post ultraviolet irradiation incubation of stationary phase cells in saline and nutrient media A primary factor encountered in attempting to study the fate of ultraA violet-induced pypy in mitochondrial DNA was the apparent loss following such post treatments of a considerable portion of the mitochondrial DNA capable of banding at a density of 1.685 g/ml in a CsC1 density gradient (Fig. 4). This loss is made more apparent if one re-runs the pooled "mitochondrial D N A " fractions in a second similar gradient, thus affording a separation of any residual nuclear DNA that had " c o n t a m i n a t e d " the original mitochondrial DNA peak (Fig. 5). An analysis of the percentage of p:~py in the remaining mitochondrial DNA banding at a density of 1.685 g/ml following 72 h liquid holding in saline, 24 h in nutrient medium, or 72 h in saline followed by 12 h in nutrient medium, revealed no change in the percentage of py~py as compared to that originally induced (Fig. 6). This latter treatment gave 20% removal of p~py from the nuclear DNA [19]. However the number of recuperable cpm in the

--

A

0.8

0£

G4

(12

~o

i [ l l l l i l l l i i l l l l l l l [

I l l l l l l l l [ l l J l l l l l l l D

C

OJ~-

OA

0.2 m

Oil

i l l l l a l l a l [ l l l l l l l t l 401 FRACTION

NUMBER

Fig. 4. The a b s o r b a n c e o f f r a c t i o n s f r o m cell e x t r a c t s r u n in CsCI gradients w h e r e " n " d e n o t e s t h e n u c l e a r D N A p e a k ( d e n s i t y 1 . 7 0 g/rul) a n d " m " t h e m i t o c h o n d r i a l D N A p e a k ( d e n s i t y 1 . 6 8 5 g / m l ) . A, u n i r r a d i a t e d cells; B, i r r a d i a t e d w i t h 5 0 0 0 e r g s / m m 2 o f u l t r a v i o l e t , i m m e d i a t e e x t r a c t ; C, 5 0 0 0 e r g s / m m 2 o f u l t r a v i o l e t f o l l o w e d b y 72 h in saline p r i o r t o e x t r a c t i o n ; D, 5 0 0 0 e r g s / m m 2 o f u l t r a v i o l e t f o l l o w e d b y 24 h in n u t r i e n t m e d i u m .

247 Q2 •

!n

m

B

n

i lt '

0.1

E=

~a2 I I I I I I I I l | l l l l l l l l l l mE

i l l l l l l l l l l l l l l l l | l l C

O

n

0.1

ix,,..= .... J ~11 I I i I i i I I I i i I I I 2O

I I i i

I I I I I l i l l l l l l l l l l l l l 401 20

4O

FRACTION HuMaER

Fig. 5. T h e a b s o r b a n c e o f f r a c t i o n s c o l l e c t e d f r o m CsCI g r a d i e n t s c o n t a i n i n g t h e r e s p e c t i v e m i t o c h o n d r i a i p e a k s , " n " d e n o t e s n u c l e a r D N A a n d " m " m i t o e h o n d r i a l D N A . A, B, C a n d D as Fig. 4.

G3

Ct2

O.1

0

0.5

I 0.4

| 0.3

I 0.2

I 0.1

I 0

tl

Fig. 6. T h e p e r c e n t o f r a d i o a c t i v i t y as a f u n c t i o n o f R F f o u n d in t h e d i m e r s r e g i o n o f c h r o m a t o g r a m s o f m i t o c h o n d r i a l D N A . N o n - i r r a d i a t e d cells (o); u l t r a v l o l e t - i r r a d i a t e d w i t h 5 0 0 0 e x g s / m m 2 , i m m e d i a t e ext r a c t ( o ) ; 5 0 0 0 e r g s / m m 2 o f u l t r a v i o l e t f o l l o w e d b y 72 h in saline p r i o r t o e x t r a c t i o n ( a ) ; 5 0 0 0 e r g s / m m 2 of u l t r a v i o l e t f o l l o w e d b y 24 h in n u t r i e n t m e d i u m (o); 5 0 0 0 e r g s / m m 2 of u l t r a v i o l e t f o l l o w e d b y 7 2 h in saline a n d s u b s e q u e n t i n c u b a t i o n in n u t r i e n t m e d i u m f o r 12 h (~).

248 T A B L E II A THE REMOVAL OF ULTRAVIOLET-INDUCED PYPY FROM THE NUCLEAR AND MITOCHONDRIAL D N A s O F S. C E R E V I S I A E O N P O S T U L T R A V I O L E T I R R A D I A T I O N D A R K L I Q U I D H O L D I N G IN NUTRIENT MEDIUM OR SALINE Treatment

% Removal from nuclear DNA

% Removal from mitochondrial DNA

% p-/survivors

5000 ergs/mm 2 of ultraviolet U l t r a v i o l e t + 24 h n u t r i e n t m e d i u m U l t r a v i o l e t + 72 h saline U l t r a v i o l e t + 72 h saline + 12 h nutrient medium

0 8 10

0 3 0

0 0 0

0 0 0

60 56 66

15

17

0

0

60

mitochondrial DNA was diminished 5--10-fold f ~ l o w i n g such post-treatments. Table II gives the total percentages of TT and CT removed from nuclear and mitochondrial DNAs following these treatments. Discussion

By measuring ultraviolet-induced p~?py in total DNA extracts of S. cerevisiae, it has been shown that these lesions are photoreactivable in vivo [22,23]. We demonstrate here that this event is c o m m o n to both the nuclear and mitochondrial DNAs in this yeast, the proportion of py~py photoreactivated by a given dose of visible light being approximately the same in each type of DNA (Table I and Figs. 2 and 3). This removal is accompanied by an increase in survival and a decrease in the percentage of ultraviolet-induced p- as compared A to non-illuminated cells. Hence, as for lethality, p y p y are at least partially responsible for the p- induction by ultraviolet light. Secondly a controlled excision mechanism, allied to that already reported for yeast nuclear DNA [ 1 9 ] , does not appear to operate on mitochondrial ultraviolet-induced p:~py (Table II). This result is similar to those reported for the fate of ultraviolet-induced p y p y in chloroplast DNA [24] and mammalian mitochondrial DNA from cells grown in vitro [25]. It is also in accord with the observation that an excision defective nuclear mutant such as radl_ 3 [4] did not demonstrate a difference in the dose response curve for ultraviolet induction of p- as compared to the original R A D wild type [4]. Thirdly a considerable loss of mitochondrial DNA was seen to occur following the dark holding of ultraviolet-irradiated stationary phase cells in both saline and nutrient media. The enhancement in p- frequency during dark liquid holding in saline of ultraviolet-treated stationary phase cells and the concomittant loss of photoreactivability of the cytoplasmic damages [ 17] are then likely to be related to such a degradative process. A progressive loss of mitochondrial DNA is also encountered when cells are treated by ethidium bromide [26,27], a potent inducer of the cytoplasmic p- mutation [1 ]. Ultraviolet light perhaps has the same effects, especially as py~py formation and intercalation of ethidium bromide have been shown to alter similarly the structure of the bacteriophage ¢ X174 replicative DNA [ 2 8 ] . The precise kinetics of ultraviolet-induced

249 degradation of mitochondrial DNA are being determined by measuring the ratio of mitochondrial over total DNA in analytical density gradients and by the loss of radioactivity from labelled mitochondrial DNA. The p- mutation is by no means comparable to a single nuclear mutation as the spontaneous and ultraviolet-induced p- frequencies are much higher than the rate of induction of recessive nuclear mutations for a given dose. Moreover, the cytoplasmic respiratory deficiency has been shown to be accompanied by a total loss or altered base composition of mitochondrial DNA [26,27,29]. Although the actual experiments give no evidence for a repair mechanism other than photoreactivation, acting on mitochondrial DNA, various biological data indicate that the fate of the respiratory character is not irrevocably determined immediately after mutagenic treatments [13,15--17,30,31]. Some mechanism responsible for curing mitochondrial DNA damages is suggested by such observations. In principle the recovery of the p÷ p h e n o t y p e may occur by at least two main mechanisms, one being the maintenance and subsequent selective multiplication of undamaged mitochondrial DNA molecules, the other being an enzymatic repair of damaged mitochondrial DNA molecules. The first suggestion would not appear to be applicable to the ultraviolet doses used in these experiments as an average of 60--200 dimers per mitochondrial DNA molecule would be induced by a dose of 1000--3000 ergs/mm 2 assuming 50 mitochondrial DNA molecules of a size of 21 • 106 daltons occur per cell [32]. Hence the second possibility of a repair mechanism remains. The mitochondrial genome has been shown to exhibit a recombination of genetic markers [ 3 3 , 3 4 ] , and more recently both p* and p- mitochondrial DNA appear to be capable of recombination [35,36]. Hence in an eucaryotic organism in which the mitochondrion is indispensable a recombinational process may well be involved in determining whether an intermediary state of the mitochondrial genome following ultraviolet irradiation is expressed as p* or p-. A prerequisite of this suggestion is that mitochondrial DNA is the primary target for p- induction. Although a nuclear master c o p y of mitochondrial DNA cannot be completely refuted, many experiments have been unable to demonstrate the existence of such a copy [ 3 7 ] . The fact that ethidium bromide at low concentrations is a p o t e n t p- inducer and is unable to induce nuclear DNA alterations also supports the idea that the mitochondrion is the main target. The absence of an excision process acting on the mitochondrial DNA of ultraviolet-treated cells indicates that any possible repair mechanisms suggested by the previously cited biological data [13,16,17,19,27,30,31] should involve both degradative and recombinational events. Acknowledgements The authors wish to thank Madame R. Guilbaud for skilled technical assistance. One of us (R.W.) is indebted to the European Molecular Biology Organization for supplying a long term fellowship, hence allowing this work to be undertaken. This investigation was supported in part by the Commissariat h l'Energie Atomique (Saclay, France) and the Ddldgation Gdndrale ~ la Recherche Scientifique et Technique).

250

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

Slonimski, P.P., Perrodin, G. and Croft, J.H. (1968) Biochem. Biophys. Res. Commun. 30, 232--239 Raut, C. (1954) J. Cell. Comp. Physiol. 44, 4 6 3 - 4 7 5 Wilkie, D. (1963) J. Mol. Biol. 7 , 5 2 7 - - 5 3 3 Moustacchi, E. (1969) Mutat. Res. 7 , 1 7 1 - - 1 8 5 Setlow, J.K. (1966) Radiat. Res. Suppl. 6, 141--145 Setlow, R.B. and Carrier W.L. (1964) Proc. Natl. Aead. Sci. U.S. 5 1 , 2 2 6 - - 2 3 1 Boyce, R.P. and Howard-Flanders, P. (1964) Proc. Natl. Acad. Sci. U.S. 5 1 , 2 9 3 - - 3 0 0 Rupp, W.D. and Howard-Flanders, P. (1968) J. Mol. Biol. 31, 291--304 Sarachek, A. (1958) Cytologia 23, 143--149 Pittman, D. and Pedigo, P.R. (1959) Exp. Cell Res. 1 7 , 3 5 9 - - 3 6 7 Jagger, J. and Stafford, R.S. (1962) Photochem. Photobiol. 1, 245--257 Harm, W. and Hillebrandt, B. (1962) Photochem. Photobiol. 1, 271--272 Maroudas, N.G. and Wilkie, D. (1968) Biochim. Biophys. Acta 166, 681--688 Moustacchi, E. and Enteric, S. (1970) Molec. Gen. Genet. 109, 69--83 Moustacchi, E. (1971) Molec. Gen. Genet. 114, 50--58 Moustaechi, E. ( 1 9 7 3 ) J . Bacteriol. 115, 805--809 Heude, M. and Moustaechi, E. (1973) C.R. Acad. Sci. 277, 1 5 6 1 - - 1 5 6 4 Chanet, R., Williamson, D.H. and Moustacchi, E. (1973) Biochim. Biophys. Acta 324, 290--299 Waters, R. and Moustacchi, E. (1974) Biochim. Biophys. Acta 353, 407--419 Bernadi, G., Carnevali, F., Nicolaieff, A., Piperno, G. and Tecce, G. (1968) J. Mol. Biol. 37, 493--505 Mehrotra, B.G. and Mahler, H.R. (1968) Arch. Biochem. Biophys. 128, 685--703 Unrau, P., Wheatcroft, R. and Cox, B.S. (1971) Mol. Gen. Genet. 113, 359--362 Resnick, M.A. and Setlow, J.K. (1972) J. Bacteriol. 1 0 9 , 9 7 9 - - 9 8 6 Swinton, D.C. and Hanawalt, P.C. (1973) Photochem. Photobiol. 1 7 , 3 6 1 - - 3 7 5 Friedberg, E.C., Minton, K., Durphy, M. and Clayton, D.A. (1974) S umma ry of Squaw Valley Meeting Goldring, E.S., Grossman, L.I., Krupnick, D., Cryer, D.R. and Marmur, J. (1970) J. Mol. Biol. 52, 32 3--355 Perlman, P.S., Mahler, M.R. (1971) Nat. New Biol. 231, 12--16 Denhardt, D.T. and Kato, A.C. (1973) J. Mol. Biol. 7 7 , 4 7 9 - 4 9 4 Mounolou, J.C., Jakob, H. and Slonimski, P.P. (1966) Biochem. Biophys. Res. Commun. 24, 218--222 Mahler, H. (1972) J. Supramol. Struct. 1, 105--124 Schenberg-Frascino, A. (1972) Molec. Gen. Genet. 1 1 7 , 2 3 9 - - 2 5 3 Goldring, E.S., Grossman, L.I. and Marmur, J. (1971) J. Bacteriol. 107, 377--381 Thomas, D.Y. and Wilkie, D. (1969) Bioehem. Biophys. Res. Commun. 30, 368--372 Coen, D., Deutsch, J., Netter, P., Petrochilo, E. and Slonimski, P.P. (1970) Symp. Soc. Exp. Biol. 24, 449--496 Michaelis, G., Petrichflo, E. and Slonimski, P.P. (1973) Molec. Gen. Genet. 123, 51--65 Sena, E.P. (1972) Ph.D. Thesis, University of Wisconsin Borst, P. and Flavell, R.A. (1972) Mitochondria/Biomembranes, pp. 1--19, North Holland, A m s t e r d a m