Molec. gen. Genet. 130, 113--125 (1974) © by Springer-Verlag 1974
A Nuclear Mutation Affecting Structure and Function of Mitochondria in Paramecium Annie Sainsard and Maurice Claisse Centre de G6n6tique Mol6culaire du CNI~S, Gif-sur-Yvette, France Monique Balm6fr6zol Laboratoire de Biologic Cellulaire IV, Universit6 de Paris XI, Orsay, France I~eceived January 4, 1974
Summary. A slow growing mutant of Paramecium, el-l, deficient in type-a cytochrome is described: it differs from wild type by a recessive nuclear mutation and also by its mitochondria. Mitochondria of the mutant are "compatible" with gene cl-1, whereas mitochondria of the wild type are "incompatible" with this gene: they are spectacularly altered in the presence of a cl-1/cl-1 nucleus. However this incompatibility is temporary and mitochondria of wild type origin slowly become compatible with el-1. The molecular basis of the incompatibility and the mechanism of its disappearance are discussed. Introduction
The formation of mitochondria in eucaryotic cells involves the cooperation of two separate but interdependent protein synthesizing systems (Rabinowitz and Swift, 1970; Boardman, Linnane and Smillie, 1971). A striking example of this interdependence is provided by the fact that the synthesis of a single enzyme requires the two systems. This is the case for cytochrome oxidase (Weiss, Sebald and Bficher, 1971; Mason and Schatz 1973; Sebald, Weiss and Jackl, 1972) and for ATPase (Tzagoloff and Meagher, 1972). In this intricated network of interactions, it can be expected that the mutational alteration of a molecule will modify also the function of other interacting molecules of either mitochondrial or nuclear origin. The phenotypic effects of such mutations may be difficult to trace either to a single nuclear or to a single mitochondrial mutation (Avner and Griffiths, 1973; Davis, 1972). Such an example may be provided by the mutant el-1 of Paramecium aurelia described in this paper. I t is a slow growing mutant deficient in type-a eytochrome. I t differs from wild type by a nuclear mutation and also by its mitoehondria. Mitochondria from the mutant are "compatible" with gene el-l, while mitochondria from wild type are " i n c o m p a t i b l e " with this gene: in presence of a cl-1/cl-1 nucleus, they are spectacularly altered showing very few or no cristae. The data raise numerous questions concerning the nature of the difference (genetic or not) between mitoehondria from mutant and wild type strains, the function of the nuclear gene el-1 and the molecular basis of the incompatibility between gene el-1 and mitoehondria from wild type. Materials and Methods
Strains and Cultures. From Paramecium aurelia, stock d4-2, species (syngen) 4, were derived the nuclear mutants: cl-1, ts401 (Beisson and Rossignol, 1969) and the mitochondrial
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Fig. 1A and B. Schematic representation of a cross between two strains of Paramecia homozygous for two different nuclear alleles (A/A and a/a). The cytoplasmic properties of the two strains are symbolized by . and ©. A: Conjugation without cytoplasmic exchange. The two F i clones derived from each of the ex-conjugants are heterozygous (A/a) and differ only by their cytoplasm, • or ©. After autogamy, each cx-conjugant clone comprises 50% A/A and 50% a/a Fe horn©zygotes. B: Conjugation with cytoplasmic exchange. Both F i clones are heterozygous A/a and both possess a mixture of cytoplasms ( . and 0). Under certain conditions the mixed state of the cytoplasms can be perpetuated and recovered in F 2 clones
mutant C~, resistant to ehloramphenicol (Adouette and Beisson, 1972). The growth medium was a grass infusion ("Scotch Grass") inoculated the day before use with Aerobaeter act©genes (Sonneborn, 1970). Unless otherwise stated, all experiments were carried out at 27°C. At this temperature, the wild type strain multiplies at the rate of four to five fissions per day. Genetic Analysis. The principle of this analysis is illustrated in figure 1. In a cross between two different homozygous strains, conjugation that consists in a reciprocal exchange of nuclei between the two conjugants, yields two exconjugants F 1 of identical heterozygous genotype. The ex-conjugants retain their original cytoplasm (Fig. 1A), unless an easily detectable cytoplasmic bridge has been formed. In this ease, each exconjugant receives some cytoplasm from its partner and contains a mixture of two cytoplasms (Fig. 1 B). About 20 generations after conjugation, autogamy is induced by starvation of the F 1 clones. During autogamy, cells undergo a nuclear reorganization which renders them homozygous for all their genes. When autogamy occurs in a clone heterozygous for a couple of nuclear alleles, 50% of the cells become homozygous for one allele and 50% for the other. Consequently a 1 : 1 ratio of the two homozygous genotypes is observed among the F 2 crones (Fig. 1). Practically, as described by Sormeborn (1970), cells of complementary mating types (VII, VIII) were mixed, couples isolated and the two exconjugants of each pair separated. F 1 phenotypes (growth rate, thermosensitivity, mating t y p e . . . ) were assayed for the clones derived from the exconjugants. The cytoplasmic origin of each F 1 clone was determined by a mating type test, since the mating type is cytoplasmically inherited in species 4 (Sonneborn,
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1954). After autogamy, 30 cells from each exconjugant were isolated and the homozygous F 2 clones derived fl'om these cells were studied. Thermosensitivity Test. The thermosensitivity of different clones was tested b y comparing their growth rate a n d survival a t 27°C a n d 36°C. Wild type cells multiply at the rate of five fissions a day at 36°C; homozygotes ts~O1/ts401 die within 48 hours at 36°C, after a few fissions. Growth Rate Determination. The growth rate of a strain is estimated by counting the n m n b e r of cells derived from a single cell isolated on the previous day. A more precise determination can be made b y counting the n u m b e r of cells in samples t a k e n from a n exponentionally growing mass culture at various times (for each time, six 1 ml independent samp!es were counted). Electron Microscopy. The loose pellet of centrifuged cells was fixed in 0.25 % glutaraldehyde ( E a s t m a n K o d a k Co., Rochester N.Y.) in 0.05 M cacodylate buffer, pH 7-7.2, for 30 ran, rapidly washed in the buffer, a n d postfixed in 2% osmium tetroxide in the same buffer. After 1 hr in the second fixative, the cells were washed in 0.05 M phosphate buffer, pI-I 7-7.2, preembedded in a fibrin clot (Charret a n d Faur6-Fremiet, 1967), dehydrated in alcohol, propylene oxide, and embedded in Epon (Luft, 1961). Sections prepared with a Sorvall MT. 1 ultramierotome were stained with saturated uranyl acetate followed b y lead citrate, carbon coated and examined with a Siemens electron microscope, model 1A Elmiskop. Respiration. 02 consumption was measured b y polarography (Gilson oxygraph). The measurements were carried out at 27°C on exponentially grown cells, in 0.01 M HC1-Tris buffer, p H 7.1. Protein was determined b y the Biuret method with bovine serum albumin as the standard (Stickland, 1951). The protein concentration was about 1.5 mg/ml in the sample chamber. Spectrophotometric Study o/ Cytochromes. A Cary 15 spectrophotometre, fitted with a high power xenon light (Claisse, P6r~-Aubert, Clavillier and Slonimski, 1970) was used in this work. The spectra were obtained at the temperature of liquid nitrogen, directly on dense suspensions of early stationary phase cells. The protein contents of these suspensions were about 80 mg/ml. The recordings were made after reduction of the sample with sodium dithionite. Neutral filters of known absorbance were placed in the reference spectrophotometric chamber for light-scattering correction. I n the measure chamber, the sample was in a cuvette 1 m m thick (light p a t h for clear solutions). Estimation o/the Contents o/Cytochromes. The content of type-a cytochrome was estimated b y the difference of absorbance between the m a x i m u m of the u absorption peak and a baseline joining the points of the spectrum located at 595 and 630 n m or 595 and 622 nm. The 595-622 n m baseline gives a b e t t e r estimation in the case of the deficient m u t a n t . The sensitivity of the spectrophotometre was increased ten fold in certain measurements (absorbance 0 to 0.1 a t full scale). The content eytoehrome c was estimated on the one hand, b y the difference of absorbance between the m a x i m u m of the ~ peak and the point of the spectrum located at 535 n m and, on the other hand, by the calculation of the v~ e value (Claisse et al., 1970). This value was obtained from the differences of absorbances between the maxima of the and fl peaks with respect to the two baselines joining the points of the spectrum located respectively a t 500 a n d 555 n m and 500 a n d 630 nm.
Results
I. Phenotypic Characterization o/the Mutant cl-1 This mutant obtained by U.V. treatment (4000 ergs/mm2), was selected for i t s slow g r o w t h . (cl-1 s t a n d s f o r , , c r o i s s ~ n c e ] e n t e " . Growth Rate. Fig. 2 s h o w s t h e g r o w t h c u r v e s of w i l d t y p e a n d of t h e m u t a n t a t 2 7 ° C . T h e g e n e r a t i o n t i m e of t h e f o r m e r is 6 h o u r s (4 t o 5 f i s s i o n s a d a y ) , of t h e l a t t e r 10 h o u r s (2 t o 3 f i s s i o n s a d ~ y ) . M o r e o v e r , i t m u s t b e n o t i c e d t h a t t h e g r o w t h c u r v e of t h e m u t a n t a p p e a r s t o b e b i p h a s i c w h e r e a s t h a t of t h e w i l d t y p e is m o n o p h a s i c . T h e s i g n i f i c a n c e of t h i s p h e n o m e n o n h a s n o t b e e n a n a l y z e d as yet.
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Fig. 2. Growth curves of wild type (-.-.-.-) and eL1 mutant (-o-o-o-)strains. Abscissa: hours; ordinate: cell number/ml
Thermosensitivity. Mutant c1-1 is weakly thermosensitive. During the 24 hr following transfer from 27°C to 36°C, it undergoes 2 to 3 fissions, then divides very slowly (0 to 1 fission a day). After a few days at 36°C, about 50% of the cells die. Mitochondrial Morphology. Mitochondria of m u t a n t cl-1 (Fig. 3 B) are morphologically identical with those of the wild type strain (Fig. 3A); some of them however, contain discrete irregular membranous formations (arrow) which are never observed in wild type mitochondria. Spectra o/ Cytochromes. Fig. 4 shows low temperature spectra of wild type and m u t a n t whole cells. The spectrum of the former displays several absorption bands, in particular peaks at 548 nm and 608 nm. These wavelengths can be attributed to the ~ bands of type-e and type-a cytoehromes respectively, and are very similar to those observed by Kung (1970) on the same organism (550 nm and 609 nm respectively). The broad shoulder at 555 nm probably corresponds mainly to a type-b eytochrome since it is also observed on isolated mitochondria, a condition which should eliminate hemoglobin which is present in notable amounts in Paramecia and has a broad absorption m a x i m u m at this same wavelength (Smith, Georges and Freer, 1962). The peaks at 520 and 527 nm are in the position of the maxima of the fl peaks of type-c and type-b cytochromes. The broad shoulder at 570 nm corresponds to a cytoplasmic pigment since it is not observed in spectra of isolated mitochondria. We also have reproducibly recorded a small shoulder at 545 nm which probably corresponds to an % band of cytoehrome c, revealed ~t low temperature. The spectrum of the m u t a n t is very similar to t h a t o f wild type except for the peak corresponding to the type-a eytoehrome which is very reduced as compared to that of wild type. Absorbances of cytochromes e and a in both strains in two independent experiments are compared
Fig. 3A and B. Morphological aspects of mitochondria of wild type cells and cl-1 m u t a n t cells. A: Wild type: the mitochondrial cristae are numerous, curved and tubular. ×30000. B: M u t a n t el-1. The only abnormalities occasionally observed are some irregular membranous formations (arrow). x 30000. M mitochondria; T trichocyst; C Y cytoplasm full of glycogen granules
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Fig. 4A and B. Absorption spectra of whole ceils of wild type (1) and el-1 mutant (2) of Paramecium aurelia frozen in liquid nitrogen. A: Normal sensitivity, Absorbance 0 to 1. B: High sensitivity, Absorbance 0 to 0.1
Table 1. Contents of cytochromes a and c in wild type and cl-1 strains of Paramecium Strain
WT WT cl-1 cl-1
AAc baseline 548 535 nm
Oc
0.193 0.237 0.203 0.208
0.115 0.134 0.129 0.102
AAa baseline baseline 595-630 nm 595-622 nm 0.030 0.048 0.003 --
0.030 0.051 0.004 0.003
In this table, the absorbances AAc, 0c, AAa obtained for two independent cultures of wild type (WT) and for two independent cultures of the mutant el-1 are given. For calculation of absorbanees, see Materials and Methods.
in Table 1. W h i l e t h e c o n t e n t of c y t o c h r o m e e is t h e same in t h e m u t a n t a n d wild t y p e , t h e c o n t e n t of t y p e - a c y t o c h r o m e is 7 to l 0 times lower in t h e former. Respiration. P o l a r o g r a p h i c m e a s u r e m e n t s of o x y g e n u p t a k e show t h a t t h e r e is no correlation b e t w e e n t h e decrease of t y p e - a c y t o c h r o m e a n d r e s p i r a t o r y r a t e in t h e m u t a n t strain. A l t h o u g h it is quite reasonable to assimilate t h e t y p e - u c y t o c h r o m e to c y t o c h r o m e oxidase as K u n g (1970) does, t h e m u t a n t s t r a i n has t h e same endogenous r e s p i r a t o r y r a t e as t h e wild t y p e s t r a i n (the QO 2 is a b o u t 30-35 mm30~/hour/mg p r o t e i n for b o t h strains). Moreover, this s t u d y has r e v e a l e d t h a t a b o u t 20 % of endogenous r e s p i r a t i o n is cyanide-insensitive in wild t y p e , a n d t h a t t h e cyanide-insensitive fraction is m u c h higher in t h e m u t a n t (ca. 80%). A m o r e extensive s t u d y of t h e r e s p i r a t o r y chain of b o t h strains is under w a y (Sainsard et al., in p r e p a r a t i o n ) .
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I I . Genetic Analysis Table 2 shows the results of a cross between the m u t a n t cl-I a n d the strain homozygous for el-1 + a n d ts401.
Segregation o/ el-1. I n all 30 couples studied, the e x c o n j u g a n t derived from the m u t a n t p a r e n t was f o u n d to acquire a growth rate of 4: fissions a d a y after a few post c o n j u g a t i o n divisions. I t m u s t be p o i n t e d out however, t h a t the e x c o n j u g a n t derived from the m u t a n t p a r e n t always r e m a i n e d slightly slower t h a n the e x e o n j u g a n t derived from the wild t y p e p a r e n t (4 to 5 fissions a day). The analysis i n F 2 of three couples revealed a 1 : 1 segregation for ts401 (95 401 : 82 401+), as well as for cl-1 (95 el-l: 84 cl-l+). Moreover, the d a t a show t h a t the four genotypes el-1 401% c1-1+401, cl-1+401, e1-1+401+ segregate i n a 1 : 1 : 1 : 1 ratio. M u t a n t el-1 therefore differs from wild t y p e b y a single recessive nuclear gene, u n l i n k e d to ts401. Interaction between Gene el-1 and Wild T y p e Cytoplasm. This cross reveals a striking difference b e t w e e n the two types of homozygotes cl-1/cl-1 o b t a i n e d i n F 2 depending on their cytoplasmic origin (Table 2). Cells i n which gene el-1 is recovered after a u t o g a m y i n eL1 cytoplasm, have a growth rate a n d mitochondriM morphology identical with those of the original el-1 m u t a n t , whereas cells in which gene el-1 becomes homozygous i n wild t y p e cytoplasm, have a m u c h reduced growth rate (one fission a day) a n d their m i t o c h o n d r i a are m a r k e d l y altered. Figure 5A shows these alterations: no m i t o c h o n d r i o n displays a n o r m a l morphology; the most conspicuous modification is the r e d u c t i o n of the n u m b e r of cristae. There is a great heterogeneity a m o n g m i t o c h o n d r i a in size, shape, m a t r i x density, c o n t e n t s a n d disposition of eristae: some of t h e m have practically r e t a i n e d only their envelope.
Table 2. Genetic analysis of the cross VII el-1 401+/c1-1 401+ × c1-1+401/c1-1+401 Parents Genotype Phenotype Growth rate
d-1 401+ d-1 401+
Thermoresistance
F1 Phenotype Growth rate
F~ Phenotype
Thermoresis- Growth tance rate
3
+
4
+
el-1+ 401 4-5 d-1 + 401
--
4-5
+
No of clones
Thermoresistance
4-5 4 5 3 3
-+ --[-
25 17 27 20
4-5 4-5
-+
1 2 1 2
-+
20 22 25 23
In the upper half of the table are given the characteristics of the progeny derived from the el-1 parent; in the lower half, those of the progeny derived from the wild type parent. Growth rates are expressed in numbers of fissions per day at 27 ° C. + indicates phenotypically 401+ cells, -- phenotypically 401 cells. In the last column, the F 2 from 3 different couples have been grouped.
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The difference observed between the two categories of F2cl-1/el-1 homozygotes depending on their cytoplasmic origin, reveals a cytoplasmic difference, stable during at least 20 cellular generations (time elapsed between conjugation and isolation of the F 2 clones) between the m u t a n t and the wild type strains. The reciprocal combination (homozygous cl-l+/cl-l+ nucleus in a cytoplasm of cl-1 origin) does not result in any detectable interaction: the cl-l+/cl-1 + homozygotes derived from either parent cl-1 or wild type parent are identical (Table 2).
Specific Interaction between Gene el-1 and Mitochondria o] Wild Type Origin. The interaction between gene el-1 and wild type cytoplasm which results in mitochondria] disorganization, represents an interaction between gene cl-1 and mitochondria and not some other component of wild type cytoplasm. This is shown by the following experiment. Mutant cl-1, sensitive to chloramphenicol was crossed with a chloramphenicol resistant el-1 + strain (C~). In some pairs, cytoplasmic bridges resulting in transfer of mitochondria between the two conjugants were obtained. By exposing the sensitive ex-cL1 exconjugant to chloramphenicol, it was possible to substitute C~ mitochondria from the c1-1+ parent to its own C s mitochondria (Adoutte and Beisson, 1972). Under these conditions, the ex-cl-1 exconjugant has only its mitochondria derived from the wild type parent, while the rest of its cytoplasm is derived from the el-1 parent. The cl-1/cl-1 homozygotes obtained in F~ from such a heterozygote were found to be quite similar to the cl-1/cl-1 homozygotes carrying wild type cytoplasm: they had a growth rate of 1 to 2 fissions a day (in non selective medium as well as in chloramphenico]), and their mitochondria were very disorganized. Consequently, the cytoplasmic elements of wild type which interact with the nuclear gene el-1 are the mitochondria (or mitochondrial products), and the cytoplasmic difference between the m u t a n t and the wild type strains is a mitochondrial one: mitochondria from the m u t a n t strain which m a y be designated M cl, are "compatible" with the homozygous cl-1/cl-1 nucleus whereas mitochondria from the wild type strain (M+) are not. This mitochondrial difference m a y explain the fact that in the above cross (el-1/cl-lC s × el-l+/el-l+C~), only a small fraction of the ex-sensitive conjugants involved in cytoplasmic bridge formation, became chloramphenicol resistant, in contrast to the cross cl-l+/cl-l+Cs× cl-l+/cl-l+C a, in which the efficiency of transformations is 100% (Adoutte and Beisson, 1972). Moreover, when it occurs, the transformation is very slow. This is quite understandable if there is "incompatibility" between M + mitochondria and the gene cl-1, since the transformation requires a selective replication of M + mitochondria in a cell which is still phenotypically mutant.
I I I . Evolution o/Homozygous el-1 Cells Containing Mitochondria o/ Wild Type Origin The two phenotypic characteristics of cl-1/cl-lM+ cells (very reduced growth rate, disorganization of mitochondrial structure) are not definitive. Through vegetative growth, these cells revert to the original m u t a n t phenotype (3 fissions a day, normal mitochondrial structure) after 15-25 generations. This evolution is progressive; although it has not been systematically studied as yet, there appears to be a correlation between the morphological improvement of mitochondrial structure and the increase of growth rate.
Fig. 5A and B. F 2 cells homozygous for the gene cl-1 and with a cytoplasm of wild type origin (cl-1/cl-lM+). A: Cell fixed 10 to 15 generations after autogamy. No mitochondria display normal morphology. The less altered ones have a very reduced number of eristae (Mr), many are practically devoid of eristae and matrix (My), others have a very dense matrix (Md). Very irregula.r membranous formations can also be observed within some mitochondria (arrow). I n all mitochondria, both the outer and the inner mitochondriM membrane persist. × 15000. B: Cell fixed 20 to 25 generations after autogamy. The alterations of mitochondrial structure are not identical with those observed earlier (A). Mitochondria without cristae (My) are less numerous, but new types of alterations can be observed: mitochondria often contain a rigid plate (P) and wavy cristae (W). × 15000
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Figure 5B shows a cl-1/cl-lM + cell ca. 20 generations after autogamy, at which time its growth rate was 2 fissions per day (i.e. intermediate between the initial one and that to be attained later). The disturbance of the mitochondrial structure is less extreme. I t is worth noting that the new types of anomalies are identical with those observed when mitochondrial protein synthesis is blocked by antibiotics (Adoutte, Balm~fr6zo], Beisson and Andr6 1972). This holds, in particular, for the rigid structure seen in some mitochondria, and for the position of some wavy tubular cristae; but it remains unknown whether these anomalies represent a specific response to a block of mitochondrial protein synthesis. The study of the mechanism of this evolution is under way: it is important, indeed, to establish whether the loss of incompatibility, i.e., the "transformation" of M+ into M cl mitochondria, is a physiological or a truly genetic transformation. Discussion The m u t a n t eL1 described in this paper differs from wild type by a nuclear recessive mutation as well as by its mitochondria. However the nature of the mitochondrial difference remains to be established. The two types of mitochondria can be distinguished both b y their eytochromic spectrum and by their response to gent el-1. First with respect to spectrum differences, two facts must be pointed out. (1) The peak of the type-a cytoehrome is 7 to 10 times lower in the m u t a n t than in wild type, but this deficiency is not apparently accompanied by a deficiency in type-b eytochrome as it is most of the time observed in other organisms (Sherman and Slonimski, 1964). (2) The spectroscopic deficiency in type-a cytochrome is not correlated with a reduction of the respiration rate. The fact that about 80% of the respiration is cyanide-insensitive in the m u t a n t (instead of 20 % in wild type), might account for this result. Since respiration in paramecia seems to involve two pathways: a "classical" cytochrome chain sensitive to cyanide and an alternate oxidase resistant to cyanide, it is possible that in m u t a n t el-l, the alternative pathway compensates a reduced activity of the cytoehrome chain. Such a situation has been demonstrated in Neurospora crassa for cytochrome oxidase-deficient mutants: the cytoplasmic m u t a n t poky (Lambowitz, Smith and Slayman, 1972 ; Jagow, Weiss and Klingenberg, 1972) and the nuclear m u t a n t cni-1 (Edwards and Kwieeinski, 1973). The two types of mitochondria can also be distinguished by their "compatibility" (M ol) or their "incompatibility" (M+) with the cl-1/cl-1 genotype. The manifestations of the incompatibility are two fold: (a) "difficulty" for C ~ (or E~)M + mitochondria to multiply in the cytoplasm of el-1 exconjugants, (b) structural alterations of M+ mitochondria in the presence of the cl-1/cl-1 genotype. These results are reminiscent of other phenomena of incompatibility between nucleus and cytoplasm: in Paramecium, mitochondria of a given species, injected into recipient cells of another species have a great difficulty to multiply (Knowles, 1974); in Oneothera, some interspeeific crosses result in a profound disorganization of the chloroplast membrane system (Schatz, 1970). However, in contrast to what occurs in our mutant, these eases of incompatibility have been observed only in interspecific hybrids.
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As a consequence of the incompatibility, the M + mitochondria slowly evolve into "compatible" mitoehondria in cl-1/cl-1 homozygotes. I t must be pointed out that this "evolution" requires a relatively long time (15 to 25 generations). This shows that the M+ mitochondrial type is rather stable even in the presence of the el-1/cl-1 genotype. Concerning the stability of the two mitochondrial types, M cl and M+, it must also be kept in mind that they are both stable in heterozygous cl-1/el-l+F 1 cells during at least 20 generations. To account for these facts, two main types of hypotheses can be put forward. (1) The difference between the two types of mitoehonch-ia has a genetic basis: the incompatibility results from the absence of fitness between the products of the M + mitochondrial and of the cl-1/cl-1 nuclear genomes. In this hypothesis the disappearance of the incompatibility results from mutation to and selection for M cl genome. The mitochondrial mutation M d may favour the establishment of the alternative pathway as the principal respiratory chain: these mitoehondria would therefore be selected for in mutant cells deficient in cytochrome oxidase. (2) Alternatively, the difference is physiological. If so, two possibilities must be considered. (a) The first (Beisson, Beale, Knowles, Sainsard, Adoutte and Taft, 1974), assumes that the difference resides in the pattern of organization of the mitochondrial membrane, and that the latter is endowed with a certain "structural inheritance" similar to the cortical inheritance described in Paramecium by Sonneborn (1963) and Beisson and Sormeborn (1965). In this hypothesis, the el-1 mutation would be responsible for the IV[cl mitochondrial organization pattern, different from that of M+ mitochondria, and the mutant product would not be easily inserted in the wild type organization pattern; this could explain the fact that the conversion from M+ to ~cl is a long and difficult process. (b) The second possibility assumes that the difference is correlated with mitochondrial metabolism. As suggested above, if respiration proceeds mainly through the cytochrome chain in M+ mitochondria while it proceeds mainly through the alternative oxidase in M ¢1 mitochondria, only the latter are compatible with the mutant nuclear genome responsible for a reduced activity of the cytochrome oxidase. But in this hypothesis, the transformation of one mitochondrial type to another would result not from a mutation-selection mechanism, but from a metabolic modification involving all the M+ mitochondria. This modification would consist in the transition from a predominantly cyanide-sensitive to a predominantly cyanide-insensitive respiration. To explain the slowness of this transformation, it must be supposed moreover that this metabolic transition requires mechanisms slow to be set up (biosynthesis, complex regulations...). Experiments are under way to test the different hypotheses.
Acknowledgments. The authors wish to express particular gratitude to Dr. J. Beisson for advice throughout this work and for many fruitful discussions of the manuscript. We also thank Pr. J. Andr~ for his collaboration, Pr. B. Ephrussi for critical reading of the manuscript and Ms. S. Chevais for preparing the drawings. This work was supported by grants (L. A. 86, E R A 174, ATP 4304 and RCP 284) from the Centre National de la Recherche Scientifique, and contract 72/778 from the Direction des Recherches et Moyens d'Essais. 9 Molec.gen. Genet. 130
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Monique Balm6fr6zol Laboratoire de Biologie Cellulaire IV Universit~ de Paris XI 91405 Orsay France
