Mitochondrial genomes

MITOCHONDRIAL GENOMES" A PARADIGM OF ORGANIZATIONAL DIVERSITY

Linda Bonen

I. II.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

416

H u m a n Mitochondrial D N A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

422

A. III. IV.

V.

VI.

Gene Organization and Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

422

B. H u m a n Mitoch0ndrial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial G e n o m e s of Other Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fungal Mitochondrial G e n o m e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

425 426 428

A.

Unicellular A s c o m y c e t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

428

B. C. D.

Filamentous A s c o m y c e t e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial G e n o m e s of Other Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . Fungal Mitochondrial Dysfunction and Senescence . . . . . . . . . . . . . . . . . .

431 432 432

Protist Mitochondrial G e n o m e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Ciliate Protists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Chlorophytic and Rhodophytic Algae . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

433 433 434

C. D. E. E

437 438 439 440

A m o e b o i d Protists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . T r y p a n o s o m a t i d Protozoa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A p i c o m p l e x a n Protists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondrial G e n o m e s of Other Protists . . . . . . . . . . . . . . . . . . . . . . . . . .

Plant Mitochondrial G e n o m e s . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

440

A.

B r y o p h y t e Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

440

B.

Flowering Plant M i t o c h o n d r i a . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

442

Advances in Genome Biology Volume 5B, pages 415-461. Copyright 9 1998 by JAI Press Inc. All rights of reproduction in any form reserved. ISBN: 0-7623-0079-5

415

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C. Plant Mitochondrial Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . ....... VII. Novel Features of Mitochondrial Genetic Systems . . . . . . . . . . . . . . . . . . . . . . . A. ModifiedGenetic Code and Unusual tRNAs . . . . . . . . . . . . . . . . . . . . . . . . B. RNA Editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mobile Introns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Scrambled Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Evolution of Mitochondrial Genomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Concluding Remarks . . . . . . . . . . . . . . . . . ..............................

445 446 446 448 449 451 451 453

The mitochondria of eukaryotic cells contain their own distinctive DNA, with essential information for energy production through oxidative phosphorylation. To this end, mitochondrial genes encode a small subset of respiratory chain proteins, as well as components of the machinery needed to synthesize them. Although all mitochondria share a common genetic function, different eukaryotic lineages display enormous diversity and plasticity in the size, structure and organization of mitochondrial genomes, in mechanisms of mitochondrial gene expression, and in the rate/mode of mitochondrial DNA evolution. The contrast between the compact 16 kbp human mitochondrial DNA and those of some plants where complex multipartite genomes are estimated to be as large as 2400 kbp, is striking. Recent sequence data suggest that the number of mitochondrial protein-coding genes in some protists and plants is at least five-fold higher than in animals or fungi, and that this can be accompanied by strong traces of a bacterial endosymbiotic heritage. This review presents a survey of mitochondrial genome organization in animals, fungi, protists and plants. It highlights recent advances in our understanding of the structural and genetic diversity displayed by mitochondria from different eukaryotes. It also outlines some of the more unusual aspects of mitochondrial gene expressionmsuch as altered genetic codes, aberrant tRNAs, RNA editing, mobile introns, and scrambled genes--features which further illustrate the multiplicity of evolutionary pathways taken by mitochondrial genomes.

I.

INTRODUCTION

It was well established that mitochondria are the site of energy production in eukaryotic cells, long before it was appreciated that they contain their own distinct genetic information and protein-synthesizing systems, without which respiration would not be possible. That discovery first came through the genetic analysis of yeast "petite" mutants in the 1950s by Ephrussi's laboratory. Respiration-deficient mutants were shown to display non-Mendelian inheritance patterns, indicative of the transmission of genetic material by the cytoplasm. That work was soon followed by the physical demonstration of nucleic acids in the mitochondria of animal and plant cells using biochemical and electron microscopic techniques. The

Mitochondrial Genomes

Table 1.

417 Mitochondrial Genome Sizes a

Animals

Homo sapiens (human)

16.6 kbp

Xenopus laevis (clawed toad)

17.6

Drosophila yakuba (fruit fly)

16.0

Caenorhabditis elegans (soil nematode)

13.7

Strongylocentrotus lividus (sea urchin)

15.7

Katharina tunicata (black chiton)

15.5

Metridium senile (sea anemone)

17.4

Fungi Saccharomyces cerevisiae (budding yeast)

68-85

Schizosaccharomyces pombe (fission yeast)

17-25

Neurospora crassa

60-73

Podospora anserina

87-100

Allomyces macrogynus

57.5

Protists Paramecium aurelia

40.5

Chondrus crispus (red alga)

25.8

Chlamydomonas reinhardtii (green alga)

15.8

Prototheca wickerhamii (colourless green alga)

55.3

Acanthamoeba castellanii

41.6

Trypanosoma brucei Plants Marchantia polymorpha (liverwort) Arabidopsis thaliana

22 187 372

Brassica hirta (white mustard) Cucumis melo (muskmelon)

2400

Triticum aestivum (wheat)

430

208

Zea mays (maize) 570 Note: aDataare summarized from refs. 1, 2, and 112. All of these mitochondrial genome sizes are derived from complete, or nearly complete sequencing data, except for those of flowering plants.

presence of DNA in the mitochondrion was well accepted by the late 1960s and it raised a number of interesting questionsmwhat specific genetic information do mitochondria possess?; how is it organized and expressed?; how is the mitochondrial genetic material passed on to daughter cells?; where did mitochondrial DNA come from? The combination of genetic, biochemical, and, more recently, powerful molecular approaches, has led to significant advances in our understanding of these small but essential genetic systems. There have been numerous recent reviews on this subject; of particular note are very comprehensive compilations by Gillham 1 and Wolstenholme and Keon 2 and references given therein.

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One of the first surprises to come from the molecular analysis of mitochondrial DNA (mtDNA) was that even though mitochondria all share the same fundamental function in respiration, they display a great diversity in genome size, gene organization, mode of expression, and rate/mode of evolution among various eukaryotic lineages (reviewed in refs. 3-7). A comparison of mitochondrial genomes reveals that their sizes vary from about 14 to 18 kbp in most animals (or as low as 6-7 kbp in several parasitic protists) to values of 187-2400 kbp in plants. The number of predicted proteins encoded within the mitochondrion varies from only three in the malarial parasite, Plasmodiumfalciparum, to over 60 in the liverwort, Marchantia polymorpha. These genomes are organized differently among eukaryotes; in most animals, the mtDNAs are circular, whereas in certain protists they are linear and in flowering plants they show a complex multipartite organization. Even more unusual are those of trypanosomatid protists, where large numbers of "minicircular" and "maxicircular" mtDNA molecules are concatenated in a complex network. In Table 1, the sizes of a number of completely-sequenced mitochondrial genomes are shown, as well as several from flowering plants where data are as yet incomplete. The number of mitochondrial genomes which have been entirely sequenced currently exceeds 60 and is growing rapidly. A more comprehensive, up-to-date listing is compiled in GOBASE, an Organelle Genome Database, accessed through the World Wide Web site (URL http://megasun.bch.umontreal.ca). Almost all mitochondrial genomes share a similar "core" set of genes for the two ribosomal RNAs (LSU rRNA and SSU rRNA ofthe large and small subunits, respectively) and transfer RNAs, as well as about a dozen protein-coding genes for subunits of the respiratory chain. The large and small ribosomal RNA genes have invariably been found in the mitochondrial genomes characterized to date. The tRNA gene collection is in some cases sufficient to decode the mitochondrial mRNAs; however, in some protists and plants a number of tRNAs are nuclear-encoded and must be imported into the mitochondrion for their role in translation. The "classical" mitochondrial protein-coding genes encode subunits of the respiratory electron transport chain--NADH dehydrogenase subunits of complex I, cytochrome b of complex III, cytochrome oxidase subunits of complex IV, and ATPase subunits of complex V. Certain mitochondrial DNAs, particularly within the plant and protist lineages, have a larger set of genes, such as ones for ribosomal proteins, cytochrome c biogenesis machinery, or succinate dehydrogenase subunits of respiratory complex II. Moreover, DNA sequence analysis has established that most of the larger mitochondrial genomes possess a number of open reading frames (ORFs, usually named according to number of codons present), which have the potential of encoding proteins of, as yet, unknown function. To be designated as such a putative gene, the reading frame must fulfill criteria such as exceeding the length which could be expected to occur by random chance (typically 60-80 codons), having a codon usage pattern typical of known genes in that mitochondrial genome, and being actively transcribed. Even stronger support is provided by the presence of homologous (and functionally constrained) ORFs among different organisms.

Table 2.

Mitochondrial Gene Content of Selected Organisms

Homo sapiens a

Saccharomyces cerevisiae b

LSU

+

+

SSU

+

+

22

25

Ribosomal

Chlamydomonas reinhardtii c

Acanthamoeba caste llanii d

Marchantia polymorpha e

+

+

+

+

+

+

3

16

27

+

+

+

+

+

+

-

+

+

+

+

+

-

+

+

RNA

5S Transfer RNA Respiratory Complex

~D

Chain:

I

nadl ( N D 1) nad2 ( N D 2 ) nad3 ( N D 3) nad4 ( N D 4 ) nad4L(ND4L) nad5 ( N D 5 ) nad6 ( N D 6 ) nad7 nad9 (orf212) nadll Complex

+

+

+

+

+

+

-

+

(+)

-

+

+

+

+

+

II

sdh2 (orf137) sdh4 (orf86a) Complex

cob

III

+

+

(continued)

T a b l e 2.

(Continued)

sapiens a

Saccharomyces cerevisiae b

Chlamydomonas reinhardtii c

Acanthamoeba castellanii d

Marchantia polymorpha e

coxI

+

+

+

+

+

coxlI coxlIl

+ +

+ +

-

+ +

+ +

Homo

Complex IV

Complex V atpA atp6 atp8 atp9

Ribosomal proteins rps l rps2 rps3 rps4 rps7 rps8 rps l O rps l l rps l 2 rps13 rps14 rpsl 9

rp[2

-

-

rpl5

-

-

rpl6

-

-

rplll

-

-

-

-

rpll

4

rpll6

-

-

varl

-

+

orf509

-

-

+

orf322

-

-

+

orf277

-

-

orf228

-

-

+

orf169

-

-

+

-

-

+

-

-

+

-

-

21

Cytochrome c biogenesis

ORF25 ORFB

(orf183) (orf172)

other orfs

Intronic O R F s group I

-

5

-

3

2

group II

-

2

-

-

8

free-standing

-

3

1

-

1

RNase

-

+

9

9

9

Notes:

P RNA

aData from ref. 81 NADH dehydrogenase genes are designated as ND genes in animal mitochondria. bData from ref. 9. The "free-standing" intron-type ORFs are related to group I (RNA maturase/endonuclease)ones. CData from ref.11. The "free-standing intron-type ORF is related to group II (RNA maturase/reverse transcriptase) ones. dData from ref. 12. coxl and coxll are fused ORFs. eData from ref. 10. ORFs shown in parentheses represent Marchantia polymorpha mitochondrial homologues. There are 2 copies of tRNA-fMet and tRNA-Tyr genes and nad7 is a pseudogene. The "free-standing" intron-type ORF (orf732) is related to group II (RNA maturase/reverse transcriptase) ones.

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In Table 2, the mitochondrial gene content is shown for a few organisms selected from the different eukaryotic kingdoms: human (animal); 8 the budding yeast Saccharomyces cerevisiae (fungal); 9 the bryophyte Marchantia polymorpha (plant), 10 and two protists, the green alga Chlamydomonas reinhardtii 11 and the amoeboid protozoon Acanthamoeba castellanii. 12 The latter two were chosen to illustrate the extreme variation in mitochondrial gene content among organisms grouped within the protozoan lineage. These and other mitochondrial genomes will be discussed in detail in the following sections. It is notable that although there is a specific evolutionary history shared by the chlorophytes (represented by Chlamydomonas reinhardtii) and land plants (represented by Marchantia polymorpha), their mitochondrial genomes are at opposite ends of the spectrum with respect to gene content and size. The Chlamydomonas reinhardtii mtDNA, which is about the same size as that of humans, has one of the smallest set of genes, whereas the Marchantia polymorpha mitochondrial genome has the largest number identified to date; namely, a total of 94 (not including genes within introns). Virtually all of the known protein-coding genes, whether for respiratory chain components or expression machinery, are subunits of large complexes which also contain nuclear-encoded proteins. Therefore, mitochondrial biogenesis and function requires precise coordination of the expression of nuclear and mitochondrial genes, as well as the proper targeting of polypeptides synthesized in the cytosol to the correct mitochondrial locations. An estimated 90% of the proteins located within the mitochondrion are encoded by nuclear genes. This is in keeping with the wellaccepted hypothesis of the endosymbiotic origin of mitochondria, one facet of which is the transfer of genes from the endosymbiont to the nucleus of the host cell. Present-day eukaryotes contain multiple DNA copies within each mitochondrion, and typically there are many mitochondria per cell. Inheritance of mtDNA is non-Mendelian and almost invariably uniparental. For example, in mammals, there are estimated to be 2-10 mtDNA copies in each mitochondrion, and hundreds (or even thousands) of mitochondria within each cell. Moreover, normally the population of these mtDNA molecules is effectively homoplasmic. This review will present an overview of some of the important structural features of mitochondrial genomes of animals, fungi, protists, and plants, and it will highlight recent advances that have been made in our understanding of the organization of mitochondrial genomes from diverse eukaryotes. Because of the wealth of information on this topic, this review aims to serve as a survey of current knowledge and views.

!i.

HUMAN MITOCHONDRIAL

DNA

A. Gene Organization and Content The human mitochondrial genome was one of the first DNA molecules chosen by Sanger and coworkers in the late 1970s for DNA sequence analysis, 8 favored in

Mitochondrial Genomes

423

Figure 1. Gene organization and transcriptional map of the human mitochondrial genome of 16,569 bp. The two inner circles show the positions of the two rRNA genes (16S and 12S), tRNA genes (black circles, in one-letter amino acid nomenclature) and protein-coding genes transcribed from the heavy (H)-strand and light (L)-strand. The outer curved bars represent the identified functional RNA species (other than tRNAs) after processing of polycistronic transcripts from the H-strand (black bars) and L-strand (hatched bars). Unstable transcripts are represented by open curved bars. The origins of replication for the H-strand and L-strand are represented by O H and OL, respectively. Reprinted with permission from ref. 13.

part because of its small size and relative ease of isolation. The complete nucleotide sequence of 16,569 bp revealed a remarkably compact organization of genes, in most cases with no, or only very short, spacers between genes (Figure 1). 13 There are a total of 37 genes: thirteen protein-coding genes, two ribosomal RNA genes and 22 tRNA genes tightly packed around the covalently closed circular molecule. Seven of the protein-coding genes, initially designated as URF1-6 and URF4L (for unidentified reading frame), were renamed as ND genes (or nad in plants) after it was determined that they encode subunits of the NADH dehydro-

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genase complex. The economical organization of genetic information even results in slightly overlapping genes, as for the ND4L/ND4 genes and the ATP8/ATP6 genes. Virtually the only noncoding region in the human mitochondrial genome is a stretch of about 1100 bp between the tRNA-Pro and tRNA-Phe genes. This "control" region, where signals for replication and transcription are located, has also been designated the D-loop (displacement loop) because of the three-stranded nature of the replication intermediate structure. The two human mtDNA strands differ in base composition so they can be separated as the H-strand (heavy) and Lstrand (light) by density gradient centrifugation. The origin of replication of the Hstrand is within the D-loop region, whereas replication of the L-strand is initiated on the opposite side of the genome within a cluster of tRNA genes (Figure 1); replication is unidirectional and asymmetric. 14 As can be seen in Figure 1, most of the genes are transcribed from the H-strand; however, the genes for eight tRNAs and ND6 are in the opposite orientation and transcribed from the other DNA strand. The two major transcription initiation sites are located within the D-loop region and are separated by about 150 bp. Both promoters share a 15 bp consensus motif, and neighboring elements are also important for efficient transcription. 13-15 An additional minor H-strand promoter is located slightly downstream of the major one, within the tRNA-Phe gene. 13 Long transcripts initiating from these promoters are precisely processed into monocistronic transcripts (or bicistronic ones, in the case of overlapping genes). As can be seen in Figure 1, tRNA genes are interspersed with the protein-coding and ribosomal RNA genes, and this arrangement contributes to what has been described as the "punctuation model of transcript processing". The tRNAs appear to serve as RNA processing signals to liberate mRNAs, rRNAs and tRNAs from long precursor transcripts. The mitochondrial mRNAs therefore lack 5' or 3' nontranslated extensions. In some cases, the termination codon UAA is not genomically encoded but is generated by the addition of poly A to the 3' termini of the mRNAs, again illustrating the economical nature of genome organization. The set of tRNA genes in the human mitochondrial genome, although only totaling 22, is large enough to support protein synthesis because of "two-out-ofthree" extended wobble codon-anticodon pairing. The human mitochondrial tRNAs are smaller than bacterial or cytosolic ones and lack certain conventional features (discussed in section VII.A). Another surprise to come from sequence analysis was that the human mitochondrial genetic code differs from the "universal" one; one example being that UGA specifies tryptophan rather than termination of translation. This too will be discussed in a later section. The ribosomal RNAs (16S and 12S in size) are also much smaller than those of bacteria (23S and 16S, respectively); however, core domains of conserved primary/secondary structure are present, illustrating the universal nature of ribosomal structure and function (reviewed in ref. 3).

Mitochondrial Genomes

425

Various aspects of the expression and evolution of human (and other animal) mitochondrial genomes have received intense study and been the subject of numerous recent reviews (refs. 1-7, 13-18). There is also extensive literature regarding the use of mitochondrial DNA as a molecular marker in population studies and phylogenetic analyses (cf. ref. 19). Human (and other mammalian) mitochondrial genes accumulate nucleotide substitutions very rapidly, at a rate estimated to be about 10-fold higher than in nuclear genes 2~ and a strong bias in the number of transitions to transversions (particularly in silent codon positions) is also typically seen. Moreover, certain regions of the mitochondrial genome, such as the D-loop "control" region, are evolving more rapidly than others. These features, in conjunction with the absence of recombination in animal mtDNA and the non-Mendelian (maternal) mode of inheritance, have led to the use of mtDNA polymorphisms in addressing issues such as the population flow of early humans (cf. ref. 21).

B.

Human Mitochondriai Diseases

Significant advances have been made over recent years in the identification and molecular characterization of numerous mtDNA mutations which are associated with maternally inherited degenerative neurological and muscular disorders. Such information has been compiled in the MITOMAP database 22 which can be accessed through the World Wide Web site (URL http://www.gen.emory.edu/mitomap.html). Sensitive techniques to rapidly pinpoint mutations in mtDNA have been developed (compiled in ref. 23) and the nature of observed alterations includes deletions, point mutations within tRNA/rRNA genes, and missense mutations, all of which are consistent with defective mitochondrial gene products and thus respiratory dysfunction. The mitochondrial DNA population in an affected individual or tissue type will be a heteroplasmic mixture of normal and mutated molecules, and the relative ratios between these two forms in some cases have been correlated with the severity of the clinical symptoms. Among the first human mitochondrial mutations to be characterized at the molecular level were ones phenotypically described as: MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like symptoms), MERFF (myoclonic epilepsy and ragged red fiber syndrome), and LHON (Leber's hereditary optic neuropathy) (reviewed in refs. 24,25). In a number of MELAS and MERFF patients, point mutations were detected within structural genes for tRNAs, and in the case of many LHON patients, missense mutations within genes such as ND4 were observed. Compelling evidence that the mitochondrial mutations indeed cause the observed clinical disorder have come from experiments using mitochondrion-less cultured human cells into which defective mitochondria (e.g. from MERFF patients) have been introduced (cf. ref. 26). Deletions, rearrangements, or duplications of regions of the mitochondrial genome have also been correlated with different human diseases, and there is evi-

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dence that defective (deletion-type) molecules are present in higher proportions in successive generations of affected individuals, consistent with a "replication race" model in which smaller mitochondrial genomes are replicated more efficiently than normal ones. 27 It has long been suspected that defective respiratory function, due in part to the accumulation of mtDNA mutations from oxidative free radical damage, contributes to the natural process of aging (reviewed in ref. 27). Evidence is accruing that in normal individuals there is an age-related increase in somatic cell mtDNA deletions and that this is correlated with a decrease in oxidative phosphorylation capacity (cf. ref. 28). The possibility that defects in the mitochondrial genetic system may play a key role in certain neuromuscular disorders associated with aging (like Parkinson's disease and Alzheimer's disease) is a topic of active investigation.

II!.

MITOCHONDRIAL GENOMES OF OTHERANIMALS

Over recent years, the mitochondrial genomes have been completely sequenced from a wide variety of animals including mammals, birds, amphibians, echinoderms, nematodes, molluscs, and cnidarians (reviewed in refs. 1,16,17). The list currently surpasses 40 (compiled in GOBASE at World Wide Web site (URL http:/ /megasun.bch.umontreal.ca) and has grown rapidly, in part, because of the small size (typically 14-18 kbp) of most animal mtDNAs. Almost all animal mtDNAs examined are circular in form, with notable exceptions being certain classes of cnidarians. 29 For example, some Hydra species have 14-16 kbp linear genomes, and others have a genome comprised of two 8 kbp linear molecules. 29'3~The base compositions of mtDNAs from different animals are typically in the 40% G+C range, but as low as 21% and 24% in Drosophila yakuba and Caenorhabditis elegans, respectively. The size differences seen among animal mtDNAs usually do not reflect changes in gene content or intergenic spacers, but rather are due to length variation within the "control" region. Tandem repeats are seen to vary in copy number and alterations are presumably caused by errors during DNA replication. For example, in the root knot nematode, Meloidogynejavanica, a 7 kbp stretch within the "control" region has three types of repeats, each comprised of 5 to 36 copies. 31 In some cases coding sequences have been duplicated, as in the lizard Cnemidophorus exsanguis, where a tandem duplication spanning the D-loop and rRNA sequences increases the mtDNA size from 17.4 to 22.2 kbp among individuals. 32 Exceptionally large mtDNAs have been found in the sea scallop, Placopecten magellanicus, where tandem duplications result in molecules of 32.1 to 39.3 kbp length. 33 Gene content is highly conserved among all animals, and gene order is, for the most part, very stable at least within phyla. Most vertebrates have an identical order of mitochondrial genes. Two notable exceptions are birds, where a differ-

Mitochondrial Genomes

427

ence from placental mammals, amphibians and fish can be explained by a single transposition event, and secondly, marsupials, where a tRNA gene cluster is rearranged (reviewed in refs. 1,16,17). The mitochondrial protein-coding and rRNA genes in echinoderms, such as sea urchin and starfish, show only a few differences relative to the gene order in vertebrates; however, tRNA genes are highly rearranged and 13 of the tRNA genes are clustered together in echinoderm mitochondria. 34 The analysis of changes in mitochondrial gene order during evolution has been used to obtain phylogenetic information about distantly related animals, such as echinoderms 35 or arthropods, 36 rather analogous to the use of mtDNA length polymorphisms exploited in studies of closely related mammals, as mentioned above. It should be noted that although mammalian mtDNA evolves rapidly, that is not the case throughout the animal kingdom. For example, in Drosophila, evidence suggests that nucleotide substitution rates are similar in mitochondrial and nuclear DNAs (cf. ref. 37). Also, although there is no evidence for recombination in animal mtDNA, it is clear that rearrangements affecting gene order do occur over evolutionary periods of time. Even though mitochondrial sequence data from lower invertebrates are relatively limited, it is evident that there is greater variation in gene order relative to those of higher animals. Several mollusc mitochondrial genomes have been completely sequenced: those of the black chiton, Katharina tunicata (15.5 kbp), 38 and two terrestrial gastropods, Albinaria coerulea (14.1 kbp), 39 and Cepaea nemoralis (14.1 kbp), 40 or nearly completely sequenced as for the blue mussel Mytilus edulis (about 20 kbp). 41 The Mytilus edulis mtDNA displays virtually no similarity in gene order to those of the other three molluscs. The mollusc mtDNAs contain the conventional mitochondrial genes except that Mytilus edulis appears to lack the ATP8 gene. It, however, was reported to have an extra tRNA-Met gene, and in Katharina tunicata mitochondria two extra "tRNAlike" genes have also been identified. 38 When the Cepaea nemoralis mtDNA was sequenced, a complete set of tRNA genes was not found. 4~ However it has recently been demonstrated that in the mitochondria of land snails, certain tRNAs undergo RNA editing 42 (discussed in a section VII.B) and hence may escape detection in DNA sequence analysis. A very unusual form of mtDNA inheritance is found in the blue mussel Mytilus genus 43 where one type of mtDNA is transmitted through the females and another through the males; this has been designated as "doubly uniparental inheritance". Female mussels have just one type of mtDNA, which they transmit to all progeny, whereas male mussels are heteroplasmic having received different types from their male and female parents and they appear to transmit only the male type to their male progeny. It appears that the mtDNA in the Mytilus lineage is evolving exceptionally rapidly, and it has been proposed that this might be related to the unusual mode of mtDNA inheritance 44.

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The mitochondrial genomes of nematodes are among the most compact known; the Caenorhabditis elegans and Ascaris suum mtDNAs are 13.8 and 14.3 kbp, respectively. 45 Spacers are virtually nonexistent and the ATP8 gene is absent. The putative "control" regions in these nematodes are only 109 and 117 bp in length, respectively, and all genes are transcribed from the same DNA strand. The tRNAs are exceptionally small, the TUC arm and variable loop being replaced with a very short loop, 46 and mRNAs have nonconventional initiation codons such as UUG. The C. elegans and A. suum mitochondrial genomes share a similar gene order and some conserved linkages with Drosophila mtDNA. On the other hand, mtDNA from the root knot nematode, Meloidygne javanica 31 shows relatively little similarity in gene order with those of the two other nematodes. A very unexpected finding has come from the molecular analysis of the mtDNA from a cnidarian, the sea anemone, Metridium senile. 47 It has a circular mitochondrial genome of 17.4 kbp containing the genes typically found in other animals. However, two of the genes, COXI and ND5, contain introns. This is the first report of introns in animal mitochondrial genes. Both introns have been categorized as group I (see section VII.C) although they possess several unusual features. Moreover, the former contains an ORF identified as a group I-type RNA maturase/endonuclease and the latter intron contains the genes for ND 1 and ND3. The Metridium senile mtDNA also appears unusual in that only two tRNA genes, tRNA-fMet and tRNA-Trp, have been found. A surprising observation has also come from the analysis of the mtDNA from another cnidarian, the coral Sarcophyton glaucum. 48 It contains a 3.0 kbp gene related to the bacterial mutS gene, whose product is a component of a DNA mismatch repair system. In certain eukaryotes, such as humans and yeast, a homologue of this gene has been identified in the nucleus. Examination of mtDNAs from other lower invertebrates may well uncover additional instances of exceptions to "conventional" animal mitochondrial genome features.

IV.

FUNGAL M I T O C H O N D R I A L GENOMES A. Unicellular Ascomycetes

The budding yeast, Saccharomyces cerevisiae, has long been a powerful model system to study mitochondrial biogenesis and function (reviewed in refs. 9,49). Because yeast is able to produce energy through fermentation and so survive without functional mitochondria, it has been possible to generate mitochondrial mutants defective in various aspects of respiratory function. These "petite" mutants include ones designated as rho- (deleted mtDNA), rho ~ (no mtDNA), and mit- (defective in a specific protein-coding gene). It is estimated that there are about 50 mtDNA molecules per wild type yeast cell (reviewed in ref. 1), and in some cases, a single multi-lobed mitochondrion per cell has been observed.

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Another feature which differs from animal mtDNA is that yeast mtDNA is highly recombinogenic. In rho mutants, the remaining mtDNA is amplified (usually as tandem repeats) so that there is about the same amount of DNA as in wild type cells. It has been proposed that replication follows a rolling circle model and there have been 7-8 potential origins of replication identified. By electron microscopic analysis and more recently, moving pictures in conjunction with pulsed-field gel electrophoresis, 5~ the vast majority of molecules appear as long linear forms. As mentioned earlier, yeast mtDNA shows non-Mendelian inheritance (reviewed in ref. 1, 51); however there is biased transmission of certain loci, such as the intron (omega locus) within the LSU rRNA gene which is transmitted with very high frequency to intron-less strains (discussed in a section VII.C). Nucleotide sequence analysis of the yeast mitochondrial genome has revealed a number of striking differences to those of animals. Its physical size is much larger, and varies among laboratory strains from about 68 to 85 kbp. The genes are not tightly packed around the circular genome but are in most cases separated by long noncoding spacers, which are very A+T rich. The overall base composition of the yeast mitochondrial genome is only 18% G+C content. The set of genes present in yeast mitochondria is given in Table 2, and notably absent from the list of expected respiratory chain genes are those for NADH dehydrogenase subunits. Nor have these genes been identified in the nuclear genome, which has been completely sequenced. Yeast, however does have a rotenoneinsensitive NADH dehydrogenase encoded by the nuclear DNA (reviewed in ref. 9)' On the other hand, yeast mitochondria do contain several genes not present in animal mtDNA: the ATP9 gene, a ribosomal protein gene (varl) and an RNase Plike RNA involved in tRNA processing. The two ribosomal RNA genes are far apart on the genome and independently transcribed. There are 25 tRNA genes, of which 16 are clustered in one region. They comprise the full set needed for translation; one additional tRNA is imported from the cytosol but it is not essential for protein synthesis and its function is unknown. 52 All yeast mitochondrial genes except for the tRNA-Tyr gene, are transcribed from the same DNA strand. Yeast mitochondrial genes show several deviations from the universal genetic code. As in animal mitochondria, UGA specifies tryptophan, and AUA is read as methionine. An additional change, namely, the use of the CUN codon family to specify threonine rather than leucine, is specific to yeast (discussed in a section VII.A). Several yeast mitochondrial genes (COXI, COB, LSU rRNA) contain introns and the number varies among laboratory strains. They have been designated as "optional" because their presence varies and no phenotypic difference is seen in their absence. These introns have been categorized as group I and II based on distinctive secondary structural features 53 which are important for splicing, and some have been shown to be self-splicing in vitro (reviewed in refs. 54,55). Many of the introns encode polypeptides which are essential for RNA splicing, as first determined by mutational analysis. Moreover, these introns also have properties of mobile genetic elements (reviewed in ref. 56). Some of the group I intronic

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ORFs have been demonstrated to have "homing" endonuclease activity, and those of group II, reverse transcriptase activity (discussed in section VII.C). In addition, there are several free-standing ORFs related to the intronic ones (reviewed in ref. 9). The yeast mitochondrial genome also contains a family of several hundred short repeated sequences, called "GC clusters" which have hairpin structures of 20--50 bp. They are usually located within spacers or occasionally within introns, and contribute to the size variation among laboratory ' strains. In this regard, the ribosomal protein gene, varl, has an interesting structure. 57 All laboratory strains share one GC cluster within the coding sequence, and some strains have a second copy in inverted orientation. A variable number of tandem repeats of AAT (specifying asparagine) also contribute to length variation of the VAR 1 protein. Recombination between GC clusters appears to play an important role in genome restructuring, another feature very different from the stable animal mitochondrial genomes. Such recombination contributes to the high spontaneous frequency (estimated to be 1-2% per cell per generation, ref. 1) of petite mutants which are deleted for regions of the mitochondrial genome. Yeast mitochondrial gene expression has been an area of intensive investigation for many years, and the reader is directed to recent reviews on this topic. 1'9'49 A few illustrative examples will be discussed here. In contrast to the case of animal mitochondria, where a single promoter directs the transcription of all the genes on one DNA strand, there are many independent transcriptional units in yeast mitochondria. About 20 potential transcription initiation sites have been identified and all the characterized promoters share the motif ATATAAGTA (reviewed in refs. 9,15,49). The mRNAs (most of which are monocistronic) lack poly A tails, but they do have long nontranslated extensions, which are known to be important for RNA stability and translational control. It is known through genetic analysis that the machinery required for RNA splicing, RNA stability and translational control involves a large number of nuclear-encoded components (reviewed in ref. 9). Interestingly, some appear to be multifunctional, having been recruited for a regulatory role in addition to other cellular functions. For example, leucyl tRNA synthetase has been shown to be essential for the splicing of several group I introns in yeast (reviewed in ref. 9). Recent advances in mitochondrial transformation technology using microprojectile bombardment with reporter genes (cf. ref. 58) are opening up new avenues in the study of regulation of gene expression in yeast. The mitochondrial genome of another unicellular ascomycete, the fission yeast, Schizosaccharomyces pombe (reviewed in ref. 59), shows a sharp contrast to that of S. cerevisiae. Although they have a very similar set of genes, the S. pombe mtDNA is much smaller. Its 19.4 kbp circular genome is tightly packed with genes in a order differing from that of S. cerevisiae or animals. All genes are transcribed from one DNA strand. Two genes do have introns: the COXI gene contains two group I introns and the COB gene has a single group II intron; all three possess ORFs. There is one additional gene not found in S. cerevisiae mitochondria, an

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ORF of 227 codons (orfa), which has been correlated with a mitochondrial mutator phenotype. 60 Unlike S. cerevisiae, the S. pombe mitochondrial genes appear to use the universal genetic code, except that UGA triplets within intronic ORFs have been proposed to specify tryptophan rather than being termination codons.

B. FilamentousAscomycetes Neurospora crassa and Podospora anserina are two filamentous fungi whose mitochondrial genetic systems have been particularly well studied (reviewed in refs. 1, 59,61). They have circular mtDNAs of about 65 and 100 kbp, respectively. As in S. cerevisiae, differences in mtDNA length are seen among strains in part because of optional introns. The Neurospora and Podospora mtDNAs share a very similar gene content, with the set of respiratory chain genes typically found in animal mitochondria (including the ND genes not found in unicellular ascomycetes). Mitochondrial gene order is for the most part not conserved between the two fungi, several notable exceptions being linkages between the ND2/ND3 genes and ND4L/ND5 genes. One difference in mitochondrial gene content between these two fungi is the absence of an ATP9 gene in Podospora mitochondria. 62 The functional gene is located in the nucleus, 63 presumably the result of gene transfer during evolution. Interestingly, in Neurospora there is an ATP9 gene in the mitochondrion as well as one in the nucleus. Throughout most of the Neurospora life cycle, the mitochondrial gene is "silent" and the nuclear ATP9 gene generates the protein found in the mitochondrion. However, the mitochondrial ATP9 gene has been shown to be actively expressed in germinating spores. 64 Aspergillus nidulans also has both mitochondrial and nuclear ATP9 gene copies, suggesting that parallel genes have been retained in both compartments over evolutionary time. 65 With respect to translational machinery, both Neurospora and Podospora mtDNAs possess the two rRNA genes and a full set of tRNA genes; namely 25 in Podospora and 27 in Neurospora. In addition, a ribosomal protein gene (designated $5) is located within an intron in the mitochondrial LSU rRNA gene in both fungi. This $5 gene, however, is unrelated to either the yeast varl or bacterial rps5 genes. The mitochondrial genome of Podospora anserina is considerably larger than that of Neurospora crassa, and this is primarily due to a very large number of introns. In fact, introns comprise about 60% of the 100 kbp mitochondrial genome. 62 In race A of Podospora anserina, there are a total of 33 group I introns and 3 group II introns, with many containing ORFs. The COXI gene alone has 16 introns and extends over 24.5 kbp, which is longer than the whole human mitochondrial genome. Short repeated sequences in noncoding regions of the mtDNA in filamentous fungi also contribute to mitochondrial genome size variation and to DNA rearrangements through recombination. The Neurospora crassa mtDNA has a family

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of repeats designated as "PstI palindromes", and those in Podospora anserina mtDNA include short elements which have been implicated in major rearrangements associated with senescence 66 (see section IV.D). It should be noted that mitochondrial plasmids have been observed and characterized in a number of filamentous fungi, particularly Neurospora (reviewed in refs. 1, 67,68).

C. Mitochondrial Genomes of Other Fungi The mitochondrial genome of the chytridiomycete fungus, Allomyces macrogynus, has recently been completely sequenced. 69 This organism branches very deeply within the fungal lineage, and was selected because of the potential presence of "ancestral features" in its mitochondrial genome. The Allomyces mitochondrial circular genome of 57.5 kbp was found to contain the conventional set of mitochondrial genes (including nadl-nad6, nad4L, and atp9), many of which contain introns. In fact, there are a total of 26 group I introns and 2 group II introns, and they comprise about one-third of the mitochondrial genome size. The Allomyces mtDNA also contains five "extra" genes. One has been identified as the homologue of the bacterial ribosomal protein rps3, the first such appearance in a fungal mitochondrial genome. Presumably, this gene was retained from the original endosymbiont set of ribosomal protein genes, rather than having been transferred to the nucleus. Three other ORFs appear related to the intronic groupI/endonuclease ORFs (orf360, orfl07, orf211) and orf204 is as yet unidentified. As in many other fungal mtDNAs, there are short GC rich clusters in noncoding spacers and introns, as well as several within variable regions of the ribosomal RNA genes. The secondary structural models of Allomyces rRNA sequences more closely resemble those of eubacteria than do ascomycete mitochondrial ones, consistent with the retention of eubacterial-type ancestral features in Allomyces mitochondria. An interesting note about the mitochondrial genomes of basidiomycetes is that they include the largest known fungal mtDNAs with sizes up to 176 kbp in various Agaricus species. 7~ Another category of organisms, which will be discussed in a later section, are the oomycetes. They were formerly classified as fungi, but have now been placed with the protists, based on molecular phylogenetic analysis (reviewed in ref. 3).

D.

Fungal Mitochondrial Dysfunction and Senescence

Numerous mitochondrial mutations leading to respiratory deficiency in fungi have been correlated with deletion/rearranged forms of mtDNAs due to the presence of short repeated sequences and the recombinogenic nature of these genomes. In aerobic fungi, these exist in heteroplasmic states and the levels of defective molecules can be correlated in some cases with degree of dysfunction. These features are analogous to certain types of human mitochondrial DNA mutations which lead to respiratory dysfunction and disease (discussed in section II.B).

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Particularly well-studied types of fungal mitochondrial dysfunction are ones leading to senescence, and this phenomenon has been extensively studied in Podospora anserina and Neurospora crassa (reviewed in ref. 1, 59, 67). In the case of Podospora, the juvenile organisms possess the typical circular mtDNA of about 90-100 kbp, whereas in the senescent state there is a massive accumulation of small circular molecules (senDNA) derived from the mitochondrial genome. These molecules are tandem repeats of specific regions of the mitochondrial genome, particularly group II intronic sequences which encode reverse transcriptase-related proteins. The various senDNAs characterized to date have at least one intronic ORF and there are short repeats near the end points of the amplified regions, 66 consistent with their role in recombination. The senescence phenotype has also been observed in natural isolates of Neurospora crassa, which unlike laboratory strains cannot sustain indefinite growth (reviewed in refs. 1, 67). Senescence is correlated with the presence of linear mitochondrial plasmids (such as kalilo and maranhar) which have protein-associated terminal inverted repeats and encode proteins homologous to T7 RNA polymerase and DNA polymerase. When plasmid sequences become integrated into the mitochondrial genome, large inverted repeats of Neurospora mtDNA are generated and their proportion relative to wild type molecules increases over time, leading to cell death. Other Neurospora plasmids (such as Mauriceville and Varkud) are circular DNAs with group I features and reverse transcriptase-like ORFs (discussed in section VII.C).

V.

PROTIST M I T O C H O N D R I A L

GENOMES

The protists constitute a very diverse group of organisms, branching deeply within the eukaryotic lineage (cf. refs. 3,71,72), so in light of the preceding discussion it is perhaps not surprising that they exhibit great diversity in mitochondrial genome features. As yet relatively few have been examined at the molecular level; however, those which have been completely sequenced range from having among the smallest set of genes (such as Chlamydomonas reinhardtii 11 and Plasmodium falciparum 73) to among the largest (such as Prototheca wickerhamii 74 and Acanthamoeba castellanii). 12 The latter however do not have mtDNAs as large as some fungal ones. This is primarily because non-coding spacers are shorter, fewer introns are present and some genes are tightly clustered. Indeed operon-like organization with the same gene order as in bacterial homologues is seen in some cases.

A. Ciliate Protists Two ciliate protists with a long history of mitochondrial analysis are Paramecium aurelia and Tetrahymena pyriformis. 75'76 They have mitochondrial genomes of 41.5 and approximately 50 kbp, respectively. Unlike the mitochondrial

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genomes from most other organisms examined, both of these are linear molecules. Replication in Paramecium mitochondria initiates at one end of the molecule in an A+T rich region and proceeds unidirectionaUy so that head-to-head dimer intermediates (observed by electron microscopy) are formed before being processed to monomers. In Tetrahymena mitochondria, the mode of replication is quite different. It is believed to be bidirectional starting in the midregion of the molecule and proceeding to both ends. The termini have inverted subterminal repeats (which contain copies of the LSU rRNA gene, see below)followed by direct G+C rich telomeric-type repeated sequences. The mitochondrial gene content (and to a lesser extent gene order) is very similar in Paramecium and Tetrahymena. In Paramecium aurelia mitochondria, the protein-coding set includes most of the classical ones, but somewhat surprisingly the COXIII and ATP6 genes appear to be absent. 76 These genes have presumably been transferred to the nucleus, although it is worth noting that mitochondrial genes in these ciliates are very divergent and there are a number of as yet unidentified ORFs in the mtDNA. The "extra" mitochondrial protein-coding genes fall in the category of additional components of respiratory chain and translational machinery. 76 They include four ribosomal protein genes (rpsl2, rpsl4, rpl2, and rpll4) and several NADH dehydrogenase subunits: ND7 (formerly called orf400) and ND9 (originally designated as psbG because of its homology to the chloroplast counterpart which was thought to be a component of photosystem II). An additional 17 ORFs have been identified. In Paramecium mtDNA, all of the genes, except ND7 and rpsl4 are encoded on the same DNA strand. Interestingly, the collection of tRNA genes in the mitochondrion is very low; there are only 3 in Paramecium and 8 in Tetrahymena mitochondria, so that most of the mitochondrial tRNAs must be imported from the cytoplasm (discussed in section VII.A). In Tetrahymena mitochondria, perhaps the most unusual feature is the organization of the ribosomal RNA genes. The LSU and SSU rRNA genes are each split in two segments, and the LSU segments (which are also duplicated because they are located on the subterminal inverted repeats) are in the reverse order to that of transcription of intact genes and there is a tRNA gene between them. 77 Abundant RNAs corresponding to the sizes of these rRNA gene pieces are present in the mitochondria and secondary structural models indicate potential basepairing between the pieces. In Chlamydomonas and Plasmodium mitochondria, more extensive scrambling of rRNA gene segments is observed (see sections V.B and V.E). In contrast, the Paramecium mitochondrial rRNA genes (which are not located on subterminal repeats) have a conventional structure.

B. Chlorophyticand RhodophyticAlgae Although the phylogenetic relationships of many protists are uncertain, it is well accepted that the unicellular chlorophytes (green algae) are representatives of the

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lineage which led to land plants, and it is believed that the rhodophytes (red algae) are a sister clade to the green algal/land plant group. The mitochondrial genomes of several green and red algae have been completely sequenced; namely those of the red alga Chondrus crispus (25.8 kbp), 78 the colorless nonphotosynthetic chlorophytic alga, Prototheca wickerhamii (55.3 kbp), 74 and the green alga Chlamydomonas reinhardtii (15.8 kbp). 11 The circular mitochondrial genome of Chondrus crispus is not markedly larger than the animal mitochondrial genomes, yet it contains about twice the number of protein-coding genes. The "extra" genes encode additional components of the respiratory chain and translational machinery. Of particular note are several ORFs with homology to bacterial succinate dehydrogenase subunits (sdh) of the respiratory complex II, which had not previously been identified in the mtDNA of any eukaryote. Two of these genes, designated as sdhB and sdhC by Leblanc et al., 78 have homologues in the mitochondria of two other red algae, Cyanidium caldarium 79 and Porphrya purpurea, 80 as well as that of a heterotrophic zooflagellate, Reclinomonas americana. 80 Burger et al. 80 have also identified a potential gene for a third subunit (sdhD) of complex II (although with lower sequence similarity to bacterial counterparts) in the mtDNAs of Porphyra, Reclinomonas, and Chondrus (the latter designated as orf84 in ref. 78). Comparisons with the liverwort Marchantia mitochondrial genome, 1~ which has the largest gene set characterized to date (discussed in section VI.A) revealed that it too has homologous ORFs corresponding to sdhB and sdhD, namely orf137 and orf86a, respectively. 78'8~The presence of these genes in not only red algal mitochondria, but also that of Reclinomonas, a jakobid flagellate, which is believed to be a very early diverging eukaryote, is consistent with an ancestral (eubacterial) nature of these mitochondrial DNAs. 8~ Additional support for this view is provided by the presence of other eubacterial-like genes: namely, a 5S rRNA gene and four ribosomal protein genes (rps3, rps11, rps12, rpll6) in Chondrus crispus mitochondria. The 5S rRNA is an essential component of bacterial (and cytosol) ribosomes, yet no 5S rRNA mitochondrial genes have been found in the mtDNA of animals, fungi, or certain protists. However, a 5S rRNA gene is invariably present in land plant mitochondria (and in Prototheca mitochondria); this too has been cited as an example of the retention of an ancestral character in these lineages. In addition, three other ORFs are homologous to ones first identified in plants: namely, orf25 (orf183 in Marchantia), orfB (orf172 in Marchantia), and orf244 in Marchantia. Chondrus crispus mitochondria have two other ORFs (off94 and orf172) which as yet are unidentified. The only deviation observed from the universal genetic code is that UGA specifies tryptophan. The Chondrus crispus mtDNA is tightly packed with very little noncoding spacer sequence. Only one intron is present: a group II intron within the tRNA-IIe gene. The genes are organized such that two transcriptional units of about equal length and opposite orientation could accomodate RNA synthesis of all the genes.

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The colorless, nonphotosynthetic chlorophyte, Prototheca wickerhamiL which is closely related to the green alga, Chlorella, has a circular mitochondrial genome of 55.3 kbp. 74 It has a considerably larger set of genes than that of the red alga, Chondrus crispus; 78 however, it does not possess any succinate dehydrogenase genes. Most of the "extra" genes encode ribosomal proteins, including 10 small subunit ones: rps2, rps3, rps4, rps7, rpslO, rpsll, rpsl2, rpsl3, rspl4, and rpsl9, and three large subunit ones: rpl5, rpl6, and rpll6. A 5S rRNA gene is also present in the mitochondrion. In addition, there are "extra" respiratory chain genes (nad7, nad9, atpA), conserved ORFs of unknown function (orf244, orfB, and orf25), and two additional reading frames (off304 and orf174), whose products have not yet been identified. It is worth noting that orf304 (although shorter) shows sequence similarity with the NADH dehydrogenase gene, nadll present in the mitochondria of protists such as Acanthamoeba 12 and Dictyostelium 81 (see section V.C). Sequence analysis suggests that the universal genetic code is used. Two Prototheca mitochondrial genes (LSU rRNA and COXI) contain a total of five group I introns, and two have intronic ORFs. The mitochondrial genes are encoded on both strands, arranged to be consistent with two transcriptional units, each encompassing about one-half the genome; polycistronic transcripts, which undergo processing, have been characterized. 82 Short tandem repeats are present in noncoding spacer regions and have been suggested to be involved in gene regulation. Another unicellular chlorophyte, Platymonas subcordiformis, has also been found to have a circular mitochondrial genome of about 43 kbp. 83 It too has "extra" genes, such as rpsl9 and rpll6, and the universal genetic code appears to be used in the mitochondria of this green alga. The mitochondrial genome of another chlorophyte, Chlamydomonas reinhardtii, presents a sharp contrast to those of Prototheca and Platymonas. This 15.8 kbp linear mitochondrial genome, 11 contains only a subset (Table 2) of the genes almost invariably found in mitochondria. Notably it lacks the COXII gene and has no genes for ATPase subunits. Moreover the genome contains only three tRNA genes, so that import of nuclear-encoded tRNA species from the cytosol is required. On the other hand, it does contain an ORF 84 related to the reverse transcriptase type found in group II introns, although curiously no introns are present in Chlamydomonas reinhardtii mitochondrial genes. The termini of the Chlamydomonas reinhardtii mitochondrial genome contain inverted repeats of about 0.5 kbp with short 3' single-stranded extensions. 85 This structure does not resemble that of either Paramecium or Tetrahymena linear mtDNA termini (discussed above). Two possible replication models, a recombination-mediated one involving site-specific endonuclease cleavage and a reverse transcriptase-mediated one, have been proposed. 85 The most unusual feature of the Chlamydomonas reinhardtii mitochondrial genome is the fragmented and scrambled organization of the ribosomal RNA genes. 86 The LSU rRNA gene is segmented into discrete segments, eight of which

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show homology to bacterial counterparts, and the SSU rRNA gene is divided into four pieces. The coding segments are interspersed with each other and with other genes, and are in an order which shows no similarity to that of conventional rRNA genes. They are, however, transcribed into abundant stable small RNAs. The rRNA pieces have the ability to base pair and reconstruct the core structure of conventional rRNAs. In other Chlamydomonas species, such as C. eugametos, the ribosomal RNA genes are also discontinuous and scrambled 87 and, in the case of C. eugametos, the small rRNA segments have been demonstrated to be associated with ribosomes. 88 Interestingly, different breakpoints in rRNA gene pieces are seen among Chlamydomonas species, but in all cases, base pairing between segments is predicted from secondary structural models. The mitochondrial genome of Chlamydomonas eugametos is circular and considerably larger (24 kbp) than that of C. reinhardtii (15.8 kbp); moreover, unlike C. reinhardtii, it possesses introns. 87 These features have led to the proposal that the mtDNAs in the Chlamydomonas lineage have rapidly evolved in directions very different from those of chlorophytes such as Prototheca.

C.

Amoeboid Protists

The nonphotosynthetic amoeboid protozoon, Acanthamoeba castellanii, appears to branch near the radiation point of the animal/fungi and chlorophyte/ land plant lineages in evolutionary trees based on rRNA sequence data (cf. ref. 12). It has a circular mitochondrial genome of 41.6 kbp 12 and a very similar, but slightly larger, gene set (Table 2) than in Prototheca mitochondria. 74 Most notable is the large collection of ribosomal protein genes: 10 for the small subunit and 6 for the large subunit of the ribosome. Interestingly, they exhibit the same gene order as seen in the E. coli str-S10-spc-alpha operons (although fewer genes are present in the mitochondrial clusters). 12 Again this is strong support for the retention of an eubacterial (endosymbiotic) organization. It is notable however, that Acanthamoeba mtDNA does not have a 5S rRNA gene, nor has a 5S rRNA species been found in the mitochondrion. 12 The LSU and SSU rRNAs are predicted to have conventional eubacterial secondary structures, and the rRNA genes have an organization similar to that of S. pombe, with the SSU rRNA gene downstream of the LSU rRNA gene and separated by a cluster of tRNA genes. The Acanthamoeba castellani mtDNA contains genes for 17 subunits of the respiratory chain, including nadll, which has been identified in only a few other mtDNAs, such as Dictyostelium discoideum 81 and Phytophthora infestans. 89 As in the case of ribosomal protein genes, a number of the Acanthamoeba mitochondrial NADH dehydrogenase genes (designated as nad) also show eubacterial-like operon organization. Of the eight additional ORFs, off25 and orfB are homologues of ones in land plants (and certain other protists), whereas three others are intronic group I RNA maturase/endonuclease ORFs (located in the LSU rRNA gene which has the only three introns in the genome). The universal genetic code

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appears to be used in Acanthamoeba mitochondria. All genes are transcribed from the same strand and there are several slightly overlapping genes. Interestingly, the COXI and COXII genes are fused into one long reading frame, and the gene products appear to be proteolytically processed (based on protein sizes from Western blot analysis). 9~ The set of 16 tRNA genes in Acanthamoeba castellani mitochondria is insufficient to support protein synthesis, and as in several cases cited above it is believed that tRNAs are imported from the cytoplasm. Interestingly, several of the mitochondrial-encoded tRNAs undergo RNA editing 91 (see section VII.B).

D. Trypanosomatid Protozoa The trypanosomatid (kinetoplastid) protists represent a very early diverging branch on the eukaryotic tree, and their mitochondrial genomes are among the most bizarre examined yet (reviewed in refs. 92,93). Many of these flagellated protozoons are parasites of clinical importance, notably for various tropical diseases. The most extensively studied are those of Trypanosoma brucei, Leishmania tarentolae, and Crithidia fasciculata. Even before they were characterized at the molecular level, it was recognized that the kinetoplasts (a distinctive structure within the single mitochondrion in each cell) have large amounts of DNA (about 7% of the total cellular DNA), and electron microscopy revealed massive networks of concatenated circular molecules. The two distinct classes have been named "minicircles" (0.5-2.5 kbp) and "maxicircles" (22-38 kbp). The latter carry all of the genetic information for respiratory chain components and translational machinery, whereas the minicircular DNAs encode only guide RNAs for RNA editing (see below). The mitochondrial "maxicircle" sizes in Trypanosoma brucei, Leishmania tarentolae, and Crithidia fasciculata are approximately 22, 31, and 38 kbp, respectively (reviewed in refs. 92, 93). DNA sequence analysis did not immediately lead to the identification of all the expected trypanosomal mitochondrial genes, and ones that were detected by homology often appeared to have frameshift mutations. When the corresponding RNA sequences were examined, it was seen that they differed at specific positions relative to the DNA; namely the mature mRNAs had additional uridine residues. In extreme cases (such as COXIII transcripts in Trypanosoma brucei) more than 50% of the coding sequence results from the addition of U residues to the precursor transcript. 94 The process whereby RNAs are specifically modified prior to translation has been called RNA editing (see section VII.B), and genomically encoded information designated as "cryptogenes" in trypanosomes. RNA editing requires guide RNAs (usually less than 60 nt in length) to specify where uridines are to be added. In cases of extensive editing, a large number of guide RNAs are required and their genes are encoded on both maxicircular and minicircular DNAs. " The mitochondrial genomes of Trypanosoma brucei, Leishmania tarentolae, and (7.rithidia fa.~ciculata (reviewed in refs. 92. 93~ share the same basic ~ene

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order and content, but differ in the degree of editing which is required to generate functional mRNAs (cf. ref. 95). Genes are clustered within a 15-17 kbp region and the remaining sequence is of unknown function. Gene content is similar to that of fungi and animals, although the trypanosomal mitochondria possess ND7, ND8, and ND9 genes not found in animals. Sequence similarity is very low with counterparts in other organisms, and genes for ND3 and ND6 have not been found; however, there are three ORFs (MURF1, MURF2, MURF5) of unknown function. Two other ORFs have low sequence similarity to atp6 and rpsl2 genes. The trypanosomal mitochondrial LSU and SSU ribosomal RNAs are among the smallest known (1150 nt and 610 nt, respectively). They can be folded with only limited success into the core secondary structural models. No tRNA genes have been identified in trypanosomal mtDNA and it has been demonstrated that tRNAs are nuclear encoded (cf. ref. 96) and imported from the cytosol into the mitochondrion (cf. ref. 97). Certain mitochondrial genes have been observed to undergo regulation at the RNA level during different stages of the trypanosomal life cycle. For example, Trypanosoma brucei has a life cycle divided between two hosts. During the procyclic form in the insect gut aerobic respiration occurs, whereas in the mammalian bloodstream form energy is produced through glycolysis (reviewed in refs. 92,93).

E. Apicomplexan Protists An intriguing picture is emerging from the molecular analysis of the mitochondrial DNAs from a number of apicomplexan parasites, among which is the malarial Plasmodium falciparum (reviewed in ref. 98). The complete mitochondrial genome appears to be a linear DNA molecule of only 6 kbp linked in tandem arrays of 2-5 copies. Replication has been proposed to involve a rolling circle mechanism with intermediates similar to those in T4 bacteriophage. 99 The presence of the COB, COXI, and COXIII genes identifies the 6 kbp molecule as mitochondrial DNA and the COXIII gene is encoded on the strand opposite to the other two genes. Although the standard genetic code appears to be used, the COXI and COXIII genes lack ATG initation codons. The 6 kbp molecules also contain LSU rRNA and SSU rRNA sequences. However, the rRNA genes are discontinuous and the segments appear scattered in a random order. These short segments (30-200 nt in length) are transcribed and present as abundant stable RNAs. This organization is reminiscent of the case described above for Chlamydomonas mitochondria. In the cattle parasite, Theileira parva, a 7.1 kbp linear molecule, 1~176 existing as monomers with terminal inverted repeats, contains the same three mitochondrial protein-coding genes as in Plasmodium, although in a different order. Fragmented LSU and SSU rRNA gene pieces have also been identified. The status of mitochondrial tRNA genes is as yet unclear; it was reported that the Theilervia mtDNA contains some regions of sequence similarity to tRNAs. 1~176

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A number of these apicomplexan parasites contain an additional extrachromosomal DNA element. In Plasmodium falciparum it is a 35 kbp circular molecule. This DNA contains genes for translational and transcriptional machinery (such as ribosomal proteins, RNA polymerase, tRNAs, and rRNAs). Unlike the fragmented rRNA genes in the 6 kbp mitochondrial genome, these rRNAs have normal eubacterial-like structures. Such features, analogous to ones of the plastid genomes of parasitic nonphotosynthetic plants (which have a depleted gene content) have prompted the suggestion that the 35 kbp molecule is the remnant of a chloroplast genome. 101

F. Mitochondrial Genomes of Other Protists The oomycetes, which formerly were grouped with the fungi, have now been classified as "primitive algae" based on molecular phylogenetic analysis (reviewed in refs. 3, 71). Sequence analysis of the mitochondrial genome of the oomycete, Phytophthora infestans, indicates that it is a circular molecule of 37.9 kbp. 89 It contains the conventional mitochondrial genes for respiratory chain components and translational machinery. In the latter category is a particularly large set of ribosomal protein genes. There are a total of 16 ribosomal protein genes, consisting of the entire set listed in Table 2 except for rpsl and rplll. The mitochondrial genomes of a number of other oomycetes, such as Achyla (water mold) are also of interest because they have an organization very similar to those of land plant chloroplast genomes. 102 Their circular mitochondrial DNAs have inverted repeats containing copies of the LSU and SSU rRNA genes. Several mitochondrial genes have been examined in the slime mold, Physarum polycephalum (cf. refs. 103-105). Transcripts of these genes undergo a novel form of RNA editing and this will be discussed below. In the slime mold, Dictyostelium, genes which have been characterized include nadll, 81 one which is not located in the mitochondrion of most organisms examined. RNA editing has not been predicted to occur in Dictyostelium mitochondrial transcripts.

VI.

PLANT M I T C H O N D R I A L GENOMES A.

BryophyteMitochondria

In 1992, the major achievement of determining the complete sequence of the 186, 608 bp mitochondrial genome from the bryophyte, Marchantia polymorpha, was reported by Ohyama's group, l~ 106 This circular genome (as confirmed by electron microscopy) contains the largest set of mitochondrial genes identified to date: a total of 103 (including 10 intronic ORFs); (Table 2). They are rather evenly distributed around the genome and located on both DNA strands (Figure 2). Of the protein-coding genes, 30 encode known components of the respiratory chain or translational machinery, and 32 are ORFs with unidentified products.

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Figure 2.

Gene organization of the liverwort, Marchantia polymorpha mitochondrial genome of 186,608 bp. The two circles show the positions of the rRNA genes (rrn 18, rrn26, rrn5), tRNAs (black circles) and protein-coding genes. Exons are shown by black bars and introns by shaded bars. Asterisks indicate introns containing ORFs. Genes shown on the outer circle are transcribed anticlockwise and those on the inner circle in clockwise direction. Reprinted with permission from ref. 10.

Several of these ORFs were noted to be conserved in flowering plant mitochondria (such as orf25, orfB) and others have since been identified; for example nad9 (orf212), several succinate dehydrogenase subunits (orf137, orf86a) (as discussed above), and ones involved in cytochrome c biogenesis (orf509, orf322, orf277, orf228, orf169, first discovered in plant mitochondria; see below). There are a total of 32 introns, seven in the group I category and 25 group II ones. Ten of the introns have intronic ORFs and there is also a free-standing ORF (orf732) related to the

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group II RNA maturase/reverse transcriptase ORFs. A full-length nad7 gene which is actively transcribed was identified but because it contains six termination codons, it has been designated as a pseudogene. RNA editing has been observed in some bryophytes (see section VII.B) but not in Marchantia polymorpha, and comparative analysis indicates that it would not be needed to generate the correct RNA sequence. Almost all Marchantia polymorpha mitochondrial genes are present in single copy. However, among the set of 29 tRNA genes, two (tRNA-fMet and tRNA-Tyr) are duplicated copies. Interestingly, two other species of tRNA genes (tRNA-Ile and tRNA-Thr) which are needed for a complete translational set were not identified in the genome. As mentioned in a previous section, in addition to the LSU and SSU rRNAs, Marchantia polymorpha mitochondria has a 5S rRNA gene as do those of flowering plants and Prototheca. Moreover, certain genes, particularly ones for ribosomal proteins, are organized in bacterial-like operons with a gene order similar to that in respiting bacteria. The genome does not contain any large repeats or recombinationally active sequences as found in flowering plant mitochondria (see below); however there are short repeated sequences. Also, unlike the mitochondrial genomes of flowering plants, no "promiscuous" chloroplast DNA sequences are present in Marchantia polymorpha mtDNA.

B. Flowering Plant Mitochondria The mitochondrial DNAs from flowering plants have long been recognized for their very large size and recombinogenic nature (reviewed in refs. 107-112). Sizes range from 208 kbp in Brassica hirta (white mustard) to 2400 kbp in Cucumis melo (muskmelon), with wide variation even among very closely related plants. For example, a seven-fold range in mtDNA size has been observed within the cucurbit family (340 kbp-2400 kbp). 113 Restriction enzyme profiles' have a nonstoichiometric nature, and physical maps are for the most part consistent with a circular "master chromosome" possessing a number of recombinationally active repeated sequences. These repeats give rise to a heterogeneous population of subgenomic physical forms. The number of such repeats varies among plants; in maize mitochondria, there are seven. Brassica hirta, which has the smallest known mtDNA among flowering plants, is the only one reported to lack such repeats. It should be noted, however, that molecules of the predicted "master chromosome" size have not been detected by electron microscopy, although very long linear forms have been seen (cf. ref. 50). In addition, some forms of the mtDNA are present at very low levels and have been termed "sublimons". Under certain conditions, such as passage through tissue culture, they appear to undergo amplification. These features have complicated physical mapping and sequencing studies, and at the time of this writing, no flowering plant mitochondrial genome has been completely sequenced. Although the inventory of genes is therefore incomplete, it

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is clear that the large size of flowering plant mtDNAs does not reflect a corresponding increase in number of genes. The genes identified thus far are virtually all a subset (albeit a large subset) of those found in Marchantia polymorpha mtDNA. The translational machinery includes the LSU rRNA, SSU rRNA, and 5S rRNA genes, an incomplete set of tRNA genes, and a number of ribosomal protein genes. Both the latter categories vary in number among plant species (discussed below). In flowering plant mitochondria, 15 respiratory chain protein-coding genes have been identified to date (all of which are present in Marchantia mitochondria) and five ORFs with homology to bacterial genes involved in cytochrome c biogenesis (cf. refs. 114,115). Moreover, several ORFs (such as off25 and orfB), first identified as conserved reading frames among plants, have since been identified in some protist mitochondrial genomes (discussed in section V.B). In the case of several plant mitochondrial ORFs, the presence of a gene product has been corroborated by immunocytochemical analysis (cf. ref. 114). A number of plant mitochondrial genes contain introns and all have been categorized as group II except for a coxl intron in Peperomia, which is believed to be the consequence of recent horizontal transfer of a group I intron from a fungal source. 116 Of the 25 group II introns identified to date, 19 reside within genes encoding NADH dehydrogenase subunits, but surprisingly, only one (namely nad2 intron 3) is present at the homologous position in the Marchantia polymorpha mtDNA. This is consistent with the movement of introns since the bryophytes and flowering plants shared a common ancestor. The terminal intron of the nadl gene contains the only intronic ORF (mat-r) found in flowering plant mitochondria. It is transcribed and edited, but curiously it lacks a typical initiation codon. 117 Several genes (namely nadl, nad2, and nad5) have undergone DNA rearrangements within intron sequences so that coding segments are dispersed around the genome. Remarkably, these fragmented genes are functional because the segments are transcribed and mRNA is generated through trans-splicing (reviewed in refs. 118, 119). Interestingly, not all flowering plants share exactly the same set of genes. Certain genes found in the mitochondrion of a particular plant are located in the nucleus of others. Data suggest that gene transfer from the mitochondrion to the nucleus is a recent and even ongoing process (reviewed in ref. 120). This has been most thoroughly studied in the case of legume coxll genes. 121'122 Although the coxll gene had been regarded as a "core" mitochondrial gene and is present in the mitochondrion of such legumes as pea and soybean, it is absent from cowpea mtDNA. The functional coxll gene in cowpea has been shown to be located in the nucleus. Moreover, in soybean, the mitochondrial copy appears to be transcriptionally inactive, with a nuclear gene being the functional one. On the other hand in pea, the situation is reversed with the mitochondrial copy being active and the nuclear one being silent. It is believed that these represent transitional states of an RNA-mediated transfer of the coxll gene to the nucleus during evolution. A num-

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ber of ribosomal protein genes (e.g., rpslO, rpsl2, rpsl3, and rpsl9) also differ in their intracellular location among plants (reviewed in refs. 108,109,120). For example, in Arabidopsis the gene encoding the mitochondrial RPS 19 protein is in the nucleus, and the mitochondrion contains a pseudogene. 123 Moreover, the nuclear copy contains an extra RNA-binding domain which has been suggested to substitute for rpsl3, since no gene has been found for that protein in either the mitochondrion or nucleus in Arabidopsis. In contrast, in certain other plants (such as tobacco and maize) an apparently functional rps13 gene is in the mitochondrion, !24 and in yet other plants (such as wheat), the mitochondrial rpsl3 gene appears silent or low-expressed. 125 Even though flowering plant mtDNAs are large, they surprisingly do not contain genes for the full set of tRNAs needed for translation (compiled in ref. 126; reviewed in ref. 127). A number of them are nuclear-encoded and imported into the mitochondrion (cf. ref. 128). Differences in the particular set of mitochondrially encoded tRNA genes are seen among plants suggesting that numerous independent events have occurred during evolution (reviewed in ref. 3). Moreover, those encoded within the mitochondrion fall in two categories based on comparative sequence analysis. Some are "native" mitochondrial tRNA genes and others are "chloroplast-derived" tRNA genes which have recently been acquired from the chloroplast (cf. ref. 129). The latter have replaced the resident mitochondrial homologues and are essential for mitochondrial translation. Interestingly, in maize, the single copy of a tRNA-Trp gene is located on a plasmid DNA. 130 The occurrence of chloroplast sequences integrated into the mitochondrial DNAs of flowering plants is common, and their distribution among plant species suggests that such events are frequent and ongoing (reviewed in ref. 120). Although most plant mitochondrial genes are present as single copies in the genome, occasionally genes are present on recombinationally active repeats and they show plant-specific differences in copy number. This plasticity in genome structure is also reflected in a general lack of conserved gene order among plants; several exceptions are the SSU rRNA/5S rRNA, nad3/rpsl2, and rps3/rpll6 gene linkages. Even regulatory sequences often appear not to be conserved among closely related plants, with mtDNA rearrangements occuring very close to or even within coding sequences. DNA sequence analysis has also shown that spacer regions often contain short copies of coding sequences, which can undergo recombination (over an evolutionary time frame) and hence contribute to the major rearrangements seen among mtDNAs of even very closely related plants. As discussed below, DNA rearrangements can also create novel chimeric ORFs. Moreover, nuclear-derived retrotransposon sequences have been identified in Arabidopsis mtDNA and it is estimated that they comprise more than 5% of the mitochondrial genome. TM It is evident that flowering plant mtDNA is evolving rapidly through rearrangements; however, the rate of nucleotide substitution within genes is very low, estimated to be only about one-sixth that of plant nuclear genes. 132

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The expression of flowering plant mitochondrial genes is less well understood than those of animals or fungi, but as in other systems it is clear that post-transcriptional events play an important regulatory role (reviewed in refs. 107, 108, 133). A number of transcriptional initiation sites have been identified (for genes within the same plant and for several different plants) (reviewed in refs. 15, 133) but they do not all share a motif as conservative as in yeast mitochondria. Most plant mitochondrial transcripts are monocistronic (although some are cotranscribed and then processed) and they usually have long nontranslated extensions but lack poly A tails. Other post-transcriptional events include RNA cis-/trans-splicing and C-toU type RNA editing to generate the correct amino acid sequence (discussed in a later section). It is also of interest that, as in filamentous fungi, certain flowering plant mitochondria contain DNA and/or RNA plasmids and ORFs with homology to bacteriophage-type RNA polymerase and DNA polymerase have been identified (reviewed in ref. 134).

C.

Plant Mitochondrial Dysfunction

Plant mitochondrial mutations associated with the phenotype of cytoplasmic male sterility (CMS) have received great attention, in part because of their value in hybrid seed production. Although affected plants appear otherwise normal, they do not produce viable pollen. This phenomenon has been studied in a variety of plants, including maize, petunia, Brassica, and bean (reviewed in refs. 108,111,135-139). The Texas cytoplasm (cms-T) of maize first attracted widespread interest because of its vulnerability to the pathogen Bipolaris maydis race T. This resulted in a severe corn leaf blight in the southern United States in 1969 and 1970. Analysis at the molecular level has established that rearrangements in the maize mtDNA have given rise to a novel chimeric gene composed of short pieces of other genes. The encoded 13-kDa polypeptide (T-URF13) is located in the mitochondrial membrane and has been shown through transgenic experiments to have a deleterious effect. In fertility restorer lines of maize, the T-URF13 polypeptide levels are reduced, and a nuclear restorer gene has been recently been identified as an aldehyde deydrogenase gene. 14~ However, its mode of action is as yet unknown. The nature of specific mtDNA alterations varies among types of CMS in different plants; however, underlying themes involve the creation of novel genes through mtDNA rearrangements, or incompatibility between the nucleus and mitochondrion, so that mitochondrial gene expression is aberrant and respiratory function is compromised. Another interesting category of maternally inherited mutations in maize are those designated as nonchromosomal stripe (NCS) mutants (reviewed in ref. 141). In this case, respiratory dysfunction has been correlated with deletion mutations within essential electron transport chain genes (which are maintained in a heteroplasmic state). In several instances, the mtDNA rearrangements involve intron

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sequences. For example, the NCS2 mutant has a chimeric nad4/nad7 gene, which appears to have arisen through recombination across a short repeat in the introns of the wild-type nad4 and nad7 genes. 142

NOVEL FEATURES OF MITOCHONDRIAL GENETIC SYSTEMS

VII.

Mitochondrial genome analysis has led to the identification of a number of novel features involved in the expression of mitochondrial genes. This section will present a brief overview of several of the more unusual aspects, most of which had not previously been observed in nature before their discovery in mitochondria. In this regard, they have been valuable in providing new insights into fundamental aspects of gene expression.

A.

Modified Genetic Code and Unusual tRNAs

In the late 1970s, with the first DNA sequence data from mitochondrial proteincoding genes, it was suspected that their codon usage differs from what had been considered the "universal" genetic code. For example, in the yeast mitochondrial atp9 gene, a CUA codon (specifying leucine in the conventional code) was found at the position corresponding to threonine in the sequenced protein. Moreover, UGA triplets which are normally termination codons were found within other yeast and animal mitochondrial protein-coding sequences, at positions expected to encode tryptophan. Now that mitochondrial gene sequences from many organisms have been analyzed, additional deviations from the standard genetic code have been found. As shown in Table 3, the use of UGA to specify tryptophan is widespread in the animal and fungal kingdoms (although notably not within Schizosaccharomyces pombe respiratory chain genes nor in Allomyces mitochondria). The UGA codon is also read as tryptophan in the mitochondria of some protists (such as Trypanosoma and Acanthamoeba) but not others (such as Chlamydomonas or Prototheca). From the distribution of codon usage patterns among phylogenetic lineages, it appears that at least some of the alterations have occurred independently during evolution. For example, the use of CUN to specify threonine rather than leucine appears localized to the yeast lineage. A detailed discussion of the evolution of the genetic code, including aspects of "codon capture," is given in a review by Osawa. 143 One frequently noted consequence of an altered mitochondrial genetic code is that such genes are "frozen" in the mitochondrion, rather than being able to successfully move to the nucleus because they would be incorrectly decoded by the cytosol translation machinery. In the mitochondria of land plants and chlorophytic protists, no deviations from the universal genetic code have been observed. It should be noted, however, that

Table 3.

Codona Specification in "universal" code Animals Homo sapiens Xenopus laevis Strongylocentrotus lividus Drosophila yakuba Katharina tunicata Caenorhabditis elegans Metridium senile Fungi Saccharomyces cerevisiae Schizosaccharomyces pombe Neurospora crassa Podospora anserina Allomyces macrogynus Protists Paramecium aurelia Chondrus crispus Chlamydomonas reinhardtii Prototheca wickerhamii Acanthamoeba castellani Trypanosoma brucei Plants Marchantia polymorpha Flowering plants

Examples of Variations In M i t o c h o n d r i a l G e n e t i c C o d e

UGA Stop

A UA lle

A GA Arg

A GG Arg

Trp Trp Trp Trp Trp Trp Trp

Met Met Met Met Met Met

Stop Stop Ser Ser Ser Ser

Stop not present Ser not present Ser Ser

Trp

Met

AAA Lys

CUN Leu

Asn u

N

Thr

"universal"

Trp Trp "universal"

Trp Trp

m

"universal" "universal"

Trp Trp

m

m

q

"universal" "universal"

Note: aTheorganisms listed are the same ones as in Table 1. Data are updated from compilations in refs.l,2,6 to includeAIIomyces (69), Chondrus (78), Prototheca (74), Acanthamoeba (12). In Schizosaccharomyces pombe mitochondria, TGA triplets within intronic ORFs have been proposed to specify Trp.

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before the discovery of RNA editing, it was believed that CGG specified tryptophan rather than arginine in the mitochondria of flowering plants. It is now known that C-to-U type RNA editing converts such codons to UGG, the conventional codon for tryptophan (reviewed in refs. 119, 133). Early sequence data of animal mitochondrial tRNA genes also indicated that they are shorter than normal tRNAs and lack certain features of the conventional cloverleaf structure, particularly within the TUC and dihydrouridine loops (reviewed in refs. 1, 6, 16, 17). In lower animals such as nematodes, deviations are even more pronounced, with the TUC arm and variable loop being replaced by a short loop in most tRNAs. 76 It is also of interest that in some protists and plants, not all the tRNAs used in mitochondrial protein synthesis are encoded within the organelle (reviewed in ref. 1,127). The trypanosomes are the most extreme case in that all tRNAs are apparently nuclear encoded and imported from the cytosol. Moreover, in flowering plants, the mitochondrial-encoded set includes chloroplast-derived tRNA genes, which have been transferred to the mitochondrion, indicating that they are similar enough to be interchangeable in the translational machinery.

B.

RNA Editing

The discovery that the nucleotides in a mature RNA sequence do not always correspond identically with the DNA sequence which encodes them first came in the mid-1980s through studies of trypanosomal mitochondria (reviewed in refs. 92, 93, 144). It was found that at specific positions within trypanosomal mitochondrial transcripts there were uridines not present (as thymidines) at the corresponding sites in the DNA sequence. The process whereby certain specific nucleotides are inserted, deleted, substituted, or converted is called RNA editing. During the last 10 years a variety of forms of RNA editing have also been found in the mitochondria of other organisms, including the slime mold, Physarumpolycephalum, land plants, and several animals and protists. In trypanosomes, uridines are inserted (and occasionally deleted) from precursor mRNAs under the direction of small antisense "guide RNAs" which determine the positions at which uridines are to be added (or deleted). The process is polar, proceeding from the 3' to 5' direction, and the mechanisms and machinery of RNA editing have been extensively studied (reviewed in refs. 92, 93, 144). The extent of editing varies among different trypanosomatid protists (cf. ref. 95), as well as among different mitochondrial genes within a particular organism. It is estimated that hundreds of guide RNAs are required for the maturation of trypanosomal mitochondrial mRNAs in some cases. In 1989, it was discovered that plant mitochondrial transcripts undergo C-to-U type RNA editing (reviewed in refs. 119, 133, 145), predominantly within proteincoding sequences and more specifically at positions which alter the amino acid specified. The changes usually result in the polypeptide being more similar to

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counterparts from other organisms. In some cases, an initiation codon is created by the conversion of ACG to AUG, so RNA editing is clearly important. C-to-U type RNA editing has also been observed at lower frequency in tRNAs and noncoding sequences. Although the mechanism of RNA editing in plant mitochondria is unknown, data are consistent with C-to-U conversion through base modification (such as cytidine deamination). RNA editing appears widespread among plant mitochondria, and has been observed in mitochondrial transcripts of certain bryophytes, such as mosses, hornwort, and the liverwort Pellia epiphylla. 146 However, RNA editing has not been observed (nor predicted to be needed based on DNA coding sequences) in the mitochondria of the liverwort Marchantia polymorpha. It is of interest that RNA editing with similar features has also been found in plant chloroplasts, although it occurs at a lower frequency (cf. ref. 147). Yet another type of mitochondrial RNA editing was reported in 1991 for mitochondrial transcripts in the slime mold, Physarum polycephalum. 1~ Transcripts of the atpA gene were found to have a number of cytidines inserted at rather regular intervals along the RNA molecule. RNA editing has since also been found in other mRNAs, tRNAs, and rRNAs in Physarum mitochondria (cf. refs. 104, 105). Although the changes are predominantly the insertion of cytidines, occasionally other nucleotides are inserted, or cytidines are converted to uridines. Various forms of RNA editing of mitochondrial tRNAs have recently been reported in a variety of organisms. In Acanthamoeba mitochondria, single nucleotide conversions were observed within the 5' region of the acceptor stem of tRNAs, and they improve base pairing within the stem. 12 In the mitochondria of the land snail, Euhadra herklotsi, 42 mismatches within the 3' region of the acceptor stem of tRNAs were seen to be corrected to classical base pairing by RNA editing. This involves nucleotide changes to adenosine residues, raising the possibility that the process is one of polyadenylation. In the mitochondria of the marsupial, Didelphis virginiana, a tRNA-Asp gene with the anticodon GCC was seen to be corrected to GUC at the RNA level. 148 From the preceding discussion, it is evident that these various forms of RNA editing appear to represent very different biochemical processes, consistent with RNA editing having arisen at independent times during evolution. One model for the origin of RNA editing proposes that it might arise if DNA mutations could be corrected at the RNA level by the recruitment of preexisting machinery within the mitochondrion (such as cytidine deaminase activity in the case of C-to-U type editing). Then such mutations would be neutral and accumulate by drift. Subsequently, with increasing numbers of sites requiring editing, the process would be maintained by natural selection. 149

C.

Mobile Introns

More than 15 years have passed since DNA sequence analysis of fungal mitochondrial genes revealed the presence of introns. They were categorized as group

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I or group II, based on distinctive primary and secondary structural features. 53 In the intervening years, members of these families of introns have been found in the mitochondria of other fungi, plants, some (but not all) protists, and even within the animal kingdom (as represented by two group I introns in sea anemone mitochondria, ref.47). Group I introns also occur in plastid and bacterial/bacteriophage genes, as well as nuclear genes of lower eukaryotes; group II introns have also been found in chloroplast and bacterial genes. Comparative sequence analysis of such introns, as well as in vivo and in vitro functional studies (reviewed in refs. 54-56) have corroborated and extended models of intron folding. Members of both classes of introns have been shown to be self-splicing in vitro, and structures of catalytic core regions are being studied in detail (reviewed in refs. 54-56). Some mitochondrial genomes (such as yeast, Marchantia, Prototheca) also have "free-standing" ORFs homologous to intronic ones (Table 2), and such a reversetranscriptase-type one is present in the mitochondrial genome of Chlamydomonas reinhardtii, although it has no introns. In addition, small circular DNA plasmids, such as Mauriceville and Varkud in Neurospora mitochondria, contain ORFs homologous to the group II intronic ones (reviewed in ref. 56). Certain of the yeast mitochondrial introns had been predicted from genetic analysis to contain trans-acting information for splicing. Indeed, DNA sequence analysis confirmed the presence of long open reading frames and the intron-encoded proteins were called "RNA maturases". Subsequent studies have shown that group I intronic ORFs also can have properties of site-specific "homing" endonucleases, and those of group II show homology to reverse transcriptases (of the non-LTR retrotransposon type) and such activity has been demonstrated. Although both classes of introns can self-splice in vitro, machinery for splicing in vivo is complex (reviewed in ref. 9). Moreover, similarities between the group II intron excision mechanisms (two-step transesterification involving intron lariat formation) and those of spliceosomal nuclear pre-mRNA has raised the possibility that they share a common origin (reviewed in refs. 54-56). In self-splicing group II introns, all the information for splicing is provided by the (autocatalytic) intron structure, whereas in spliceosomal introns that information is provided by numerous small RNAs and proteins (snRNPs). The "optional" nature of yeast mitochondrial introns and sequence similarities among them, first suggested that they might be "infectious elements" which can spread around genomes. This is supported by comparisons of the distribution of mitochondrial introns among organisms. For example, the mtDNAs of Marchantia and any flowering plant together have over 50 group II introns, yet only one is located at the same site in homologous genes (namely nad2 intron 3). This is consistent with the movement of introns since the time that bryophytes and flowering plants shared a common ancestor. As in fungi, the loss of"optional" introns appears to be RNA-mediated in plant mitochondria (cf. ref. 153). Members of group I and group II intron families have indeed been shown to behave as mobile genetic elements and much is being learned about the molecular nature of these processes. The

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first one extensively studied was a group I intron (designated the omega locus) within the yeast mitochondrial LSU rRNA gene. It shows biased inheritance in genetic crosses such that the intron is transmitted to a specific site in an intron-less strain with high frequency. This process is dependent on the activity of the intronic "homing endonuclease", and follows a double-strand-break repair pathway (reviewed in ref. 56). Group II intron mobility has recently been shown to involve a ribonucleoprotein particle containing intron RNA with catalytic (reverse splicing) activity and the intronic ORF protein with reverse transcriptase and site-specific DNA endonuclease activities. 150This remarkable "retrohoming" pathway of group II introns resembles the activities of non-LTR retrotransposons and telomerases, raising the possibility that they share a common origin (reviewed in refs. 151,152).

D. ScrambledGenes The impact of the rearranging nature of certain mitochondrial genomes on gene structure is perhaps most strikingly illustrated by the fragmented ribosomal RNA genes in a number of protists, particularly in Chlamydomonas species 86'87 and parasites such as malarial Plasmodium (reviewed in ref. 98). Even though the segments are not organized in the mitochondrial genome in the normal transcriptional order, small discrete RNAs are generated, and they have the potential to interact through base pairing to form the typical ribosomal RNA structure. In plant mitochondria, "gene scrambling" has also been seen in the case of certain NADH dehydrogenase (nad) genes (reviewed in refs. 118,119). Group II introns within these genes have undergone rearrangements so that coding segments are at distant locations in the genome. Base pairing between the intronic halves is postulated to allow the correct RNA folding for splicing, analogous to the assembly of protist mitochondrial rRNA pieces. Novel chimeric genes have been observed in mitochondria, perhaps most notably those associated with cytoplasmic male sterility in plants (discussed in section VI.C). Normal respiratory chain genes can also be affected by mtDNA rearrangements. For example, the atp6 genes of different plants are preceded by fused, inframe ORFs of various lengths and sequences (reviewed in ref. 108). The recombinogenic nature of plant mtDNA and the presence of trans-splicing illustrate the potential of new proteins being created through exon shuffling during evolution.

Viii.

EVOLUTION OF M I T O C H O N D R I A L GENOMES

The debate over whether mitochondria arose through intracellular compartmentalization of functions within a primitive eukaryote, or from a respiring bacterial endosymbiont has been settled largely on the strength of molecular evidence. Comparative sequence data convincingly support the latter hypothesis, and indicate that the closest living relatives of mitochondria are within the alpha-subdivi-

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sion of purple bacteria (Proteobacteria). The analysis of ribosomal RNA sequences has been instrumental in this determination and these topics are thoroughly discussed in reviews by Gray. 3'5 Because mitochondrial genetic systems exhibit extreme diversity among present-day eukaryotes, this raises other questions: did mitochondria all arise from a single endosymbiotic event or are they polyphyletic?; what type of events led to the stable association between the nucleus and organelle?; why have the mitochondrial genomes of different eukaryotic lineages (sometimes even very close ones) evolved in such different directions? An implicit tenet of the endosymbiotic hypothesis is that early events included the massive transfer of genes from the organelle to the nucleus. In present-day eukaryotes, it is estimated that about 90% of the mitochondrial proteins are encoded in the nucleus. Moreover, mitochondrial genome analysis indicates that gene transfer events have occurred at different times during evolution, and that some lineages (such as liverwort and certain protists) have retained a larger set of mitochondrial genes than others (such as animals). These "extra" genes, such as ones for 5S rRNA and ribosomal proteins, have strong bacterial features (including, in some cases, the same gene order as in homologous bacterial operons). This has been suggested to reflect the stable and "ancestral" nature of these mitochondrial genomes. Because virtually the same collection of about 18 ribosomal protein genes (which is only about one-quarter of a full bacterial set) is found in the mitochondrial genomes of organisms as diverse as Marchantia, Acanthamoeba, Prototheca, and Phytophthora, it has been argued that this reflects the common (monophyletic) origin of these mitochondria. 7'12 The alternative (less likely) scenario would be that mitochondria, derived from separate endosymbioses, underwent random, independent transfers of almost exactly the same large set of ribosomal protein genes to the nucleus. Moreover, from the complete yeast nuclear DNA sequence 154 (Yeast Protein Database URL http://www.proteome.com/YPDhome.html), 44 genes have been identified as mitochondrial ribosomal protein genes, and 16 of them are detectably homologous to bacterial ones. Interestingly however, only five (namely rps7, rpsll, rpsl3, rpsl4, rpll6) correspond to ones located in the mitochondrion in plants/protists. Thus, a set of 29 identified ribosomal protein genes appear to reflect components of the bacterial (endosymbiont) ribosomal machinery. Because the transfer of functional genes from the mitochondrion to the nucleus appears to be an ongoing process in flowering plants (reviewed in ref. 120), their study provides insight into potential transitional states involved in successful movement: events such as duplication of the mitochondrial gene (and in plants, this step must involve an RNA-edited intermediate), physical movement of the gene to the nucleus, integration in the nuclear genome and acquisition of associated expression signals, and subsequent degeneration/loss of the mitochondrial copy. Experimental systems to study the process of mtDNA movement to the nucleus in yeast are also proving informative. It has been estimated that events are

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occurring at the rate of approximately 2 x 10-5 per cell per generation, whereas movement in the opposite direction (from the nucleus to the mitochondrion), if any, is below the level of detection (reviewed in ref. 155). It is evident that many different evolutionary forces are shaping the mitochondrial genomes of various eukaryotes. Animal mitochondrial genomes are relatively stable in gene order, whereas those of flowering plants are very recombinogenic and dynamic. Marked differences in nucleotide substitution rates are also observed, raising questions about DNA replication/repair mechanisms and the fixation/transmission of mutations. Why, for example, are plant mitochondrial genomes evolving very slowly with respect to nucleotide substitution, yet rapidly with respect to DNA rearrangements? Moreover it appears that unusually rapid evolution may be occurring within particular lineages as well: two examples being Chlamydomonas (compared to other green algae) and Mytilus (compared to other mussels), as discussed in earlier sections. The mosaic nature of some mitochondrial genomes also complicates tracing their evolutionary history. Flowering plant mtDNAs accept and integrate foreign sequences, such as chloroplast and nuclear DNAs. In fact, certain transferred chloroplast tRNA genes have been recruited as essential components of the mitochondrial translational machinery. Moreover, the presence of mobile genetic elements (as represented by group I and II introns and their resident genes) can have a profound effect on genome structure and even gene structure (as illustrated by the trans-spliced nad genes in flowering plant mitochondria). The group I and group II introns (and various plasmids found in plant/fungal mitochondria) carry viraltype genes for activities such as reverse transcriptase, site-specific DNA endonuclease, and DNA/RNA polymerases (as well as their own catalytic RNA activities), all of which can play a major role in genomic restructuring. In this regard, it is interesting that the fungal and animal mitochondrial RNA polymerases are related to T3/T7 bacteriophage RNA polymerases, rather than to the eubacterialtype, multi-subunit RNA polymerase (expected from the bacterial endosymbiont).

IX.

CONCLUDING REMARKS

During the 15 years since the complete sequence of the human mitochondrial genome was reported, there has been a explosion of mitochondrial DNA sequencing data. More than 60 mitochondrial genomes from a variety of eukaryotes have been completely sequenced, and this has provided a wealth of new information: it has been seen that in some organisms the number of proteins synthesized inside the mitochondrion greatly exceeds that of human mitochondria; clusters of mitochondrial genes with the same order as in homologous bacterial operons have been observed; and new, seemingly complex, strategies of gene expression have been uncovered. As more is learned about early diverging eukaryotic lineages, it seems likely that this will provide additional insight into the nature of "ancestral" types

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of mitochondrial genomes. On the other hand, as information accumulates from rapidly evolving lineages (particularly from organisms with specialized life styles), further novelties in gene organization and expression are likely to be seen. Although significant advances have been made in our understanding of the genetic information inside mitochondria, relatively little is known about the nature of communication between the mitochondrion and nucleus. How are events coordinated between the two compartments? What nuclear-encoded machinery is involved in mitochondrial gene regulation and how did it evolve? What impact does the mitochondrial genetic system have on other (nonrespiratory) cellular functions? The complete yeast nuclear genome sequence (reported in the spring of 1996) will be a valuable resource in addressing such questions. A total of 274 nuclear genes encoding mitochondrial proteins were identified, 154 a conservative estimate considering that approximately one-third of the yeast nuclear genes are of as yet unknown function. The study of mitochondrial genomes will no doubt continue to provide insight into fundamental processes of mitochondrial function, as well as mitochondrial dysfunction. At the same time, such studies also provide us with glimpses into the many possible solutions nature has devised to store and handle genetic information.

NOTE ADDED IN PROOF The number of fully sequenced mitochondrial genomes has continued to grow since this review was written, and two are of exceptional note. The A r a b i d o p s i s thaliana mitochondrial genome, the first completed from a flowering plant, is 366,924 bp in length and by far the largest sequenced to date. 156 It is estimated to encode 57 genes that comprise only about 10% of the genome. In contrast, the flagellate protozoon R e c l i n o m o n a s a m e r i c a n a has a mitochondrial genome of 69,034 bp containing 97 genes, the largest set yet identified. 157 Its eubacterial-like features, including the presence of four bacterial-type RNA polymerase genes, suggest that it is more similar to the ancestral protomitochondrial genome than are other characterized mtDNAs.

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