nucleus cll/cl 1 is able to distinguish between M r and ..... 110 fissions. 130 fissions. 2R 8R(2-3) 5S 10R(2-3) 14R 14R(2-3) 2R 7R(2-3) 1R 8R(2 3) 5R 10R(2-3).
[ -R3
Molec. gen. Genet. 145, 23-30 (1976)
© by Springer-Verlag 1976
Gene Controlled Selection of Mitochondria in Paramecium
-k
Annie Sainsard-Chanet Centre de G~n~tique MoI~culairedu C.N.R.S., F-91190 Gif s/Yvette (France)
Summary. The slow growing mutant cl1 of Paramecium, previously described (Sainsard, Claisse and Balmefrezol, 1974) differs from wild-type by a single recessive nuclear mutation and by a particular mitochondrial phenotype (M d) that gene cl 1 distinguishes from the wild-type mitochondrial phenotype (M+). A further analysis of these nucleo-mitochondrial interactions was carried out by confronting the genes cla and cl~ with mixed populations of M + and M c~ mitochondria obtained after cytoplasmic exchange at conjugation. The following results were obtained: 1. M + and M ~ mitochondria introduced respectively into mutant and wild-type cells do not multiply easily; 2. when a mixed population ( M + + M ~l) is established, both mitochondrial types are maintained during the growth of the F 1 heterozygous Cll/cl ~ clones; 3. when the nuclear segregation occurs in F2, the formation of homozygotes cll/cl 1 or c l ( / c l [ is soon followed by the segregation of the two mitochondrial types, M cl or M +, reconstituting the two parental nucleo-mitochondrial associations.
if this phenomenon clearly indicates that the mutated nucleus cll/cl 1 is able to distinguish between M r and M c~ mitochondria, the basis of the difference (genetic or not) between these two mitochondrial types is not known and is not clarified by the fact that the cll/cl 1M ÷ cells slowly evolve towards a cll/cl~M °1 phenotype, as if M + mitochondria were replaced by or transformed into Md-like ones. In order to analyze further these nucleo-mitochondrial interactions, we have studied cl~/cl[ heterozygotes containing the two types of mitochondria and followed the evolution of these mixed mitochondrial populations in the F 2 cla/cl 1 and cl~-/cl~ homozygotes obtained after autogamy of these heterozygotes. These experiments have led to two main conclusions: 1. the two mitochondrial types M + and M ¢1 are stable and can coexist in F~ heterozygous clones; 2. in F 2, the cI1/cl 1 and cl~-/cl[ homozygotes obtained from mixed (M ÷ + M cl) heterozygous clones become rapidly pure for M °l or M r mitochondria respectively, that is each homozygous nucleus selects "its" own mitochondria. The significance of this active gene-controlled selection of mitochondria is discussed.
Introduction A strong incompatibility between a nuclear mutation (cll) and wild-type mitochondria has been described in Paramecium (Sainsard et al., 1974). This incompatibility is regularly observed in the progeny of crosses between the cl 1 mutant and wild-type. In the F 2 of such crosses, homozygous nuclei cla/cll can be recovered associated with mitochondria from either wild-type (M r) or m u t a n t (M cl) origin. While clx/cl 1 M ¢1 cells have the parental cl 1 phenotype, the cll/cllM ÷ cells display a severe disorganization of their mitochondria and a very reduced growth rate. However, * This paper is dedicated to Professor T. M. Sonneborn on the occasion of his 70th birthday
Materials and Methods
Strains All the strains used in these experiments originated from the wildtype strain (stock d4-2) of Paramecium tetraurelia, according the new nomenclature (Sonneborn, 1975) and formerly Paramecium aurelia, syngen 4. clI is a slow growing mutant; its growth rate at 27°C is 3 fissions a day instead of 4-5 for wild-type. When associated with M+ mitochondria, the growth rate of the cll/cl 1 cells falls down to 1-2 fissions a day. ts 401 is a thermosensitive nuclear mutant (Beisson and Rossignol, 1969); cells homozygousfor ts 401 die at 36 °C within 48 hours whereas wild-type cells grow as well at this temperature as at 27 °C.
24
A. Sainsard-Chanet: Gene Controlled Selection of Mitochondria in Paramecium
E~, ER02, and ER01 are mitochondrial erythromycin resistant mutants. Mutations E R and ERo2 were selected from wild-type (Adoutte, 1974), E2aol from the cl 1 mutant.
cll /c11,
I
Growth Conditions Growth conditions have already been described (Sonneborn, 1970). Cells were cultured at 27 °C in a "Scotch grass" infusion bacterized with Aerobacter aerogenes. Their resistance or sensitivity to erythromycin was tested by putting them in bacterized medium containing 150 7/ml erythromycin: the multiplication of sensitive cells is blocked whereas resistant ones grow normally.
®
®
® E
Crosses Crosses and genetic analysis were performed as previously described (Sonneborn, 1970; Sainsard et al., 1974). Pairs that remained united by a cytoplasmic bridge after fertilization were selected for study. The presence of a bridge indicates that cytoplasm, including mitochondria, is exchanged between the mates.
N
N
1/
\x
11
ii/
\\
clones
~
[
V
~\
autogamy
]
---F
....
T ---
........
0
Results
As previously pointed out, mitochondria from mutant and wild-type cells ( M cl and M +) differ by their "compatibility" or their "incompatibility" with the cll/cl 1 genotype. Besides this feature, mitochondria can also be labelled by mutations giving resistance to antibiotics such as erythromycin (ER). Therefore, four categories of mitochondria, phenotypically distinguishable, are available: M + E s, M + E R, MolE s, MOlER. Two types of crosses were carried out: cll/cl~MdE s x cl+/cl+M+ER a n d Cll/CllMClg R x cl~/cl+M+E s and all the pairs having shown a cytoplasmic bridge were analyzed as illustrated in Figure 1. The two ex-conjugants were first allowed to divide once in normal medium and then, one cell was put in erythromycin, the other one in normal medium. The cell in erythromycin indicated if the E R mitochondria received by the sensitive ex-conjugant could transform its progeny into E R cells. The sister cell grown in normal medium yielded a F 1 clone from which, after autogamy, the F 2 cells were tested for their mitochondrial composition. This study revealed two original phenomena: 1. the low efficiency of transformation from erythromycin sensitivity towards resistance of the Cll/cltMclE s exconjugant t by M+E R mitochondria; 2. the specific recognition and selection by each allele, cl~ and cl +, of "its" own mitochondria, M cl or M +. 1 At the end of conjugation between two strains, the two members of each pair separate and are called ex-conjugants. By convention, to make clear the parental origin of each of them, they are still named by their parental genotype. For instance in the cross cl+/cl+M+ERxelJcllM~lEg, the two ex-conjugants (both heterozygous cll/cl~) are respectively called cl+/cl~ M + E R ex-conjugant and cll/cl ~ MClEs ex-conjugant
r
x
/
,,
\x
~,dor,%,
t
~
x
/
\
',\/ /
\
Fig. 1. Analysis of a cross with cytoplasmic exchange between the
cl1 mutant and wild-type. The two homozygous nuclei, Cll/Clt and cl~/cl~, are respectively represented by the symbols o and o, the heterozygous nucleus by e and the two types of mitochondria, M d and M +, by o and o. At the end of conjugation, some pairs develop a cytoplasmic bridge through which cytoplasmic material is exchanged between the two mates. After a variable time, the bridge is broken and the two conjugants separate. After they had undergone one post-conjugal fission, one cell of each ex-conjugant was placed into erythromycin (E), the other kept in normal medium (N) in which it gave rise to a F1 clone. About 20 generations after conjugation, autogamy was induced by starvation of the Ft clones and 50 % of homozygous cl~/cll, 50% of homozygous cll+/cl~ Fz cells were obtained from each ex-conjugant. These F 2 clones were maintained in normal medium and tested repeatedly for their resistance or sensitivity to erythromycin; for that, one cell of each clone was subcloned in normal medium, an other cell placed in erythromycin.
I. Low Efficiency of Transformation of the Cll/Cl1MOlEs Ex-conjugant by M +E R Mitochondria The process of transformation from erythromycin sensitivity to erythromycin resistance of sensitive cells receiving some E R mitochondria has been extensively studied. It has been shown that, in crosses between two st~rains of wild-type nuclear genotype, whenever a sensitive ex-conjugant undergoes a cytoplasmic exchange with its resistant partner and is placed in a medium containing erythromycin, it becomes resistant after a lag of 2-4days (Adoutte and Beisson, 1972; Perasso and Adoutte, 1974).
A. Sainsard-Chanet: Gene Controlled Selection of Mitochondria in Paramecium
25
Table 1. Efficiency of the transformation from erythromycin sensitivity to resistance of the E s ex-conjugant in crosses with cytoplasmic exchange between E Rand E s strains. Each o f the 3 types of crosses has been carried out several times and all the pairs studied in each of them have been grouped. The determination of the ability of the E s ex-conjugant to become resistant in erythromycin is explained in the text. The figures in parentheses in the lower line of the table indicate the lag in days before the sensitive cells became resistant
Cross
cl~/cl 1MO~ER x cl+/cl; - M + E s
cll/cll M~lEs x cl[ /cl~- M + E ~
R M ¢1E2m Cross No. No. of pairs with cytoplasmic bridge No. of E s ex-conj u g a n t s which transformed to resistance
1
2
3
4
5
6
16
47
29
4
6
2
3
0
0
0
(I0)
(I2)
cll/cll M~lE~ol x cll/cll M¢lE s
MeiER02
Total
1
2
3
1
Total
13
115
5
10
38
8
61
0
5
54
2
9
36
7
(3-4)
(3-4)
(3-4)
(3-4)
1
2
Total
21
15
36
8
7
15
(7-8)
(7-8)
Table 2. Analysis of the F 2 progeny of crosses without cytoplasmic exchange between cll/cllM~E s and cl+/cl+M+E R and between Cll/cl~M~lERo~ x cl~/cl+M+E s. For each of the two crosses, the two columns derived from the cl I ex-conjugant, represent the segregation in F2 within this exconjugant clone, of the two homozygous genotypes, clffcl~ and cl~/cl~, respectively designated by cl~ and cl +. In the same way, the two columns derived from the cl~ ex-conjugant represent the segregation in F 2 of the two h o m o z y g o u s genotypes within this ex-
conjugant clone. The growth rate is expressed in number of fissions per day in normal medium at 27°C, S=erythromycin sensitivity, R = erythromycin resistance Cross
cll/cl~MO~Es x cl[/cl?M+E~
Cytoplasmic origin of the ex-conjugant
cl~
cli/cl~M~'E~o~ x cl~-/cl~-M+E s
cl~
cl~
cl~
F 2 genotypes
cl~
cl 1
el~
cl 1
cli~
cl 1
cl~
cl 1
N u m b e r of cells
16
14
14
16
16
20
12
18
Growth rate 5 fissions after a u t o g a m y Phenotype in erythromycin 5 fissions after a u t o g a m y Phenotype in erythromycin 130 fissions after a u t o g a m y
4-5 S S
3 S S
4-5 R R
1-2 R R
4-5 R R
2-3 R R
4-5 S S
1-2 S S
The same process was studied for the 3 following crosses: ell/cllMClE s x cl[/cl[ M + E R, cll/cllMClE R x cl[ /cl~ M + E s cll/cllMClESx Cll/cllMClE R. The results are given in Table 1. The first type of cross between the cl 1 mutant whose mitochondria are E s with a wild-type strain carrying E R mitochondria was carried out 6 times and altogether 115 cll/cl~MdE s ex-conjugants which had received some M ÷ EaR mitochondria from their partner were placed in erythromycin: 110 remained sensitive and only 5 became resistant. Moreover, an unusually long lag (10-12 days) was necessary for these 5 cells to resume growth in erythromycin and no correlation between the acquisition of the resistance and the duration of the cytoplasmic exchange was observed: the 5 cells which became resistant had undergone a short bridge (1 min), whereas some of the ll0cells which remained sensitive underwent a much longer exchange. The second type of cross between an erythromycin resistant cl~ strain and an erythromycin sensitive wild-type strain was carried out with two different cll/cI 1 strains: one carried the E~o~ mutation, the other, the E~o2 one. 6 1 c l ? / c l ? M + E s ex-conjugants
which had received some MC1ER mitochondria were studied: 54 became resistant after a lag of 3-4 days, only 7 remained sensitive. The third type of cross between two Cll/Cl1 strains, one sensitive, the other resistant to erythromycin, was carried out twice. Out of the 36 clt/cllMdE s ex-conjugants which had received MdE R mitochondria, 15 became resistant in erythromycin after a lag of 7-8 days. In summary, Table 1 shows that in a cl~/cl~ exconjugant, M°IER mitochondria can multiply (as M ÷ E R ones do) whereas in a cll/cl 1 ex-conjugant, MolE R mitochondria multiply only in 50 % of the cases and M + E R mitochondria practically never. It seems therefore there is a correlation between the efficiency of transformation and the nuclear background into which E R mitochondria are introduced: the transformation of an ex-cl~/cl~- cell is easy, that of an exCll/cl I one is difficult and this difficulty increases when the "invading" mitochondria are M ÷.
II. Selection by Each Allele ( cl I and cl +) o f " I t s " Own Mitochondria M cl and M + This phenomenon was revealed by the study of the progeny of the F 1 cells grown in normal medium
26
A. Sainsard-Chanet: Gene Controlled Selection of Mitochondria in
of the Cll/claM °l ones, 3 or 2-3 fissions depending on the E s or E R genotype of M ~ mitochondria. These results clearly show that the four mitochondrial types are easy to identify in F 2 clJcl2 cells on the basis of the two criteria: resistance or sensitivity to erythromycin and growth rate as summarized in Table 3. These different phenotypical characteristics may therefore allow the identification of the type(s) of mitochondria present in F 2 cells derived from conjugants which have exchanged cytoplasm and whose mitochondrial composition after this exchange (pure for M ÷ or for M d, or mixed) cannot be predicted a
(Fig. 1). These F a clones gave rise after autogamy to 50~o of cll/cl I and 50~o of cl~/cl~ cells. Before describing the results obtained when a cytoplasmic exchange has occured between the two conjugants, those obtained in the absence of exchange are given in Table 2. All the F z clones, regardless of their genotype, derived from the resistant parent were resistant and remained resistant over 130 generations of growth in non-selective medium; all the F 2 clones derived from the sensitive parent were sensitive. The growth rate of the cl~/claM + cells was 1-2 fissions a day, that
priori.
Table 3. The phenotypes of cl~/cl~ cells according to the mitochondrial type they contain. The growth rate is expressed in number of fissions per day in normal medium at 27°C; S = e r y t h r o m y c i n sensitivity, R = erythromycin resistance Cellular phenotype
Growth rate Phenotype in erythromycin
a) Studies on Autogamous Cells Containing the Two Types of Mitochondria. The heterozygotes cll/cl +, formed in the crosses cll/cllMclESxcl~/cl~M+E~ and cljcllMClE~ol x cl~/cl~M+E s, may contain the
Mitochondrial type MalE s
M~IER
M+E s
M+E k
3 S
2-3 R
1-2 S
1-2 R
Paramecium
two types of mitochondria (MdES + M + E~ or ]~[clFR ~,~ ~201 + M + E s) if cytoplasmic exchanges have occured. Regarding these two categories of mitochondria M + and M d, it cannot be predicted whether they will be
Table 4. Evolution in F 2 of mixed mitochondrial populations M d E s + M + E~R obtained in crossing cll/cl~M~lEs with cl~/cl~M+E~. In this table are given two phenotypic characteristics (resistance or sensitivity to erythromycin and growth rate) of the F 2 cells derived from 6 cl~/cl~E R ex-conjugant clones. Each column represents the segregation in F z within a same ex-conjugant F~ clone, of the two homozygous genotypes, cl~/cl~ and cl+/cl~, respectively designated by cl~ and cl~. R indicates the erythromycin resistance; S, the erythromycin sensitivity; r indicates cells rift sensitive and becoming resistant after a lag of 5-7 days in erythromycin. Growth rates (in number of fissions per day at 27 °C, in normal medium) are indicated in parentheses. The duration in minutes (min) of the cytoplasmic bridge at conjugation is indicated
cl~/cl~ ex-conjugant
no. 1
no. 2
no. 3
no. 4
no. 5
no. 6
Duration of cytoplasmic bridge
1 min
1 min
1 min
1 rain
1 min
1 min
F 2 genotypes
cl~
cl I
cl~
cll
cl~+
cl 1
cl~-
cl 1
cl~
cll
cl~-
cl~
No. of F 2 clones studied
6
8
13
6
20
22
25
19
16
14
30
20
6R
4r(3) 4S(3)
13R
3r(3)
20R
25R
3R(1 2) 6r(3) 10S(3)
16R
14S(3)
30R
26S(3)
8S
13R
25R
3R 16S
16R
14S
20R 3r 7S
26S
Phenotype after: 5 fissions
3S(3)
8R(1-2) 9r(3)
5S(3) 15 fissions
6R
6S
20R
8R 14S
30 fissions
50 fissions
7R 5r 18S 6R
8S
70 fissions
13R
6S
25R
20R
3R 16S
16R
14S
8R 14S
4R lr 25S
90 fissions
1R 29S
110 fissions 130 fissions
5R lr 24S
25R 6R
8S
3R 16S
30S 16R
14S
A. S a i n s a r d - C h a n e t : G e n e C o n t r o l l e d Selection o f M i t o c h o n d r i a in Paramecium
maintained in the F~ clones until recovery in the F 2 cells. Regarding the E R and E s markers, it has been shown previously that in the absence of erythromycin, the E s mitochondria have a selective advantage over the E R ones (Adoutte and Beisson, 1972). The E Rexconjugants kept in normal medium were therefore expected to be enriched in E s mitochondria and to be mixed when autogamy occurs, about 20 fissions after conjugation. Mixed autogamous cells were easily obtained from the cross cll/claM~lERoixcl~/cl~-M+ES" out of 15 clones derived from the cll/cllM d E2o R 1 ex-conjugant, 13 were mixed (M d E R 2 0 I + M + E s) and 2 were pure for MdvR ~ 2 0 1 mitochondria. In contrast, very few mixed a u t o g a m o u s cells were obtained from the cross cI~/cllMdE s x cl+/cl+M+ER" out of 58 clones derived from the cl/-/cl~M+E~ ex-conjugant, only 6 were mixed (M~1ES+M+E~), the 52 others were pure for M + E~ mitochondria. The significance of this difference will be discussed later.
b) Fate of the Two Mitochondrial Types, M + and M cz, in F2 Homozygotes. The analysis of the F 2 progeny derived from the 6 clones mixed for M+E~ and M~E s mitochondria and from the 13 clones mixed for M + E s and M ~'~ ¢~FR ~ 2 0 1 mitochondria is given in Tables 4 and 5. The significant results are the following: 1. Both E R and E s mitochondria coexist till autog a m y in these 6 and 13 ex-conjugant clones since both E R and E s cells are recovered in the F 2 progeny from each of them. 2. Both M + and M *~ mitochondrial types coexist till autogamy in the ex-conjugants clones nos. 3 and 4 of Table 4 and no. 10 of Table 5 since two types of Cll/cl I homozygotes are recovered in their F 2 progeny: the ones with a growth rate of 3 or 2-3 fissions a day are identical to that of cla/cl~M °l cells; the others with a growth rate of 1-2 fissions a day are identical to that of cla/cllM + cells. This result suggests that both M c~ and M + mitochondria can coexist and maintain their respective characteristics in the same cytoplasm through 20 fissions, in presence of a heterozygous nucleus cll/cl ~. 3. The parental associations between the mitochondrial type M ÷ o r M ~1 and the E R or E s marker are quite stable until autogamy. This is shown by the phenotype of the cl~/cl~ cells: when the parental mitochondria were respectively M d E s and M+E~R (Table 4), the mitochondria found in the F 2 cll/cl 1 cells were either E s and M cl (3 fissions a day) or E R and M + (1-2 fissions a day); when the parental mitochondria were respectively ~'~ M°IF ~ 2R 0 1 and M + E s (Table 5), the mitochondria found in the F 2 cl~/cl 1 cells were either E R and M ~1 (2-3 fissions a day) or E s and M ÷ (1-2 fissions a day).
27
The o n l y a p p a r e n t exceptions are the few cla/cl t clones in Table 4 which had a growth rate of 3 fissions a day and were not erythromycin sensitive; they are designated by " r " in the table. In the first test (5 fissions after autogamy), the cells of these clones were initially blocked in erythromycin but became progressively resistant after a lag of 5-7 days, which is typical behaviour for cells containing a mixture of E R and E s mitochondria. In a second test (15 fissions after autogamy), the cells were pure for MC~Es mitochondria. Therefore, these clones r did not contain "'recombin a n t " MClE~ mitochondria but a mixture of the two types, M + E R 1 and MOlEs Although only the E a or E s character and not the M d or M ÷ mitochondrial type is detectable in the F 2 cl~/cl~ cells, it is reasonable to extrapolate from the results obtained with the cll/cl 1 clones and assume that the parental associations between the two mitochondrial characters ( M + / M ~1 and ER/E s) are also maintained in the F 2 homozygous cl+/cl~ cells. 4. The segregation of the nuclear genotypes cll/cl~ and cl+/cI~ is followed by the segregation of the mitochondrial markers E s and E R. Table 4 shows that as soon as the 5th post-autogamous fission, all the cl~-/cll~clones were erythromycin resistant (R = 110/110). 80 of these were still resistant after 130 generations in normal medium and were therefore pure for E R mitochondria; only 30 clones, all derived from the same ex-conjugant no. 6, became sensitive between the 15 th and the 110 th generation and therefore contained some E s mitochondria which were selected through growth in normal medium. Conversely, 5 fissions after autogamy, the majority (62/95) of the cll/cl 1 clones were pure for sensitive mitochondria or contained a few resistant mitochondria which were lost 10 generations later (r=22/95). Only 11 cll/cl 1 clones out of the 95 ones were pure for resistant mitochondria (they remained resistant after 130 generations in normal medium). Similarly, Table 5 shows that by the 5th postautogamous fission, 54 out of 92 cl~/cl( clones were pure for sensitive mitochondria, the 38 others still contained some E R mitochondria (35 R, 3 r), but they lost them during the 10 following divisions. Therefore by the 15 th postautogamous fission, all the cl/-/cl~clones (92/92) were pure for sensitive mitochondria. Conversely, 5 fissions after autogamy, 108 cla/cl 1 clones out of 112 were erythromycin resistant, 106 of which were pure for E R mitochondria, only 2 (derived from the ex-conjugant no. 9) were mixed E R + E s. They became sensitive between the 7 0 th and the 9 0 th generation. Only 4cll/cl 1 clones were pure for sensitive mitochondria as soon as the 5th postautogamous fission. Since, as previously discussed, the parental associa-
A. Sainsard-Chanet: Gene Controlled Selection of Mitochondria in Paramecium
28
R I + M+E s obtained in crossing cll/cllMdE~oa with cl+/cl(M+Es for Table 5. Evolution in Fz of mixed mitochondrial populations M c~Ezo no. 2
no. 3
no. 4
no. 5
no. 6
Duration of cytoplasmic bridge 45 rain
40 rain
30 rain
30 rain
30 ruin
10 rain
F2 genotypes
Cll+ clI
el(
cl1
cl(
clI
Cll+ cl1
cl+
cl~
cl+
el1
No. of F2 clones studied
7
8
5
10
15
14
8
7
7
8
5
10
2R 5S
8R(2-3)
5S
10R(2-3)
14R lr
14R(2-3)
7R(2-3)
1R 6S
8R(2 3)
5R
10R(2-3)
7S
8R
5S
10R
15S
14R
2R lr 5S 8S
7R
7S
8R
5S
10R
cll/cl I ex-conjugant
Phenotype after: 5 fissions
15 fissions 30 fissions 50 fissions
no. I
8R
10R
14R
7R
8R
10R
70 fissions 90 fissions 110 fissions 130 fissions
7s 8R
5S
10R
tions between the mitochondrial characters ( M + / M ~1, ER/E s) are m a i n t a i n e d in F 1 a n d r e c o v e r e d in 172, the r a p i d s e g r e g a t i o n of the E R a n d E s m a r k e r s in the F 2 cells c o r r e s p o n d s to a s e g r e g a t i o n of the M ÷ a n d M ~ m i t o c h o n d r i a l types. A l t o g e t h e r , these results i n d i c a t e therefore t h a t in b o t h crosses a n d in the large m a j o r i t y of the cases, a c y t o p l a s m c o n t a i n i n g in F 1 the t w o types of m i t o c h o n d r i a , M el a n d M +, evolves differently a c c o r d i n g to the nucleus r e c o v e r e d in F 2. In presence of a cl~/cl I nucleus, it b e c o m e s p u r e for M °~ m i t o c h o n d r i a , in presence of a cl~/cl~ nucleus, it b e c o m e s p u r e for M + m i t o c h o n d r i a regardless of their
E s or E g character. Discussion
T h e p u r p o s e of this s t u d y was to a n a l y z e further the p h e n o m e n o n o f r e c o g n i t i o n by gene cll o f the two m i t o c h o n d r i a l types M .1 a n d M ÷. The m u t a n t a n d w i l d - t y p e alleles clI a n d cl~- were c o n f r o n t e d with m i x e d p o p u l a t i o n s of M ÷ a n d M c~ m i t o c h o n d r i a a n d the s u b s e q u e n t n u c l e o - m i t o c h o n d r i a l i n t e r a c t i o n s s t u d i e d in F 1 h e t e r o z y g o t e s cll/cl + a n d in F 2 h o m o zygotes cll/cl t o r cl~/cl~. T h e s e e x p e r i m e n t s h a v e s h o w n t h a t 1. the m i t o c h o n d r i a l exchanges b e t w e e n the cl 1 m u t a n t a n d wild-type, necessary for the o b t e n t i o n of m i x e d m i t o c h o n d r i a l p o p u l a t i o n s ( M ÷ + MC~), a r e difficult; 2. once a m i x t u r e of the two types of m i t o c h o n d r i a is o b t a i n e d in F1, b o t h types a r e m a i n t a i n e d over 20 cell g e n e r a t i o n s in a h e t e r o z y g o u s
15S
14R
8S
7R
7S
8R
5S
10R
clx/cl ~ c o n t e x t ; 3. when a u t o g a m y occurs in these m i x e d clones ( M d + M+), the n u c l e a r s e g r e g a t i o n into h o m o z y g o t e s cla/cl a a n d cl+/cl~ is generally a c c o m p a n i e d b y the s e g r e g a t i o n of the two m i t o c h o n d r i a l types, s o o n yielding the reconstitution of the original parental nucleo-mitochondrial associations. These results will be discussed successively.
1. Difficulty of the Mitochondrial Exchanges between the cllMutant and Wild-type. I n two instances, the " i n v a d i n g " m i t o c h o n d r i a d i d n o t easily succeeded in m u l t i p l y i n g in the r e c i p i e n t e x - c o n j u g a n t : M + E R m i t o c h o n d r i a succeeded in m u l t i p l y i n g in the Cll/cl 1 MOlEs e x - c o n j u g a n t o n l y in 5 cases out of 115 d e s p i t e the selective pressure of e r y t h r o m y c i n a n d M°IE s mitochondria succeded in m u l t i p l y i n g in the cl+/cl+M+E R e x - c o n j u g a n t o n l y in 6 cases o u t of 58. This is r e m i n i s c e n t of the difficulty of m i t o c h o n d r i a l e x c h a n g e s b e t w e e n different species of P a r a m e c i a r e p o r t e d by K n o w l e s (1973) a n d Beale a n d K n o w l e s (1976). In c o n t r a s t , M~IE k m i t o c h o n d r i a were o b s e r v e d to i n v a d e in m o s t o f the cases (54/61) a cl+/cl+M+E s e x - c o n j u g a n t p l a c e d in e r y t h r o m y c i n a n d M + E s m i t o c h o n d r i a were o b s e r v e d to m u l t i p l y in m o s t o f the cases (13/15) in a cl~/cl~M~lE R ex-conjugant. These results seem c o n t r a d i c t o r y a n d no simple h y p o t h e s i s c a n a c c o u n t for all o f them. H o w e v e r , it m u s t be p o i n t e d o u t that w h e n the c y t o p l a s m i c exchanges occur, the m i t o c h o n d r i a are transfered into a cell which still retain its p a r e n t a l p h e n o t y p e . If, as
A. S a i n s a r d - C h a n e t : G e n e C o n t r o l l e d Selection o f M i t o c h o n d r i a in
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Paramecium
legends, see T a b l e 3. no. 7
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discussed below, the cl~/cl( and cl~/cl 1 nuclei respectively counterselect M ¢1 and M ÷ mitochondria, it can be expected that exchanges of these two types of mitochondria are difficult. However, the counterselection of the "invading" M cl mitochondria by the cI~/cl~ nucleus might be overcome in erythromycin if these ones carry an E R mutation. In contrast, the negative effect of the cll/cl I nucleus upon "invading" M ÷ mitochondria would be still greater if these ones carry an E R marker since any E R mutation has a deleterious effect per se on the cll/cla strain (reduced growth rate of the cljcll M~E R strains, low efficiency of transformation of the Cll/cl 1 MdEs cells even by M*IER mitochondria). However, the mechanisms which govern the mitochondrial exchanges between the cl a mutant and wild-type are probably more complicated than the only conterselection of the "invading" mitochondria because this simple fact does not explain the frequent recovery of M + E s mitochondria in the cll/cllM¢lE R ex-conjugants.
2. Maintenance of the Two Mitochondrial Types in Heterozygous cl~/cl~ Clones. A detailed study of the stability of the two mitochondrial types in heterozygotes will be reported elsewhere but the results reported here already show that once a mixed population is established in F1, it is generally stable over 20 cell generations. Only a few exceptions to this rule have been observed: out of the 205 clones mixed for M + E~R and MolES mitochondria, llcl~/cl 1 clones
5S
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appeared immediately pure for M + E IR and out of 204 clones mixed for M + E s and l,~ IDIclFR ~ 2 0 1 mitochondria, 4 cl~/cI~ clones appeared immediately pure for M + E s mitochondria. It seems therefore that these 15 cl 1/cl I clones derived from a u t o g a m o u s cells already pure for M ÷ mitochondria, that is to say the M d mitochondria had been eliminated during the F 1 growth. However, these cases are rare and it can be concluded that the two types of mitochondria are generally maintained in heterozygous cells.
3. Selection by Each Homozygous Nucleus, Cll/Cla and cl[ /cl~, of "Its "' Own Mitochondria M d and M +. This phenomenon is clearly established from the results reported in this paper and in this case also, only rare exceptions to the rule have been observed. Out of the 205 clones containing a mixture of M + E R and M~IEs mitochondria, 30 cl~/cl~ clones (all derived from the same ex-conjugant) became erythromycin sensitive whereas they were expected to become rapidly pure for M + E R mitochondria. O f the 204 clones containing a mixture of M + E s and M°IER o 1 mitochondria, 2 Cll/cll clones (derived from the same ex-conjugant) also became sensitive whereas they were expected to become rapidly pure for M°I• l,~ ~ 2R 0 1 mitochondria. This could be due to an incomplete elimination of the counterselected mitochondria during the first F 2 generations. Indeed it is known that the M c' mitochondria can be "transf o r m e d " into M + ones in cI+/cl + cells (unpublished results) and the M + into M cl ones in cll/cl 1 cells
30
A. Sainsard-Chanet: Gene Controlled Selection of Mitochondria in Paramecium
(Sainsard et al., 1974). Therefore, if the elimination of the MC~Es mitochondria in mixed cl(/cl~- cells (M°1ES+M+E ~) is not complete during the first few F2 post-autogamous fissions, these mitochondria may be transformed into M ÷ E s. These cells, now mixed for M + E R and M ÷ E s mitochondria, are expected then to evolve towards erythromycin sensitivity in normal medium. Similarly, cll/cl 1 cells, initially mixed for MclER and M + E s mitochondria, will evolve towards erythromycin sensitivity if M+E s mitochondria are transformed into MalE s ones. The two ex-conjugants from which derived the 30cl+/cl( clones and the 2cll/cl 1 ones perhaps contained respectively more MdE s and M + E s mitochondria than the others. Two other hypotheses could account for these few exeptions: a reversion of the E R mutation or a recombination between M+E~ and M~JEs yielding M + E s mitochondria in the first case, between M+E s and M~E R yielding MalE s mitochondria in the 2 "d case. These last two hypotheses cannot be ruled out but they are unlikely because 1. the mutation E~ and EZROl have never been observed to revert, and 2. recombinant mitochondria, easy to detect during the first F 2 generations in the Cll/cl I cells, have never been observed. Anyway, these few exceptions do not challenge the general rule of selection by each homozygous nucleus of "its" mitochondria. This phenomenon is remiscent of that described by Tilney-Basset (1973) in Pelargonium where two alleles of a nuclear gene respectively favour the replication of wild-type and mutant chloroplasts.
4. The Mechanism of the Gene-controlled Selection. The rapid elimination of the "incompatible" mitochondrial type M d or M + in cl+/cI + or Cll/Cl~ homozygotes opposed to the stability of these two types in cll/cl ~ heterozygotes indicates (1) that the maintenance of the mitochondrial phenotypes M ~ and M + is dependent on the expression of the cl~ and cl( genes and (2) that the products of these two alleles cl~ and cI + are active in heterozygotes. The simplest hypothesis to account for these results is to assume that the genes cl~ and cl( act directly on the mitochondria in providing products involved either in replication, transcription, translation or methylation of the mitochondrial D N A or in the specificity and (or) organization of the membranes as previously suggested (Beisson et al., 1974). However,
it is not possible to exclude an indirect control of the two genes on the mitochondria, for instance by modification of the physiological conditions. In any case, the actual mechanism of this nuclear control cannot be understood until the basis (genetic, structural or only physiological) of the difference between M ÷ and M cl phenotypes is clarified. Experiments aiming at solving this point are now in progress. In any case, it is interesting to point out that the phenomenon described here of gene-controlled selection of mitochondria does not need to involve any complex regulatory mechanism but could simply result from the fact the nuclear gene provides a mitochondrial building-block. Acknowledgements. I express special thanks to Dr. J. Beisson for advice throughout this work and for many helpful comments on the manuscript.
References Adoutte, A.: Mitochondrial mutations in Paramecium: phenotypical characterization and recombination, in: The biogenesis of mitochondria, pp. 263-271. New York: Academic Press, Inc. 1974 Adoutte, A., Beisson, J.: Evolution of mixed populations of genetically different mitochondria in Paramecium aarelia. Nature (Lond.) 235, 393-396 (1972) Beale, G., Knowles, J.: Interspecies transfer of mitochondria in Paramecium aurelia. Molec. gen. Genet. 143, 197-201 (1976) Beisson, J, gossignol, M.: The first case of linkage in Paramecium aurelia. Genet. Res. 13, 85-90 (1969) Beisson, J., Sainsard, A., Adoutte, A., Beale, G.H., Knowles, J., Tait, A.: Genetic control of mitochondria in Paramecium. Genetics 78, 403 -413 (1974) Knowles, J.: Genetics of protozoa specially in relation to mitochondria. Ph.D. Thesis, University Edinburgh (1973) Perasso, R., Adoutte, A.: The process of selection of erythromycin resistant mitochondria by erythromycin in Paramecium. J. Cell Sci. 14, 475 497 (1974) Sainsard, A., Claisse, M., Balmefrezol, M.: A nuclear mutation affecting structure and function of mitochondria in Paramecium. Molec. gen. Genet. 130, 113 125 (1974) Sonneborn, T. M.: Methods in Paramecium research. In: Methods in cell physiology 4, 241-339 (1970) Sonneborn, T. M.: The Paramecium aurelia complex of fourteen sibling species. Trans. Amer. micr. Soc. 94, 155-178 (1975) Tilney-Bassett, T.A.E.: The control of plastid inheritance in Pelargonium. II. Heredity 30, 1 13 (1973)
Communicated by W. Gajewski Received January 8, 1976
