Mitochondrial Genetics of Paramecium aurelia

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 71

Mitochondrial Genetics of Paramecium aurelia G. H. BEALEAND A. TAIT Institute of Animal Genetics, Edinburgh, Scotland I. Introduction . . . . . . . . . . . . . . 11. The Mitochondrial Genome . . . . . . . . A. DNA Characteristics . . . . . . . . . B. Replication . . . . . . . . . . . . . C. Restriction Enzyme Map . . . . . . . . D. Chromatin Structure . . . . . . . . . . E. Species Differences . . . . . . . . . . III. Inheritance of Mitochondrial Characters . . . . A. Introduction . . . . . . . . . . . . B. Drug Resistance . . . . . . . . . . . C. Modifiers of the c l , Mutant . . . . . . . D. Interspecific Differences . . . . . . . . E. Complementation and Recombination Involving Genes . . . . . . . . . . . . . . . 1V. Biochemical Products of Mitochondrial Genes . A. Introduction . . . . . . . . . . . . B. Ribosomal RNA and Ribosomal Proteins . . C. Respiratory Proteins and ATPase . . . . . D. Other Mitochondrial Constituents . . . . . V. Conclusion . . . . . . . . . . . . . . References . . . . . . . . . . . . . .

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Mitochondrial

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I. Introduction Ciliate protozoa belonging to the Paramecium aurelia complex have long been known to be very suitable organisms for the study of extranuclear genetics (Sonneborn, 1947, 1974; Beale, 1954). Therefore, after the development of research on the genetics of mitochondria, mainly in yeast, it was natural to start work on this subject with Paramecium also (Beale, 1969). In recent years the yeast work has undergone a huge expansion, and many novel features have been brought to light. However it is important to establish which of these findings apply to mitochondria in general, and which to yeast, or other fungi, alone. It is evident that mitochondria1 genetic systems in different groups of organisms show considerable diversity (Borst, 1972; Beale, 1979; Mahler and Perlman, 1979; Borst, 1980), in regard both to the organization and the functions of the 19 Copyright @ 1981 by Academic Press. Inc All rights of reproducuon in any form reserved. ISBN 0-12-364471-2

20

G . H . BEALE A N D A . TAIT

mitochondrial genome. Hence it is desirable to obtain more information from organisms other than yeast. In this article we will draw attention to the differences between yeast, Paramecium. and other organisms. Admittedly the data from Paramecium are far less extensive than those from yeast, but on the other hand Puramecium provides certain types of information not readily available from other organisms. First we outline a few relevant features of Paramecium. Further details are given in earlier reviews (Beale. 1954; Sonnebom, 1974, 1975). A single cell or individual belonging to the P . aurelia complex of species, as denoted by Sonneborn (19751, normally contains two diploid micronuclei, a single macronucleus containing about 400 haploid genomes, and very approximately 5000 mitochondria. Unlike the mitochondria of Saccharomyces cerevisiae, which under certain conditions may fuse into a single large mass (Hoffman and Avers, 1973). the mitochondria of Paramecium appear to remain separate at all times. They vary in length between 1 and 12 pm, with a mean of 2-5 p m at the beginning of the cell cycle, and 4-8 pm about one-quarter of the time through the cycle, when most mitochondria divide (Perasso and Beisson, 1978). Paramecium replicates by binary fission, and mitochondria are presumed to be distributed randomly among the daughter cells. Electron microscopy shows that Puramecium mitochondria are bounded by a characteristic double membrane, and contain large numbers of tubular cristae. In general the mitochondria have the same basic structural features as are found in other organisms. During conjugation between two paramecia, haploid gamete nuclei pass from each conjugant to the other, but cytoplasmic constituents of the size of mitochondria are not usually transferred. Under special conditions however such cytoplasmic exchange can be induced (see Fig. 1). In nature conjugation occurs periodically between individuals belonging to different mating types of one species (formerly denoted variety, or syngen) of the P . aureliu complex. It is unlikely that any appreciable mixing of mitochondria from different stocks (i.e., genetically distinct individuals) of a single species occurs in nature and it is clear that mixing of mitochondria from different species does not take place at all, under natural conditions. However, heterogeneity among mitochondria within a single cell may arise as a result of mutation, and it has been shown that in the absence of selection such heterogeneity may persist for long periods (see Section 1II.E). In the laboratory mixtures of diverse mitochondria may be produced by cytoplasmic exchange at conjugation between different stocks, or by microinjection, thus making it possible to cany out mitochondrial genetic analysis. In this article, work on the mitochondria of Paramecium is considered in three sections: the mitochondrial genome (Section II), the inheritance of mitochondrial characters (Section In), the biochemical products of the mitochondrial genes (Section IV). In the final section the known facts of the Paramecium mitochondrial genetic system are compared with those of yeast and other organisms.

21

MITOCHONDRIAL GENETICS OF P. AURElJA

ER

/

ER

ES

/

ER

No cytoplasmic exchange

ES

Cytoplasmic exchange

ES

ER

ER

FIG. 1 . Diagram showing conjugation of Paramecium aurelia with and without cytoplasmic exchange. (Details of macro- and micronuclei not shown.) ER, Erythromycin-resistant; EE, erythromycin-sensitive cells.

11. The Mitochondria1 Genome

A. DNA CHARACTERISTICS The density of the mitochondrial DNA of P . aurelia (stock 513, species 1) is 1.699 gm cm-3and is clearly different from that of macronuclear DNA ( p = 1.686 gm ~ m - ~on) ,cesium chloride gradients (Flavell and Jones, 1971; Goddard and Cummings, 1975). Examination of isolated mitochondrial DNA by electron microscopy shows linear molecules of length 13.8 pm (Cummings et af., 1976).

22

G . H. BEALE AND A. TAIT

Except for another ciliate, Tetrahymena pjrijiirmis, which also has linear mitochondrial DNA (Suyama and Miura, 1968; Flavell and Jones, 1971), all organisms studied so far have circular mitochondrial genomes. This clearly makes these ciliates very exceptional, though the significance of this difference is unknown. The mass of the Paramecium mitochondrial DNA is 27.1 X lofidaltons, as determined by electron microscopy (Goddard and Cummings, 1977) or restriction enzyme digestion (Maki and Cummings, 1977). The agreement between the results obtained by these two methods of determining the mass, taken together with the value of 30-35 x lo6 daltons for the kinetic complexity (Flavell and Jones, 197 I ) , indicates that only a single type or sequence of mitochondrial DNA is present in a given stock of P. aurelia. The total quantity of DNA per mitochondrion has been estimated at 3.7 x lo-’” p g (Suyama and Preer, 1965), which is equivalent to a molecular weight of 2.4 X lo8. Hence the number of genomes per mitochondrion is of the order of 8-10. Calculation of the GC content of this DNA gives a value of 40%, which is high by comparison with that found in yeast. This value, taken together with the results of partial denaturation studies (Cummings et al., 1976), suggests that the mitochondrial genome of P . uureliu does have AT-rich regions, but these are small by comparison with those in Drosophila, yeast, and other organisms. Recently, studies on the mitochondrial DNA of Paramecium aurelia species 4 have shown results similar to those described above for species 1 (Findly and Gall, 1980).

B. REPLICATION Electron microscopy of the mitochondrial DNA isolated from actively growing cultures reveals predominantly linear molecules of length 13-15 pm, but in addition small proportions of two other types of molecule, namely, “dimers” of length 25-28 p m and “lariats” (i.e., closed circles each with a tail). Treatment of cultures with ethidium bromide results in a large increase in the percentage of lariat molecules, and since ethidium is known to inhibit DNA replication in other organisms, this suggests that these lariats are replication intermediates. Furthermore, treatment of cultures with chloramphenicol results in a considerable increase in the proportion of dimer molecules. Fiom these results a replication scheme has been proposed (Goddard and Cummings, 1975), as shown in Fig. 2. At the start, the two polynucleotide chains in the linear duplex undergo ligation at one end, after which replication begins at a site close to the ligation point, and proceeds unidirectionally. This results in the formation of the lariat. When DNA replication is complete, a “head-to-head’’ linear dimer is formed, and the latter is cleaved at the junction of the two monomers. Since chloramphenicol treatment causes accumulation of dimers, it has been suggested that their cleavage is brought about by a mitochondrially synthesized enzyme.

MITOCHONDRIAL GENETICS OF P. AUREUA

23

Linear parent molecule ~

c

~

~

I

Ligation of one end of molecule

c

Initiation of replication near site of ligation

Replication o f , both strands

I

Linear dimer

.c

-Parental

heavy strand

-- Newly formed

-Parental

light strand

--Newly

heavy strand

formed light strand

FIG.2. Replication of rnitochondrial DNA of Paramecium aureliu (after Gcddard and Cumrnings , 1975).

Several experiments of Cummings and co-workers (Cummings et af., 1976, 1979a,b; Cummings, 1977) have provided strong evidence in favor of this scheme of replication. Snap-back renaturation of monomers yields a high percentage

24

G. H. BEALE AND A. TAIT

of double-stranded monomers, suggesting that they are covalently linked at one end, and the abolition of this renaturation by prior treatment with S , nuclease further suggests that this link is single stranded. Measurements of the size of the lariat molecules in the electron microscope shows that they fit the relationship tail YZ loop = 13.5 p m which would be predicted by the scheme in Fig. 2. Evidence for the structure of the dimer molecules has come from partial denaturation, snap-back renaturation, restriction enzyme digestion, and BUdR labeling, which all suggest a head-to-head linking of the dimers followed by a splitting of this linkage and production of two monomers. Although the detailed mechanisms of the ligation of monomers and the cleavage of dimers are not known, sequence analysis of the initiation end of the molecule will doubtless throw light on these mechanisms. A mitochondrial replication complex, comprising mitochondrial DNA and some proteins, has been extracted from mitochondria by treatment with sarkosyl, implying attachment of the DNA to the mitochondrial membrane (E. Olszewska and A. Tait, unpublished). This complex has been shown to incorporate labeled nucleotides into complete length molecules and could provide an in v i m system for the analysis of the mechanism of replication and the proteins involved in this process.

+

C. RESTRICTION ENZYME MAP Restriction enzyme maps of the mitochondrial genome have been constructed for several restriction enzymes using mitochondrial DNA from stock 513, species 1 and stock 87, species 5. These maps are not, per se, of great interest unless related to the biological function of the DNA; some of these latter aspects will be considered in Sections II1,D and IV,B. The restriction maps for species 1 are presented in Fig. 3. These maps have been obtained by analysis of double and partial digests together with analysis of some cloned fragments. D. CHROMATIN STRUCTURE Unlike most other types of mitochondrial DNA, which are usually represented as being typically prokaryotic, i.e., devoid of histones, that of Paramecium forms a beaded, chromatin-like structure (see Fig. 4).This was demonstrated by electron microscope observations of the replication complex mentioned above. Electrophoresis of the basic proteins in this complex shows some four or five histone-like proteins, which are not identical with the nuclear histones of Paramecium. Endonuclease treatment confirmed the histone-like nature of these proteins (Olszewska and Tait, 1980). In view of the apparent discrepancy between these observations, and those with other organisms, the conclusion should

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FIG. 3 . Restriction endonuclease maps of mitochondria1 DNA of Paramerium aurelia species I , stock 513, showing location of 12 S and 20 S rRNA genes. (a) From P. Kelly, D. Anott, J. 0. Bishop, and A. Tait (unpublished). (b) From Cummings era!. (1980). The fragments labeled E and B refer to the restriction enzymes EcoRI and BamI. I = end of molecule at which replication is initiated, T = end of molecule at which replication is terminated.

26

G. H. BEALE AND A. TAIT

FIG. 4. Electron micrograph of the mitochondrial chromdtin-like structures of Paramecium aurelia (from Olszewska and T i t , 1980). Three different regions are observed: (A) region of regular nucleosome-like structures; (B) thin filament lacking any structures; (C) more condensed large particles.

be considered tentative at the present time, but it should be noted that somewhat similar observations of chromatin-like structures have been reported in Xenopus (Pinon et al., 1978) and Physarum (Kuroiwa et al., 1976). In yeast, on the other hand, Caron et al. (1979) have reported only a single histone-like protein which binds mitochondrial DNA, closely resembling the situation in Escherichia coli.

MlTOCHONDRlAL GENETICS OF P. AUREWA

27

E. SPECIESDIFFERENCES Differences between the mitochondrial DNAs of species 1, 4,5 , and 7 of P . aurelia ( P . primaurelia, P . tetraurelia, P . pentaurelia, P . septaurelia) have been studied by Cummings et al. (1979b). Each species shows characteristically different restriction enzyme patterns with the enzymes EcoRI and HindIIl. DNA-DNA hybridization in solution of the entire mitochondrial genomes shows that species 1,5, and 7 are closely homologous, with 100%hybridization, while species 1 and 4 show only 40% hybridization. Comparisons based on hybridization of the separated restriction fragments (by Southern transfers) of the part of the genome concerned with initiation of replication also showed homology between species 1, 5 , and 7, but none between species 1 and 4. On the other hand the DNA segments containing the rRNA genes (see Section IV, B) were homologous in all species. It is interesting to note that these mitochondrial homologies between different species closely parallel homologies of the paramecia based on enzyme electrophoretic variations, which are determined by nuclear genes (Tait, 1970). Thus there has been a parallel development of nuclear and mitochondrial divergencies between the different species.

111. Inheritance of Mitochondria1 Characters

A. INTRODUCTION The variants of P. aurelia which are controlled by mitochondrial genes can be classified in three groups: (1) drug-resistant mutants; (2) modifiers of the c l , mutant, and (3) interspecies differences. Variants of mitochondria which are controlled exclusively by nuclear genes have also been described (Tait, 1970; Ruiz and Adoutte, 1978), but will not be discussed here. Drug resistance work has been concerned mainly with erythromycin and chloramphenicol, two antibiotics which inhibit protein synthesis on bacterial and mitochondrial ribosomes, but not on the cytoplasmic ribosomes of eukaryotes. Resistance to two other antibiotics-spiramycin and mikamycin, having effects similar to those of erythromycin and chloramphenicol-is also known in Paramecium, but tests with some 20 other inhibitors of mitochondrial functions have failed to yield any useful mutants (Adoutte, 1977; A. Tait, unpublished). This failure may be due in part to the capacity of Paramecium to adapt to changes in the environment by nongenetic means, as a result of which selection of genuine genetic variants is technically difficult. Moreover, since Paramecium is an obligate aerobe, mutants like the “petite” yeasts are unavailable. All this

28

G . H. BEALE AND A. TAIT

severely limits the number of mitochondrial mutants available for study in Paramecium. However, as a compensation, it is possible to utilize interspecific variation, by the technique of microinjection of mitochondria from one species of the P . aurefia complex into another. This is a very unusual and advantageous feature of the Paramecium material. B. DRUGRESISTANCE Spontaneous erythromycin-resistant mutants of P . uureliu (species 1) can be obtained by placing the organisms in medium containing 0.25 mg/ml erythromycin (Beale, 1969). At this concentration, the paramecia remain alive for 2 to 4 weeks, but do not divide after one to three residual fissions. Electron microscope observations show that erythromycin causes marked effects on the mitochondria, which eventually lose nearly all their internal structures (Knowles, 1972; Adoutte et a l . . 1972). A small number of paramecia in erythromycin-containing medium may however change abruptly, regaining the normal mitochondria1 structure and resuming growth and division. These are drug-resistant mutants and remain permanently resistant after prolonged growth in either drug-containing or drugfree media, and after passage through autogamy. Similar erythromycin-resistant mutants have been obtained in P . aurelia species 4, 5 , and 7 (Adoutte and Beisson, 1970; Beale et al., 1972; Beale and Knowles, 1976). The spontaneous mutation rate to erythromycin resistance was roughly estimated at one per thousand cells, or one per 5 x lofimitochondria (Beale, 1969). Mutations can be produced at higher frequencies by growing paramecia first in subinhibitory erythromycin concentrations (0.02 mglml) and gradually raising the antibiotic concentration (Quieroz and Beale, 1974). Moreover some mutagens (e.g., acriflavine, manganese, nitrosoguanidine) increase the mutation frequency. The erythromycin-resistant mutants which have been obtained are phenotypically diverse, in regard to concentration of erythromycin tolerated, cross-resistance to other drugs (Quieroz and Beale, 1974), and temperature sensitivity (Adoutte, 1974). Mutants giving resistance to chloramphenicol, mikamycin, and spiramycin have been obtained by similar methods (Beale et a f . , 1972; Beale, 1973; Adoutte, 1974, 1977). By comparison with erythromycin resistance, however, chloramphenicol resistance is relatively hard to obtain in Paramecium, usually requiring chemical mutagen treatment (Beale et af., 1972). Mutants simultaneously resistant to two drugs (e.g., erythromycin and chloramphenicol, or erythromycin and mikamycin) have been obtained by two-step selection procedures. Table I summarizes the different classes of mutant which have been obtained. Inheritance of resistance to erythromycin, chloramphenicol, and mikamycin has been shown to follow the cytoplasm at conjugation (Beale 1969, 1973;

29

MITOCHONDRIAL GENETICS OF P . AURELJA TABLE I DRUG-RESISTANT MUTANTS I N Paramecium aurelia, SPECIES I A N D 4m.b.r.n ~-

~

~

~~

Growth in antibiotics Mutant

Species

Growth at 36°C

I

+

4

1 4 4 4 1 4 4 4 4

r

+ +

nd

ER (high)

ER (low)

SPIR

CAP

+ + + + + 2

+

+

+

+

2 t

+

MM

+

"The various classes of drug-resistant mutant are denoted according to their ability to grow in the antibiotics indicated. Temperature sensitivity is measured by placing cells at 36°C and measuring their growth rate compared to wild type which grows at four to five fissions per day. bER (high), erythromycin at 250 pg/ml; ER (low), erythromycin at 100 pglml; MIK, mikamycin at 250 pg/ml; SPIR, spiramycin at 600 pg/ml; CAP, chloramphenicol at 200 pg/ml. In all these concentrations of antibiotic, wild type cell growth is blocked after one to three residual fissions. ' + , Normal growth rate; 2, slow growth; -, no growth. "Data from Beale (1973). Adoutte (1977). and unpublished results of the authors.

Adoutte and Beisson, 1972) (see Fig. 1). Direct proof that the cytoplasmic factors controlling drug resistance are located within the mitochondria was obtained by injection of mitochondria from drug-resistant into drug-sensitive paramecia and exposure of the recipients to the appropriate drug (Beale et al., 1972; Beale, 1973; Knowles, 1974). Under favorable conditions, such injection results in 90- 100% transformation of the recipients, and of their cellular descendants, to resistance. Even a single injected mitochondrion is sufficient to bring about eventual transformation (Knowles, 1974). Such transformation is about 1000 times more frequent than that occurring spontaneously-Le.. in the absence of injection. Moreover, by injecting mitochondria from doubly resistant paramecia (i.e., those resistant to both erythromycin and mikamycin) into sensitive paramecia, it was shown that there is an exact correspondence between the phenotypes of the donor and the transformed recipients, whether the recipients were submitted to the selective action of one or other, or both, of the two drugs (Beale, 1973). Thus it is clear that transformation results from a replacement of mitochondria of the sensitive cells, by the mitochondria from the resistant, donor cells, and not by induced mutation, recombination, or other genetic process. The

30

G . H. BEALE AND A . TAIT

detailed steps have been followed by electron microscopy (Knowles, 1972), from which it was shown that the transformed paramecia pass through an intermediate stage containing a mixture of sensitive and resistant mitochondria in individual cells. A similar process of mitochondrial substitution was observed by Perasso and Adoutte ( 19731, by studies of paramecia containing mixtures of two types of cytoplasm following conjugation with cytoplasmic exchange. E\ idence that it is the DNA wilhin mitochondria which determines erythromycin resistance was obtained by Maki and Cummings ( 1977). who showed that the mitochondrial DNA in transformed paramecia resembled that of the cells from which the donor mitochondria were obtained, rather than that of the original mitochondria in the recipient cells. These experiments involved injection of mitochondria from one species of P . ciureliu into another, in which the mitochondria1 DNA could be distinguished by restriction endonuclease analysis (\ee Section 1I.E).

c. MODIFIERSOF T H E ( , I ,

MUTANT

A class of mitochondria1 mutants whose effect is the partial suppression of the action of a nuclear mutant gene ( c l , ) has been described by Sainsard et al. (1974).Sainsard (1975. 1976, 1978), and Sainsard-Chanet and Knowles (1979). The slow growing mutant cl ,, which has severely disorganized mitochondria, lacks cytochrome aa:,. and is thermosensitice. was first obtained by ultraviolet treatment and shown to be due to a mutation in the nuclear genome ( d , +-+ cI ) . After growth of this mutant for about 20 cell generations, a partial "reversion" was observed. whereby the fission rate increased somewhat (though not to the normal rate of wild-type cells), and the mitochondria regained their normal structure, though the cytochrome aa:, deficiency and a slight thermosensitivity remained. Genetic analysis showed that the "reversion" was due to mutation in the mitochondrial genome. Several different mitochondrial mutations ( M ( " , ,W".M * ) were found, producing different degrees of suppression of the nuclear gene c.1 ,. The mutant mitochondria, once formed, retained their altered properties indefinitely. even after being introduced into cells containing the wild-type nuclear allele c / ~ However, ~ . where there was a mixture of MC" and M + mitochondria, selection of the M"' type occurred in cells containing the nuclear gene c l , . and of the M' type in cells containing the nuclear gene el,+. These mitochondrial mutations, which arise spontaneously and are subject to positive selection in presence of the defective nuclear gene cl ,, are presumed to produce some reaction which compensates for the cytochrome defect produced by the gene c l , . In combination with the wild-type allele d I + the , mitochondrial gene M" slightly reduces the cellular growth rate and cytochrome aa3 content, in cells in the "stationary" growth phase (Sainsard-Chanet, 1978).

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MITOCHONDRIAL GENETICS OF P. AURELIA

D. INTERSPECIFIC DIFFERENCES Artificial transfer of mitochondria by microinjection can be carried out not only between paramecia belonging to the same species of the P . aurelia complex, but also, to a limited degree, between different species (see Table II). Successful transfers have been made between species 1, 5, and 7, though not between these and any other species (e.g., species 4) in the aurelia complex, or into more distant species (e.g. P . caudarum) (Beale and Knowles, 1976). Interspecies transfers make possible a rudimentary genetic analysis of interspecific variations, even though absence of recombination (see below) prevents the carrying out of any detailed mapping work. As an illustration of the method, the work of Knowles and Tait (1972) on inheritance of electrophoretic variations of fumarase may be considered (Fig. 5). Species 1 and 7 show different electrophoretic forms of this enzyme, while hybrids containing species 1 mitochondria and species 7 nuclei were found to produce a fumarase variant like that of species 7, and unlike that of species 1 . This shows that variation of this enzyme is controlled by nuclear genes, for if the enzyme were to be determined by mitochondrial genes one would expect to observe the species 1 variant in the hybrid cells. The method has been applied to variations affecting the following mitochondrial constituents: ( I ) mitochondrial DNA; (2) mitochondrial ATPase; (3) mitochondnal ribosomal proteins, and (4) mitochondrial membrane proteins. It has

INTERSPECIES

TABLE 11 TRANSFER OF MITOCHONDRIA BY

~~

MICROINJECTION”’b’P

~

Species of donor cells Nucleus A 1

Species 1

Species 5

Species 7

1

+ +

+ +

+ +

5 7

B I

1 5 1

I 5

Recipient cells

Mitochondria

5

7

~~

2 2

+

-

+

+ + +

“The donors are the erythromycin-resistant cells from which mitochondria were prepared for microinjection into the recipient cells. In section A the donor cells are strains from species 1, 5, and 7, respectively; in section B they are artificially constructed “hybrids” containing the nucleus of one species and the mitochondrial genome of another. b + , High rate of transformation to erythromycin resistance; 2, very low rate of transformation to erythromycin resistance; - , no transformation. ‘Data from Beale and Knowles (1976).

32 NUCLEUS MITOCHONORIA

G . H. BEALE AND A. TAIT SP 1

SP?

SP7

SPlE"

SP?ES

SPIE"

Selection

*

mitochondria

ELECTROPHORESIS

I t FIG. 5 . Diagram showing nuclear control of interspecies variation in fumarase (from Knowles and Tait. 1972). SPIER. species I cells. erythromycin-resistant mitochondria; SP7Es, species 7 cells. erythromycin-sensitive mitochondria. The lower portion indicates the electrophoretic mobility of fumarase after starch gel electrophoresis 0. Origin.

been shown that variations of all of these constituents are controlled partly, or sometimes completely, by the mitochondrial genome. Further biochemical details are given in Sections IV,B,C,D. The compatibility of mitochondria from one species with nuclei from others has been studied by the microinjection technique (Beale and Knowles, 1976). This study involved P. aureliu species 1, 5, and 7. From previous work (Sonne born, 1975; Tait , 1972, 1978), based on characters presumed to be determined by nuclear genes, it was known that species 1 and 5 are closely related, while species 7 is relatively distant. Microinjection experiments showed that erythromycin-resistant mitochondria could be readily transferred from species 1 and 5 into all three species, while species 7 mitochondria, which could be transferred to other species 7 cells, usually failed to develop in cells containing species I or 5 nuclei (see Table U). Moreover mitochondria from hybrid cells containing species 7 nuclei and species 1 or 5 mitochondria were also not transferable to species 1 or 5 recipients. It was concluded that the mitochondria in the hybrid cells, even though they contained mitochondrial DNA from species 1 or 5, were modified by the nuclear genomes of species 7. The following sequence of events is envisaged to take place when erythromycin-resistant mitochondria are transferred by injection from one species to another (Beale and Knowles, 1976). The mitochondrial DNA is transferred intact to the recipient cell, and characters controlled by this DNA, such as erythromycin resistance, are acquired by the recipient. The majority of mitochondrial constituents are however determined by the nuclear genome. Consequently the mitochondria which are introduced into cells of a different species may have to undergo changes in membrane and other components controlled by

MITOCHONDRIAL GENETICS OF P. AUREUA

33

the nuclei of the recipient cells, and these changes may provoke disturbances in the growth processes of the introduced mitochondria. Various detailed hypotheses have been suggested (Beale and Knowles, 1976), and the recent data on DNA homologies of restriction fragments (see Section I1,E) provide a further hypothesis to explain the limitation of successful interspecies transfers by injection to species 1 , 5, and 7. In these three species, the restriction fragments containing the initiation end of the molecule shows strong homology, but no homology at all to the equivalent fragment from species 4 (Cummings er al., 1980). Thus the crucial factor for successful interspecies transfer of mitochondria might be the ability of the enzymes involved in replication to recognize the initiation point of the mitochondrial DNA.

E. COMPLEMENTATION A N D RECOMBINATION INVOLVING MITOCHONDRIAL GENES Interactions between diverse mitochondria within a single paramecium have been studied by observations on cells containing mixtures of different types of mitochondria, obtained by abnormal cytoplasmic exchange at conjugation, or by microinjection. It has been found that a cell containing both erythromycinresistant and chloramphenicol-resistant mitochondria is resistant to either antibiotic, but only when each is added separately. If erythromycin is added, the chloramphenicol-resistant mitochondria are eliminated, leaving the erythromycin-resistantones to multiply; if chloramphenicol is added the situation is reversed and only the chloramphenicol-resistant mitochondria grow; if both antibiotics are added, no growth occurs. Thus there is no complementation between diverse mitochondria, so far as drug resistance is concerned (Beale er al., 1972; Adoutte and Beisson, 1972). In the absence of either antibiotic, mixed populations of mitochondria may persist for long periods, though with some combinations of mutants one type may have a selective advantage over another. This varies according to individual mutants, which can be ranked according to their relative selective advantage over others. Attempts have been made to detect recombination between different mitochondrial drug resistance genes, by establishing paramecia containing mixed populations of mitochondria, allowing growth to take place, and later attempting to select for doubly resistant cells. The latter have never been demonstrated to occur by this procedure (Beale et al., 1972; Adoutte and Beisson, 1972; Beale, 1973), though, as already mentioned, such doubly resistant cells can be obtained by two successive mutations. A thorough search for recombination of mitochondrial genes, including those controlling resistance to various antibiotics, temperature sensitivity, and suppressors of the e l , mutant, has been made by Adoutte et al. (1979), again with negative results. The reasons for this absence of recombination in the mitochondria of

34

G.

H.BEALE AND

A. TAlT

Purcitneciion. which in this respect behave strikingly differently from those of yeast. are not known. One possible reason is that mitochondrial DNA in P~irrrt~ieciutn is always confined within the bounds of single organelles, which do not fuse. in contrast to the situation in yeast. The demonstration of variation in restriction enzyme patterns in different stocks of a single species, as shown by Findley and Gall ( 1980) now makes it possible to examine this question more thoroughly.

1V. Biochemical Products of Mitochondria1 Genes A. INTRODUCTION

In yeast only a small fraction of the biochemical activities of mitochondria is known to be controlled by mitochondrial genes: the two kinds of rRNA, a set of tRNAs. and a few protein components-subunits of cytochrome oxidase, oligomycin-sensitive ATPase, cytochrome b. and one ribosomal protein (VARI ) (Borst and Grivell. 1978). It would be interesting to determine whether the same. and no other, products are encoded in the mitochondrial genome of P~ir~aneciiitn. but this is not possible, due to the lack of mitochondrial genes so far identified. Nevertheless, even with the limited data available, some differences between Parumeciiini and yeast are apparent. Before describing what is known about the functions of the mitochondrial genome of Purcitneciurn, we give a few details about its respiratory system. Several enzymes of the tricarboxylic cycle, e.g., fumarase, isocitrate dehydrogenase, and malate dehydrogenase, have been shown to be present, and the mitochondria found to carry out oxidative phosphorylation using the respiratory chain (Doussiere ef c d . . 1976). However, there appear to be marked differences between the spectral and redox properties of Puruniecium mitochondria, and those of yeast or mammalian mitochondria. In Purameciurn a branched respiratory chain has been demonstrated (Doussiere ef a / . . 1976) involving a typical cytochrome oxidase and a cyanide-insensitive, SHAM-sensitive alternative oxidase. The presence of one or more alternative respiratory pathways may partially explain the difficulties in isolating mutants resistant to inhibitors of the "normal" respiratory chain (e.g., antimycin A), and raises the possibility that at least some of the mitochondrially determined proteins of Purumeciuni are dift'erent from those of yeast or mammalian mitochondria. B.

RlBOWMAL

R N A A N D RIBOSOMAI.PROTEINS

In all organisms so far studied it has been shown that mitochondria contain a protein-synthesizing system distinct from the main cytoplasmic system whose components are all controlled by nuclear genes. This is true also in Parumecium. The mitochondrial ribosomes of P. nurelia sediment at 80 S in a sucrose

MITOCHONDRIAL GENETICS OF P . AURELIA

35

gradient, and in this respect resemble the cytoplasmic ribosomes, but the two classes can be distinguished by other criteria (Tait and Knowles, 1977). The mitochondrial ribosomes dissociate into a single class of 55 S particles on treatment with low Mg2+ concentrations or EDTA. The 55 S fraction almost certainly contains the two subunits of the 80 S monomer and although these subunits are morphologically distinct they are similar in size (Tait and Knowles, 1977; Stevens et al., 1976). Attempts to separate the 55 S particles into two fractions have been unsuccessful but electron microscopy clearly shows that they are not monomers, and analysis of their RNA on sucrose gradients shows that they contain both the 14 S and 20 S rRNA species. Similar results have been obtained with Tetrahymena (Chi and Suyama, 1970) and thus the mitochondrial ribosomes of these two ciliate protozoa form a class different from those of fungi and higher animals (see Borst, 1980, for review). As regards the 80 S sedimentation value, Paramecium mitochondrial ribosomes resemble those of higher plants. In sensitivity to erythromycin and chloramphenicol, and insensitivity to cycloheximide, as shown by the effect of these substances on incorporation of labeled amino acids into nascent polypeptides, the mitochondrial ribosomes of Paramecium resemble those of yeast, higher animals, and other groups of organisms (Tait and Knowles, 1977). The two mitochondrial rRNAs, like those of other organisms, are coded by mitochondrial genes, as shown by hybridization of labeled 14 S and 20 S rRNAs with fragments of mitochondrial DNA produced by restriction endonucleases (see Fig. 3). The two genes are not immediately adjacent on the mitochondrial DNA. From the size of the Hind111 fragments which hybridize to the rRNA, it is believed that there is only a single 20 S rRNA gene. Cummings et a / . (1 980) have also shown that there are no extensive introns in the 20 S rRNA gene. The mitochondrial ribosomes in erythromycin-resistant paramecia are different from those in sensitive cells, since binding of radiolabeled erythromycin to the ribosomes is less in the resistant than in the sensitive paramecia (Tait 1972; Spurlock et al., 1975). From preliminary studies of the ribosomal proteins by acrylamide gel electrophoresis of crude extracts, it has been shown that erythromycin resistance is associated with an alteration in a single mitochondrial ribosomal protein (Beale et al., 1972), though this finding needs to be confirmed by studies on purified ribosomes. Further evidence showing that the mitochondrial genome has some control over the mitochondrial ribosomal proteins has been obtained by studies with interspecific hybrids. The mitochondrial ribosomal proteins of species 1 and 7 of the P . aurelia complex have been shown, by immunodiffusion and quantitative immunoprecipitation tests, to differ. By comparing the proteins from these two species with proteins from the “hybrids” containing species 7 nuclei and species 1 mitochondria, it was found that both nuclear and mitochondrial genomes were involved in the determination of these differences (Tait er al., 1976b). More re-

36

G . H . BEALE AND A . TAlT

cently it has been shown by 2D gel electrophoresis that eight ribosomal proteins are determined by the mitochondrial genome (A. Tait, unpublished). The conclusion is that a considerable proportion of these proteins are controlled by mitochondrial genes, but it remains to be proved that these are structural rather than regulatory genes. It also appears that Terrahyrnena is similar in this respect since several mitochondrial ribosomal proteins have been shown to be synthesized on mitochondrial ribosomes (Chi and Suyama, 1970; Millis and Suyama, 1972). C. RESPIRATORY PROTEINS A N D ATPASE

Little is known about the genetic control of cytochrome oxidase and cytochrome b in P . aurelia. From the work of Sainsard (see Section III,C), however, it is known that both nuclear and mitochondrial genes affect cytochrome oxidase (aa:,) in some way. Moreover Ruiz and Adoutte (1978) have identified a number of nuclear mutants affecting the respiratory system. With regard to mitochondrial ATPase there is evidence that in Paramecium. as in yeast, both nuclear and mitochondrial genomes are involved. ATPase from P . uureliu species 1 differs from that of species 7 in the degree of inhibition by oligomycin. inhibition of ATPase in the “hybrid” containing species 7 nuclei and species 1 mitochondria was found to be intermediate between that of ATPase from the two parent species. Gel electrophoresis experiments showed that out of 15 polypeptides, three were different in species 1 and 7, one being controlled by the nuclear genome and two by the mitochondrial genome (A. Tait and J . Hunter, unpublished).

D. OTHERMITOCHONDRIAL CONSTITUENTS Species 1 and 7 of P . aurelia also differ in regard to some chloroform/ methanol soluble mitochondrial proteins, as shown by immunological techniques. Studies with hybrids produced by microinjection of mitochondria have shown that these differences are determined partly by mitochondria genes (Tait et (11.. 1976a). From studies in yeast (Tzagoloff and Akai, 1972) and higher animals (Kadenbach, 1971) it has been shown that this fraction contains a high proportion of mitochondrially synthesized proteins, including (in yeast) one of the proteolipid components of the ATPase but none of the components of cytochrome oxidase (Schatz and Mason, 1976).

V. Conclusion The general conclusion from this study of P . aurelia is that there are substantial differences between the mitochondrial genetic systems of these organisms, and corresponding systems in yeast and other groups. In Table I11 we have

TABLE I11 COMPARISON OF MITOCHONDIUAL GENETICSYSTEMS OF Paramecium, YEAST, A N D HIGHERANIMALS" Yeast

Higher animals

Circular 50 x loe daltons (25 p m ) Absent (except for single E. colilike basic protein)

Circular 10 X lo6 daltons ( 5 p n ) Absent in HeLA Present in Xenopus

Absent Absent in rRNA genes

Present Present in rRNA and other genes

Absent Absent

Absent Rarely, or under abnormal conditions

Present Present

Absent Absent

80 S

20 S and I4 S

72 S 50 S and 40 S 23 S and I6 S

55-60 S 40 S and 33 S 12 S and 16 S

Sensitive Sensitive Resistant

Sensitive Sensitive Resistant

Sensitive Sensitive Resistant

Yes Not known Several Part Pan

Yes Yes Probably one only Part Pal7

Yes Yes Not known Part Part

Paramecium I . Genome characteristics a . Structure b. Size C . Histone-like proteins andor beaded chromatin structure d. Extensive AT-rich regions e . lntrons

2. Genetic mechanisms a. Recombination b. Mixing of paternal and maternal mitochondria in zygote 3 . Mitochondrial protein synthesizing system a . Ribosomes, S value b . Ribosomes, subunits c. rRNAs d. Sensitivity of mitochondria1 ribosomes to Chloramphenicol Erythromycin Cycloheximide

Linear

27. I x Present

55

lo6 daltons (14 g m )

s

4. Biochemical end products of

mitochondrial gene activity a. rRNAS b. tRNAs c. Ribosomal proteins d. ATPase e. Respiratory proteins

~~

~

"Data from Borst and Gnveil (1971, 1978). Beale and Knowles (1978), and Cummings et al. ( 1 9 7 9 ~ ) .

38

G . H. BEALE AND A. TAIT

attempted to make clear these differences by comparing the main features of the systems in Pcilntn~ciim,yeast, and higher animals. Among the more noteworthy points of difference are: (1) the absence of recombination i n Ptrrclmeciuni (and possibly also in higher animals) compared to its frequent occurrence in yeast; ( 2 ) the linearity of the mitochondrial genome in Purcrmeciutti and Tetrahjmena, as contrasted with the circular mitochondrial genomes in all other organisms so far studied: (3) presence of several histone-like proteins. and a beaded chromatin-like structure, in the mitochondrial genome of Ptrrimecium (somewhat resembling the mitochondrial chromatin of Xenopus), as contrasted with the E . cdi-like structure in yeast, and apparent total absence of histones in HeLa cell mitochondria; (4) differences in the mode of replication of mitochondrial DNA in the different groups, and (5) the larger number of mitochondrial ribosomal proteins which are controlled by mitochondrial genes in Purcimeciurn as compared with other organisms. However, as regards the biochemical end-products of mitochondrial gene activity (apart from the mitochondrial ribosomal proteins), these seem to be not clear14 different in the different groups of organisms, so far as can be judged by the limited data available. These conclusions justify our belief iI1 :!ie need to study mitochondrial genetic systems in a variety of different groups of organisms.

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