Molec. gen. Genet. 162, 1--8 (1978) © by Springer-Verlag 1978

Selection and Characterization of Nuclear Mutations Affecting Mitochondria in Paramecium Frangoise Ruiz and Andr6 Adoutte Centre de G6n6tique Mol6culaire du C.N.R.S., F-91190 Gif-sur-Yvette, France

Summary. A screening m e t h o d , b a s e d u p o n resistance to a t e t r a z o l i u m salt (TTC), is d e s c r i b e d which p e r m i t ted the i s o l a t i o n in P a r a m e c i u m o f 28 m u t a n t s resist a n t to T T C . These m u t a n t s d i s p l a y e d v a r i o u s defects in m i t o c h o n d r i a l f u n c t i o n s ( c y t o c h r o m i c content, cyanide insensitive respiration). S o m e m u t a t i o n s seemed to affect directly the r e s p i r a t i o n c h a i n while others s e e m e d to cause indirect m o d i f i c a t i o n s , p o s s i b l y altering m i t o c h o n d r i a l p r o t e i n synthesis. G e n e t i c analysis o f four m u t a n t s s h o w e d in all t h a t the resistance to T T C was o f n u c l e a r origin.

t o r y c h a i n a n d the p r o b l e m o f n u c l e o - m i t o c h o n d r i a l interactions. T h e screening is b a s e d on the use o f a t e t r a z o l i u m salt k n o w n to stain specifically r e s p i r a t o r y c o m p e t e n t colonies o f yeast (Nagai, 1955) a n d used successfully in N e u r o s p o r a c r a s s a to d i s c r i m i n a t e r e s p i r a t o r y deficient m u t a n t s (Gillie, 1970; E d w a r d s et al., 1973; Bertr a n d et al., 1977). T h e p h e n o t y p e s o f 28 m u t a n t s o b t a i n e d by this p r o c e d u r e are d e s c r i b e d a n d the possible m o l e c u l a r basis o f these m u t a t i o n s is discussed.

Material and Methods Introduction Like a n u m b e r o f o r g a n i s m s , in p a r t i c u l a r vegetals (reviewed by H e n r y a n d N y n s , 1975), P a r a m e c i u m t e t r a u r e l i a possesses a b r a n c h e d m i t o c h o n d r i a l respir a t o r y c h a i n consisting o f a relatively " c l a s s i c a l " cyanides-sensitive p a t h w a y a n d a cyanide-insensitive " a l t e r n a t e " p a t h w a y ( D o u s s i & e et al., 1976). In wildtype, u n d e r u s u a l c o n d i t i o n s , electrons are essentially c h a n e l l e d t h r o u g h the " c l a s s i c a l p a t h w a y " a n d the cells d i s p l a y only a b o u t 2 0 % K C N - i n s e n s i t i v e respiration. A slow g r o w i n g m u t a n t , strongly deficient in c y t o c h r o m e oxidase, n a m e d cll, p r e v i o u s l y d e s c r i b e d by S a i n s a r d et al. (1974) retains a r e s p i r a t i o n rate e q u i v a l e n t to that o f wild-type b u t d i s p l a y s a highly i n c r e a s e d (up to 100%) level o f K C N - i n s e n s i t i v e respiration. These results o p e n e d the possibility o f isolating m u t a t i o n s affecting the t e r m i n a l p o r t i o n o f the " c l a s s i c a l " r e s p i r a t o r y c h a i n in a strictI.y a e r o b i c o r g a n i s m . Such m u t a t i o n s m a y p r o v i d e useful i n f o r m a t i o n c o n c e r n i n g b o t h the structure o f P a r a m e c i u m respiraAddress offp'rint requests to : Fran~oise Ruiz, Centre de G6n6tique Mol~culaire du C.N.R.S., F-91190 Gif-sur-Yvette (France)

Strains and Culture Procedures. The reference wild-type strain from which all the mutants were obtained was a line of stock d4-2 of Paramecium tetraurelia, formerly Paramecium aurelia syngen 4, according to the new nomenclature proposed by Sonneborn, 1975. Cells were of either mating-type 7 or 8 and are named thereafter 7 W.T. and 8 W.T. 7 cll is a nuclear mutant described in Sainsard et al. (1974). E,R, E~0~, C4R, CE~-b are mitochondrial antibiotic-resistant mutants. Their full description is given in Adoutte and Doussi6re (1978). The culture methods used were those described by Sonneborn (1970). Cells were grown in a Scotch grass infusion bacterized with Klebsiella pneumoniae; cultures were carried out at 28°C or at 18°C or 36°C for particular purposes. Growth-rates were measured as indicated in Adoutte and Doussi6re (1978). Antibiotic Resistance Tests. Antibiotic-containing media were pre-

pared by adding a concentrated solution of the antibiotic to the usual bacterized medium just before utilization so as to reach a final concentration of 50, 75 or 1000 txg/mi for erythromycin (from Roussel), 375, 500 or 1000 gg/ml for spiramycin (from Rh6nePoulenc), 75, 100 or 200 ~tg/ml for chloramphenicol (from Roussel) and 50 or 100 btg/ml for oligomycin (from Sigma). Resistance tests were carried out by transferring individually a sample of cells in depression slides into a range of concentrations of the various antibiotics and recording daily their growth-rate and survival. All tests were repeated at least twice. Tetrazolium Resistance Tests. These were performed either on living cells or on crude extract from sonicated cells. 2, 3, 5 triphenyltetra-

0026- 892 5/78/0162/0001/$01.60

2

F. Ruiz and A. Adoutte: Selection of Nuclear Mutations Affecting Mitochondria in Paramecium

zolium chloride (TTC), from Merck, was dissolved just before use in sodium phosphate buffer 0.01 M p H 6.5 and added to the cells or to the extracts as described in results, to a final concentration of 0.2%.

Mutagenesis and Crosses. Mutagenesis was carried out with NMethyl-N'-nitro-N-nitrosoguanidine (NG), from Aldrich-Chemical Co Inc, at a final concentration of 50 gg/ml for 30 min on exponentially growing preautogamous cells. Such cells contain a highly polyploid macronucleus and two diploid micronuclei. After mutagenesis, autogamy was induced by starvation. This leads to the breakdown of the old macronucleus and to the formation of new macro and micronuclei originating from only one haploid product of the meiosis of micronuclei. A u t o g a m y therefore yields homozygous cells which can express the mutations that have occurred in vegetative micronuclei. Such cells were grown for a few generations to allow the expression of the new genotype and were then challenged with TTC, according to the procedure described in the text. T T C resistant cells were isolated and those displaying stable characteristics were further analyzed. Four T T C resistant m u t a n t s were crossed to wild-type strains and the crosses analyzed according to the classical methods described by Sonneborn (1970).

Respiration Measurements on Whole Cells. The oxygen consumption of whole cells was measured polarographically in a Clark electrode fitted on a Gilson oxygraph. For all respiration measurements, cells were either in full exponential phase (500-800 cells/ml) or in stationary phase (2000-3000 cells/ml). 0.2 ml of the loose pellet of cells obtained by centrifugation were transferred into a chamber containing 1.5 ml of 10 -2 M Tris-HC1 buffer, p H 7.2. Molar K C N (from Prolabo) was prepared in molar MOPS (morpholino propane snlfonic acid) buffer (from Sigma) and adjusted to p H 7.2 with concentrated HC1. Molar S H A M (Salicyl-hydroxamic acid, from Aldrich) was prepared in dimethylformamide. K C N and S H A M were added respectively to a final concentration of 1 to 4 r a M and 2 to 4 raM. A mild stirring of the cells was maintained in the respiration chamber by use of a voltage regulator. This was found to be of critical importance for the quality and reproductibility of the oxygraphic traces. Increased KCN-insensitive respiration of the wild-type strains was periodically observed over a 2 year period. Such increases appear to be grouped during certain periods and m a y correspond to seasonal variations. The values that will be given correspond to sets of experiments carried out under strictly controlled conditions, at periods of m i n i m u m KCN-insensitive respiration in the wild-type strain.

Spectroscopy of Whole Cells. Spectra were recorded in a Cary 15 spectrophotometer equipped with a high intensity xenon light according to the method described by Sainsard et al. (1974). Using a pellet of whole cells reduced by sodium dithionite spectra were recorded at the temperature of liquid nitrogen. The ratio of cytochrome c over cytochrome a, 0c/a, was calculated as described in Sainsard et ah, according to the method of Claisse et al. (1970). 0c/a is a ratio of the height of cytochrome c over cytochrome aa 3 calculated by taking into account variations in the baseline.

Results

1. Description of the Screening Method To devise a screening method for respiratory deficient strains, advantage was taken of the existence of the cl~ mutant that is strongly deficient in cytochrome

Table 1. Reconstitution experiment This table gives the results of reconstitution experiments. The first two columns indicate the number of W.T. and cll cells mixed in the test tube containing 0.2% TTC. For the first experiment, 7 tubes were studied and surviving cells in each tube were isolated after 6 h of treatment. In the second experiment, the number of cells per tube was half that in the first experiment and the surviving cells were isolated after 3h. No of cells WT

ell

No of surviving cells

Genotype of surviving cells WT

1st experiment

3000 3000 3000 3000 ~3000 3000 0

2nd experiment

1600 1600 1600 1600 1600 1600 1600 0

0 0 5 10 20 50

1 0 2 15 11 27

~3000

~1500

0 1 5 10 20 40 50

2 1 2 1 9 15 27

~1600

cll

1 1 10 4

1 5 7 27

2 1

1 3

2 1 8 15 24

~800

oxidase (Sainsard etal., 1974). Conditions were sought under which T T C discriminates this mutant from wild-type. The following conditions were found to be optimal: 0.25 ml of T T C dissolved in 0.01 M phosphate buffer, p H 6.5 are added to cells growing exponentially in tubes containing 4 ml of bacterized medium, yielding a final concentration of T T C of 0.2%. The tubes are kept in the dark at r o o m temperature (20 22 ° C). Under these conditions wild-type cells become red and die after a few hours while most cll cells remain white or slightly pink and survive. It must be pointed out that the duration of survival of wild-type cells is proportional to the number of cells in the tube: for example, 1600 cells/ml will survive for about 3 h while 3000 cells/ml survive for about 6 h. Reconstitution experiments were carried out as /follows. F r o m 1 to 50 c11 cells were mixed with about 1600 or 3000 wild-type cells and surviving cells were recovered and identified. The results are reported in Table 1. It can be seen that the screening is very efficient: about 0.1% wild-type cells survive while 50% of survivors are of cll, genotype. It can be noted however that a fraction of cll cells die. This defines a mutant category which will be called "intermediate T T C resistant". To ascertain whether the preferential survival of cl~

F. Ruiz and A. Adoutte: Selection of Nuclear Mutations Affecting Mitochondria in Paramecium Table 2. Selection of TTC R mutants (after N G mutagenesis) The 28 TTC R cells recovered after this selection experiment were isolated from different tubes. Nitrosoguanidine treated cells

Control cells

No of treated cells

No of No of surviving TTC R cells cells

No of treated cells

No of surviving cells

~310,000

88

,~130,000

0

28

3

cells was not due to a decreased of cellular permeability to TTC, TTC was also added to crude sonicated extracts of wild-type and cl~ cells; the wild-type extract developed a red colour while the cll one remained slightly pink.

2. Mutagenesis and Isolation of Mutants

50,000 pre-autogamous exponentiallygrowing 8 W.T. cells were treated with nitrosoguanidine. After wash-

Table 3. Phenotypic characterization of TTC R strains The first column indicates the level of TTC resistance. + + + means strong resistance, + + intermed!ate resistance, + weak resistance, - means sensitivity to TTC. The second column shows the intensity of coloration of the sonicated extract after TTC treatment. + + + corresponds to a strong positive reaction (red coloration), + + to an intermediate reaction (pink coloration), + to a weak reaction (slightly pink) and - to absence of reaction (colorless). The fourth column indicates the thermosensitivity (ts) or cold sensitivity (cs) : high thermosensitivity or cold sensitivity is represented by + and intermediate thermosensitivity by +. The fifth column gives 0c/a values for each strain. Each value is the mean of at least 2 experiments. In the last column are indicated the levels of the KCN-insensitive respiration. For these last two parameters (0c/a and % KCN-insensitive respiration), the limits of their variation for the wild type are indicated above the mean value. Strains

WT TTC~2 TTC~2 TTC~6 TTC6R5 TTC6Ro TTC~6 TTC§7 TTC§5,c TTC~4_2 TTC~s TTC~ TTC~4 TTC~ TTCvR6F TTC6Rs TTC~o TTC~0 TTCsRgF TTC~3 TTC~9 TTC~5 TTC6R6 TTC~6p TTC~s TTC6R9 TTC~4.~ TTC~7 TTC~2

cl~

TTC resistance

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ÷ + Jr + + + + + + +++ + + + + +

a Deficient in cytochrome c

Reaction of sonicated extract with TTC

Generation time (in hours)

+ + + + + + + + + + + + + + + +

6 9 13 10 19 7 8 14 9 8 6 16 6 10 21 7 14 10 22 18 8 13 10 10 22 9 10 9 24 9

+ ---

+ + + + + + + @ +++ + +

ts

cs

+ +

+ + +

0c/a

% KCN-insensitive respiration

Exponential phase

Stationary phase

2.9-6.1 4.2 (48) b 4.4 4.0 4.3 3.6 4.2 7.3

2 6.6 3.9 (29) b

(10) "'b >11 (5) b

10.7 29.5 20.1 10.1 11.1 13.5 14.6 15.4 19.0 19.5 19.5 20.3 22.8 24.6 26.2 28.1 30.8 32 33.3 33.5 40.3 48.2 54.1 55.1 68.5 84 91 100 100 100

> 23.5

100

4.2 5.8 3.7

+ + +

5.6 3.8 3.7 3.5 3.2

+ +

_+ +

+

b Number of independent measurements

3.4 3.7 7.2 7.7 3.7 8.2 9.7 (9) b =>14 9 (20) a'b 4.7 (5) b 5.6 >27.8

Exponential phase

2.7

9.1 (8) u

Stationary phase 11-27.1 18.8

25.2

28.4 33.3 70

4

F. Ruiz and A. Adoutte: Selection of Nuclear Mutations Affecting Mitochondria in Paramecium

ing, the cells were distributed in 90 test tubes and fed with enough medium to enable them to undergo 2 to 3 fissions on the average before getting starved and undergoing autogamy. 42 tubes of untreated control cells were examined in parallel and handled in exactly the same way except for the N G treatment. Samples from several tubes indicated that the percentage of autogamy was 97% in controls and 85% in treated cells and the survival rate of 100% in controls and 50% in treated cells. After autogamy, fresh medium was added to the tubes to enable the cells to undergo 3 to 4 fissions and express their new genotype. Then a sample of 0.25 ml from each tube was tested with T T C in the conditions indicated above. The recovery of surviving N G treated cells was started when the great majority of cells in control tubes were dead. These surviving cells were grown into clones and tested several times for their resistance to T T C after two successive autogamies. Experiments were also carried out to isolate conditional (thermosensitive) TTC-resistant mutants but since mainly nonconditional mutants were obtained, they will not be described. The results have been grouped in Table 2. The following points can be stressed. About 30% of the surviving cells from the N G treated culture yielded stable T T C resistant strains (TTC R) while no clearly resistant strains could be isolated from the control tubes. T T C R strains were obtained from about 300,000 screened cells. The 28 strains originated from different tubes and therefore correspond to independent mutations. A precise mutation rate is difficult to calculate since cellular multiplication has occurred between autogamy and screening and only a sample of cells were screened. A rough estimation taking these factors into account gives a mutation rate of the order of 10 . 4 per mutagenized cell.

W

i

l

d

- type

Fig. 1. KCN-insensitive respiration of one T T C R mutant. Oxygraphic traces were obtained from a suspension of whole wild-type and TTC~4.1 cells. 0.2 ml of a dense cell suspension (ca. 4.10 5 cells) were transferred into the chamber containing 1.5 ml of 10 -2 M. Tris-HC1 buffer p H 7.2

450

500

nm

550

600

3. Phenotype of the T T C g Strains Several phenotypic characteristics were recorded for the 28 T T C R strains. (1)Measurement of the level of resistance to TTC. Three levels were distinguished: weakly resistant mutants, for which more than half of the cells die under the standard conditions of testing ; intermediate resistant mutants, for which approximately half of the cells die; and strongly resistant mutants, for which virtually all cells survive in the presence of TTC. (2) Measurement of growth-rates at 18 °, 27 ° and 36°C, to identify slow growing, coldsensitive or heat-sensitive strains. (3) Whole cells low temperature spectra in exponential and, in some cases, in stationary phase were recorded and for each spectrum, the 0c/a value calculated (see Material and

I

2

1 wild typ 2 mutantCll 3 mutantTTCR 64-1 Fig. 2. Low temperature spectra of whole cells (dithionite reduced)

F. Ruiz and A. A d o u n e : Selection of Nuclear Mutations Affecting Mitochondria in Paramecium

5

c (Fig. 2). As for b-type cytochromes, no definite conclusion can be drawn from whole cell spectra because of the partial overlaps of b and c type cytochromes. However, preliminary data from study of isolated mitochondria suggest that some mutants may be deficient in a b-type cytochrome. For two strains (TTC~7 and TTC6Rs), the resistance was due to a lack of penetration (through either the cell membrane or the mitochondrial membrane) since the sonicated extracts of these strains were clearly stained by TTC, all the others being either non stained or very weakly stained. No systematic correlation between these various parameters can be adduced. In particular no clear relationship is found between the level of resistance to TTC and either spectroscopic deficiencies or extent of KCN-insensitive respiration. There is however a general trend for cytochrome aa3 deficient strains to display a high level of KCN-insensitive respiration as shown in Figure 3.

Methods). (4) Measurement of the amount of KCNinsensitive respiration in exponential phase and, in some cases, in stationary phase. Sensitivity of respiration to SHAM which can be measured reliably on isolated mitochondria was also recorded but study of whole cells yielded irreproducible results and are not reported. (5) Staining of a crude cellular sonicated extract by TTC. The results are reported in Table 3. Several comments can be made. The various mutants differ by (1) their level of resistance to TTC, (2) their generation time (ranging from 6 h to 22 h) and (3) their level of KCN-insensitive respiration ranging from 20% i.e. equivalent to W.T., to 100%. An example is shown in Figure 1. Half of the strains display spectral deficiencies. 10 strains display partial deficiencies in cytochrome aa3 which can be considered as significant in view of the regularity of 0c/a values obtained in many independent experiments on the wild-type strain. One strain, TTC~4_I, is strongly deficient in cytochrome

64-1 @

100

c[i

@

90 80 70

~o60 85

IZ Z

59

u 50

@

40

5O 68 76F

30

74 WT

3

20 65 10

O--

2

60 56 62 52

.I.L-

I

I

[

i

I

I

I

3

4

5

6

7

8

9

1©

zJ

P

>30

ec/a Fig. 3. Correlation between % KCN-insensitive respiration and 0c/a values. Vertical dashed lines delineate the variations of the 0c/a values of W.T. Horizontal dashed lines delineate the variation of the KCN-insensitive respiration of W.T. The numbers correspond to the various T T C R m u t a n t s described in the text. [] Strains hypersensitive to erythromycin and spiramycin. © Strains hypersensitive to erythromycin, spiramycin and chloramphenicol

6

F. Ruiz and A. Adoutte: Selection of Nuclear Mutations Affecting Mitochondria in Paramecium

Relationships between T T C Resistance and Mitoribosomal Alterations

A range of mitochondrial mutants suspected to be altered in mitochondrial protein synthesis, as well as erythromycin treated wild-type cells display the same correlated properties as the TTC R mutants, namely high KCN-insensitive respiration and deficiency in cytochrome aa3. It was therefore interesting to verify (1) whether TTC resistance was observed for mitochondrial mutants and erythromycin treated wildtype cells and (2) whether the TTC R mutants showed any mitoribosomal abnormalities. (1) The range of mitochondrial mutants C4R, g lRo 2 , E~ and CRE~-b described by Adoutte and Doussi6re (1978), were found to display various levels of resistance to TTC well correlated with the extent of their alterations. For example C] which has a nearly wild-type phenotype with respect to K C N insensitive respiration and cytochromes aa3 content is weakly resistant to TTC, while cRER-b which is deficient in cytochrome aa3 and presents a 100% KCN-insensitive respiration in exponential phase, is highly resistant to TTC. (2) Wild-type cells treated in exponential phase with 100 gg/ml erythromycin, a dose which affects only slightly the growth rate showed after few fissions a KCN-insensitive respiration and a deficiency in cytochrome aa3 (Adoutte and Doussi6re, 1978). Under these conditions wild-type cells were found to be highly resistant to TTC. Three inhibitors of mitochondrial protein synthesis (erythromycin, spiramycin, chloramphenicol) were tested on 27 TTC R mutants to detect possible differences with W.T. that might be indicative of mitoribosomal modifications. In addition, oligomycin, an ATPase inhibitor not affecting ribosomes, was also tested. Several strains display differences in sensitivity, being either slightly more resistant to one or several of these drugs or slightly more sensitive than W.T. Experiments were repeated at least twice with a range of concentrations of each antibiotic and the rate of growth of the strains in normal medium was taken into account for these determinations. The major results are indicated in Figure 3. The results are particularly clear for erythromycin and spiramycin: strains displaying modifications are grouped in a region corresponding to deficiencies of cytochrome aa3 and increased KCN-insensitive respiration that is a phenotype similar to that of mitochondrial mutants supposed to have altered mitoribosomes (Adoutte and Doussi~re, 1978). Genetics Analysis of Some T T C R Mutants

Four mutants have been analyzed genetically: TTCRg, TTCRT, TTC~6 p and TTCR4_I. The last three of these

are particularly interesting since they yield highly pleiotropic effects. Each of the four mutants was shown to correspond to a single nuclear mutation. Furthermore TTC~6p and TTC6R4_I were shown to belong to unlinked loci.

Discussion

This paper describes a screening method aimed at isolating mutations affecting mitochondria in Paramecium. The method, which is based on a positive screening resistance to a tetrazolium salt, appears to be very efficient since it permits the easy isolation of a large number of mutants out of which over 50% display respiratory chain abnormalities. The four mutants which have been genetically analyzed thus far, correspond to nuclear mutations and this is presuma b l y the case for the other mutants. This fact is not surprising in view of the conditions under which mutagenesis and selection were carried out. The mutagenic treatment was applied just before nuclear reorganization (autogamy) allowing the expression of new nuclear mutations. Selection in TTC was applied a few generations after mutagenesis and lasted only a few hours: there was therefore not enough generations nor enough time of treatment in TTC to permit the purification of possible mitochondrial mutations conferring TTC-resistance since the number of mitochondria is extremely large in Paramecium. 28 mutants were isolated from about 3.10 s cells. Although a precise mutation rate is difficult to calculate, it can be considered as high and of the order of 10 _4 . In view of this high mutation rate and knowing the mutagenic efficiency of nitrosoguanidine, it is quite likely that some of the mutants obtained correspond to multiple mutations. This imposes some caution towards the conclusions that will be drawn, except for the four mutants that have been analyzed genetically and for which all the phenotype traits were shown to be due to a single nuclear mutation. Some of the possible mechanisms leading to TTCresistance will now be discussed on the basis of the phenotypic parameters that have been recorded for the 28 mutants analyzed. Since the particular TTC salt used has been reported to interact preferentially with the terminal portion of the respiratory chain (Slater, 1963) because of its oxydo-reduction potential, we expected a particular enrichment in cytochrome oxidase deficient mutants. This is indeed the case since about 50% of the mutants are deficient in cytochrome aa3. Since death of wild-type cells in the presence of TTC is accompanied by their progressive staining in red, while resistance is correlated with the fact that the cells remain unstained or only slightly stained,

F. Ruiz and A. Adoutte: Selectionof Nuclear Mutations Affecting Mitochondria in Paramecium resistance corresponds to the failure to reduce the dye intracellularly. This could be achieved a priori, by mutations corresponding to several distinct molecular mechanisms such as : a) lack of TTC penetration of the cell, i.e. cell membrane permeability mutations; b) lack of TTC penetration into mitochondria, i.e. mitochondrial membrane permeability mutations; c) failure of the respiratory chain to reduce TTC through lack or decrease of a component (s) necessary for the transfer of electrons to TTC, such as one of the cytochromes. In turn, such defects might be due to a direct or indirect cause (through alteration of the mitochondrial protein synthesis). The phenotypical analysis c,arried out suggests that indeed several of these distinct types of mutants were obtained. (1) Strain TTC~7 may correspond to a permeability mutation since, in contrast to most of the other strains, its sonicated extract is stained by TTC. While this test does not discriminate cellular from mitochondrial permeability mutations, a number of arguments point to a mitochondrial alteration in this strain: it is resistant to oligomycin, hypersensitive to a series of antibiotics, it displays a 10(7% KCN-insensitive respiration in exponential phase and 30% in stationary phase. Finally, it shows a decrease in cytochrome aa3 in stationary phase. This last property is in marked contrast with that of a set of mutants affecting the mitoribosomes, which will be described below. It is, on the contrary, similar to that of a mitochondrial mutation suppressing a nuclear gene affecting mitochondria (Sainsard-Chanet, 1978). On the whole, this strain may tentatively be considered as altered in mitochondrial membranes. (2) Strain TTC6R4.1 may be directly affected in the respiratory chain. It is strongly deficient in cytochrome c and displays 100% KCN-insensitive respiration in exponential phase. In this strain, TTC resistance may therefore correspond to the impossibility for TTC to receive electrons because of the absence of a " t r a n s m i t t e r " of suitable oxido-reduction potential. In stationary phase, this strain recovers a KCNinsensitive respiration like wild-type without any spectral modifications. This puzzling result may mean that additional defects, not detected spectroscopically, are responsible for KCN-insensitive respiration and that these defects appear only in exponential phase. Alternatively, a small amount of cytochrome c may still be present in the mutant which would be functional in stationary but not in exponential phase. (3) Two strains TTC6R6.v and TTC~9 may be affected in mitochondrial ribosomes. They are partially deficient in cytochrome aa3, hypersensitive to spiramycin and erythromycin and display very high levels of KCN-insensitive respiration in exponential phase. The phenotype of these strains (strongly

7

TTC resistant, slight decrease in growth-rate, cytochrome aa 3 deficient and KCN-insensitive respiration) ressembles that of a set of mitochondrial mutants having altered mitoribosomes as well as that of wildtype cells treated with low concentrations of erythromycin (Adoutte and Doussi~re, 1978). It is therefore possible that a number of tetrazolium resistant mutants correspond to strains having altered mitochondrial protein synthesis. The fact that both strains TTC~6_p and TTC~9 display abnormal sensitivity to anti-mitoribosomal inhibitors fits well this hypothesis. Abnormal mitochondrial protein synthesis has been demonstrated in the cni-1 mutant of Neurospora (Klein et al., 1975) which has been selected by the use of a TTC method (Edwards et al., 1973) and displays many similarities (Edwards et al., 1974) with this class of Paramecium mutants. The fact that perturbations in mitochondrial protein synthesis lead to TTC resistance provides one explanation for the high mutation frequency observed since most of the proteins of the mitochondrial protein synthesizing machinery are probably coded by nuclear genes (see Schatz and Mason, 1974, for a review) therefore providing numerous targets for mutations. In these strains, TTC resistance may result from a mechanism similar to that described for mutants of type c: electrons would flow mainly in the alternate KCN-insensitive pathway and TTC would therefore be much less reduced that in wild-type. Since SHAM is reported to be a specific inhibitor of the alternate pathway (Schombaum et al., 1971) it would have been useful to test the above hypothesis which implies that many TTC resistant strains characterized by KCNinsensitive respiration should display increased levels of S H A M sensitive respiration. Unfortunately the action of SHAM on whole cells was found to be irregular and no definite conclusion could be drawn. However, preliminary data obtained on isolated mitochondria (Ruiz and Doussi6re) indicate that several TTCresistant strains display high levels of SHAM-sensitive respiration, in agreement with the expectations. (4) Finally a number of strains do not appear to belong to any of these three classes. In particular, we have at the moment no explanation concerning a set of mutants characterized by a high TTC resistance and a virtually normal KCN-insensitive respiration (TTC§4, TTC~4, TTC~o, TTC6R4_2 ...). In conclusion, the screening of TTC mutants in Paramecium has been quite successful and permitted the isolation of a number of mutations affecting the respiratory chain. Although the molecular basis of the mutations remains uncertain in most cases, it is clear that several classes of mutations were obtained which affect the respiratory chain directly or indirectly probably through perturbation of mitochondrial protein synthesis. Such mutations should prove

8

F. Ruiz and A. Adoutte: Selection of Nuclear Mutations Affecting Mitochondria in Paramecium

useful for two purposes. Firstly, some of the mutations may constitute useful tools for the analysis of nucleo-mitochondrial interactions at the mitoribosome level. In particular, the nuclear mutations tentatively classified as affecting the mitoribosomes will be interesting to combine with mitochondrial resistant mutations known to modifiy mitoribosomes (Tait, 1972). Secondly these mutations should provide further information on the structure and regulation of the branched respiratory chain of Paramecium. Acknowledgments. We would like to thank Dr. J. Beisson for continuous support and many helpful comments. We also thank Dr. L. Belcour, Dr. A. Sainsard-Chanet, Dr. J.K.C. Knowles and especially Dr. D. Cummings for critical reading of the manuscript.

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Communicated by W. Gajewski Received February 18, 1978