RESTORATION OF NUCLEO-MITOCHONDRIAL ... - Genetics

RESTORATION OF NUCLEO-MITOCHONDRIAL COMPATIBILITY I N PARAMECIUM ANNIE SAINSARD-CHANET

AND

JONATHAN KNOWLES*

Centre de G6nBtique Molkculaire du C.N.R.S., 91190 Gif-sur-Yvette, France Manuscript received March 17, 1979 Revised copy received May 21, 1979 ABSTRACT

I n Paramecium, as previously described, the nuclear mutation cl, is incompatible with wild-type mitochondria ( M + ) ; however, all cZ,/cZ,M+ cells eventually overcome this incompatibility ( SAINSARD, CLAISSEand BALMEFREZOL 1974). We have studied the kinetics and genetic basis of the spontaneous restoration of harmony between nucleus and mitochondria. W e also studied the modification of these kinetics following microinjection of compatible mutant mitochondria into cZ,/cl,M+ cells. We demonstrate that nucleomitochondrial readjustment is always achieved by mitochondrial changes that fall into two classes. The first class corresponds to spontaneous mitochondrial mutations affecting the amount of cytochrome aa, and is similar to the previously described McJ and Msu mutations ( SAINSARD-CHANET 1978; SAINSARD 1975). The nature of the second class of modification is not yet understood; it may correspond either to a mitochondrial “adaptation” or to an unusual type of mutation arising and reverting at high frequency.

is now well established that biogenesis of mitochondria depends upon an ‘Tntimate interaction between the products of both nuclear and mitochondrial genomes. A particularly striking illustration of the specificity of this interaction is provided by the properties of the monogenic nuclear mutation cl, and its mitochondrial suppressors in Paramecium. As previously described ( SAINSARD, CLAISSEand BALMEFREZOL 1974), mitochondria of wild-type genotype ( M + ) are incompatible with a homozygous cl,/cl, nucleus. When such homozygotes arise from cl, +/cl, heterozygotes carrying M + mitochondria, the mitochondria undergo a severe desorganization, resulting in a very reduced cellular growth rate. However, all cl,/cl,M+ cells eventually overcome this nucleo-mitochondrial incompatibility; the mitochondrial structures and the growth rate improve. We analyze here the kinetics of this recovery and the genetic basis for the readjustment of the mitochondria to the cl,/cll nucleus. Two different mitochondrial mutations, M c z and Msu, have been previously described (SAINSARDCHANET1978; SAINSARD 1975). They suppress to different extents the incompatibility between cl, and the mitochondria. In this paper, we have attempted to answer the following questions: does the recovery of cl,/cZ,M+ cells always result from the selection of mitochondrial * Present address. Department of hfedtcal Chemistly, University of Helsinki, Siltavuorenpenger 10, SF 00170 IiELSIYKI 17, (Finland). Genetic. 93: 832-859 December, 1979.

834

A. SAINSARD-CHANET A N D J. KNOWLES

mutations suppressing the effect of cl,? Can the exceptionally long lag (about 20 cell generations) observed before recovery be explained by the frequency of mitochondrial mutations appearing randomly among the mitochondrial genomes of cl,/cl,M+ cells and by the time required for the selection and expression of such mutations? To answer these questions, we have used the technique of microinjection of mitochondria (KNOWLES 1974) in the following way: M C I E R mitochondria (i.e., mitochondria compatible with the cl,/cl, genotype and carrying a mutation for erytliromycin resistance) were injected into cl,/cl,M+ETerythromycin-sensitive cells. The evolution of the injected cells was followed and compared to that of noninjected sister cells. The main results are the following. In noninjected control cells, restoration of nucleo-mitochondrial compatibility always occurs by mitochondrial modifications that fall into two types. The first type corresponds to mitochondrial mutations ( M ” ) . similar to the previously described M c Lor Msu mutations. The second type corresponds to an apparently unique class of reversible mitochondrial “change.” These modified mitochondria ( M A )* are less compatible with gene cl, than mutated M Bmitochondria, and they do not retain this compatibility when grown in a cl,+/cl,+ wild-type cell. I n injected cl,/cl,M+ cells, the MclERmitochondria are actively selected in 5 to 8 fissions, if the injection is made at an early stage of their spontaneous evolution (after 2-3 divisions). At later stages, their selective advantage decreases, and the cells injected at 6-7 and 14-15 fissions either select the M r z E Rmitochondria or modified type A mitochondria pre-existing prior to the injection or type B mutations occuring from type A mitochondria. Two main conclusions can be drawn from these results. First, the restoration of nucleo-mitochcndrial compatibility can indeed result from the selection of mitochondrial mutations randomly occuring at a frequency of about 5 X IO-‘ per mitochondria throughout the vegetative growth of cl,/cl,M+ or cl,/cl,M.L cells. Second. if such a mutation has not been selected. a minimal restoration of compatibility is regularly observed towards the 20th fission. This seems to be due iiot to a clear-cut mutation, but to some kind of mitochondrial adaptation occuring between 2 and 15 gentrations. The possible nature of this phenomenon will be discussed. MATERIALS A N D M E T H O D S

Strains and growth conditions All strains used originated Erom the wild-type strain (stock d4-2) called Paramecium tefraurelia, according to the new nomenclature (SONNEBORN 1975). The strain cl, is a double nuclear and mitochondrial mutant. It carries a nuclear recessive mutation cl, and a mitochondrial mutation I l l c l . Its growth rate at 27” is 3 fissions per day (fpd) instead of 4-5 for wild-type. It is weakly thermosensitive at 36” and spectrally deficient in cytochrome QQ,, (SAINSARD, Cr.41ss~. and BALR’IEFREZOL 1974). * The5e iiiilocliondiia will he repreaenled by to n genetir or a nongenetic change.

although, as discussed helow, it is no1 clear mlicllier the>- correspond

N U C L E O - M I T O C H O N D R I A L COMPATIBILITY

835

t s l i l is a thermosensitive, recessive nuclear mutation (BEISSONand ROSSIGNOL 1969). Homozygotes for t s l l i die within 24 h r at 36”, whereas wild-type cells grow normally at this temperature. ER,,, is a mitochondrial erythromycin-resistant mutation selected in the original cl, strain 1976). from McL mitochondria. Growth rate of the ER,,, strain is 2 to 3 fpd. (SAINSARD-CHANET En87is a mitochondrial erythromycin-resistant mutation selected in the wild-type strain from wild-type mitochondria ( M + ) . The growth rate of the ER,, strain is approximately the same as that of wild type (ADOUTTE 1974). Cells were cultured at 27” in a “Scotch” grass infusion or a Cerophyll infusion supplemented with 1 pg/ml of /3 sitosterol. This infusion was inoculated the day before use with Klebsiella pneumoniae (SONNEBORN 1970) and is referred to as “bacterized’ medium. Thermosensitivitg was tested at 36”. Phenotypic tests Erythromycin resistance: The erythromycin resistance or sensitivity (ER or ES phenotype) was tested by placing the cells in “bacterized” medium containing 100 or 200 pg/ml erythromycin. This test not only permits the identification of the ER or ES phenotype of the cells, but also the estimation of the relative proportion of ER to E” mitochondria in a cell (ADOUTTE and BEISSON1972; PERASSO and ADOUTTE 1974; KNOWLES 1974). If a cell grows i n erythromycin without a lag, most ‘or all its mitochondria are ER. If it grows in erythromycin only after a lag of some days, it contains a mixture of both E R and E S mitochondria. To some extent the duration of the lag is inversely proportional to the number of E R mitochondria. If a cell fails to grow in erythromycin, it contains only E S mitochondria. Growth rate: The growth rate of a clone was generally determined by daily reisolation of single cells and daily recording the number of cells produced. For instance, 4 cells produced over a 24 hr period are recorded as 2 fpd, 6 cells as 2-3 fpd, etc. For some strains, growth curves were determined on mass cultures grown in 1 1 flasks of “bacterized” Cerophyll medium. The generaation times were calculated from the slopes of the linear regression lines. Respiration and cytochrome spectra: The percentage of cyanide-insensitive respiration of whole cells was measured polarographically with a Gilson oxygraph in 0.01 M HC1-Tris buffer, pH 7.2. KCN was added to a final concentration of lo-” M. The ratio of cytochrome c over cytochrome aa, (ec/a) was calculated as previously described (SAINSARD, CLAISSE and BALMEFREZOL 1974) from low-temperature spectra recorded directly on dense suspensions of cells with a Cary 15 spectrophotometer. Crosses Crosses and genetic analysis were performed as previously described (SONNEBORN 1970). In order to obtain cells with a cl,/cl, nucleus and M + mitochondria, the cl,/cl, MclEs strain was crossed with a cl,+/cZ,+M+ES str-in carrying the t s l l l mutation. A reciprocal exchange of nuclei between the two conjugants yields two F, clones of identical heterozygous genotype c l , t s l l l + / c l , + t s l l l , which differ only by their cytoplasm. About 20 generations after conjugation, autogamy is induced by starvation of the F, clones. Half homozygous cl,/cl, and half homozygous cl,+/cZ,+ cells are obtained from both F, lines; cl,/cZ,M+ES cells constitute, therefore, half of the F, progeny derived from the wild-type parent (SAINSARD, CLAISSEand BALMEFREZOL 1974). Microinjection Individual erythromycin-sensitive paramecia were injected with cytoplasm derived from the erythromycin-resistant cl,/cl,MclER,,, strain, according to the method described by (1974) and KOIZUMI(1974). About 5000 pms of cytoplasm was injected into each KNOWLES paramecium; this corresponds to ca. 100 mitochondria or 2% of the mitochondrial population of the cell. After microinjection, the cells were transferred into nonselective medium.

836


RESULTS

As previously described (SAINSARD, CLAISSEand BALMEFREZOL 1974), the nuclear mutation cl, is incompatible with wild-type mitochondria ( M +) . This incompatibility is observed as a very slow growth rate (1-2 fpd) and disorganized mitochondria in all cl,/cZ,M+ cells. However, all cl,/cl,M+ cells evolve from this initial phenotype into a final stable state characterized by a faster growth rate (2-3 fpd on average) and the reappearance of organized mitochondrial structure. The aim of these experiments WSS to analyze the mechanism (s) of this regular evolution and more precisely to examine to what extent this evolution might be due to the selection of mitochondrial mutations of the M L ztype, compatible with the cl, mutation ( SAINSARD-CHANET 1978), arising among the original M + mitochondrial population. If this were the case, the early injection of compatible M c l mitochondria into cl,/cl,M+ cells would be expected to speed up the regular, but slow, spontaneous evolution of such cells and lead more rapidly to the final stable phenotype identical to that of cll/cllMczcells. The experiments were carried out in the following way. From a single cl,/cl,+ F, clone carrying M+ES mitochondria. 150 autogamous cells were isolated, among with 74 were cl,/cl,. These cells were injected with cytoplasm derived from the cl,/cl,MCIE,,,Rstrain at three different stages of their evolution. The first stage was at 2-3 post-autogamous fissions (paf) , when the interaction between the cl,/cl, nucleus and the M + nlitochondria was strong, and the later stages at 6-7 and 14-15 paf as the interaction began to disappear. Each injected cell received about 5000 pm3 of cytoplasm from cl,/cl,MczER,,I cells, (about 100 MCIERIol mitochondria). The use of the ER,,, mitochondrial marker means that the injected mitochondria can be distinguished from those of the recipient cells by their resistance to erythromycin. The evolution of the injected cells was followed on the basis of two criteria, growth rate and erythromycin resistance. This evolution was compared to that of the noninjected sister cells of the same clone. Figure 1 shows the pedigree of injected and noninjected control cells within each cl,/cl,M+ES clone studied. (1) 24 hours after their isolation, each autogamous cell had produced a clone of 6-8 cells from which one (or two) was injected and one reisolated as control (c-1). The injected cells (i-I) was allowed to undergo one fission in normal medium after injection; then one fission product (i-1-2) was placed in erythromycin-containing medium (E), while the other (i-1-1) was left in normal medium. After 24 hours, three cells were reisolated from the i-1-1 subclone (lines I,), and three cells from the control subclone c-1 (lines C,) . Thereafter, the growth in normal medium of the three experimental and the three control subclones was recorded daily. Periodically, samples from the experimental subclones were tested for their erythromycin resistance. (2) When the control cells of this first series had reached their 6-7th paf, one or two of these cells were injected (i-2), and the same procedure described above was repeated for this second series. From the injected cell, one daughter

837

NUCLEO-MITOCHONDRIAL COMPATIBILITY

cell (i-2-2) was put in erythromycin, the other (i-2-1) in normal medium. Then, three subclones from i-2-1 (lines I*) were grown in parallel with three new subclones from the control lines ( C , ) . Their growth rate in normal medium was recorded daily, and periodically the experimental subclones were tested for erythromycin resistance. (3) When the control cells of the second series had reached their 1 6 1 5 t h paf, a third series of microinjections was carried out and their effects followed according to the same procedure (lines I, and C , ) . Additional controls were provided by injecting MCIERlol cytoplasm into both wild-type cl,+/cl,+M+ES and cl,/ cl,MCzEScells. The early test (one fission after injection) in erythromycin permitted one to ascertain the efficiency of the microinjection (see MATERIALS AND METHODS: erythromycin resistance test). Results are given in Table 1. The efficiency of injection does not depend on the cl,/cl, or cl,+/cl,+ genotype, nor on the age of the cells. Only the injected lines whose i-1-2, i-2-2 and i-3-2 cells were capable o€ growth in erythromycin, that is only those injected clones that had received MC1ERlol mitochondria, were followed. Altogether. 20 different cl,/cl,MfES clones were studied: 13 clones as shown on Figure 1 (first series of injections); the seven others were injected at 6-7 and 14-15 paf. In some cases, two cells of one clone were injected, though only one is shown on the figure for simplicity; altogether 19 cells were injected at 2-3,28 a t 6-7 and 22 at 14-15 paf. The subclones derived from these cells and the control subclones were followed for at least 150 fissions.

Phenotypic evolution of the cl,/cl,M+ cells Evolution of the noninjected cells: Figure 2 illustrates the characteristic evolution of the growth rate of cl,/cl,M+E" cells compared t o the growth rate of the original cl,/cl,M"ES strain. The growth rate of all the cl,/cl,M+ES cells, initiTABLE 1 Efficiency of the injections Age

Genotype

2-3

6-7

cl,/cl,M+ES

14-15

cl, +/cl,+M+ cl,/clIMC1Es

ES

2-3 13-14 30-35

No. of injected cells

No. of cells developing erythromycin resistance

Lag (in days)

19 28 22

17 28 22

5-6 4 5 5-6

25

18 5 4

5-6 5 5

5 5 10 ~~~

5-6

9 ~~~

~

Cytoplasm from cl,/cl,MC~ER,,,, cells was injected into sensitive cl,/cl,M+ES, e l l + / cl,+M+ES and cl,/cllMczES cells. The age of the injected cells is measured by the number of post-autogamous fissions. The test of the injected cells in erythromycin was made 24 hours after injection, that is, 1 to 2, 4 to 5 and 2 to 3 generations after the injection of the cl,/cl,M+ES, cl,+/cl,+M+ES and cl,/cl,MClES cells, respectively.

838

01

A. SAINSARD-CHANET A N D J. K N O W L E S

2.3

6.7

14-15

1

qenerations

FIGURE 1.-Experimental protocol for the analysis of c l , / c , M f E s cells injected with MCLEISS, mitochondria. The experimental procedure is explained in the text. The injected cells are indicated by an arrow. The cell lines (Il, I?, 13) derived from these injected cells are shown in black and the control lines (C,, C,, C,) in white. The "abcissa" corresponds to the time scale of the experiment expressed as number of cell generations counted from time "0" in the association between the cl,/cl, nucleus and wild-type mitochondria.

FIGURES 2 and 3.-Evolution in nonselective medium of the growth rate of control and injected cl,/cl,lLffES cells. These figures show the evolution of the growth rate of cl,/cllAi+Eq cells. The time axis starts at autogamy of the F, clone, i.e., the time when M + E s mitochondria first become associated with a cl,/cl, nucleus. On the first day, all the cells underwent 2 to 3 fissions (phenotypic lag). The characteristic growth rate of cl,/cl,M+ES cells was observed only from the second day on. For the control cl,/cl,MC~ESand cl,/cZ,MCLER, the curves represent the mean growth rate of 9 subclones. Abcissa: hours, ordinate: total number of fissions. FIGURE 2.-Noninjected cells; 39 subclones of line C, were followed. Curve A represents the mean growth rate of all the type A subclones. Curve B represents the growth rate of one type B subclone (No. 8-c-1). The study of all the subclones was interrupted for 10 days (//). FIGURE 3.-Cells injected with MCIERpoJmitochondria. The first series (Figure 3a) was injected at 2-3 postautogamous fission (paf), the second series (Figure 3b) was injected at 6-7 paf, the third series (Figure 3c) was injected at 14-15 paf. I, represents the mean growth rate of the 51 subclones, C, the mean growth rate of all the noninjected type A subclones of line C, (curve A of Figure 2). I, represents the mean growth

839


i

rate of 81 subclones injected at 6-7 generations that selected MCtER2,,1mitochondria (Table 3 ) . C, shows the niean growth rate of all the noninjected type A subclones of line C,. i-2 represents the growth rate of the 2 subclones derived from the injected cell 16-i-2. I, represents the mean growth rate of the 58 subclones injected at 14-15 generations that selected MclER,,, mitochondria, C, the mean growth rate of all the non-injected type-A subclones of line C,. The first part of curves of Figure 3b and 3c are redrawn from the corresponding part of curves of Figure 3a and 3b, respectively.

840

A.

SAINSARD-CHANET A N D J. KNOWLES

ally 1-2 fpd, progressively improved and reached a stable growth rate: 2 to 2-3 fpd for the majority of the clones (Group A), but a few grew at 3 to 4 fpd (Group

B). For the clones of the first category (referred to as group A), this stable state was reached by the 16-17th generation (note the change of slope in the curve) and all acquired about the same growth rate, which was slower than that of the cl,/cl,MclESstrain. This evolution is represented in Figure 2 by curve A; the experimental points correspond to the mean growth rate of all the type A subclones. In group B clones, the stable state sometimes appeared earlier. One example of such a clone detected at the 13-15th generation is shown (curve B). Although the growth rate varied from 3 to 4 fpd between different clones of this group, it always was equal or greater than that of the cl,/cl,PIclES strain. All clones were followed f o r over 100 cellular generations. No further change in the growth rate of the group B clones was ever observed. In contrast, about 20% of the group A clones changed to grmp B phenotype at some later stage of growth. Table 2 TABLE 2 Number of group -4and group B subclones obtained from noninjected cl,/cl,M+ES cells

Clone no.

1 2 3 4

5 6 7 8 9 10 11 12 13 14

15 16 17 18 19 20

Group A

2 3 2 1 3 1 3 3 3 2 3 3 2

c,

c2

GroupB

Group A

Group B

Group A

1 (70g) 0 1 (14g)

3 2 1 2 3 1 3 3 2

0 1 (5Od 2 (IOW 1 (75d 0 2* ( 1 5 d 0

2 3 3 3 0 0 1

0 1 (45d

3

2 (90,) 0

2* ( 1 5 d

-

0 0 0 1 (559) 0 0 1 (4%)

-

lost at 40g 3 0 2 1 (35d 3 0 2 1 (6Og) 2 1 (35e) 3 0 2 1 (75g) 3 0 2 1 (35d

2 0 3 1

c3

-

Group B

1 (5%) 0 0 0 3 (80d 3*(15g) 2 (25d 0 1 (100g) 3t(25g) 0

-

2 (25d

-

3 3 3 3 3

0 0 0 0 0

This table gives the number of group A and group B subclones that appeared over 100 cellular generations in the control noninjected C, C, and C, series. The figure in parentheses indicates the time of B phenotype appearance (in number of post-autogamous fissions). In each series, 3 subclones per clme were studied. -indicates that the clone was not tested. * indicates subclones that were derived from the same initial c-I cl,/cZ, M f E S cell and acquired at the same time the same B phenotype. t indicates that the 3 subclones were derived from the same c-2 cl,/cl,M+ES cell.


841

indicates the total number of group A and group B clones obtained during the 100 cellular generations followed in the three series C,, C, and C,. I t also indicates the time of appearance of group B clones. This study shows that the growth rates of all the cl,/cl,M+ESclones increased, but in different ways. Group A clones all evolve towards similar final growth rates (intermediate between that of cl,/cl,M+E” and that of cl,/cllMCIES clones) , which is reached simultaneously towards the 16-1 7th generation. In contrast, group B clones evolve towards different final growth rates, similar to or higher clones, with the kinetics of this evolution varying than that of the cll/cl,MCzES from clone to clone.

Evolution of the injected cells Evolution of the injected control cells: ‘The fate (selection or elimination) of the MC1ERZol mitochondria injected into nine cl,/cl,MclESand 10 c l l + / c l l + M + E S cells (in nonselective medium) was determined by testing the erythromycin resistance of these injected cells after some growth in normal medium. Table 3 and 30 from the shows that the 27 subclones derived from the nine cl,/cl,MLIES 10 cll+/cl,+M+EScells tested 5 and 8 generations after injection had completely mitochondria that they contained at the second and the lost the injected MCIEReol fourth post-injection fission (pif) . Thus, MclE2201 mitochondria are not selected for in either cll/cl,MCIESor cl,+/cl,+M+ES cells, in agreement with previous 1976). results (SAINSARD-CHANET Evolution of the first series of injected cl,/cl,M+ES cells: Of the 19 cll/cllM+ES cells injected at 2-3 pa€, 17 received MCzERdol mitochondria and were studied as indicated in Figure 1. The 51 subclones were followed to the 45th pif. Beyond 45 fissions, only the 24 subclones derived from 8 injected cells were followed to the 150th pif. The evolution of the growth rate of these injected subclones (I-i) is shown in Figure 3a, as well as that of the control C, and of the cl,/cl,M“ES and c ~ ~ / c reference ~ ~ M strains. ~ ~ EThe~ fate ~ of ~ the ~ injected mitochondria was followed by testing their resistance or sensitivity to erythromycin at regular intervals (Table 3 ) . Figure 3a shows that the injected subclones acquired a growth rate of 2 to 2-3 fpd, similar to that of the cl1/cl,Me1EReol cells as early as the 7-8th pif, whereas the control cells still grew at 1 to 2 fpd. Table 3 shows that all 51 injected subclones also become erythromycin resistant by the fifth pif. Furthermore, of the 24 subclones derived from eight injected cells and followed to the 150th pif, 23 retained their erythromycin resistance and their growth rate of 2 to 2-3 fpd; only one subclone (IO-i-1) became erythromycin sensitive between the 35th and the 50th pif, at the same time showing a faster growth rate of 3 fpd. These results indicate that MCzERrol mitochondria, when introduced into cl,/ cl,M+ES cells at 2-3 generations, have a very strong selective advantage with respect to the mitochondrial population of the host cell since practically all the subclones derived from the injected cells became pure for this marker. Evolution of the second series of injected cl,/cl,M+ES cells: Twenty-eight cl,/cllM+ES cells were injected at 7-8 paf and 84 subclones were followed to the 430th pif. Beyond 40 generations, only 28 subclones derived from 12 injected

842

A . SAINSARD-CHANET A N D J. K N O W L E S

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845

cells were followed to the 180th pif. The growth rate of the injected subclones (Iz) is shown in Figure 3b and their resistance to erythromycin in Table 3. AS in the first series, it can be seen that 81 out of 84 injected subclones selected initially the MCZERIolmitochondria in nonselective medium within 7 to 8 generations. They acquired a growth rate of 2 to 2-3 fpd, similar to that of ell/ c ~ ~ cells, M and ~ became ~ E erythromycin ~ ~ ~ ~resistant. Only the three subclones derived from cell 16-i-2 (curve i2) rapidly lost these mitochondria and conespondingly acquired a growth rate similar to that of cl,/cllMczEScells and more rapid than that of the other subclones (Figure 3b). However in contrast to the first series, 10 of the 28 subclones followed for 180 generations did not retain their erythromycin resistance. Of 12 injected cells, only six gave rise to three subclones that retained the characteristic phenotype of c ~ ~ / c to ~ the ~ 180th M ~ pif. ~ From E ~ each ~ ~of ~the six other cells, one to mitochondria and became erythromycin sensithree subclones lost the MC1ERBol tive. Two types of sensitive subclones were observed: those that became sensitive while retaining a growth rate of 2 to 2-3 fpd and those that became sensitive and concomitantly improved their growth rate up to 3 or 4 fpd. The first type is similar to the group A subclones, the second to the group B subclones described in the previous section. Before becoming sensitive, the first group behaved as mixed for MCzEReot and sensitive mitochondria for a considerable period (from the 25th till the 90th pif in the case of subclone 7-i-2). Two subclones derived from cell 17-i-2 remained mixed even until the 180th pif. The results indicate that MczER,o,mitochondria, when introduced into cl,/ cllM+ES cells at 6-7 generations, no longer possessed a clear-cut selective advantage over the mitochondrial population of the host cell, since only about 60% of the subclones derived from the injected cells became pure for MCzERpol mitochondria. Evolution of the third series of injected cl,/cl,M+ES cells: Twenty-two cll/ CZ,M+E~cells were injected at 14-15 paf. and the 66 subclones were followed during 180 pif. ‘ f i e growth rate of these subclones is shown in Figure 3c. No difference in growth rate was observed between the injected (I3) and the noninjected (C,) cells, since the injection took place approximately when the cells reached a growth rate of 2 to 2-3 fpd. Table 3 shows that, out of the 66 submitochondria in nonselective medium. clones, 58 initially selected MCZERZOI These cells, when tested in erythromycin 8-10 pif, grew after a short lag o r with no lag at all. Only three subclones derived from cell 6-i-3 and two subclones derived from cell 9-i-3 rapidly lost the injected mitochondria. As in the second series, the injected cells gave rise to four types of subclones: ( 1 ) resistant; (2) sensitive with a type A growth (2 to 2-3 fpd); (3) sensitive with a type B growth (3 to 4 fpd) ; and (4)mixed. However, their relative frequencies were quite different from those of the second series. After 180 pif, only 10 subclones remained resistant, while 32 were sensitive with a type A growth, 17 were sensitive with a type B growth and seven were mixed subclones. Furthermore, it should be noted, no injected cell showed all three subclones pure

846


for MCzER201 mitochondria (with the possible exception for cell 3-i-3, where it is not clear whether two subclones were still mixed at the 110th generation). These results indicate that MCzEReol mitochondria when introduced into cl,/ cl,M+ES cells at 14-15 generations had a much reduced probability of being selected with respect to the mitochondrial population of the host cell. Only about 15% of the subclones derived from the injected cells became pure for the injected MrLER201 mitochondria (in some clones after a long period with a mixed mitochondrial population). Further characterizalion of the group A and group B growth rates: From the results presented in Figures 2 and 3, it can be seen that group B subclones grow like the cl,/cl,McLESstrain or faster and that group A subclones grow more slowly, like the cl,/clfMCzER201 strain. In order to ascertain the significance of the results, growth curves were determined f o r mass cultures of six subclones representative of group B (four derived from noninjected and two from injected cells), six subclones representative of group A (four derived from noninjected and two from injected cells) and two injected subclones that had selected ML1ER201 mitochondria. The generation times of these strains were compared to those of and cl,/cl,MxuESstrains. Results are given in the cL,/cl,M'lES,clI/cllMC1ER201 Table 4. It can be seen that the generation time of a given strain may be different in different experiments, 11 hr and 14 hr for c ~ ~ / c ~ , for M instance. ~ ~ E ~Each ~ ~ ~ , individual value should be considered therefore only as indicative. However, when different strains are compared repeatedly, the relative values of the generation times generally vary in the same way (ADOUTTE and DOUSSIBRE 1978). Therefore, only the generation times recorded in the same experiments (same day. same batch of culture medium, etc.) that are given in the same column of Table 4 can be compared directly. Under these conditions, it can be seen that in the same experiment, group A subclones have a generation time similar to that of the cl,/cl,MCIER,,, strain, while group B subclones have a generation time similar to that of the cl,/cllMCzESstrain (subclones no. 10-c-3, 3-c-1, 16-i-2, 13-c-1,13-c-3) or to that of the cll/cl,M8uE'strain (subclones no. 6-c-3 and 9-i-3). These results confirm the differences in growth rate between group A and group B subclones. They show that the growth of the injected subclones that have selected the MCzER201 mitochondria is similar to that of subclones that have lost these mitochondria and acquired a type A growth (cf., subclones 7-i-3-1 and 7-i-3-2). This indicates that MCIERsol mitochondria and the sensitive mitochondria of group A subclones have approximatively the same replication rate (ADOUTTE and DOUSSIBRE 1978). It is not surprising. therefore, that the simultaneous presence of these two types of mitochondria in one cell is maintained for a very long time, as observed in the second and third series of injected cells. In summary, these results have revealed two important facts: (1) Noninjected cells evolve towards two types of stable states, A and B, and (2) Injected cells either retain the injected MCIEKBol mitochondria and show the characteristic o l or they loose the MCIER;lol mitochondria and phenotype of c l l / c l l M c l E R ~cells evolve towards the same A or B phenotypes found in noninjected cells. The

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848


later the W z E R 9 0mitochondria 1 are introduced into the cl,/cl,M+ES cells, the less chance there is of their being selected.

Genetic basis of the A and B phenotypes In order to determine the genetic basis of the final stable A and B phenotypes of cZ,/cZ,M+ cells, three strains representative of group B (no. 3-c-1, 20-c-2, 13-c-1) and four strains representative of group A (no. 3-c-2, 5-c-2,4-c-2, 2-i-3) were crossed to a strain with a wild-type nucleus and M + mitochondria carrying an erythromycin resistance marker (cl,+/cZ,+M+ER,,). These crosses all gave the same type of result; therefore, only those concerning one group A strain, no. 24-3, and one group B strain, no. 20-c-2. are given in Table 5. For each or the two strains, the results obtained with (couples no. 1 ) or without (couples no. 2) cytoplasmic exchange between the two parental strains are represented. TABLE 5

F , analysis of t h crosses of one group B (no. 20-c-2) and o m group A (no. 2-i-3) strain by the cl,+/cl,+M+ER,, strain Cytoplasmic origin of the Flclone F, genotypes

Tested strain Cll/Cl,

C1,+/Cl1+

Wild-type strain cl,+/c~+

Cl,/Cll

No. of clones Growth rate (3g) Erythromycin resistance (log)

10 3 4

11

9

12

4

1-2

4-5

-

-

+

+

No. of clones Growth rate (3g) Growth rate (log) Erythromycin resistance ( 1Og)

8 3-4 3-4

6 4 4

3-4

-

-

-

+

No. of clones Growth rate (3g) (I) Erythromycin Strain 2i3Es resistance (log) (group A) X No. of clones cl,+/cl,+M'ER,, Growth rate (3g) (2) Erythromycin resistance (log) (20g) (5%)

6 2-3

15

10 1-2

11 4-5

+

+

5

12 4-5

(1) Strain 20c2ES (group B) X cl,+/cl,+M+ER,7 (2)

8 $3

4

12 4

5 1-2

2-3

10

4-5 44-5

For each cross, two pairs of conjugants were studied. In one (l), no cytoplasmic exchange occurred between the two conjugants; in the other (2), exchange did occur, yielding F, clones containing a mixture of E R and ES mitochondria. Growth rate is expressed in number of fissions per day in normal medium at 27". Erythromycin resistance or sensitivity is represented by or -. The number in parentheses indicates after how many post-autogamous fissions the growth rate and the phenotype in erythromycin was determined.

+


849

The following conclusions can be drawn from the results of Table 5: (1) The stable A or B phenotype is not based upon a nuclear change; only the cl, and cZI+ alleles segregate in F, and the cl, gene retains its incompatibility with M + mitochondria, as shown by the growth rate (1 to 2 fpd) of the cZl/cl,M+ER37F2 clones. (2) The stable A or B phenotype is cytoplasmically inherited, since in the absence of cytoplasmic exchange during conjugation, the F, homozygous cl,/cl, clones display different phenotypes in the two cytoplasmic lines, those deriving from the tested strain showing the same A or B phenotype as their parent. (3) The stable A or B phenotype results from a mitochondrial modification as shown by the F, generation derived from the couples that had undergone cytoplasmic exchange and carried ER and ES mitochondria in Fl. The F2 cll/cZl clones selected the Es mitochondria and became pure for this mitochondrial type in less than 10 generations. This selection was accompanied by the recovery of the parental A (2-3 fpd) or B (3-4 fpd) phenotype. In contrast, the F, cll+/cZl+ clones did not actively select the sensitive mitochondria, but remained erythromycin resistant during the first post-au togamous fissions. These results show therefore that in all cases the evolution of the cl,/cZ,M+ cells results from a mitochondrial modification (-MAor M B ) that is responsible for the stable phenotype of the two groups of cZ,/cZ, cells.

Analysis of the MAand MBmitochondria Genetic analysis: An important question raised by these results is whether the mitochondrial modifications, M Aand MB, actually result from a mutation of the mitochondrial DNA similar to the M c l or Msu mutations, which have been shown to retain indefinitely their distinctive properties (and in particular their compatibility with a cZ,/cZ, nuclear genome) in the absence of the cl, gene, i.e., in cZl+/cZl+MCZ orcZI+/clI+MSU cells (SAINSARD-CHANET 1978; SAINSARD 1975). The stability of the M A and M B mitochondria was examined in the following way. First, C Z ~ + / C Z ~ +and M ~C Z ~ + / C Z ~ +clones M ~ were chosen from theF, progeny E ~cl,/cl,MBEs ~~ X of the seven previous crosses c l , / ~ l , . M - ~XEC~I , + / C Z ~ + M + or cZ1+/cZl+M+ERs7. Then, these seven clones were recrossed to a cZl/clIMclstrain after 25, 50 and 100 cellular generations. If the M A or M Btypes corresponded to mitochondrial mutations, at least to mitochondrial mutations similar to M c l o r Msu, none of the cll/cZ, F, clones obtained from these crosses would display the nucleo-mitochondria1 incompatibility expressed by the characteristic 1-2 fpd growth rate. The F, phenotypes observed in the progeny of these crosses are shown in Table 6. Since all four crosses of cZl/cZ1+M"EsX C Z , / C Z , M ~ ~ Eand ~,,~ all three crosses of cZ,+/cZ,+MBE"X c ~ ~ / c Z ~gave M identical ~ ~ E ~results, ~ ~ ~only one example of each category is given. The following conclusions can be drawn from the results shown in Table 6: ( 1 ) The M B mitochondria (Maoczin the table) retain their compatibility with gene cl, (growth rate: 3 to 4 fpd) even after 100 cellular generations in association with a wild-type nucleus, and they reduce the growth rate of c2,+/cll+ cells (3-4 fpd instead of 4-5 fpd for cZ,+/cl,+M+Es cells). (2) In contrast, the M A mitochondria lose their compatibility after 25-50 generations in association with

850


TABLE 6

F , analysis of the crosses between two cll+/cll+ strains containing MB (no. 20-C-2) or (no. 2-i-3) mitochondria and the cll/cllMC'ER,,l Cytoplasmic origin of the Fl clone F2 genotypes

(1) cl,+/cll+M20C~E~ X cl,/cl,MC1 ER,,,

(2)

(1) cl,+/cl,+M*~~E~

No. of clones Growth rate Erythromycin resistance

(2)

9 2-3

Cl~+/Cl1+

Tested strain cl,+/cl,+

Cll/Cl1

5

15

3-4

3-4,4

+

-

+


48 34,4

41 3-4,4


22 2-3

23 3-4

20 1-2

25 4-5

+

+

-

-


21 1-2

27 4-5

24 1-2

4-5

-

-

-

-

x cll/cl, MCIER,,,

cll strain

cl,/cl,

MA

strain

20

Legends as in Table 5. The erythromycin sensitivity or resistance and the growth rate were determined 5 fissions after autogamy.

a wild-type nucleus and interact again when reassociated with a cl,/cl, nucleus (growth rate 1-2 fpd). The interaction seemed slightly less strong (1-2 to 2 fpd) at 25 generations than at 100 generations (1-2 fpd). ( 3 ) When a cytoand MAESor MBES plasmic exchange has occurred and a mixture of MCIEReol mitochondria is established in a heterozygous clone, the sensitive mitochondria are actively selected for during the 20 generations of heterozygosity, and no resistant mitochondria are recovered in the F,. Physidogicul properties of MA and MB mitochondria: It is known that the M" and M 8 umitochondrial mutations alone (when associated with a wild-type nucleus) are responsible for reduction in the amount of cytochrome auS (SAINSARD-CHANET 1978; SAINSARD 1975). It was, therefore, of interest to study the spectral and respiratory properties of c l l f / c l , + M ' and cl, + / c l ,+ M B strains. Cytochrome spectra and the extent of cyanide (CN)-insensitive respiration were recorded on whole cells for three cl+/cll+MB strains: (cl1+/c1,+Meocr ,Cl,+/ cl, +MSr', ~ 1 ~ + / c l ~ + Mand ' ~ ~for ' ) four cll+/cll+M" strains: ( ~ 1 ~ + / c l , + M ~ ~ ~ , ). strains were studied ~ l ~ f / c l ~ c+ l M~ ~+ ~/ ~~ l, ~and + Mc l~l +~/ c~l l + M 2 a ?These at different ages. The results are given in Table 7. The @c/a value estimates the CLAISSE and BALMEFREZOL ratio of cytochrome c to cytochrome ua3 (SAINSARD, 1974). Wild-type cells have a @ c / avalue between 3 and 4, and 20-30% of their respiration is CN insensitive, as previously described (ADOUTTE and DOUSSIBRE

851


TABLE 7 Respiratory characteristics of MA and MB mitochondria Strain

Age

Phase

%KCNE

3.8 3.7 3.6

30% 30%

-

251% 30% 23%

(expo Istat stat stat stat

5 9.5 9 11 5

341% 36% 26% 23% 37%

stat stat

10.5 6

29% -

stat

8

stat stat stat

3.4 3.8

50% 35% -

stat late expo stat

5.3 8 2.8

641% 17% -

7.2 3.7

32% 28% 13% 42% 53% 58% 30%

stat stat expo expo expo stat

Wild-type

@/a

-

%%

M.4 mitochondria c11+/c11+M3CP

60g 80g 1log

25g 35g 1oog 11og

stat stat stat

{:z

: :I

5.5 3.4 3.2 3 2.5 10

stat stat

5 5 7 3

2.5

31% 28% 45 % 351% 59% 26%

The age represents the number of generations of association between the mitochondria and a wild-type nucleus. The &/a values correspond to the ratio of cytochrome c over cybchrome aa,. They were calculated from whole-cell spectra according to SAINSARD, CLAISSE and BALMEFREZOL (1974). The percentage of CN-insensitive respiration (% KCNR) was determined after addition of KCN to a final concentration of 1 miv. -: not tested. {: aliquots derived from the same culture were studied i n exponential and stationary phases.

852


1978; RUIZ and ADOUTTE 1978). The three cZ1+/cZ1+MBstrains have higher ~ c / values a (5 to 11) even when tested after 100 cellular generations and are

deficient in cytochrome an3. The extent of their CN-insensitive respiration is also slightly higher (often between 30 and 40%) than that of wild-type cells. These two phenotypic characteristics are similar to those of the M C zmutation (SAINSARD-CHANET 1978). The four cZI+/cZ1+MAstrains that have lost their compatibility with gene cl, (cf., previous paragraph) have a more complex phenotype. For a given strain, the @c/a varies from wild-type (2.5) to mutant (> 4) values, as does the percentage of CN-insensitive respiration (35 to 50% for Msc2, 17 to 64% for M5", 13 to 58% for Mhca,26 to 59% for MZi3.It does not seem that these variations depend only on the phase of growth or on the age of the strain. Most often, an inverse relationship is observed between the amount of cytochrome mSand the extent of CN-insensitive respiration: a mutant @c/a value is correlated with a wild-type percentage of CN-insensitive respiration, and vice versa. However, this relationship is not absolute, and a completely wildtype phenotype is sometimes observed. These results show that M B mitochondria carry a mitochondrial mutation belonging to the same category as the M c l and Msumutations and display the same clear-cut physiological properties as these mutations, in particular compatibility with cl, and a reduced amount of cytochrome m3.I n contrast, it is not clear that M A mitochondria result from a mitochondrial mutation. Their phenotype is unstable and they lose their compatibility with cZ, in the presence of a dl+/ cl,+ genome, while displaying variable physiological properties. DISCUSSION

A strong incompatibility between a cZ,/cl, nucleus and wild-type mitochondria ( M + ) has been described previously. It is expressed in particular by a severe disorganization of mitochondria and a markedly reduced growth rate. However, this incompatible state is not final and, in all cells, the mitochondrial structure and growth rate improves towards the 20th generation, reaching finally a stable, compatible state (SAINSARD, CLAISSE and BALMEFREZOL 1974). This paper reports the study of this recovery phenomenon and its genetic basis. We show that the nucleo-mitochondrial readjustment is always brought about by mitochondrial, not nuclear, modifications and that these modifications fall into two types. The first type, MB, corresponds to a mitochondrial mutation 1978; SAINSARD event similar to the M c zand Msumutations (SAINSARD-CHANET 1975), while the second type, MA, corresponds to reversible mitochondrial changes that are difficult to explain in terms of clear-cut mutations or simple physiological transformations. These two types of modifications differ in their kinetics of appearance and in their level of compatibility with a cZ,/cZ, nucleus. An extensive study of the evolution of the growth rate of cl,/cl,M+ cells has shown that the cZ,/cZIMAstate is reached in all cells (except for the few that selected M B mitochondria) about at the same time towards the 17th generation. It is expressed by a growth rate of 2 to 2.3 fpd for all the cells. However, the


853

cll/c2,MB state is not reached in all cells. It can appear any time after the 13th generation during vegetative growth in cells still expressing the cl,/cl,M+ phenotype or in cl,/cl,MA cells, and it is typified by a higher growth rate ranging from 3 to 4 fpd. After 100 generations of vegetative growth, the proportions of the two states were found to be 80% cl,/cl,MA and 20% cl,/cl,MB cells. mitochondria at differThe study of cl,/cl,M+ES cells injected with MCZER2,,, ent stages of their spontaneous evolution showed that the MCzEReol mitochondria were systematically selected in the absence of erythromycin in 5 to 8 fissions when the injection was performed at an early stage (2-3 fissions). When the injection was carried out later, the selective advantage of these mitochondria decreased. Although all cells injected at 6-7 fissions and most of those injected mitochondria during the first at 14-15 fissions became enriched in MCzERrol seven pif, only 60% of the subclones injected at 7-8 fissions and 15% of those injected at 14-1 5 fissions eventually became pure for MCZEReol mitochondria. All the others retained a mixture of MCzEReI), and sensitive mitochondria over 150 cell generations or became erythromycin sensitive and either cl,/cl,MA or cl,/cl, MB. All these results show that the readjustment between mitochondria and the cl,/cl, nucleus does not result from a unique mechanism, but from at least two types of events whose frequency, kinetics of selection and genetic basis are discussed below. Since the evolution of the injected cells containing a mixture of two types of genetically different mitochondria provided additional information about the absence or rarity of mitochondrial recombination in Paramecium (ADOUTTE, KNOWLESand SAINSARD-CHANET 1979), this point will be briefly considered. Properties of the MBmitochondrial mutations As previously suggested (SAINSARD-CHANET 1978), the recovery of compatibility in cl,/cl,M+ cells could result from the selection of mutant mitochondrial genomes compatible with a cl,/cl, nucleus. Two questions are raised. First, is the frequency of M Bmutations compatible with that of spontaneous mutations? Second, can the long lag observed before their expression at the cell level be explained by the time required for their selection? The frequency of type B mitochondrial mutations can be estimated from the data of Table 2. Since each subclone is derived from only one cell, the analysis of 39 (1st series), 54 (2nd series) and 51 (3rd series) control subclones during 100 generations amounts to the observation of 3900,5400 and 5100 cells, respectively. From the number of independent cl,/cl,MB subclones that appeared ( 7 in the first series, 11 in the second and 11 in the third) and on the rough assumption that there are about 5000 mitochondria per cell, the frequency of M B mutations appears to be of the order of per mitochondrion per cell generation. This frequency is similar to that o€ the spontaneous mitochondrial mutations ER in Paramecium (QUEIROZ and BEALE1974). Therefore, M B mutations are very likely to be spontaneous mitochondrial mutations selected in a cl,/cl, nuclear

854

A. SAINSARD-CHANET

A N D J. KNOWLES

context. However, in a wild-type nuclear context, such mutations are eliminated 1976). when in competition with wild-type mitochondria ( SAINSARD-CHANET The time required for the expression of compatible mitochondrial genomes can be estimated from the evolution of the injected cells. Injection of about 100 Mr1ER2,,,mitochondria into cll/cl,M+ES cells required 5 to 7 cellular generations before improvement of the growth rate and expression of the cZ,/ellMr' ERgol phenotype. It has been shown for mutatioiis conferring resistance to erythromycin that a cell expresses the E" phenotype only when it contains a substantial 1972; PERASSO and ADOUTTE 1974). It is majority of E R mitochondria (KNOWLES reasonable to assume that a cZ,/cZ,M+ cell phenotype will change only when a majority of its mitochondria became compatible M A or M U .Since 5 to 7 generations are required for 100 MclERmitochondria to become the major type (3000 to 5000). it can be predicted that at least 12 to 13 generations would be required for one M C I E R pmitochondrion ol to affect the cell phenotype. The selective advantage of sensitive MU mitochondria is higher than that of MC1ERZOI mitochondria, but probably some lag is required Between the occurrence of the mutational event and its expiession at the level of the individual mitochondrion. It is therefore likely that even if a M Umutation occurs during the first few post-autogamous fissions, it will not be expressed at the cellular level before the 10th-15th fission. This is verified by the evolution of the subclones derived from clone no. 6 (Table 2). All of them derived from the same initial 6-c-1 cell isolated at 2-3 fissions and all of them expressed the same cI,/cIIMB phenotype by the 15th generatim. The MU mutation therefore probably occurred before 2-3 fissions, but was expressed only 10 to 15 fissions later. The mutations of the M U type studied in this paper ( M s c l ,MIsc1,M Z o r 2and ) five other mutations selected independently in another set of experiments (results not shown) have properties quite similar to the M c Land M"'&mutations described previously ( SAINSARD-CHANET 1978; SATNSARD 1975). These mutations suppress the incompatibility between mitochondria and a cZ,/cl, nucleus to different extents, and all of them, when associated with a wild-type nucleus. are responsible for a decreased cellular growth rate and decreased amounts of cytochrome aa3 in stationary phase cells. This phenotype has been discussed in a previous paper, and the hypothesis that both the nuclear gene cl, and the MU mitochondrial mutations alter mitochondrial products has been considered. The suppression would then result from functional interactions between two modified products ( SAINSARD-CHANET 1978). Unfortunately, the absence of mitochondrial recombination in Paramecium (ADOUTTE, KNOWLESand SAINSARD-CHANET 1979) does not permit us to determine whether the independently selected mutations from M + or M A mitochondria arc different alleles of the same gene or are different mutant genes. Similarly, because the inaccuracy of generation time estimation makes a precise determination of the efficiency of suppression difficull, it is not possible to say whether all these 'hew'' mutations are in fact reisolations of Me' and M 8 u or whether mutations at numerous different loci can act as suppressors of the gene el,.

NUCLEO-MITOCHONDRIAL. COMPATIBILITY

855

Possible nature of the MAmodification The behavior of the injected cells provides information concerning the properties of the M4modification. As shown in RESULTS. the selection or the loss of MCIERZol mitochondria injected into cl,/cl,M+ES cells depends on the age of the recipient cells. The later the injection takes place, the larger is the proportion of injected cells losing the M C z E z ~mitochondria pOl and becoming erythromycin sensitive. The origin of ES mitochondria could be a priori either a reverse mutation of E",,,, a recombinational event between the original M+ES and the injected MC1E'22,,, mitochondria, or a M I or M B modification of the original M+ES mitochondria that gave them a selective advantage over M C I E R pmitochondria. Ol The first two hypotheses imply that the sensitive subclones derived from the injected cells had the same growth rate as that of the original cl,/cllMczESstrain (3 fpd) . This was not, however, the case for the majority. Furthermore, the backmutation frequency of ER,,, was found to be low. In the 150 clI/cl1MC1ERpol cells that were regularly subcloned in normal medium for 100 cellular generations, no reversions were observed. In addition this strain, first isolated 5 years Etgo. has never been observed to revert in stock tubes. It is now established that the irequency of mitochondrial recombination in Paramecium is extremely low KNOWLESand SAINSARD-CHANET 1979). Th'is conor nonexistent (ADOUTTE, clusion is confirmed by these results where only one case (cell 164-2, discussed below). could have resulted from recombination. In all other cases, the sensitive subclones (even those whose phenotype was similar to clI/clIMczES cells) appeared at a time when none of the original unmodified M+ES mitochondria would still be present. The simplest hypothesis to account for these sensitive subclones is to assume that the injected cells from which they derived contained, prior to the injection, modified mitochondria or M + mitochondria about to be modified. Except for one cell (cell 6-i-3) that contained M B mitochondria earlier than 14-1 5 generations and rapidly eliminated the injected mitochondria, all the other injected cells that gave rise to sensitive subclones most likely contained ii4" mitochondria prior to injection. From the data of ADOUTTE and DOUSSI~RE (1978), such mitochondria would be expected to have approximately the same mitochondria since clI/cll.MAand clI/cllMCzER201 replication rate as MC1ER2,, cells have a similar growth rate. This would explain both their stability in the ol and also why a mixture of M A and MCzER201 presence of M C I E R z mitochondria would be maintained for such a long time (over 150 to 180 generations). Such a mixture could evolve randomly since the same injected cell (e.g., cell 7-i-3) can give rise to both cll/cl,MAESand cl,/c1,MC1ER,,, subclones and since some mixed subclones eventually selected MAES mitochondria ( 17-i-3,18-i-3),whereas others selected MCIERzOl mitochondria (8-i-3, 4-i-3). The cl,/cl,MB subclones that appeared after 50 pif probably result from a mitochondrial mutation GCcurring in M 1 mitochondria and not from pre-existing M B mitochondria that would h a w been more quickly selecbrd. Nor is it likely that recombination between MC7ER101 and MI E" mitochondria accounts for M B subclones, as their frequency after 100 generations was about 20% for M B compared to 80% for M', as found in control experiments. Since only one out of 8 cells injected at

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SAINSARD-CHANET A N D J. KNOWLES

2-3 fissions gave rise to an E S subclone, whereas 6 out of 12 cells injected at 6-7 h i o n s and all 22 cells injected at 14-15 fissions gave rise to one or more sensitive subclones, it can be concluded that the MI1 modification occurs between approximately two and 15 generations. Any hypothesis concerning the nature of the M A modification must account for the following facts: (1) it appears in all cl,/cl,M+ cells that have not already selected M B mitochondria; (2) it does not appear during vegetative growth, but only during the first post-autcgamous fissions and is expressed in all the cells at approximatively the same time (after 17-20 generations); (3) it is stable for at least 20 generations in a heterozygous cl,/cl,+ but not in a wild-type nuclear context. Although the M A mitochondria lose their compatibility with gene cl, when associated with a wild-type nucleus, they do not seem to return to their state. I+ initial -% These properties are difficult t o reconcile with either the hypothesis of “classical” mitochondrial mutation(s) or with the hypothesis of a simple physiological transformation of mitochondria by the product of the cl, gene. If the M Amodification is due to mutation, these mutations must have particular properties that account for the kinetics of their expression and instability. They must occur within 3 and 8 cell generations following ci,/cl, homozygote formation. If selected in 12 to 15 cell generations (as are MCzERZol mitochondria, see above), they would indeed be expressed towards the 20th generation under the conditions used. Over 1,000 cl,/cl,M+ cells have been observed during the past few years and not one has remained cl,/cl,M+. Since the M A event occurs between 3 and 8 generations. then at least one mutated mitochondrion out of 25.000 (5000 mitochondria per cell and 5 cellular generations) may appear in each of the I O 3 cells. The Poisson distribution predicts that, for a mutation rate of 5x per mitochondrion per generation, the probability of finding a nonmutated cell is per generation. This would then explain the systematic evolution of all the cells studied. This hypothesis is not unlikely, as the profound disorganization of mitochondrial structure observed during the first postautogamous fissions of cl,/cl,M+ cells (after 2-3 generations of phenotypic lag) may well induce mutations in the mitochondrial genome, for example, by abnormal replication of the mitochondrial DNA. It should be pointed out, however, that this mutation hypothesis is difficult to reconcile with the observed instability of the modification although cytoplasmic mutations that revert at high frequency have been previously reported (GOUHIERand MOUNOLOU 1973; NAGI,OCHI and NISHIMURA, personal communication). It is not known whether the stability of the M A type in a heterozygous clI/clI+ nuclear context is due to the activity of gene cl, or if this modification is stable f o r at least 20 generations even in absence of the mutant product. On the other hand, the hypothesis of a simple physiological transformation of mitochondria by the activity of gene cl, is difficult to reconcile with an altered mitochondrial phenotype retained for more than 100 cell generations in a wildtype nuclear content and with the long time required for this modification to be


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set up unless one assumes that complex metabolic regulatory mechanisms are at work (for review, see BEISSON 1977). Another type of hypothesis is based on the idea of structural heredity develet al. 1974; BEALEand KNOWLES 1976). It is oped in previous papers (BEISSON based upon the phenomenon of structural inheritance of cortical patterns in Paramecium (BEISSON and SONNEBORN 1965). According to this hypothesis, the M A modification would be a stable modification of the wild-type mitochondrial structural organization caused by the cl, gene. The mitochondrial structure reformed after disorganization during the first postautogamous fissions could possess a new pattern of organization to allow the insertion of the mutant product, thus rendering them compatible with gene cl,. I n the absence of the cl, gene, the wild-type product would replace the mutant product, and the mitochondria would lose their compatibility with gene cl,, while retaining the new pattern of organization. This hypothesis is difficult to test.

The euolution of cell 16-i-2: mutation or recombination? While it was not the object of this work, the question of mitochondrial recoinbination in Paramecium should be briefly considered here since mixtures of genetically different mitochondria were formed by microinjection and their evolution studied. No mitochondrial genetic exchange in Paramecium was detected in extensive studies designed to probe this question (ADOUTTE, KNOWLES and SAINSARD-CHANET 1979). This property distinguishes Paramecium from all other organisms so far studied (for review, see BIRKY1978; GILLHAM1978), and it is important to discover if mitochondrial recombination does not occur or is simply extremely rare. Any experiment where recombination between different mitochondria could have taken place bust be carefully studied. One case will be considered here: the evolution of cell 16-i-2. The three subclones derived from this cell were injected at 6-7 fissions; they first selected MczEReol mitochondria but rapidly lost them, acquiring towards the 10th pif a growth rate indistinguishable from that of the cl,/cl, MczESstrain. Since growth of the three noninjected control subclones (16-c-2) remained slow, the event responsible of the evolution of cell 16-i-2 occurred after the injection and before the isolation of the three subclones, that is during the first or second and the M+ES (or post-injection fission. Recombination between the MCzERpol MAES)mitochondria of the host would lead to such a result, as would also a M c z mutation of M+ES (or MAES)mitochondria occurring just after injection. This result, therefore, does not permit any conclusion concerning mitochondrial recombination in Paramecium. I n conclusion, the main result of this work was the demonstration that in all cl,/cl,M+ cells tkat display nucleo-mitochondrial incompatibility, harmony is eventually restored by either one of two types of mitochondrial changes. Other cases of incompatibility between nuclear and cytoplasmic genomes have been reported. However, they have been observed only between different species, in Paramecium (BEALEand KNOWLES 1976) and in several plants between a given chloroplastic genome and an interspecific hybrid nucleus (for review, see TILNEY-

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BASSET1975; GILLHAM1978). To our knowledge, only one case of a chloroplast mutation suppressing incompatibility with a hybrid nucleus has been described (MICHAELIS 1969). The M A type of mitochondrial change, which may correspond to an “adaptation” of the M + mitochondria to the cZ,/cl, nucleus or to a new type of mutation, is of particular interest. Whatever the molecular basis of these mitochondrial changes responsible for the restoration of harmony with a specific nuclear gene, they emphasize the very close integration of the nuclear and mitochondrial genetic systems within eukaryotic cells. W e are grateful to J. BEISSONfor advice throughout this work and for helpful comments on the manuscript. We also thank R. CHANETfor critical reading of the manuscript. LITERATURE CITED

ADOUTTE,A., 1974 Mitochondrial mutations in Paramecium: Phenotypical characterization and recombination. pp. 263-271. In: The Biogenesis of Mitochondria. Edited by A. KROON Academic Press, London. and C. SACCONE, ADOUTTE,A. and J. BEISSON,1972 Evolution of mixed populations of genetically different mitochondria in Paramecium aurelia. Nature 235 : 393-396. 1978 Physiological consequence of mitochondrial antibioticADOUTTE, A., and J. DOUSSIERE, resistant mutations i n Paramecium. Molec. Genet. 161: 121-134. and A. SAINSARD-CHANET, 1979 Absence of detectable mibchonADOUTTE, A., J. K. KNOWLES drial recombination in Paramecium. Genetics 93 : 797-831. BEALE,G. H. and J. KNOWLES,1976 Interspecies transfer of mitochondria in Paramecium aurelia. Molec. gen. Genet. 14.3: 197-201. BEISSON,J., 1977 Non-nucleic acid inheritance and epigenetic phenomena. In: Cell Biology. Academic Press 1: 375421. 1969 The Krst case of linkage in Paramecium aurelia. Genet. BEISSON,J. and M. ROSSIGNOL, Res. 13: 85-90. BEISSON,J., A. SAINSARD, A. ADOUTTE, G. H. BEALE,J. KNOWLES and A. TAIT,1974 Genetic control of mitochondria in Paramecium. Genetics 78: 403-413. BEISSON,J. and T. M. SONNEBORN, 1965 Cytoplasmic inheritance of the organization of the cell cortex in Paramecium aurelia. Proc. Natl. Acad. Sci. U.S. 53 : 275-282. BIRRY,C. W. JR.,1978 Transmission genetics of mitochondria and chloroplasts. Ann Rev. Genet. 12: 471-512. GILLHAM, N. W., 1978

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J. K., 1972 Observations on two mitochondrial phenotypes in single paramecium KNOWLES, cells. Exp. Cell Res. 70: 223-226. -, 1974 An improved microinjection technique in Paramecium aureZia. Exptl. Cell Res. 88: 79-87. KOIZUMI,S., 1974 Microinjection and transfer of cytoplasm i n Paramecium. Ekptl. Cell Res. 88: 7478. MICHAELIS,P., 1969 Uber Plastiden-Restitutionen (Ruckmutationen). Cytologia M Suppl.: 1-115.

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T. M., 1970 Methods i n Paramecium research. pp. 241-309. In: Methods in Cell SONNEBORN, Physiology. Edited by D. PRESCOTT, Academic Press, New York. -, 1975 The Paramecium aurelia complex of fourteen sibling species. Trans. Amer. Mic. Soc. 94: 155-178. TILNEY-BASSET, R. A. E., 1975 Genetics of variegated plants. pp. 266-308. In Genetics and Biogenesis of Mitochondria and Chloroplasts. Edited by C. W. BIRKY,JR., P. S. PERLMAN and T. J. BYERS.Ohio State University Press: Columbus. Corresponding editor: J. E. BOYNTON