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52 Zamore, P.D. et al. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101, 25–33 53 Plasterk, R.H. and Ketting, R.F. (2000) The silence of the genes. Curr. Opin. Genet. Dev. 10, 562–567 54 Bloom, L. and Horvitz, H.R. (1997) The Caenorhabditis elegans gene unc-76 and its human homologs define a new gene family involved in axonal outgrowth and fasciculation. Proc. Natl. Acad. Sci. U. S. A. 94, 3414–3419 55 Rougvie, A.E. and Ambros, V. (1995) The heterochronic gene lin-29 encodes a zinc finger protein that controls a terminal differentiation event in Caenorhabditis elegans. Development 121, 2491–2500 56 Furuta, T. et al. (2000) EMB-30: an APC4 homologue required for metaphase-to-anaphase transitions

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during meiosis and mitosis in Caenorhabditis elegans. Mol. Biol. Cell 11, 1401–1419 Laughton, D.L. et al. (1997) Alternative splicing of a Caenorhabditis elegans gene produces two novel inhibitory amino acid receptor subunits with identical ligand binding domains but different ion channels. Gene 201, 119–125 Kim, V.N. et al. (2001) Role of the nonsense-mediated decay factor hUpf3 in the splicing-dependent exonexon junction complex. Science 293, 1832–1836 Lykke-Andersen. et al. (2001) Communication of the position of exon–exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science 293, 1836–1839 Gingras, A.C. et al. (1999). eIF4 initiation factors: effectors of recruitment to ribosomes and

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regulators of translation. Annu. Rev. Biochem. 68, 913–963 Knight, S.W. and Bass, B.L. (2001) A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science 293, 2269–2271 Hammond, S.M. et al. (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 Parrish, S. et al. (2000) Functional anatomy of a dsRNA trigger. Differential requirement for the two trigger strands in RNA interference. Mol. Cell 6, 1077–1087 Hammond, S.M. et al. (2000) An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature 404, 293–296

Making mitochondrial mutants Howard T. Jacobs Mitochondrial DNA (mtDNA) encodes a mere 13 polypeptides, all with welldefined cellular functions in mitochondrial energy metabolism. It was first sequenced over two decades ago, yet our understanding of the wider physiological role of mtDNA is surprisingly sketchy. Partly, this reflects the fact that the mitochondrial gene products are essential for life; that is, most mtDNA mutations are expected to be lethal. The technical difficulty of engineering mtDNA mutations has been a major handicap in furthering our understanding of the mitochondrial genetic system. Recent developments now offer some possibilities for the genetic manipulation of mtDNA and for elucidating its contribution to human development, physiology and disease.

Mitochondrial DNA (mtDNA) is an independent genome within eukaryotic cells. Although human mtDNA was sequenced over two decades ago1, the fact that it has not been possible to manipulate it at the sequence level means that huge gaps remain in our understanding of the mitochondrial genetic system.

The maternally inherited mtDNA is a 16 569-bp circular molecule, organized compactly into large, overlapping, POLYCISTRONIC (see Glossary) transcription units. The primary transcripts are processed into two rRNAs, 22 tRNAs and 11 mRNAs, the last encoding 13 of the 80 or so polypeptides of the mitochondrial OXPHOS complexes (Fig. 1). The mitochondrial rRNAs and tRNAs contribute to a separate translation system within the organelle that is dedicated to the synthesis of these polypeptides. Mitochondrial protein synthesis requires, in addition, well over 100 dedicated nuclear gene products. Even though their cellular functions are known, detailed knowledge of the biology of the mtDNAencoded polypeptides and RNAs is limited. To investigate structure–function relationships in these gene products, we have needed to rely upon indirect

Glossary

Howard T. Jacobs Institute of Medical Technology and Tampere University Hospital, FIN-33014 University of Tampere, Finland. e-mail: [email protected]

Atresia: Oocyte degeneration by programmed cell death. The majority of oocytes are lost to this process during development/ageing. Biolistic transformation: Introduction of DNA using bombardment with microprojectiles coated with DNA (also known as ‘the gene gun’). Chloramphenicol: An antibiotic that inhibits the elongation step of translation on bacterial and mitochondrial ribosomes. Mutations to chloramphenicol resistance occur on the large subunit rRNA. Cybrid: A genetic hybrid containing the nuclear genome from one source but the cytoplasmic (i.e. mitochondrial or chloroplast) genome(s) from another. Homoplasmy (homoplasmic): Genetic uniformity of the cytoplasm; that is, all copies of mtDNA identical in sequence within a single cell or individual. Heteroplasmy (heteroplasmic): Genetic heterogeneity of the cytoplasm; that is, presence of two different types of mtDNA that differ in sequence within a single cell or individual. Mitotic segregation: Shift in relative amounts of heteroplasmy with eventual resolution to homoplasmy, as a result of random partition of mtDNA to daughter cells. OXPHOS: Oxidative phosphorylation, requiring the five multisubunit complexes of the inner mitochondrial membrane. Polycistronic: Containing coding regions of many different genes within a single primary transcript. Polymorphy: Genetic variation within a population of individuals. Polyplasmy (polyplasmic): Multiple heteroplasmy; that is, many different sequence variants of mtDNA present within a single cell or individual. Replicon: An autonomously replicating segment of DNA. ρ0: A cell, cell line or (yeast) strain devoid of mtDNA. ρ+: A yeast strain containing intact mtDNA, although it can carry point mutations. ρ–: A yeast strain containing massively deleted mtDNA, organized in tandem repeats of the retained portion of the mitochondrial genome. Synaptosome: Preparation of subcellular fragments of the synaptic region of neurons, rich in mitochondria.

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Fig. 1. Maps of human (a) and yeast (b) mtDNA. Gene names follow the usual species conventions. For human (NCBI accession NC_001807), protein-coding genes are subunits I, II and III of cytochrome c oxidase (COXI, COXII, COXIII), apocytochrome b (cyt b), subunits 1–6 and 4L of NADH dehydrogenase (ND1–6, ND4L) and subunits 6 and 8 of ATP synthase (A6, A8). For yeast (NCBI accession NC_001224), proteincoding genes are subunits I, II and III of cytochrome c oxidase (COX1, COX2, COX3), apocytochrome b (COB), subunits 6, 8 and 9 of ATP synthase [ATP6, ATP8 (AAP1), ATP9 (OLI1)], and mitoribosomal protein Var1p (VAR1). Ribosomal RNA genes are shown as 12S and 16S for human, 15S and 21S for yeast, plus the yeast 9S RNA subunit of mitochondrial RNase P. Note the presence of multiple introns in the yeast COX1 and COB genes, and of a single intron in 21S rRNA. Many of these contain open-reading frames (not shown) encoding proteins involved in splicing and intron mobility. tRNA genes are denoted according to the one-letter amino-acid code, with codon recognition groups as shown. In yeast mtDNA all genes except tRNAThr (CUN) are encoded in the clockwise direction as shown. In human, all protein-coding genes are encoded in the clockwise direction as shown, except for ND6, which is encoded on

the opposite strand as indicated by the arrowhead. The direction of transcription of the human tRNA genes varies: those shown in green are transcribed clockwise (H-strand-encoded), those in blue anticlockwise (L-strand encoded). Replication origins for the two strands (H and L) are shown for human mtDNA. The H-strand origin, OH, gives rise to an abundant, aborted replication product in which the region between OH and tRNAPro is maintained as a triplex (D-loop) structure. Also shown are the promoters PH2 and PL, which gives rise to full genome-length transcripts for the H- and L-strands, respectively, and PH1, which gives rise to a shorter H-strand transcript that terminates at the 16S tRNALeu (UUR) gene boundary as shown. In yeast, eight mtDNA replication origins (ori1-8) have been identified on the basis of their presence in ρ− genomes, although it is unclear which or how many operate in any given replicating wild-type mtDNA molecule. Each ori sequence is associated with a promoter. Other promoters are omitted, for clarity. Also omitted are short open reading frames that have not been shown to encode a translated polypeptide. Note that yeast mtDNA in vivo appears to be mainly composed of linear fragments representing overlapping, circularly permuted derivatives of the circular map as drawn.

methods of analysis, such as crystallographic studies of purified mitochondrial enzymes (reviewed in Ref. 2), or inferences from genetic studies in bacteria3,4 or yeast. The RNA components of the mitochondrial translational machinery are highly unorthodox in structure, and many questions remain open concerning their functions. Maintenance of mtDNA also involves a complex machinery for DNA replication, partition and copy number regulation. Little is known about signals in mtDNA that mediate these processes. Based on bacterial precedents5, a recombinational machinery should also exist for resolving genomic multimers to monomers and which should depend on sequence information in mtDNA. Remarkably, although present in thousands of copies per cell, all copies of mtDNA within a healthy individual appear to be identical in sequence, a condition described as HOMOPLASMY (although HETEROPLASMY is sometimes seen within the

hypervariable portion of the noncoding region). By contrast, mtDNA shows great variability among individuals (10–30 bp differences even between ethnically related persons). The finding of homoplasmy against a background of POLYMORPHY suggests that a genetic bottleneck operates in the female germline to purify the mtDNA population of every individual, perhaps involving genetic selection during oocyte ATRESIA. Nevertheless, some deleterious mutations are clearly not systematically counterselected6. It has been argued7 that selection during oogenesis would be an efficient method of protecting the long-term genetic fitness of mtDNA which, in an essentially asexual population, should be subject to Müller’s ratchet – the progressive accumulation of deleterious mutations that cannot be eliminated by sexual recombination. We understand little of how mitochondrial genetic fitness is maintained throughout life, or how somatic mutations affect cell phenotype. The mitochondrion is a major site of generation of reactive oxygen species

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(ROS), which can potentially damage mtDNA, thus inhibiting its replication or promoting the accumulation of mutations. The mtDNA is therefore dependent on protective enzymes for ROS detoxification, as well as on an extensive apparatus of DNA repair8. The lifetime accumulation of mtDNA damage remains a popular, though unproven mechanism to account for many of the phenomena of ageing9,10. However, at present we have no model systems available in which to assess the relationship of mtDNA mutation load to somatic ageing.

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Pathological mtDNA mutations: the beginnings of human mitochondrial genetics

Since the late 1980s, a large number of pathological mtDNA mutations, including both point mutations and genomic rearrangements, have been identified in association with diverse human disorders (reviewed in Refs 11,12). These mitochondrial disorders range from relatively mild, late-onset conditions (e.g. sensorineural hearing loss or ocular myopathy) to devastating and frequently fatal syndromes, such as MELAS (mitochondrial encephalomyopathy with lactic acidosis and stroke-like seizures), or Leigh disease (fatal necrotizing encephalopathy). Many, but not all mtDNA disease mutations are heteroplasmic; that is, they co-exist with wild-type mtDNA. However, the amount and tissue distribution of heteroplasmy is not the sole determinant of severity or clinical phenotype, which can be highly variable, even within a single family. This is particularly clear for the more commonly observed pathological mutations, such as the A3243G MELAS mutation in the tRNALeu (UUR) gene. The discovery of mtDNA disease mutations provided an opening into human mitochondrial genetics. However, it raised many new questions concerning pathological mechanisms and the relationship between mtDNA genotype and phenotype. Examination of these questions required the development of suitable model systems, such as trans-mitochondrial CYBRID cell lines, in which patient-derived mtDNA is transferred by cytoplast fusion to a tester recipient line (ρ0 CELLS) lacking any mtDNA of its own13 (Fig. 2). Cybrid cells have been used to confirm the pathogenicity of mtDNA disease mutations, to develop insights into the molecular mechanisms by which they impair mitochondrial function, and to demonstrate that a heteroplasmic threshold effect operates at the cell level. Cybrid models have also hinted at the importance of nuclear background in determining the extent and direction of segregation of heteroplasmic mutations14,15 during repeated rounds of cell division. However, their use has serious limitations. Why make mitochondrial mutants?

Despite the advances that cybrid technology provided, many fundamental questions about mtDNA disease remain unanswered, concerning, for example, MITOTIC SEGREGATION in both the germline or in somatic tissues, or the tissue-specificity of the phenotypes observed. In http://tig.trends.com

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Fig. 2. Phenotypic analysis of heteroplasmic mtDNA mutations, using cybridization to ρ0 cells (see Glossary). Cytoplasts prepared from heteroplasmic cells (e.g. from a patient with mtDNA disease) are fused to ρ0 cells from a standard tester line. Phenotypic effects due to mtDNA can then be evaluated in a control nuclear background. Output clones typically stabilize at various levels of heteroplasmy, allowing a detailed analysis of the effect on cell phenotype of different levels of heteroplasmy for a given mtDNA mutation. Cybrid clones with only wild-type mtDNA from the heteroplasmic patient, shown in blue, represent an important internal control in such studies.

addition, physiologically relevant models are needed to test strategies for therapy. Cultured-cell models are unreliable for a variety of reasons. Primary cell lines from patients (e.g. fibroblasts, myoblasts) are not immortal, but they also do not retain a fully physiological, differentiated phenotype in culture. They can also lose mutant mtDNA as a result of mitotic segregation. Cybrid cells, however, are necessarily tumour-derived, which means that they are usually aneuploid, exhibit genetic instability in longterm culture, and cannot reproduce the characteristics of differentiated cells in the body. Finally, because of strong selection for efficient growth, genetic or epigenetic changes that mask or modify mutant phenotype in cybrid cells can be unwittingly selected. Even though a careful analysis can exploit such effects, they remain a serious impediment to accepting cybrid cell models as truly physiological. Far preferable would be a system to introduce, or at least select for, specific mutations in the mtDNA of a whole organism. The ability to manipulate mammalian mtDNA would also enable a comprehensive analysis of structure–function relationships in mtDNA-encoded gene products, as well as providing, for the first time, a direct test in vivo of the role of signals in mtDNA itself in the maintenance, inheritance, expression and selection of mtDNA. It would reveal which types of mtDNA sequence confer fitness at the cell or organism level, or which are crucial for facilitating recombination. This might help us to understand better the nature of the mtDNA bottleneck in the female germline and the mechanisms that exist to protect mtDNA from Müller’s ratchet. Problems in manipulating mammalian mtDNA

Direct, DNA-based transformation of mitochondria in living cells is clearly not straightforward. Any delivery

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Fig. 3. Yeast mtDNA transformation with a desired mutation16,17 as used by Rohou et al.18 to introduce a previously characterized tRNAGly mutation (ts9) into wild-type mtDNA. Two constructs, one nuclear, one mitochondrial, are co-transformed using biolistic transformation. The nuclear construct requires an appropriate replication origin (e.g., an ARS sequence), but the mitochondrial plasmid does not need to contain a known ori sequence. Selectable markers are also needed for each compartment, for example, LEU2 for the nucleus, allowing selection in a leu2-mutant background, and a respiratory chain gene such as COX2 for the mitochondria, allowing verification of mitochondrial transformation by crossing to a tester strain carrying a point mutation in the same gene. A copy of the target gene containing the desired mutation (red cross) is also included in the mitochondrial construct. After selection for the nuclear marker, transformant colonies are tested for the presence of the artificially constructed, ρ− mitochondrial replicon (see Glossary), by crossing to a cox2-mutant tester strain. The desired transformants are the ones that can give recombinant colonies capable of growth on glycerol (gly+) after this cross, because growth on this substrate requires functional mitochondrial OXPHOS. This can only come about if the strain being tested contains the engineered, COX2-containing construct in its mitochondria. The selected transformants are then mated separately to a wild-type strain, ρ+, and progeny that contain the desired mutation, in this case ts9, are finally selected by PCR or by phenotypic screening.

system must ensure that DNA crosses not one, but three membranes (the plasma membrane, plus the outer and inner mitochondrial membranes), while preserving cell and organelle integrity and viability. The high copy number of mtDNA (thousands per cell) means either that multiple transformation events are needed, or that stringent selection must be imposed, to ensure that untransformed mitochondria and their genomes are eliminated. Moreover, our incomplete understanding of mtDNA replication and partition in mammals means that we do not know what constitutes the minimal mitochondrial REPLICON, nor can we judge the effects that bacterial vector sequences needed for cloning might have on the ability of such a molecule to replicate in vivo. In yeast, where mitochondrial genetics is better established, a tolerably efficient mtDNA transformation procedure has been developed16,17. This exploits the lower copy number (50–100 per cell), efficient mitochondrial DNA recombination, rapid mitotic segregation and good selection systems available in yeast. The engineered segment of mtDNA is incorporated within an artificial ρ− REPLICON also containing a functional copy of one of the mitochondrial http://tig.trends.com

protein-coding genes, as a selectable marker (Fig. 3). The construct is delivered by BIOLISTIC TRANSFORMATION, with initial selection for a co-transfected nuclear marker. Colonies are then tested by mating to a strain that carries a point mutation in the corresponding marker gene, allowing a simple nutritional selection, based on respiratory competence. The original mitochondrial transformant can then be mated with a wild-type strain, to engineer the desired replacement, which can be easily confirmed by PCR. Manipulation of yeast mtDNA has great potential as a system for investigating the physiological significance of human mtDNA disease mutations, especially where they affect phylogenetically conserved tRNA or protein-coding sequences. Rohou et al.18 recently demonstrated the feasibility of this approach by artificially introducing into yeast mtDNA a tRNA mutation previously demonstrated to produce a temperature-sensitive, respiratory-deficient phenotype. If this can be made to work, for example, for the common disease mutations in mitochondrial tRNALeu (UUR), yeast could provide a valuable system in which to screen for potential suppressors of the mutant phenotype. Meunier19 also used this approach to model, in yeast, the effects of several proposed disease mutations in subunits of cytochrome c oxidase. However, many features of mitochondrial gene expression and respiratory chain biogenesis in human cells cannot be reproduced in yeast (Fig. 1)20. Complex I, the proton-pumping NADH:ubiquinone oxidoreductase, is absent in yeast mitochondria, and the machinery of mitochondrial RNA synthesis operates very differently in the two organisms (yeast mtDNA having multiple transcription units, introns and endonucleolytic mRNA 3′ end processing, whereas human mtDNA has genome-length transcription units, tRNA ‘punctuation’, mRNA polyadenylation, and complex transcriptional regulation). Further potential complications arise from genetic code differences between yeast and human mitochondria, rapid mitotic segregation that precludes the maintenance of stable heteroplasmy, and the tendency of yeast to lose mtDNA altogether if mitochondrial protein synthesis is severely crippled. In addition, questions specifically relevant to multicellular organisms (e.g. differentiation and development, apoptosis, mitochondrial inheritance) cannot be addressed easily in yeast. For these reasons, it would be highly desirable to achieve direct transformation of mammalian mtDNA. However, attempts to replicate the yeast transformation scheme in mammalian cells have, thus far, met with little success, although many studies are still in progress. For example, Bigger et al. have recently developed a method for eliminating potentially toxic bacterial vector sequences from putative mitochondrial replicons21, and they and others have proposed CHLORAMPHENICOL resistance as a promising candidate for a mitochondrial selectable marker22. This has the drawback that, based on recent studies in the mouse that are discussed below, chloramphenicol resistance

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mutations in mtDNA might also give a respiratory chain enzyme deficiency. Another approach is to try to facilitate the uptake of DNA or DNA analogues into mitochondria, either by coupling to peptides that naturally target the protein import machinery23,24 or by engineering mitochondrial expression of a naturally occurring DNA-uptake system, such as that of Haemophilus25,26. Another possible strategy is to exploit and develop the natural systems for RNA uptake that exist in mitochondria of many eukaryotes, including plants and fungi27, which could be useful for both transient and permanent transformation. However, more laborious or indirect methods will probably be needed to engineer specific mtDNA mutations in mammalian cells. One possibility is to transform mitochondria in cell-free suspension, for instance by electroporation28 or liposome-mediated fusion29, then reintroduce them into ρ0 cells by microinjection or cytoplast fusion. Another strategy that has more limited applications is the use of antisense peptide nucleic acids to influence the relative replication of two different mtDNAs in heteroplasmic cells30. Although designed with gene therapy in mind, in principle this approach could also be used to drive cells to homoplasmy for rare variants. However, it has not yet worked in vivo31. Beyond mtDNA transformation in cell culture, there is the additional and daunting problem of transferring manipulated mtDNA to the whole organism. However, this has been solved in principle using the various pioneering strategies that have resulted in the creation of transmitochondrial mice (discussed below). Strategies for engineering an enhanced mtDNA mutation rate

In view of the difficulties in achieving mtDNA transformation in animal cells, indirect methods of creating new mtDNA variants are being pursued through the exploitation of natural mutation mechanisms, including systems for inducing replication errors or for promoting the accumulation of DNA damage. Once again, yeast has provided an important model. As far as we know, all mitochondrial DNA synthesis depends on a single, nuclear-encoded enzyme, DNA polymerase γ, a member of the PolA superfamily32. In both yeast and metazoans (see Fig. 4), the catalytic subunit has well-defined, phylogenetically conserved domains involved in both DNA polymerization and proofreading (3′–5′ exonuclease). Specific mutations in the proofreading domain abolish exonuclease activity33,34 and lead to the accumulation of mtDNA mutations. In yeast, the absence of proofreading activity from DNA polymerase γ generates predominantly A–T transversions, whereas mutants defective in mitochondrial mismatch repair mainly accumulate transitions35. The combination of both leads to an error catastrophe in yeast mtDNA (Ref. 35). In long-term culture, human cells overexpressing a proofreading-deficient variant of DNA polymerase γ http://tig.trends.com

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Fig. 4. The catalytic subunits of yeast and human DNA polymerase γ (Mip1p and POLG, respectively), showing the mitochondrial targeting signal (mts, orange), the three conserved regions in the N-terminal half of the protein required for 3′–5′ (proofreading) exonuclease activity (Exo1, Exo2 and Exo3, green), and the region with polymerase activity conserved among all members of the PolA family (red). Compared with the human polypeptide, the yeast enzyme contains a C-terminal extension of unknown function, including a region similar to 3′ phosphoglycerate kinase (yellow). The human protein requires a second subunit (POLGB, not shown) for processivity. The D198A substitution in human POLG, like the corresponding D171G in yeast Mip1p, abolishes 3′–5′ exonuclease activity and creates an mtDNA mutator34. Scale bar: 200 amino acids.

(POLG) also accumulate mtDNA mutations to a high level, though in this case mainly G–C to A–T transitions34. It is unclear whether these represent uncorrected replication errors or subsequent DNA damage that has not been repaired. After three months of culture, mutation loads of approximately one mutated base pair per 2 kb are reached in mtDNA. Virtually every mtDNA molecule in such cells must carry multiple deleterious mutations. Yet the effects on cell phenotype are modest, implying that the mutations are complemented by still intact copies of each gene, insufficient time having elapsed for the mutations to segregate to homoplasmy. Each individual mutation is probably present in a particular cell only at a relatively low level, a situation that can be described as POLYPLASMY. In longer-term culture, intercellular selection against continued expression of the mutator occurs, indicating that mtDNA mutations do impair cell growth or viability once they surpass a threshold level or are subject to mitotic segregation33. In principle, a mitochondrial mutator could also be created by disrupting genes encoding enzymes of mtDNA repair, allowing other classes of mutations than G–C to A–T transitions to be generated. Indeed, this might occur ‘naturally’ in some tumours. In the future, other strategies could emerge for accelerating the generation of novel mtDNA mutations. To use an mtDNA mutator as a method for isolating specific mtDNA mutants, it is necessary to ‘purify’ the mtDNA population: in effect, to impose a genetic bottleneck in cell culture, so as to isolate a clonal cell population containing a specific, mutated mtDNA. Selectable markers such as chloramphenicol resistance are only useful in this context if they are themselves the object of study. One approach is transient mtDNA depletion, using drugs that inhibit mtDNA replication, e.g. ethidium bromide36 or dideoxycytidine37, or transient expression of dominant–negative versions of POLG (Ref. 34), which

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would prevent synthesis of new mtDNA. Cytoplast fusion to ρ0 recipient cells can also achieve the same result. In each case, mtDNA rapidly re-amplifies to an apparently predetermined level, according to cell type38. Provided that the re-amplification of mtDNA is allowed to take place under non-selective conditions from a very low level, preferably of the order of one copy per cell, it is possible to generate cell clones each containing only one type of mutated mtDNA. By preselecting cells in which mutator is already inactivated, mtDNA genotype in such clones will thereafter be stable. Cell cones containing any given desired mutation can then be identified by PCR-based batch screening. This could be used, for example, to isolate a mouse cell clone containing mtDNA with a human disease-equivalent mutation, for instance A3243G. Whole organism mtDNA mutators: the problems of selection and segregation

Attempts to engineer a POLG mutator in a whole organism have had mixed success. Zhang et al.39 reported a severe cardiomyopathy in a transgenic mouse line overexpressing a proofreading-deficient Polg in the heart. However, this could be an artefact, because the mtDNA mutation load reached was far lower than that seen in cultured human cells after long-term expression of the POLG mutator, and no respiratory chain abnormalities were detected. In heart, high-level expression of other transgenes, such as greenfluorescent protein (GFP), can produce a rather similar phenotype40. A better strategy might be to knock-in a proofreading-deficient mutation to the endogenous gene and then breed to homozygosity, so that it is expressed physiologically. This could also be done tissue specifically, using Cre–loxP recombination41. Such approaches could test the predictions of the somatic mtDNA mutation theory of ageing. In Drosophila, overexpression of the catalytic subunit of the mitochondrial DNA polymerase, encoded by the tamas gene, also creates a dominant–negative phenotype42. Therefore, to create a viable tamas mtDNA mutator, it is necessary to ensure carefully regulated expression of the mutator transgene. Preliminary data suggest that more moderate overexpression of wild-type DNA polymerase γ has no apparent consequences, whereas equivalent expression of a mutator variant produces developmental defects, as well as reductions in reproductive capacity and lifespan (L.S. Kaguni and R. Garesse, pers. commun.). Whole organism mtDNA mutators suffer from similar potential limitations to cultured cell mutators. First, the mutator must be carefully regulated, or an mtDNA error catastrophe should eventually lead to a severe loss of fitness. Although the conditions under which such an error catastrophe might occur are of keen interest, it will also be important to be able to switch off the mutator. This could be done by using tightly regulated promoters or, more simply, by outcrossing or using Cre–loxP recombination41. One useful approach might be to use Cre–loxP http://tig.trends.com

recombination to restrict expression of the POLG mutator to the female germline. The effects of a specific mtDNA mutation load on different physiological processes could then be carefully evaluated, without the complication of further mutations accumulating during life. Second, as in cell lines, different mutations will accumulate at a low level in each different cell and its progeny, so that the whole organism will be polyplasmic, rather than heteroplasmic at a high level for a few, defined variants. The biological outcome will thus depend both on overall mutation load and the patterns of selection and mitotic segregation in different tissues. Analyses of artificially generated heteroplasmic mice43 indicate that segregation can operate in a tissue-specific fashion, even for apparently neutral polymorphisms44. Clearly, to avoid the complications of polyplasmy, it will be necessary to impose a genetic bottleneck on the mutator strain to purify the mtDNA population, so that defined variants can be studied. Although this occurs naturally in the female germline, it is obviously not practical to establish and then sift through thousands of mouse lines, to find those with interesting mtDNA genotypes. Moreover, the most interesting variants might already have been eliminated by germline selection. Instead, the technologies recently developed for in vitro manipulation of embryos offer the prospect of transferring defined mtDNA mutants into the mouse germline. Introduction of mutant mtDNA into animal models

Zygote microinjection43,45, nuclear transfer46, blastomere electrofusion47,48 and blastocyst injection of ES cell cybrids49,50 have each provided feasible strategies for creating heteroplasmic mice containing mtDNA from donor cell lines or tissues. Of these, ES cell cybridization, followed by blastocyst injection or electrofusion of cytoplasts to single-cell embryos, seems to be the most promising technique. Transfer can be facilitated by the use of selectable markers (e.g. chloramphenicol resistance) but can also work in the absence of selection, when recipient cells are pretreated with a mitochondrial toxin, such as rhodamine6-G. After such treatment, cybrids are found to contain mainly or exclusively donor mitochondria. The use of donor cells carrying specific mtDNA mutations of interest allows the creation of transmitochondrial mice that are heteroplasmic (or even, in principle, homoplasmic) for such mutations. The first generation mice born using this technique are chimaeric, which potentially allows mutations to be analysed that would be lethal if expressed ubiquitously. Variants of this technology49,50 have been used to transfer mtDNA carrying either of two chloramphenicol resistance (CAPR) mutations, each derived originally from a cell line, into the somatic tissues of heteroplasmic mice. In one case50, the marker was also successfully transmitted in the germline, either in the heteroplasmic or homoplasmic state. High levels of germline-transmitted CAPR mtDNA were

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Acknowledgements I thank Hans Spelbrink, Nils-Göran Larsson, Françoise Foury, Rafa Garesse, Laurie Kaguni, Monique Bolotin and Laura Frontali for useful discussions and comments on the manuscript. My research is supported by the Academy of Finland, Tampere University Hospital Medical Research Fund and the European Union.

Fig. 5. Proposal for the introduction of a specific, desired mtDNA mutation into the mouse germline. A mouse Polg mitochondrial mutator cell line (pink outline), analogous to the human mtDNA mutator cell line already established, is used to generate a high frequency of mtDNA mutations (although preferably not so high as to make it likely that more than one novel mutation is introduced into a given mtDNA molecule). Each cell in the population contains mtDNA molecules with different mtDNA mutations, denoted by the different colours. The mutator is preferably already inactive in this starting cell population. To convert this population to cells that have a high level of only one mutation (red), ethidium bromide (EtBr) or other treatments can be used to deplete the mtDNA content of the cells to ~one copy per cell. After removal of the drug, mtDNA reamplification repopulates each cell with a specific mutant mtDNA, although each cell in the population will now carry a different mutation(s). To isolate a cell clone carrying a specific mtDNA mutation of interest, cells can be batch screened using a sensitive PCR assay for the desired mutation (e.g. based on mini-sequencing or allele-specific PCR). The procedure is repeated using successively smaller pools of cells until a pure clone is isolated, whose mtDNA should be completely sequenced to ensure that no other novel mutations are present. This is then used as a cytoplast donor (Fig. 2), in a fusion to mouse ES cells (blue outline) treated with rhodamine-6-G (R6G) to eliminate endogenous mtDNA. ES cells repopulated with the mutant mtDNA of interest (red) are then injected into a recipient blastocyst and implanted into a pseudopregnant foster mother to generate chimaeric progeny (or alternatively electrofused as cytoplasts to single-cell embryos). Progeny can then be screened for maternal, germline transmission of the mutant mtDNA. Several variations could be used to generate heteroplasmy; for example, to examine mutations expected to be lethal in a homoplasmic state, such as the mouse equivalent of A3243G. These could involve a less stringent copy number depletion in the first step, or incomplete destruction of the endogenous mtDNA of the ES-cell recipient.

associated with a cluster of phenotypic features similar to those observed in mitochondrial disease in humans, including heart, muscle and retinal pathology, combined with retarded growth, cataract and early death50. However, it is not clear whether these are the direct result of translational impairment associated with the CAPR mutation, or with other point mutations carried in the donor mtDNA, which had been maintained in cell culture over many years. http://tig.trends.com

A similar approach has been used to transfer heteroplasmic, deleted mtDNA, originally derived from brain SYNAPTOSOMES of a donor mouse, through the maternal germline48. Here again, a complex phenotype was observed, including, surprisingly, renal failure, which has not often been reported in humans with mtDNA deletions. In theory, this technique offers the prospect of transferring any mutant mtDNA that can be obtained from cultured cells into a whole organism model. If combined with the Polg mutator and an appropriate regime for mtDNA purification-selection in cell culture, this could be used to create mouse models of human mtDNA disease (Fig. 5). Conclusions: the mtDNA paradigm and nuclear genomics

Although mtDNA encodes ostensibly ‘housekeeping’ functions, mutations in it can cause a bewildering range of pathophysiological effects in humans or model organisms. Indeed, many novel phenotypes associated with mtDNA defects might remain to be discovered, already suggested by the fact that unexpected features were seen among the first crop of transmitochondrial mice48. Surprisingly, some two decades after mammalian mtDNAs were first sequenced, we remain largely ignorant of how mitochondrial genotype impinges mechanistically on whole organism phenotype. In part, this reflects the continuing difficulties of establishing workable mitochondrial genetics in mammals, although work described in this article shows that we are now approaching this goal. However, it also indicates that mitochondrial gene function is far more complex than we once imagined and provides a sobering counterbalance to the euphoria generated by the

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completion of the nuclear genome sequences of humans and model organisms. Note added in proof

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