Molecular genetics of chloroplasts and mitochondria in the unicellular green alga <i>Chlamydomonas</i>

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Article

Reference

Molecular genetics of chloroplasts and mitochondria in the unicellular green alga Chlamydomonas

ROCHAIX, Jean-David

ROCHAIX, Jean-David. Molecular genetics of chloroplasts and mitochondria in the unicellular green alga Chlamydomonas . FEMS Microbiology Reviews , 1987, vol. 46, no. 1, p. 13-34

DOI : 10.1111/j.1574-6968.1987.tb02449.x

Available at:

http://archive-ouverte.unige.ch/unige:149396

Disclaimer: layout of this document may differ from the published version.

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FER 00052

Molecular genetics of chloroplasts and mitochondria in the unicellular green alga Chlamydomonas

J.-D. Rochaix

Departments of Molecular Biology' and Plant Biology', Uni~ersi(v of Gener,a, Genes,a, Switzerland Receivcd 7 July 1986

Accepted 16 September 1986

Key words: Photosynthetic mutants; DNA, chloroplast; DNA, mitochondrial; Chlamydomonas

1. INTRODUCTION

The green unicellular biflagellate alga Chlamy- domonas reinhardtii provides interesting possibili- ties for a combined genetic and molecular study of the biosynthesis and function of cellular organelles.

This is due to the fact that numerous mutations have been identified in Chlamydomonas that affect photosynthesis, chloroplast protein synthesis, flagellar structure, motility and mating. This review will focus mostly on the major organelle of Chlamydomonas, its unique chloroplast, which oc- cupies more than 40% of the cell volume.

It is well established that chloroplasts contain their own genetic system which comprises DNA, RNA, DNA- and RNA-polymerases, ribosomes and translation factors. This system co-operates closely with its homologue in the nucleocytoplasmic compartment in the biosynthesis of chloroplast components. Most prominent among these are the photosynthetic apparatus and the chloroplast protein synthesizing system. They consist of a large number of polypeptides, some of which are

('orrespondence to: J.-D. Rochaix, Depts. of Molecular Biology and Plant Biology, University of Geneva, 1211 Geneva, Switzerland.

encoded by the chloroplast genome while others are coded for by the nuclear genome, translated as precursors on cytoplasmic ribosomes and imported into the chloroplast where they assemble with their partner polypeptides into functional complexes. Little is known about the regulation of this complex interplay between chloroplast and nucleocytoplasmic compartments which results in the establishment of an active photosynthetic system. Among photosynthetic eukaryotes, Chlamy- domonas is uniquely suited for studying this prob- lem, since it can be manipulated with ease at the genetic, biochemical and molecular levels.

The aim of this article is to provide an overview of the molecular genetics of Chlamydomonas with special emphasis on photosynthesis. Only recent developments are covered, which include the organization of the chloroplast genome in Chlarnydomonas, the isolation and characterization of both chloroplast and nuclear photosynthetic mutants, the use of these mutations for studying the function and assembly of photosynthetic complexes, the improved correlation between the genetic and physical chloroplast DNA maps, and the molecular and genetic analysis of mitochondrial DNA. Several recent reviews provide more detailed coverage of various aspects of chloroplast molecular biology [1-5].

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2. GENERAL PROPERTIES OF CHLAMY- D OMONA S RE1NHA RD TII

Cells of opposite mating-type (mt) of this het- erothallic alga can be propagated vegetatively by mitosis (Fig. 1). Upon transfer into a medium deprived of a reduced nitrogen source, the vegetative cells differentiate into gametes and cells of opposite mating-type fuse to form a zygote.

Nuclear fusion is followed by chloroplast fusion, thus allowing the chloroplast genomes of both parents to mix and recombine. At the end of the meiotic cycle, haploid daughter cells are released which can initiate a new cycle. It is also possible to produce vegetative zygotes which do not enter meiosis and which divide mitotically as diploid cells [15].

As in higher plants, there are three genetic systems in Chlamydomonas, located in the nucleocytoplasm, chloroplast and mitochondria, respectively. Table 1 summarizes the major features of these systems. It is noteworthy that in contrast to higher plants, the size of the mitochondrial genome is only 16 kb, similar to animal mitochondrial DNA [12] (see section 7).

Although the complexity of the chloroplast DNA (190 kb) represents only 0.3% of the total cell DNA complexity, the amount of chloroplast DNA constitutes 14% of the cellular DNA mass [7,11].

This implies that the chloroplast DNA is present in more than 50 copies per cell. Similarly, the mitochondrial DNA also exists in numerous copies per cell. While nuclear genes are inherited accord- ing to mendelian rules, the chloroplast DNA is inherited in most cases from the mt + parent (see [15]). In rare cases, biparental zygotes occur in which the chloroplast genes of both parents are transmitted to the meiotic progeny, allowing the study of chloroplast DNA recombination. Surpris- ingly, in crosses between C. reinhardtii and Chlamydomonas smithii the mitochondrial genome of the mt ~ parent is transmitted uniparentally [14]

(see section 7).

Cells can be grown under phototrophic (minimal medium with light), heterotrophic (acetate medium in the dark) or mixotrophic conditions (acetate medium with light). Photosyn- thetic function is therefore dispensable when the cells are grown in the presence of acetate. This property allows one to isolate and maintain photosynthetic mutants that are unable to grow in the absence of a reduced carbon source such as acetate [15]. Conversely, mitochondrial respiratory function appears to be dispensable when the cells are grown in the light, but not when they are grown heterotrophically. Several obligate photoau- totrophic mutants which die in the dark have been characterized (see [15]).

gamete (-~ gamete ~ ÷

-NH~ .'"'-

^{... ~} ... "".. -NH~

'" ~ Fusion ""~'o~~

/ Vegetative ~ ~ y Vegetative \

Fig. 1. Life cycle of C. remhardtM rot, Mating type: n, nucleus. Zygote can undergo meiosis or remain diploid and divide mitotically.

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Table 1

Genetic systems in Chlamydomonas reinhardtii

Complexity (kb) % genetic % Mass Copy number Inheritance information

Nuclear DNA 7-9 x 104 [7-9] 99.7 85 1 Mendelian

Chloroplast DNA 190 [10,11] 0.3 14 50 80 Uniparental maternal [13]

Mitochondrial DNA 16 [12] 0.02 1 50-80 Uniparental paternal a [14]

a This has only been demonstrated in crosses between C. reinhardtii and C. smithii [14].

3. PHOTOSYNTHETIC APPARATUS

Since the following sections will deal with the genes and proteins involved in photosynthesis, the main features of the photosynthetic apparatus will be briefly described. The primary reactions of photosynthesis occur in the thylakoid membranes, in which five major macromolecular complexes can be recognized: photosystem II (PSII), photosystem I (PSI), the cytochrome b 6 / f complex, the ATP synthase complex and the light-harvesting system (Fig. 2). Light energy is harvested by the pigment antenna and directed to the PSII and PSI reaction centers where the primary photochem- istry results in a charge separation across the membrane. The PSII reaction center generates a strong oxidant capable of splitting water into molecular oxygen, protons and electrons. The latter are transferred on the reducing side of PSII to the primary and secondary quinone electron acceptors

QA

and QB, respectively, and channeled along the electron transport chain through the plastoquinone pool, the cytochrome b 6 / f complex, plastocyanin and finally to the PSI reaction center.

This complex generates a strong reductant capable of reducing ferredoxin and NADP, the terminal electron acceptor. The electron flow is coupled to an influx of protons into the thylakoids, creating a proton gradient which drives ATP synthesis through the ATP synthase complex. Both ATP and NADPH are fed into the Calvin cycle which results in the fixation of CO 2, catalyzed by ribulose 1,5-bisphosphate carboxylase-oxygenase (RuBisCO), and in the synthesis of carbohydrates.

The oxygenase activity of RuBisCO initiates the first step of photorespiration, a competing reaction of photosynthesis which ultimately results in the loss of CO 2 [16]. The existence of a respiratory chain in the thylakoid membranes of C. reinhardtii has been recently demonstrated [17]. This chloro-

PSII

stroma

N t DP (~

intrathylakoid space

CF1

!FO~ Ithy[ak°id

Fig. 2. Tentative structural model of the photosynthetic complexes in the thylakoid membrane. Numbers indicate the M r ( × 10 3 ) of the polypeptides of PSII. LHCP, light-harvesting chlorophyll binding proteins: PC, plastocyanin: CPI, chlorophyll-protein complex of PSI. Rieske, Rieske Fe-S protein. CF1 refers to the coupling factor (ATP synthase) and CFo to its membrane anchor.

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plast respiratory chain oxidizes NAD(P)H at the expense of oxygen and shares the plastoquinone pool with the photosynthetic chain.

4. O R G A N I Z A T I O N OF THE CHLOROPLAST G E N O M E OF CHLAMYDOMONAS

4.1. Organization, structure and function of chloro- plast genes of C. reinhardtii

The chloroplast genome of C. reinhardtii consists of 190-kb DNA circles [10,11]. Its physical map is shown in Fig. 3. As in many higher plants, the two ribosomal regions are located within two repeats that are oriented in opposite directions.

The chloroplast genes which have been identified fall into three groups. The first set encodes polypeptides of the photosynthetic apparatus, the second set codes for components of the chloroplast protein-synthesizing apparatus which include ribosomal RNA, tRNAs, RNA polymerase subunits, transcription and translation factors, and ribosomal proteins, and there may be a third set which encodes components involved in DNA replication and recombination (table 2; and Fig. 3).

While over 20 protein genes have been localized on the chloroplast genorne of C. reinhardtii (Fig.

3), the existence of at least 40 chloroplast genes of known function can be inferred based on the assumption that all the proteins synthesized within the organelle are encoded by the chloroplast DNA.

Indeed, there is no convincing evidence for mRNA transport across the chloroplast envelope. By using inhibitors specific for cytoplasmic translation, it has been shown that close to 20 ribosomal proteins are synthesized in the chloroplast of C. rein- hardtii [18]. A list of RNAs and proteins known to be encoded or synthesized by the chloroplast is given in Table 2. Sequencing of several chloroplast genes has revealed a high degree of sequence conservation between the algal and higher plant genes [3]. As an example, the rbcL and psbA genes are 77 and 80% homologous, respectively, between C. reinhardtii and spinach [19,20].

Four chloroplast genes of C. reinhardtii (out of a total of ten examined at the nucleotide level) have been shown to contain introns: the 23S rRNA gene [21,54] psbA, coding for the D1 protein of

PSII [20]; psaA1 coding for the P700 apoprotein A1 of PSI (U. Kiick, unpublished results); and rpoC, coding for the/3' subunit of an RNA polymerase (S. Surzycki, unpublished results). The ribosomal intron consists of 888 bp and can be folded with a secondary structure that is typical of group I introns of fungal mitochondrial genes [54-56]. It also has sequences homologous to the box 9 and box 2 consensus sequences in which cis-dominant mutations leading to splicing deficiency have been found in yeast mitochondrial introns [57]. The intron contains a 489-bp open reading frame coding for a potential polypeptide that is related to mitochondrial maturases (Fig. 4) [54,55]. The four introns of psbA range from 1.1-1.8 kb and appear to belong to the group I introns based on the presence of box 9 and box 2 consensus sequences [20]. Intron 3 of psbA is completely absent from C. smithii and therefore represents an optional intron [36]. Introns 2 and 3 have been entirely sequenced except for a small gap in intron 3 (J. Erickson, M. Rahire and J.D.

Rochaix, unpublished results). It is interesting to note that open reading frames of 69 and 160 codons in introns 2 and 3, respectively, prolong the upstream exons (Fig. 4). The presence of similar open reading frames in several yeast mitochondrial genes is well documented and has led to the 'maturase' concept [58]. The open reading frame of intron 2 is followed, after a frameshift, by a second open reading frame of 298 codons (Fig. 4). Recently, a 2.2-kb intron has been found in psaA1, which appears to belong to the group lI introns (U. KiJck, unpublished results).

4.2. Comparison of the chloroplast genome organiza- tion of C. eugametos, C. moewusii, C. reinhardtii and C. smithii

The genus Chlamydomonas is one of the largest genera of the green algae. The physical map of the chloroplast genomes of the two interfertile species Chlarnydomonas eugametos and Chlamydomonas moewusii have been determined and shown to consist of 242 and 292-kb circles, respectively [59,60]. The two genomes display the same arrangement of common sequences, except for two major insertions in the chloroplast DNA of C.

moewusii: a 21-kb insertion in the inverted repeat

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• boo 4

i

3o,~ o ,,"

t

L.) I1~

~ .~.

07s,6 'c

0 0

... Q'O.

~.~ ...-- --.~,aoc ~

e o _{_} ~'o .-'" . - _ _ "" ~ "-.9,>

2~.

16

,~Qt~ 8

O

F'ig. 3. Physical map of the chloroplast genome of C. reinhardtii. The inner circles represent the BamHI (Ba) and Ec, RI (R) restriction fragments [11]. The two segments of the inverted rcpcat containing the ribosomal RNA genes [21] and pshA [20] are drawn on the outside and bounded by' arrows. Introns are drawn in thinner lines relative to the coding sequences. Dark ~edgcs indicate 12 identified tRNA genes of which three are located within the inverted rcpcat [53] (scc Table 2). Characterized protein genes in the single copy region are marked: psaA1 (U. Ki~ck, unpublished results), psaA2 (M. Schneider et al., unpubli,,,hed rcsults): l~shB, p~h("

(C. Kovacic, P. Malnoe and J.D. Rochaix, unpublished results) p~hD [22], atpA [23], atpB [24,25] atpE. atpF, atptt (J. Woessncr, J.

Robertson, J. Boynton and N. Gillham, unpublished rcsuhs), rhcL [19], tufa [26], rpoA [27], q~oB, q~oC, dnuA [28] (S. Surzvcki, unpublished results) q~s-12, rps-7, rpl-2 [50]. Recently, Surzycki and collaborators have found chloroplast DNA ~,equcnccs that cross-hybridize to the E. coh genes ut,rC (Rll), re<A (RI3, R22) and the gene of rho factor (RI1. marked with *). The eight identified chloroplast ARS sequences are indicated by 01 to 08 [29 31]. The four chloroplast DNA sequences promoting autonomous replication in C. reinhardtii are marked by ARC1, ARC2, ARC3a, ARC3b [32]. Two authentic origins of replication are indicated by oriA and oriB [33]. Abbreviations for genes are explained in Table 2.

and a 5.9-kb insert in the single copy region close to the 16S rRNA genes [61,62]. Interestingly, this 5.9-kb DNA also exists as a free linear plasmid in these cells and it has been shown to be inherited uniparentally [63,64]. It has not yet been determined whether the plasmid replicates autono- mously or whether it is amplified from its copy in the chloroplast genome. Since the plasmid is absent from C. eugametos, the analysis of the trans- mission pattern of the free and integrated forms in

interspecific hybrids between C. eugametos and C.

moewusii should provide new insights into the mechanism of replication of this plasmid.

Comparison of the chloroplast genomes of C.

eugametos and C. reinhardtii, which are not interfertile, reveals a great deal of divergence in the primary sequences and in gene organization [65].

For example, the orientation of psbA relative to the ribosomal RNA genes is different, and rbcL is included in the inverted repeat of C. eugametos. In

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Table 2

Chloroplast genes and characterized mutations in Chlamydomonas reinhardtii

The location of the genes on the chloroplast genome is shown in Fig. 3. -, Not determined; P, point mutation; A, deletion; v, insertion. In addition 9 distinct tRNA genes have identified: trnT, trnP, trnF (2 genes) trnN, trnL, trnL trnA, trnH, of which the last three are located within the inverted repeat [53].

Complex Component Gene Introns Mutant Nature of mutation Reference

(,4) Photosynthetic components

ATP synthase a subunit atpA - -

/3 subunit atpB 0 FuD50

c subunit ^atpE ⁰ ^-

CI subunit atpF - -

CIII subunit ^atpH ^- ^-

Photosystem II " D1 psbA 4

Photosystem I

FUD7, 8-36C DCMU4, Dr2, Ar207, Br202 47-50-kDa protein psbB -

43-47-kDa protein psbC - FUD34, MA16?

D2 psbD 0 FUD47

A1 P700 apoprotein ^psaA1 ¹ A2 P700 apoprotein psaA 2 0

RuBisCO Large subunit ^rbcL ⁰ 10-6C

18-7G 18-5B

,x (8-10 kb) P P

x7 (46 bp)

a (4 bp)

P P P

[23,24]

[24,25,34]

(J.D. Woessner, J. Robertson, J. Boynton and N. Gillham, unpublished results)

1241

[35,36]

[37-40] (J. Erickson, L. Mets and J.-D. Rochaix,

unpublished results) (M. Kuchka, P. Bennoun and J.-D. Rochaix, unpublished results) [41]

(U. Kiick, unpublished results) (M. Schneider et al.,

unpublished results) [42,43]

[44,45]

[44,451 (B) Components involved in chloroplast transcription and translation and in DNA replication and recombination

Ribosome 23S rRNA rnaL 1 CapR, ^{EryR b} ^p?

16S rRNA rnaS 0 Spc R, Str R b p?

Ribosomal protein rpl-2 - -

rps-7 - -

rps-12 - -

Elongation factor Tu tufa - -

RNA polymerase a suburtit rpoA - - -

fl subunit rpoB - - -

13' subunit ^rpoC ¹ ^- ^-

Termination factor Rho factor rhoC c _ _ _

(C) Proteins involved in DNA replication and recombination dnaA ~ - uvrC c _

recA c

[46-48]

[48,49]

[501 [501 [50]

[26]

[27]

[281

(S. Surzycki, unpublished results)

(S. Surzycki, unpublished results) (S. Surzycki, unpublished results) (S. Surzycki, unpublished results)

" The PSII core contains in addition apocytochrome b559, whose gene has not yet been located on the chloroplast genome of C.

reinhardtii.

b The assignment of uniparental chlorampheniol (CapR), erythromycin (Eryg), spectinomycin (Spc g) and streptomycin (Str R) resistance mutations to the 23S and 16S rRNA genes is based on the location of similar mutations in the yeast mitochondrial rRNA gene [51], in the chloroplast 16S rRNA gene of Euglena gracilis [52] and on recent correlations between restriction site alterations and resistance phenotypes (see section 6 for details).

c These gene assignments are based on cross-hybridizations detected between ^{E. coil} gene probes and chloroplast restriction fragments of C. reinhardtii (S. Surzycki and S. Hong, unpublished results).

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23 S rDNA PI

,00 bc psbA

a~ oa aa

P TGA

ATG P

Fig. 4. Chloroplast introns in the 23S rDNA and pshA of C.

reinhardtii • exon sequences, [] intron sequences (not determined), [] intron regions with blocked reading frames, []

intron open reading frames that prolong the upstream exons, []

open reading frame within introns. The box 9 (zx) and box 2 (O) consensus sequences [57] characteristic of group I introns, are indicated. P1 marks a conserved dodecapeptide found in mitochondrial intron open reading frames [55]. Sizes of polypeptides encoded by the open reading frames are indicated in amino acids (aa). Numbers above psbA correspond to the amino acids contained in each of the five exons [20]. The boundary between the first and second open reading frame in the second intron of pshA is enlarged in the lower part of the figure (J. Erickson, Rahire and Rochaix, unpublished results).

Abbreviations for gcnes are explained in Table 2.

addition, transfer of chloroplast DNA sequences have occurred from one single copy region to the other during the evolution of these two species.

The variation in chloroplast DNA between them greatly exceeds that observed in the vast majority of higher plants [65,66]. Whether this variation results from a longer period of evolution separat- ing these two algae as compared to that of higher plants, or whether it reflects a high rate of DNA rearrangement in algae, remains an open question.

The latter possibility has to be considered since photosynthetic mutants induced by 5-fluorode- oxyuridine have suffered extensive sequence rearrangements principally in the region of the inverted repeat [36]. In two of these mutants the inverted repeat has been expanded from 20 kb to 63 kb [36].

5. PHOTOSYNTHETIC MUTATIONS: TOOLS

FOR UNDERSTANDING THE FUNCTION

AND ASSEMBLY OF PHOTOSYNTHETIC COMPLEXES

A large number of chloroplast and nuclear mutations affecting photosynthetic enzymes and

complexes have been identified and characterized in Chlamydomonas. Methods of mutant isolation have been covered in recent articles and will not be discussed here [67,68].

5.1. Ribulose 1,5 bisphosphate carboxvlase / oxvgenase

Among the photosynthetic complexes, RuBisCO has the most simple structure. The holoenzyme consists of eight identical large subunits (LS) and eight identical small subunits (SS), which are encoded by the chloroplast and nuclear genomes, respectively. The large subunit contains the cata- lytic sites for two competing reactions: carboxyla- tion by CO 2 and oxygenation by 02 of ribulose bisphosphate [16]. While there are between 50-100 LS genes (rbcL) per Chlamydomonas cell [69], there are only two closely linked genes of the small subunit (rbcS) in the nuclear genome [70].

Both nuclear genes contain three introns which are located at different positions within the coding sequence than in higher plants [71]. The genes encode two slightly distinct proteins which differ by four amino acids, and their expression is dif- ferentially regulated by the growth conditions [71].

The first uniparental RuBisCO mutant, 10-6C, was isolated as a light-sensitive acetate-requiring mutant by Spreitzer and Mets [42]. The mutation alters the isoelectric point of the large subunit and it greatly reduces carboxylase and oxygenase activity. Two additional mutants 18-5B and 18-7G were isolated by screening for uniparental mutations that did not recombine with the first mutation in 10-6C [44]. These two mutants do not accumulate either large or small subunit. The rbcL genes of the three mutants were isolated, partially sequenced and found to contain single base pair changes. Mutant 10-6C has a missense mutation that changes a gly (residue 171) to asp near one of the active sites of the large subunit (Fig. 5) [43].

The significance of this base change was con- firmed by isolating a revertant in which the wild- type gly 171 was found to be restored [72]. The other two mutants contain nonsense mutations near the 3' and 5' ends of rbcL (Fig. 5) [45].

Interestingly, the 18-5B mutant which produces a truncated large subunit only 25 amino acids shorter

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20

i~ ~ .~ HA

I

~0 6C G IAT °~: + --

R/. 7 OiGy;g ,l "t" "1"

18-5B TGI A ' ¢'c

TAOlcmee 18-7G

Fig. 5. Mutations in the gene of the large subunit of ribulose bisphosphate carboxylase. The rhcL coding sequence is shown with its 5'- and 3' untranslated regions. Regions I, III and IV correspond to the active sites of the large subunit of ribulose bisphosphate carboxylase and region II corresponds to the CO 2 activator region [16]. Base and corresponding amino acid changes are shown for 10-6C [43], R4-7, a revertant of 10-6C [72], 18-5B and 18-7 G [45]. +/- refer to the presence or absence of holoenzyme (H) and of enzymatic activity (A).

than its wild-type counterpart, is unable to form a stable holoenzyme [44].

Since the intact large subunit is no longer synthesized in these mutants, they allow one to study whether synthesis of the two subunits of RuBisCO is tightly coordinated. In both nonsense mutants examined, the steady-state levels of the large and small subunit mRNAs are not apprecia- bly changed relative to the wild type. Pulse-labeling of cells reveals that the small subunit is synthesized and processed to its mature size, thus strongly suggesting that it is imported into the chloroplasts. However, pulse-chase experiments reveal that the small subunit is rapidly degraded in these mutants [45]. Similar results have been obtained by Schmidt and Mishkind who inhibited chloroplast protein synthesis with chloramphenicol or used a chloroplast ribosome deficient strain [73]. It can be concluded that there is no tight coordination of synthesis of the two subunits and that the stoichiometric accumulation of the two is achieved at the post-translational level by specific degradation of the subunits present in excess. It will be of interest to examine the fate of the large subunit in mutants unable to produce intact small subunit. However, mutants of this type have not yet been reported.

One of the nonsense mutants, 18-5B, reverts at a high spontaneous frequency, 6 x 10 6 [74]. These revertants are unstable: they segregate both wild- type and acetate-requiring phenotypes in crosses

or under permissive growth conditions for acetate-requiting mutants [74]. Spreitzer et al. [74]

presented a model based on the observation that the acetate-requiring segregants were always unstable. The model postulates a heteroplasmic population of chloroplast DNA molecules in which all of them carry the rbcL mutation of 18-5B and where some fraction of these DNA molecules contains in addition a suppressor mutation capable of restoring the wild-type large subunit. The wild-type allele of the suppressor gene would be required for performing its normal function. While homoplasmicity of the wild-type allele would confer a selective advantage for cells grown under heterotrophic conditions (where photosynthetic function is not required), homoplasmicity of either allele would be lethal under phototrophic conditions. In this model, heteroplasmicity would be maintained by constant selection for photosynthetic function.

The molecular basis of this suppression has not yet been elucidated and it could involve tRNAs, ribosomal proteins, ribosomal RNAs or other factors involved in chloroplast translation.

5.2. Photosystem H (PSII)

The PSII core, which is embedded in the thylakoid membrane consists of at least five chloroplast-encoded proteins (Table 2) [76]. The genes of four of these polypeptides have been located on the chloroplast genome of C. reinhardtii [20,22]

(C. Kovacic, P. Malnoe and J.-D. Rochaix, unpublished results), psbB and psbC encode two proteins of 47-50 and 43-47 kDa, respectively, which have been shown to be chlorophyll a-binding proteins [75]. The polypeptides D1 and D2 are encoded by psbA and psbD, respectively [20,22]. The gene of the apoprotein of cytochrome b559, psbE, has not yet been located on the chloroplast DNA map of C. reinhardtii. Three nuclear-encoded polypeptides of 33, 24 and 18 kDa are involved in oxygen evolution (Table 3). Several low-molecular-weight proteins appear to be associated with the PSII core [76].

Mutants deficient in PSII activity can usually be recognized by their high fluorescence yield [68].

A striking feature of these mutants is that they all lack the core PSII polypeptides, indicating that they are unable to assemble a stable PSII complex

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Table 3

Properties of photosystem II mutants of Chlamydomonas reinhardtii

psB, pshC, psbA and psbD are the chloroplast genes of the 50-kDa, 47-kDa, D1 and D2 proteins of the PSII core. The 33-kDa, 24-kDa and 18-kDa proteins of the oxygen evolving complex (OEC) of PSII are encoded by nuclear genes. I-[ primaw lesion in the gene, + stable accumulation of protein, ( + ) reduced amount of protein, no accumulation of protein.

Mutant PSII core OEC of PSII

psbB psbC pshA psbD

(50 kDa) (47 kDa) D1 D2 33 kDa 24 kDa 18 kDa

Chloroplast

FuD7 - ~l - ( + )

FuD47 -

FuD34 ~? - - +

MA16 - ~? - +

Nuclear

BF25 + + + + +

FuD39 + + + + +

FuD44 (+) (+) (+) (+)

+ +

-t- +

I I

4- +

[35,41]. Pulse-labeling of cells with [14C]acetate in the presence of an inhibitor of cytoplasmic ribosome translation allows one to easily detect the most abundant polypeptides synthesized in the chloroplast: the large subunit of RuBisCO and several thylakoid polypeptides which include the PSII core proteins (Fig. 6). It is therefore possible to measure the synthesis of thylakoid polypeptides of mutants that are unable to stably insert these proteins into the membranes. Analysis of several PSII mutants by this method has revealed that more than 85% lack the D1 polypeptide (Fig. 6).

Surprisingly, all the mutants of this class which were examined have both copies of psbA ^deleted from their chloroplast genome [35]. It is known that the chloroplast inverted repeat of C. rein- hardtii contains numerous repetitive elements, in- terspersed throughout the chloroplast genome [6,21,77]. Since psbA is known to be surrounded by these repeats, it is possible that the deletions arise through recombination between the repeats [35,361.

Another class of nuclear and chloroplast PSII mutants unable to synthesize both the D1 and D2 polypeptides is of special interest. Genetic and molecular analysis of one of these chloroplast mutants, FUD47, has revealed that its psbA is intact and transcribed at wild-type levels [41]. The mutation was found to be a 46-bp direct DNA duplication within psbD, the gene of the D2 protein, thus causing a frameshift which results in a truncated D2 polypeptide that is highly unstable.

With the exception of D1 and D2, all the other PSII core polypeptides are synthesized and integrated into the membrane in this mutant, but never accumulate. It appears therefore that D2 is not only involved, directly or indirectly, in the stable assembly of the PSII core complex, but also regulates D1 synthesis or stability at either the translational or the post-translational level.

Two other chloroplast PSII mutants, FUD34 and MA16, also lack the PSII core (J. Girard- Bascou and P. Bennoun, unpublished results).

Analysis of the newly synthesized chloroplast pro-

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22

....

-- 0'}

LL LL LL

CFI< ^CFI<

6 Cyt.

D2 D2 E

19

34 36

n

ii%1 ¸ ~ ...

r-~ D

LL LL

L2 .... * ~- L3 ...

L4 - ~ ~ ~ ...

C F I,(::{,,p -~__ ~

5 --

6 -- ...

Fig. 6. Gel electrophoretic fractionation of thylakoid membrane polypeptides from wild-type cells and photosystem II mutants missing psbA (FUD7, FUDll2 and FUD13: see Table 2 for details) of C. reinhardtii labelled for 45 rain with [14C]acetate in the presence of anisomycin. Stained gel and corresponding autoradiogram are shown in the left and right panels, respectively. Upper part: 12-18% SDS polyacrylamide gel with 8 M urea. Lower part: 7.5-15% SDS polyac~lamide gel: only the region of polypeptide 5 is shown because this band comigrates with the a subunit of ATP synthase in the SDS urea gel shown above. PSII polypeptides are marked with dots (from [35] with permission).

teins in these mutants reveals that FUD34 specifically lacks the 43-47 kDa polypeptide, and that this protein is altered in MA16. The other PSII core polypeptides, including D1 and D2, are synthesized normally, but do not accumulate. It is likely that the primary lesion in these two mutants

is located in psbC, although no molecular evidence of this has yet been found. These results suggest that any of the PSII core polypeptides may play a role in the stable assembly of the core complex. It is interesting to note that nuclear mutations have been isolated which have the same phenotype as FUD34, i.e., these mutants are unable to synthesize the 43-47-kDa polypeptide.

Recently, a nuclear mutant has been isolated from FUD34 in which the chloroplast mutation of the latter is specifically suppressed (J. Girard-Bascou, unpublished results).

The oxygen-evolving complex of PSI! consists of at least three nuclear-encoded polypeptides of 33, 24 and 18 kDa (see [79] for review). By screening a cDNA library of C. reinhardtii (M.

Goldschmidt-Clermont, A. Shaw, P. Malnoe, S.

Mayfield, unpublished results) in the expression vector Xgtll [80] with monospecific antibodies against these proteins, it has been possible to isolate the three respective cDNA and genomic clones (S. Mayfield and J.-D. Rochaix, unpublished results). Southern hybridizations of genomic DNA with the three cDNA clones reveals that the corresponding genes are present in single copies.

Three low-fluorescent nuclear mutants deficient in oxygen evolution have been isolated [78] (P. Ben- noun, unpublished results). Two of these, FUD39 and BF25, lack the 24-kDa polypeptide, but not the 33 and 18 kDa polypeptides, while FUD44 lacks the 33 kDa polypeptide but not the 24 and 18 kDa polypeptides (S. Mayfield and J.-D.

Rochaix, unpublished results). The core PSII complex is still present in FUD39 and BF25, indicating that it can assemble independently from the oxygen evolving complex. This also holds true for FUD44, except that the amount of core complex proteins is reduced. No mRNA for the missing polypeptide is detectable in any of these mutants.

Genomic rearrangements have occurred at or near the 24-kDa and 33-kDa protein genes in FUD39 and FUD44, respectively. Revertants of FUD44, which occur at a frequency of 10-7, all display a novel, identical genomic arrangement that is different from both wild type and mutant. The molecular basis of this phenomenon, which may involve transposable elements, is under study.

A great deal of similarity exists between the

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reaction centers of PSII and of purple photosynthetic bacteria. In both cases a pheophytin acts as an intermediate electron acceptor and a very similar electron accepting quinone iron complex exists [81,82]. A structural homology between D1, D2 and the L and M subunits of the bacterial reaction center has been noted [83,84,123]. Recently the molecular structure at 0.3 nm resolution of the reaction center of Rhodopseudomonas viridis has been determined by X-ray diffraction studies on crystallized reaction centers [85]. Based on the

structural and functional homology between the two bacterial and PSII subunits, it has been proposed that D2 and D1 are the apoproteins of the stable primary and secondary electron acceptors of PSII, respectively, and that they can be folded with 5 transmembrane domains to form a core with chlorophyll, pheophytin and quinone which is very similar to the bacterial reaction center [82,85]. While this model of D1 (Fig. 7) fits nicely with data obtained from herbicide-resistant mutants (see below), it does not completely agree

5C

I

NH2

250~

F#~. ~ - -

' 1

~ G

G H

~

^COOH

Fig. 7. Membrane-spanning model of the photosystem II polypeptide DI of C. remhardtii. This model proposed by Trebst [82] is based on the homology between D1 and the L subunit of the bacterial photosynthetic reaction center [83,123] and on the X-ray structure of the latter [85]. Amino acids are represented in the single letter code. The five transmembrane domains are framed.

Stroma and intrathylakoid space are on the upper and lower side, respectively. Residues V219, F255, $264 and L275 which are individually changed in four different herbicide resistant mutants [39,40], (J. Erickson et al., unpublished results) are indicated. The region which binds azidoatrazine (residues 244-224) is marked [118]. The trypsin cleavage sites at R225 and R238 are indicated [86].

The two H215 and H272 residues together with the corresponding residues of the D2 polypeptide may be involved in iron and QB binding [82,85].

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24

with results obtained with protease digestion of thylakoid membranes which indicate that the carboxy-terminal end of D1 is on the stromal side [86].

Several uniparental mutants with different levels of resistance and cross-resistance to the herbicides atrazine, diuron and bromacil have been isolated in C. reinhardtii [37-39,87]. Analysis of psbA from some of these mutants has revealed four distinct residues on the D1 polypeptide which can be changed (Fig. 7) [39~40] (J. Erickson, L. Mets and J.-D. Rochaix, unpublished results). It is noteworthy that although the four possible mutation sites identified to date in D1 (Val 219 ~ Ser; Phe 255 ---, Tyr; Ser 264 ~ Ala; Leu 275 --* Phe) are scattered over a stretch of 56 amino acids, they are located close to each other in the above model in a region of D1 that is oriented towards the stromal side of the thylakoid membrane, close to the pre- sumed acceptor quinone-binding niche (Fig. 7).

The iron which connects the two quinone acceptors in the bacterial reaction center would also be located in the same region and held by four His residues (His 215 and His 275 from D1; Fig. 7;

and two from D2 at the same positions). It is noteworthy that the mutations affecting Val 219, Phe 255 and Leu 275 do not markedly alter electron transport and thus photosynthetic yield, in contrast to the Ser 264 mutation, which signifi- cantly retards electron transport [38,39]. Atrazine resistance in higher plants has also been shown to be due to a change at the same Ser 264 residue, but in contrast to C. reinhardtiL this serine is replaced by glycine [88,89].

5.3. Photosystem I

This photosynthetic complex consists of a large number of polypeptides: two chloroplast encoded polypeptides of apparent M r 60000-70000 (subunit I) both of which appear to be the apoproteins of the CPI chlorophyll-protein complex which comprises the reaction center chlorophyll P700 [90,91], and several smaller subunits with sizes ranging between 25 and 8 kDa [92]. Recently, the two genes of subunit I, psA1 and psaA2, of C.

reinhardtii have been sequenced (U. Khck, M.

Schneider, M. Dron, Y. Choquet, unpublished results). Whilst psaA2 is continuous, psaA1 con-

tains a large intron near its 5' end. The function of the other subunits of PSI has not yet been clearly established. Some of the smaller subunits have been tentatively identified as the apoproteins of the stable Fe-S electron acceptors of PSI [93].

Comparisons of PSI defective chloroplast and nuclear mutants of C. reinhardtii with wild-type cells have shown that all the mutants lack subunits I and the same set of low-molecular-weight thylakoid polypeptides [94]. Genetic analysis of 25 nuclear PSI mutants has revealed that they belong to 13 complementation groups scattered throughout the nuclear genome [94]. Eight chloroplast PSI mutants fall into four distinct genetic loci as defined by the inability of mutants of the same group to recombine with each other (J.

Girard-Bascou, unpublished results; see section 6). One chloroplast PSI mutant, FUD26, produces a truncated form of subunit I. Partial sequence analysis of psaA2 from this mutant has revealed that it has suffered a 4-bp deletion which results in a frameshift (M. Schneider, Y. Choquet, J. Girard, F. Galangau, M. Dron, P. Bennoun, unpublished results).

5.4. Cytochrome b 6 / f complex

As in the other photosynthetic protein complexes, the cytochrome b6/f complex consists of subunits of nuclear and chloroplast origin. It is well documented that cytochrome f, cytochrome b6 and subunit IV are encoded by the chloroplast genome whereas the Rieske protein is coded for by the nuclear genome [95]. A fifth subunit of this complex has recently been identified in C. rein- hardtii by comparing the polypeptide profiles of the purified complex with that of thylakoids from mutants lacking this complex [96]. Translation of this subunit V appears to occur on cytoplasmic ribosomes [96]. Analysis of thylakoid polypeptides of pulse labelled cells from several chloroplast and nuclear mutants affected in the function of the cytochrome b6/f complex has revealed that it is assembled in two steps. The three chloroplast-encoded subunits are inserted independently in the thylakoid membrane and assembled into a sub- complex in the absence of the nuclear-encoded Rieske protein and subunit V. These two proteins are unable to insert and assemble in the mem-

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brane without the presence of the chloroplast-encoded sub-complex. The assembly of the cytochrome b6/f complex resembles to some degree that of PSII, where the chloroplast-encoded subunits can also assemble in a core complex in the absence of the nuclear-encoded subunits. How- ever, while the latter are peripheral membrane proteins, the Rieske protein of the cytochrome b6/f complex is a transmembrane polypeptide.

5.5. A TP synthase complex

This complex consists of a peripheral component which included 5 subunits a, /3, y, 6, ~ and a membrane-embedded component which contains at least three subunits (CI, CII, CIII proteolipid) [97]. The genes of the chloroplast-encoded subunits, atpA (cO, atpB (/3), atpE (~), atpF (CI) and atpH (CIII) have been identified on the chloroplast genome of C. reinhardtii (see Fig. 3). Six- teen chloroplast mutations affecting the ATP synthase complex have been characterized [24]. Com- plementation assays in young zygotes and recombination analysis has revealed five complementation groups. Although this number agrees with the number of identified ATP synthase chloroplast genes, this correlation still needs to be proven rigorously at the molecular level. In one case it has been possible to assign mutants of one complementation group to the atpB locus based on the fact that some of the mutants have chloroplast DNA deletions in this region [34].

6. RECOMBINATION ANALYSIS OF CHLO- R O P L A S T GENES: C O R R E L A T I O N BE- TWEEN PHYSICAL AND GENETIC CHLO- ROPLAST DNA MAPS

The localization and characterization of several mutations on the chloroplast genome of C. rein- hardtii has allowed one to attempt to correlate physical distances with recombination frequencies between markers. Genetic mapping of chloroplast genes in Chlamydomonas has been performed in several ways including recombination and co- segregation analysis in pedigrees [98] and segregation and recombination analysis in the progeny of biparental zygotes [15,98]. Since these methods

have been extensively discussed (see [15]), only the zygote clone analysis, which is commonly used, will be described. One first selects for biparental zygotes and scores the frequencies of different chloroplast genotypes among the progeny of each biparental zygote, usually 64 or more randomly selected cells. Because thousands of cells need to be examined in order to obtain reproducible maps [48], the standard zygote clone analysis has been modified in order to obtain reliable mapping data with a smaller number of cells [99]. Cells are selected that carry at least one marker from the paternal mt parent, and only one cell from each biparental clone is examined, thus ensuring that each cell scored has a genotype that is established independently from the others [99].

Chloroplast gene mutations conferring resistance to erythromycin, neamine, spectinomycin and streptomycin were the first to be mapped by recombination analysis [15,48,98]. These mutations were shown to be genetically linked and their order and relative position were determined.

A first indication that these mutations may affect the chloroplast rRNA genes was provided by interspecific hybrids of C. eugametos and C.

moewusii whose chloroplast DNA restriction patterns differ. Linkage between a streptomycin sensitivity locus and a chloroplast restriction frag- ment which hybridizes to the 16S rRNA gene region of C. reinhardtii was shown by an analysis of recombinant chloroplast restriction patterns from the hybrid progeny [100]. Similarly, a correlation was found in other hybrids between the inheritance of chloroplast mutations conferring resistance to streptomycin and erythromycin and the small specific deletion/addition differences at the 5'-end of the 16S rRNA gene and the 3'-end of the 23S rRNA gene, respectively [101]. Strep- tomycin resistant mutants from Euglena gracilis have been shown to contain a single base change in an invariant position of their chloroplast 16S rRNA gene [52].

Recently, four independent mutations conferring resistance to spectinomycin have been mapped to the 16S rRNA gene of C. reinhardtii based on alterations of a restriction site in a highly conserved region of this gene (J. Boynton, E. Harris and N. Gillham, unpublished results). The same

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restriction site change has also been observed for similar mutants in E. coli [102] and tobacco [103].

A uniparental chloramphenicol resistant mutant has been isolated but its mutation has not yet been mapped relative to the other antibiotic resistance markers [46]. Based on the sequence homology between chloroplast and mitochondrial rRNAs [6] and on the localization of mutations conferring resistance to chloramphenicol and erythromycin in the yeast mitochondrial large rRNA gene [51], it is tempting to think that the homologous chloroplast mutations will be found in the 23S rRNA gene of C. reinhardtii.

Using the paternal marker selection mapping method, Mets and Geist [99] have demonstrated linkage between three photosynthetic mutations and the uniparental linkage group comprising the antibiotic resistance mutations. One of the photosynthetic mutants~ 10-6 C, was shown to have a single base-pair change in rbcL (see section 5.1) and therefore provided the first correlation site between the physical and genetic chloroplast DNA maps [43,99]. No recombinants could be recovered between the other two mutants (Dr2, which is herbicide-resistant, and 8-36C which lacks photosystem II), suggesting that the same gene is affected in both mutants [99]. Indeed, a molecular analysis of these mutants has revealed that the first carries a single base-pair change in psbA (see section 5.2) and the second has a double deletion in its inverted repeat that includes all of psbA [35,40]. Surprisingly, the results of crosses in which the same mt parent carried the herbicide and antibiotic resistance markers, were quite different, depending on whether the marker of the mt + parent was in rbcL or psbA. In the first case, the parental class accounted for two-thirds of the progeny and the data obtained with four markers could not be resolved into a single linear map. In the second case, the frequency of recombination was higher: the parental class accounted for only 1/4 of the progeny and the frequency of recombination between the same two antibiotic resistance markers was nearly twice as high as in the first cross [99]. It was suggested that the organization of the chloroplast DNA into nucleoids could lead to incomplete mixing of the parental chloroplast DNA and that the extent of mixing may depend

on the organization of the thylakoids, which is different in PSII mutants compared to wild-type and rbcL mutants [99]. It is also conceivable that the deletion in 8-36C may stimulate chloroplast DNA recombination in crosses with strains lacking the deletion. Effects of this sort are well-documented in yeast: in heteropolar crosses of yeast strains that differ by the presence and absence of the omega insertion in the mitochondrial large rRNA gene, striking perturbations occur in the recombination frequencies of nearby markers [104].

Another complication in the study of genetic linkage of chloroplast markers arises from the structure of the chloroplast genome. Since in- tramolecular recombination is known to occur within the inverted repeat [36,105], one single copy region may be inverted relative to the other. If this recombination occurs frequently, markers in each of the single copy regions would appear to be unlinked with respect to each other. On the other hand, genetic linkage would be expected between markers in the single copy region and in the inverted repeat, as has been found between markers of rbcL and psbA [99]. This can be attributed to an efficient gene conversion which appears to operate between the two segments of the inverted repeat. In at least one case this has been demonstrated for a single base pair mutation conferring herbicide resistance [40] and in several cases double symmetrical deletions have been found in the inverted repeat [34,36]. Because of this mismatch correction mechanism, intramolecu- lar recombination in this region would not disturb recombination frequencies between markers outside and inside of the inverted repeat.

The observed genetic" linkage between markers in rbcL and psbA or in the ribosomal region which are more than 10 kb distant from each other on the chloroplast genome, indicates that chloroplast markers recombine at a lower rate than yeast mitochondrial markers. In the latter case, genetic linkage is only apparent between mitochondrial markers separated from each other by less than 1 kb [104]. An alternate gene linkage analysis (J. Girard-Bascou, unpublished results) uses vegetative zygotes obtained from gametes of opposite mating-type each carrying a photosyn-

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thetic mutation and either the arg-2 or arg-7 nuclear mutation which are closely linked [106].

Vegetative zygotes remain diploid, divide mitotically and can be selected on medium lacking arginine. The rate of recombination is estimated from three parameters: the number of biparentals, usually higher than 40%, the frequency of cells able to grow on minimal medium and the differen- tial growth rate of wild-type and mutant cells.

Using this method, Girard-Bascou (unpublished results) has grouped eight different chloroplast PSI mutants into four distinct genetic loci. Mutants of the same locus recombine with a frequency of less than 5 x 10 s while recombination between mutations of presumably unlinked loci is greater than 10 1. The same method yields a recombination frequency of 2 x 10 2 between mutations in rbcL and psaA2, which are 1.9 kb apart (M.

Schneider, Y. Choquet, M. Dron, J. Girard-Bascon and P. Bennoun, unpublished results).

In conclusion, chloroplast DNA recombination has been clearly demonstrated in Chlamydomonas.

Six distinct loci on the chloroplast genome have been correlated with defined photosynthetic mutations in C. reinhardtii. The correct interpretation of recombination frequencies between chloroplast markers is not an easy task, however, since it depends on several parameters, such as the input of the parental chloroplast genomes in the cross [107,108], the extent of mixing of these two genomes in the zygotic cell, and possible alterations in recombination frequencies due to somatic mutations. Before recombination frequencies can be safely correlated with physical distances on the chloroplast genome, it is clearly important to examine more markers with different and independently expressed phenotypes and to compare care- fully the recombination frequencies between the same markers obtained with the different mapping methods. It may then be possible to use genetic mapping to locate interesting mutations on the chloroplast genome, such as those involved in the regulation of the assembly and function of photosynthetic complexes.

It is noteworthy that a complementation test has been developed for photosynthetic mutations which examines the fluorescence induction kinet- ics or the luminescence properties of young zygotes

[109]. This test works for all nuclear photosynthetic mutations examined and for the chloroplast mutations affecting D1, D2 and the ATP synthase subunits, but apparently not for chloroplast mutants blocked in the electron transport chain downstream from the plastoquinone pool (J.

Girard-Bascou and P. Bennoun, unpublished results). This observation may be related to the specific deficiency of PSI activity in gametes as compared to wild-type cells (P. Bennoun, unpublished results).

7. G E N E T I C F U N C T I O N A N D I N H E R I - TANCE OF M I T O C H O N D R I A L DNA IN CHLA M YD OMONA S

It is well documented that the mitochondrial DNA of higher plants ranges between 400 and 2000 kb [110]. In C. reinhardtii, this DNA appears to consist only of 16-kb linear molecules with unique ends and a homogenous sequence [12].

Whether this D N A represents the entire mitochondrial DNA of C. reinhardtii has not yet been proven rigorously because of the difficulty of obtaining pure mitochondria from this organism.

The genes of apo-cytochrome b, of subunit I of cytochrome oxidase and of the mitochondrial rRNAs have been mapped on the 16-kb linear DNA molecule [111]. This DNA has also been shown to contain open reading frames which are homologous to URF2 and URF5 of the mam- malian mitochondrial genome [112-114]. The products of these two open reading frames appear to be components of the NADH:ubiquinone re- ductase of the inner mitochondrial membrane [115]. From the available partial sequence of the C. reinhardtii mitochondrial genome it can be concluded that tryptophan is specified by T G G rather than by TGA, as in the mitochondrial DNAs of fungal and animal cells [104,116]. Al- though the mitochondrial genomes of C. rein- hardtii and animal cells have similar sizes, their genome organization is different [111]. No discrete DNA species around 16 kb are detectable in C.

eugametos and C. moewusii, but sequences homologous to the 16-kb DNA from C. reinhardtii have been found [111]. The size of mitochondrial