dramatic simplifications of mitochondrial translation systems, particularly in animals (6-8). This reductive tendency 1, correlated with another striking characteristic of animal mitochondrial genomes; this is their apparent inability to support genetic recornbination. These features can be brought together in a model that relates the genomic characteristics of the animal mitochondria to the striking simplifications of their translation systems, some of which are expressed as codon reassignments for a small number of amino acids.

Summary Mitochondria1 genomes are clearly marked by a strong tendency towards reductive evolution. This tendency has been facilitated by the transfer of most of the essential genes for mitochondrial propogation and function to the nuclear genome. The most extreme examples of genomic simplification are seen in animal mitochondria, where there also are the greatest tendencies to codon reassignment. The reassignment of codons to amino acids different from those designated in the so called universal code is seen in part as an expression of the reduction of the number of genes used by these genomes to code for tRNA species. The driving force for the reductive evolution of mitochondrial genomes is identifed with two population genetic effects which may also be operating on populations of parasites.

Introduction It is well known that the genetic code is not strictly speaking universal and that its most dramatic variants are found in animal mitochondria. What may not be so evident is that mitochondrial variants provide a unique opportunity to study the coevolution of the genetic code and the translation system. My thesis is that the rearrangements of the genetic code in mitochondria are driven by forces that can be identified with those that have shaped other peculiarities of the mitochondrial genome. This means that I abandon two venerable views of the nature of the genetic code. One of these is the notion that there is a necessary, structural relationship between the nucleotides that code for the amino acids and the amino acid side chains for which they code (I). The other is that the code and its variants represent ‘frozen accidents’ (*). Instead, I will follow the view developed by Osawa and Jukes (3-4) who suggest that genetic codes evolve and are still evolving under the influence of mutational bias. In addition, I want to stress the role of other forces because it is difficult to account for the observed patterns of codon reassignment by mutational bias alone (s). In effect, I argue that coding sequences (read genotypes) have a phenotype that is shaped by selective forces as well as by mutational bias. I view the codon reassignments characteristic of mitochondria as a small part of a larger evolutionary pattern. Thus, the transfer of genes to the nuclear genome in the course of mitochondrial evolution has been accompanied by

Outline of a Model Changes in the genetic code must reflect changes in the translation system because the selection of tRNA species by codon programmed ribosomes is the process that determines which amino acids correspond to which codons. Indeed, a strong correlation is observed between the most radically simplified translation systems and the most unconventional codes utilized by animal mitochondrial genomes. Here, we find that the codon reassignments are a very small part of a much larger patlern that includes a massive reduction in the complexity of the tRNA population of these mitochondria (see Table 1). This sort of linkage shifts our attention to thc forces that lead to the reductive evolution of animal mitochondria. Most often a number of mitochondria, each with at least one genome copy, replicate within a eukaryotic cell. This means that the mitochondria1 genomes of a cell make up a micro-population within which competition can take place to select the most rapidly replicating genomes. Under some conditions mitochondrial replication will be faster if some genes and their products are eliminated from the mitochondrial repertoire. In addition, defective genomes, and in particular those with deletions, can take over the micro-populations of a cell line when the intracellular genomic populations are small and random fluctuations in the proportional segregation of different genomes are common. Normally deletions of essential genetic information would be eliminated by selcction. However, if these genes are permanently transfered to nuclear genornes and if they can be expressed effectively there, mitochondria with deleted genes can survive. Even where this mechanism to transfer mitochondrial genes to nuclear genomes exists, the process will be opposed by the opportunity to recover deleted sequences through recombination. Accordingly, the net rate of deletion will be slower in cells such as those of fungi that support mitochondrial recombination and faster in animal mitochondria that cannot do so. Plant mitochondrial genoines can recombine with themselves as well as with those of chloroplasts and consequently the tendency to simplify the translation system is even less pronounced than in fungal mitochondria. Instead, plants may util yze hybrid translation systems that operate with a conventional code. Codon Usage and tRNA Usage The work of Grantham and other, W 1 ’ )established some of the orderly ways that organisms exploit the degeneracy of the

code to spccify amino acid sequences in general or to specify the sequences of some special groups of gene products. One consequence of such distinctive codon usage patterns is that homologous protein structures are often determined by quite different codon sequences in difrerent organisms. This is another way of saying that a distinctive phenotype can be specified by two differcnt genotypcs. In an organism such as Esclzerichia coli we can divide codons into two usage classes: One class corresponds to the codons most frequenlly used in the genes that are highly expressed under fast growth conditions; these are the major codons ( l l ) . The other class contains all the other codons. Now, it is has been known for a long time that the protein composition of cells varies quite markedly whcn the growth media are varied (I2). For example, the proteins of the translation system dominate the composition of rapidly growing bacteria but as thc quality of the medium decreases the translation apparatus becomes a progressively smaller part of the bacterial mass. In parallel the protein composition of the bacteria becomes more heterogeneous a1 the lower growth rates because a more complex complement of enzymes is required to deal with poorer media. This mcans that the mRNA composition of the bacteria varies in such a way that the frequency of major codons expressed in the rnRNA pool increases with the growth rate ofthe bacteria. Recently it has been found in Edzerichin coli that this growth rate-dependent variation of major codon usage is reflected in a corresponding variation of tRNA usage. Expression levels for twenty different tRNA isoacceptor species have been studied in E.coli under different growth conditions (13) (Emilsson, V., Nblund, K. and Kurland, C.G., in preparation). It is observed that tRNA species that translate the major codons tend to increase in abundance as the growth rates are increased. In contrast, tRNA species that translate other codons tend to decrease in abundance as the growth rates increase. As a consequencc, as the growth rate increases, the mRNA pool contains a progrcssively more biased representation of major codons and this is reflectcd in a correspondingly biased tRNA abundance profile. Thc twinned notions of codon usage and tRNA usage play an essential role in the present description of the evolution of mitochondrial translation systems. In the case of the major codon bias in E.coli, the selection of a prcferred subset of codons for highly expressed genes coupled with a correspondingly biased tRNA abundance has the consequence that translation rates pcr mass of translation apparatus are maxiniizcd; this in turn tends to maximize bacterial growth rates in rich media ( l 4 - l 5 ) .For our purposes the relevant conclusion is that codon usage and tRNA usage in an optimal situation are selected coordinatcly.

Minimal Translation Systems Most of the genes that specify the proteins of the mitochondrial translation system, the ribosomal proteinq and translation factors, are found in the nuclear genome. On the other hand, one gcnomic motif common to all nlitochondrial systems is the retention of the RNA components of the translation apparatus: ribosomal RNA5 and tRNAs. Systcmatic structural

variations in these RNA species reveal much about Lhe evolutionary history of mitochondria. The evolution of mitochondrial gcnomes is apparently inonophyletic, with some complications among the higher plants (8, 16). In the course of that cvolutionary development therc is a progrttssive reduction of the tRNA complement (Table I), a shrinkage of ribosomal RNA size and a minimization of total genomic sequence lenglh, all of which are quantitatively correlated (j).In particular, animal cells have undergone the most radical simplification of the mitochondrial translation apparatus that includes the discard of roughly half of thc tRNA species that normally are required for translation (17-1x) and up to a threefold reduction in the size of ribosomal RNA species (19-20). These simplifications suggest that even though they are retained within mitochondrial genomes, there is strong seleclive pressure for minimimtion of the coding sequences for the RNA components of the translation system. That the extreme minimalist tendency of animal mitochondria is also correlated with the highest frequencies of reassigned codons seems not to be fortuitous.

Hyperwobble One way to significantly reduce the size of mitochondrial genomic sequences would be to eliminate some of the tRNA sequences normally requircd to translate the standard 6 1 sense codons. The minimum would correspond to one tRNA species per amino acid. In fact, simplilications of tFWA structure such as cliniination of nucleotide modifications and shortening the tRNA sequenccs are the rule in animal mi tochondria. The functional conscquences for typical animal mitochondria is that there are twenty two tRNA species serving the translation system: up to nine tRNA spccies may each translate a box of four codons and the remaining tRNA species niay translate subsets of two codons that differ only at codon position three (I7-l87 21-22). This is illustrated in Table 1, where the tRNA populations of E. roli and the human mitochondrion are compared. It is evident that a reduction of the number of mitochondrial tRNA genes is associated with a reduction of the significance of codon position three in the tRNA sclection process. Thc most radical element in this reductive pattern is that in as many as nine instances a single tRNA species is able to translate a codon box with four triplets. Although this pattern is vaguely reniiniscent of orthodox wobble, the milochondrial decoding rules are considerablcly more flexible than Crick's 03)wobble rules. Accordingly, 1 call this unorthodox codon-anticodon matching process hyperwobblc. At first glance, the capacity to distinguish the first two nucleotides of a codon, to be indifferent to the third nucleotide, but nevertheless to advance the mRNA three nucleotides at a time niay seem to bc something of a deus e.r machina. However, afliic.ionadosof translational accuracy in bacteria have known for a long time that mutant tKNA isoacceptors tend to hyperwobble. For example, tRNATrP normally translates UGG. but a mutant form with a single subslitution in its D stem was identified as an effective translator of the UGA termination codon by D. Hirsh Cz4); this nonstandard codon recognition is observed as wcll with the wild-

Table 1. A comparison of h e tRNA populations Of'E. coli and the human mitochondrion U

E.c.

E.c.

G

A

C

Mt

Mt

E.C.

Mt

E.C.

Mt

U C A

G U C A

G U C A

G U C A G

There are no eukaryotic systems for which the lolal complement of tRNA genes have been identificd and sequcnced. Therefore, I have chosen the fully described tRNA population of E. ~ 0 1 6to~represent ~) the nuclear complement which is compared with that of the human mitochondrion"7.'x).Here each codon box contains an identification of the amino acid (s) for which it codes in both systems. The numbers of tRNA species with unique anticodons are listed both for the bacterial code (E.c.) and Tor the mitochondria1 codc (Mt). The distributions within the codon boxes describe a total of 40 tRNA species for E. coli and 22 for the human mitochondrion.

type tRNATT but at a lower frequency (25). It was subsequently shown that the Hirsh mutant could also compete wilh tRNACYs at UGU codons strengthening the interpretation that the Hirsh mutant is effectively a decoder of the first two nucleotides of codons of the form UGN (26-27). A general tendency for tRNA isoacccptor species to mistranslate codons primarily on the basis of the identities of the first two codon positions was discovered by Lagerkvist and colleagues (28-29). Here it was observed that a tRNA species with a mismatch at the third codon position often is responsible for the highest frequency of missense errors at a particular codon. Subsequent studies showed that the bacterium Mycoplasrna mnycoides has a simplified tRNA population with codon specificities that for a number of tRNA species show hyperwobble (30-31). Furthermore, the anticodon UCC artificially introduced into E. coli tRNA"'Y translates by conventional wobble rules codons of the rorm GGR, while the same anticodon in Mycuplasma mycoides tRNAG1ytranslates the codon box GGN (32). Thus, the hyperwobble functions are dependcnl on the tRNA structural context as well as on an appropriate anticodon. In summary, the capacity of tRNA species to decode a triplet codon primarily on the basis of the first two nucleotide positions is not unique to mitochondria. It is, however, heavily exploited by mitochondria apparently in response to the forces tending to minimize genomic size.

Codon Reassignment If it were advantageous to translate each amino acid family of

codons by one tRNA species, four amino acid familics would present special problems. Because they are normally coded by six or three membered codon families, Arg, Leu, Ser and Tle can not bc translated by single tRNA species that simply distinguish the first two nucleotides in the corresponding codons. For these amino acid families an additional strategem seems relevant, namely, rearrangements of the genetic code that facilitate a reduction of the number of tRNA species (33-34). The codon-tRNA usagc patterns in some invertebrates illustrate the interplay between hyperwobble and codon reassignment for the rcductive evolution of mitochondrial genomes. The mitochondria of such diverse creatures as sea urchins, round worms and flat worms can translate the genetic code with the aid of only 22 tRNA species (35-37). In these animals the mitochondria1 tRNA species are reduced to lengths close to 60 nucleotides and they lack the hypermodifications characteristic of thc cytoplasmic tRNA species. Nine isoacceptors each translate a different codon box made up of four triplets and thirteen translate the pairs of triplets within a codon box that end either with the purines or with the pyrimidines. Such a tRNA ensemble o f twenty two species is thc smallest known. The point is that codons for three of the four troublesome amino acids (i.e. Ile, Arg. and Ser) are involved in the reassignments of codons in the mitochondria of these exemplary creatures: AUA that normally codes Ile is now a Met codon that is translated by the same tRNAMctthat translates AUG. The AGR codons that are normally translated as Arg, are now Ser codons translated by a single isoacceptor that reads the AGN box. Each of these codon reassignments makes possible the elimination of one tRNA species. In particular. the acquisition of two new codons for Ser by hyperwobble allows the relevant Arg isoacceptor to be eliminated from the mitochondrial ensemble. Likewise, the acquisition of an Ile codon by the tRNAMeteliminates. in effect, one of two tRNA species needed to translale Ile codons. Thus, the reassignments of codons for these three troublesome amino acid families is a small part of a much larger pattern. This consists of the elimination of roughly twenty tRNA spccies, which is made possible by an expansion of the codon repetoire of the retained tRNA specics. In addition, the conventional UGA stop codon is reassigned to Trp and translated by thc same tRNAT" that translates UGG. This reassignment can be correlated with anothcr tendency in the codon usage of animal mitochondria. For many animal mitochondria there is very clear avoidance of G in the coding sequences. This bias is found in ribosomal RNA sequences as well as in mRNA, but it is particularly prominent at third codon positions of the mRNA's (j). The important conscquence of the reassignment of AUA to Met as well as that of UGA to Trp is that while the conventional codons for these amino acids, which end in G, may be greatly reduced in frequency, the reassignments to AUA and UGA for these two amino acids permit them to be represented at normal or even higher than normal frequencies in the corresponding proteins (5). Accordingly, the selective advantage of some codon reassignments may be that they minimize the undesirable effects of a bias to avoid certain codons. This

effect is particularly imporlant for Met and Trp since they are the only amino acids that have only one codon each in the conventional code. Finally, the expectation that codon reasignments would generate unacceptable disruptive effects on the quality of proteins has dominated previous views of the origin of variation in genetic codes (2-4). I have not been so preoccupied by this perception because 1 believe that it is based on false premises. Indeed, a review of the consequences of amino acid substitutions on the physical stability and biological functions of proteins reveals that most often these are modest. incremental effects rather than totally destructive ones (38). This means that the functional barriers to codon reassignment are considerably lower than previously thought.

When Smaller Is Better Now I want to shift the focus to the mitochondrial genomes themselves in order to identify some of the forces that are driving their evolution. Then it will be possible to inspect the genomic characteristics of the mitochondria of different groups of organisms in order to relate these to their particular codon-tRNA usage patterns. The most striking characterisitic of mitochondria is that their genomes encode only a very small fraction of the protcins that are required for lheir propagation. If mitochondria originated from endosymbiotic bacteria, then the evolution of the the modern eucaryotic cell must have involved a massive transfer of coding scquences from the tamed bacteria to the early nuclear genome. There are at least two sorts of forces that could drive this genetic transfer process One of these is the growth rate competition that automatically arises when small populations of microorganisms or organelles propagate themselves i n cells. The faster growing variants will eventually displace the morc slowly growing ones. If we assume that having shorter genomic DNA sequences to replicate and fewer gene products to be expressed would at least somctimes shorten the doubling time of a microorganism or a bacterium, then the reductive evolution of the genome would have at least one very obvious origin. A second sort of reductive e~~olutionary force operates on small genomic populations and under suitable conditions, this process can lead to the loss of important gencs from the genome of an organelle. Small populations are vulnerable to genetic fluctuations in inverse proportion to their size. This, means that a defective genome can supplant a healthy one simply on the basis of statistical fluctuations in the replication and segregation of genomes in a minute intracellular population. For deletions the back mutation rate will be negligible. Therefore, there will be an inescapable tendency to lose sequences from genomes and this will be particularly evident in very small genomic populations. This mechanism is the driving force of what has been called Muller’s ratchet (39-40). Normally, Muller’s ratchet can be compensated by recombination between healthy and defective genomes within a population. I want to suggest that for genomes such as those of organelles or parasites it can also be compensated by

recombination between the nuclear genome and the organelle or parasite. Furthermore, the transfer of genes to the nucleus expands the number of genes that can be eliminated from the mitochondrial genome to increase its propagation rate. An evolutionary scenario based on genc transfer requires that molecular mechanisms exist to support the necessary recombination events. as well as the retargeting and reverse transport of required products rrom the cytoplasm to the mitochondria (or parasite). Recent observations suggest that none of these steps presents an insurmountable obstacle to the evolutionary process. Thus, the rate of transfer of genelic sequences from mitochondria to nucleus is sufficiently great to be measured experimentally in Saccarumvces cerevisiae (41). Likewise, quite extensive sequence homologies between nucleus, mitochondria and chloroplast are found in higher plants which suggests a high degree of promiscuity for these genomes (42-44). In addition, experimental manipulation of leader sequences has shown that specific addressing signals can be attached to arbitrarily chosen genetic determinants and the corresponding gene products will be directed to the mitochondrian (45). Finally, there are clear indications of the influence of reductive evolutionary forces on the translation systems of modern organisms. For example, pathogcnic bacteria such as Myctiplusma capricolum use 29 tRNA species instead of the standard45 to translate the code and at least seven of these translate by hyperwobble (4h). Most relevant are data indicating that modern mitochondria suffer spontaneous mutations that enable defective genomes to replace healthy ones. Two diverse examples illustrate this phenomenon. First, defective respiratory mutants such as the rho mutants of yeast are associated with a mitochondrial genome that lacks essential sequences but which can so effectively propagate itself that it suppresses the expression of wild-type mitochondria in heteroplasmic cells (47). Siinilarly, both point mutations and deletion mutations in mitochondria are associated with human disease (48). Such mutants can wise during the development of an individual or they can be inherited maternally. The resulting heteroplasmic mitochondrial populations may support normal functions for long periods of time. However, in certain cell lines, the defective mutants take over and a specific adult pathology results (49-51). In summary, the speculativc scenarios for the reductive evolution of mitochondrial genomes that I envoke to explain the origins of their minimalist codontRNA usage patterns find their counterparts in modern biological phenomena.

Genomic Variations Phylogenetic analyses of ribosomal RNA as well as cytochrome sequences very convincingly point to a eubacterial origin for the mitochondria (% 52). More specifically, mitochondria arose from the a group of the purple bacteria. Many a purple bacteria are intracellular parasites so it is not surprising that mitochondria have evolved from this group. However, because of the great reduction of mitochondrial genome size, it would be difficult to establish the affinity of

lnitochondria with a purple bacteria were it not for the small core of genes that are retained in mitochondrial genonies. This COmlnon core of mitochondria1 genes consists of those for a number of respiratory proteins and thosc for the RNA molecules required for their translation. The diversity of genomic arrangements that propagate this common Core of genes is quite astonishing. On one extreme are the plant mitochondria with paradoxically the most conserved coding sequences when compared with bacteria, but with the most complex and varied genomic architectures (s3).Plant mitochondria usually have genoines occupying the large end of the spectrum with sizes in the hundreds of thousands of base pairs, but there are examplcs with very small genomes. For most plant mitochondria the coding sequences make up a small part of the total DNA complement, which may contain large repeat sequences that play an important role in the dynamics of the genome. On the other extremc are the more uniform genomes of animal mitochondria. These barebone genomes tend to be at the small end of the spectrum, with sizes in the range between 14 and 20 thousand base pairs; typically they lack introns and usually they have only one substantial noncoding sequence, which overlaps the origin ofreplication (e.g. (*I)). In the middle range of genomic sizes we find the protista and the fungi, which are also intermediate in the sense lhat they carry noncoding sequcnces but these are not quite as extensive as for the plant mitochondria. It is unclear whether or not mitochondria were acquired at an early or at a late stage rclative to the appearance of the nucleus. Nevertheless, it seems most parsimonious to assume that early in the evolution of mitochondria many ofthe genes of the bacterial ancestor were simply lost, while others that were required for the perpetration and function of these organelles were transfered to nuclear genomes. Among the genes that are particularly relevant to genomic architectures are those participating in DNA replication, DNA repair. recombination and the regulation of DNA biosynthesis. If loss or mutation of these particular genes during the evolution of the mitochondrial lineages that gave rise to the organelles of different kingdoms were to have proceeded along somewhat different paths, the subscquent evolution of distinctive genomic characteristics of these mitochondria would be understandable. Thus, the rich diversity of mitochondrial genomic architectures may itself reflect the fluctuations inherent in the reductive evolution of thcse organelles. Finally. there is one property of the nuclear genomes that seems to have been common to the evolution of almost all eukaryotes. This is the capacity of the nucleus to accept, integrate and express genes transfered from mitochondrial genomes. Genomes need not have this capacity. as the animal mitochondria have shown us. Accordingly, there is reason to wonder whether or not a successful symbiosis established between the ancestors of mitochondria and the early nuclear genomes was instrumental in selecting precisely those genomic properties of the nucleus. The implication here is that nuclei without these properties may have been lost in the competition with those that adopted the mitochondrial genetic symbiosis. In other words, did the mitochondria play a role in selecting the phenotypes of nuclei?

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C. G. Kurland is at the Dcpartnient of Molecular Biology, Biomedical Ccnter. Box 590, Uppsala. Sweden 75 1 24.