REVIEW ARTICLE

Genetic conservation versus variability in mitochondria:the architecture of the mitochondrial genomein the petite-negative yeast Schizosaccharomyces pombe

Received: 26 February 2003 / Revised: 8 April 2003 / Accepted: 12 April 2003 / Published online: 9 May 2003� Springer-Verlag 2003

Abstract The great amount of molecular informationand the many molecular genetic techniques availablemake Schizosaccharomyces pombe an ideal modeleukaryote, complementary to the budding yeastSaccharomyces cerevisiae. In particular, mechanismsinvolved in mitochiondrial (mt) biogenesis in fissionyeast are more similar to higher eukaryotes than tobudding yeast. In this review, recent findings on mtmorphogenesis, DNA replication and gene expression inthis model organism are summarised. A second aspect isthe organisation of the mt genome in fission yeast. Onthe one hand, fission yeast has a strong tendency tomaintain mtDNA intact; and, on the other hand, the mtgenomes of naturally occurring strains show a greatvariability. Therefore, the molecular mechanisms behindthe susceptibility to mutations in the mtDNA and themechanisms that promote sequence variations duringthe evolution of the genome in fission yeast mitochon-dria are discussed.

Keywords Mitochiondrial biogenesis Æ Petite-negativeyeast Æ Group I intron homing Æ Group II intronretro-homing

Introduction to the molecular geneticsof fission yeast mitochondria

The fission yeast Schizosaccharomyces pombe is the sixtheukaryote of which we know the entire sequence of thenuclear genome. The 4,824 protein-coding genes of thisgenome represent the smallest number yet knownamongst eukaryotes. The small genome within fission

yeast mitochondria is also entirely sequenced. Thisexcellent genetic background and the extensive collec-tion of molecular genetic tools make Sch. pombe an idealmodel eukaryote, although the development of fissionyeast as a model system is comparatively recent. It hasbeen used predominantly for studies of cell-cycle controland differentiation (Forsburg and Nurse 1991; Forsburg1999), although it is also becoming more popular inother fields. In particular, mitochiondrial (mt) move-ment and morphogenesis in fission yeast is more similarto higher eukaryotes than to budding yeast, whichmakes it a promising new system for studying this pro-cess in order to find out more about the molecular basisof mt disorders in human. The tendency to maintain itsmtDNA intact forms the basis for studying factorsaffecting DNA integrity. Furthermore, the presence ofmobile group I and group II introns within the small mtgenome makes investigations possible which lead to ananswer to the question on which way mobile elementshave possibly shaped the mt genome during evolution.The review covers these aspects.

Biogenesis of fission yeast mitochondria

Schematic representations of eukaryotic cells usuallyshow mitochondria as kidney-shaped organelles. Earlyserial thin-sectioning of Sch. pombe cells exhibited twolarge and two small mitochondria (Davidson and Gar-land 1977). Further 3-D reconstruction experiments ofserial-section electron micrographs of freeze-substitutedfission yeast cells revealed that the mitochondria in Sch.pombe represent a polymorphic tubular network span-ning the length of the cell rather than defined kidney-shaped organelles (Fig. 1). Similar to the situation inSaccharomyces cerevisiae, fission yeast mitochondriarapidly change size, morphology and intracellularlocalisation, depending on physiological conditions andessentially on their state in the cell cycle. The changes inmorphology are accompanied by fusion and fissionevents (Kanbe et al. 1989).

Curr Genet (2003) 43: 311–326DOI 10.1007/s00294-003-0404-5

Bernd Schafer

Communicated by M. Brunner

B. SchaferDepartment of Biology IV (Microbiology),Aachen Technical University,Worringer Weg, 52056 Aachen, GermanyE-mail: bernd.schaefer@rwth-aachen.de

Unfortunately, the mechanisms that promote mor-phogenesis, distribution and inheritance of mitochon-dria in Sch. pombe are yet poorly understood. Themajority of information on mt dynamics was obtainedfrom the budding yeast Sac. cerevisiae. These data aresummarised in some excellent review articles (Nunnariand Walter 1996; Nunnari et al. 1997; Hermann andShaw 1998; Yaffe 1999). First insights into the molecularmechanism behind these processes in fission yeastresulted from the analysis of a collection of conditional-lethal Sch. pombe mutants with aberrant cell morphol-ogy or a block in the cell division cycle at thenon-permissive temperature (Berger and Yaffe 1996).

Fluorescence microscopy recovered mutant ban5-4with aggregated mitochondria that were asymmetricallydistributed at the non-permissive temperature. Theaffected gene BAN5 is allelic to ATB2, encoding thea2-tubulin subunit. This clearly establishes that move-ment and positioning of mitochondria in Sch. pombe ismediated by microtubules in the same way as inmammalian cells. Consistent with this finding is thatcells with a conditional mutation in NDA3 (encodingb-tubulin) or treatment of wild-type cells with theantimicrotubule agent thiabendazole displayed a similarphenotype (Yaffe et al. 1996). A co-localisation of themitochondria with cytoplasmic microtubules was alsoverified by indirect immunofluorescence microscopy inSch. pombe and in Sch. japonicus var. versatilis (Yaffe etal. 1996; Svoboda and Slaninova 1997). It is worthmentioning that, in both Sac. cerevisiae cells (whichusually lack an extensive cytoplasmic microtubule array)and the filamentous fungus Aspergillus nidulans,mitochondria do co-localise with the actin cytoskeletonbut not with microtubules (Adams and Pringle 1984;Kilmartin and Adams 1984; Oakley and Rinehart 1985).

MSP1 is another essential nuclear gene involved inmorphogenesis and maintenance of fission yeastmitochondria. Pelloquin et al. (1998) found thatoverexpression of MSP1 resulted in a gross alteration ofthe mt structure and function, whereas the conditionalloss of function of the gene lead to growth arrestassociated with respiratory deficiency. In all cases, the

maintenance of mtDNA was also affected. Msp1p is adynamin-related GTPase protein homologous toMgm1p from Sac. cerevisiae. It is anchored to the matrixside of the mt inner membrane (Pelloquin et al. 1998,1999). To illustrate a possible function for msp1, it mightbe helpful to look at Sac. cerevisiae, because the role ofdynamin-related GTPases is much clearer there. Inbudding yeast, mt morphology is regulated by a balancebetween fission and fusion of mitochondria. While mtfission is regulated by Dnm1p, mt fusion requires Fzo1pand Ugo1p at the outer mt membrane. Both proteins areassociated with each other and with Mgm1p. Wonget al. (2003) proposed that Mgm1p could control thebehaviour of the four mt membranes during mt fusionby establishing a connection of the inner membrane tothe outer membrane, promoted by the protein–proteininteractions of Mgm1p with Ugo1p and Fzo1p (Wong etal. 2003). Since the dynamin-related GTPase Mgm1p isclosely related to the Msp1p from fission yeast, it isrational to assume a similar function for the MSP1protein. Similar functions were also described for otherproteins related to fission yeast Msp1p. For example, thebudding yeast dynamin DNM1 and the dynamin-relatedprotein ADL1 from Arabidopsis thaliana are known tobe involved in membrane constriction and vesicleformation. Consequently, Pelloquin et al. (1999)proposed that Msp1p could play an active role in themembrane events that occur throughout fission of amitochondrion, at the stage of generation of cristae orduring formation of the tubular mt network.

A further gene, MDM12, was originally identified byBerger et al. (1997) as an evolutionarily conservedhomologue of a gene from Sac. cerevisiae. Mdm12ptogether with Mdm10p and Mmm1p in Sac. cerevisiaebelong to a class of new outer membrane proteins whichpromote the active transmission of mitochondria todaughter cells prior to the completion of cytokinesis (mtinheritance). Although theirmolecular activity remains tobeuncovered, the location of these proteins in themtoutermembrane indicates that they may interact with cyto-skeletal elements to mediate mt distribution. Interest-ingly, heterologous expression of theSch. pombeMDM12homologue in Sac. cerevisiae conferred a dominant-neg-ative phenotype of giant mitochondria and aberrant mtdistribution, suggesting only partial functional conser-vation ofMdm12p activity between the two yeasts (Bergeret al. 1997). This is an expected finding, because (asalready mentioned) some of the central structures medi-ating movement and positioning of mitochondria aredifferent in budding and fission yeast.

In summary, recent investigations recovered newclasses of proteins involved in the maintenance offunctional mitochondria under different physiologicalconditions, in changes in morphology and intracellularlocalisation and in the inheritance of mitochondria.These proteins build up structures on the mt outer andinner membrane that interact with different cytoskeletalcomponents. Although the mitochondria in eukaryotespresumably have a monophyletic origin, there are

Fig. 1 3-D reconstruction of the tubular mitochondrial (mt)network within a log-phase cell of fission yeast. The morphologyand localisation of the mitochondria was analysed using the geneencoding the green fluorescent protein from Aequorea victoria fusedto a DNA sequence which coded for the mt import leader peptidefrom the Cox4p of Saccharomyces cerevisiae for subsequentconfocal fluorescence imaging

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striking differences in the mechanisms used and there isan unexpected low degree of conservation in theinvolved genes, even between the budding yeast Sac.cerevisiae and the fission yeast Sch. pombe.

Organisation of the mt genome

The overall DNA content within the mt networkamounts to 6% of the total DNA in cells from the logphase and increases up to 14% in stationary-phase cells(Bostock 1969). Gene content and sequence of themtDNA of the fission yeast strain ade7-50h- was deter-mined by extended Southern hybridisation experimentswith homologous gene probes from Sac. cerevisiae andsubsequent sequencing (Lang 1984; Zimmer et al. 1984;Lang et al. 1985, 1987; Trinkl and Wolf 1989). The sizeof this genome is 19,431 bp and it contains genesencoding RNA molecules of the translation apparatus(rnl, rns; 25 tRNA), the RNA component of mt RNaseP (rnpB; Bullerwell et al. 2003) and eight protein-codinggenes (cox1, cox2, cox3, cob, atp6, atp8, atp9, plus rps3encoding the ribosomal protein). In different wildstrains, cox1, cox2 and cob are mosaic genes, whereas allother genes are continuous in all strains (Fig. 2).

Replication of mt DNA

Nothing is known about the initiation of replication infission yeast mitochondria. MtDNA replication is carriedout on presumably one single origin by the DNA poly-

merase c, which is encoded by the Mip1 gene on chro-mosome III. The 116-kDa enzyme consists of1,018 amino acids, including a N-terminal mt targetingsignal and is 48% identical with the Sac. cerevisiaemtDNA polymerase Mip1p. Both DNA polymerasesbelong to a family of DNA polymerases that are stronglyconserved from yeast to man and are only loosely relatedto the family A DNA polymerases (Ropp and Copeland1995). The enzyme has sequence blocks comprising the 3¢–5¢ exonuclease and DNA polymerase-active centres.

Following 2-D pulsed field gel electrophoresis ofentire DNA molecules from fission yeast mitochondria,it was suggested that mtDNA might exist as circularmultimers with an average size of 100 kbp (Han andStachow 1994), which are replicated by a mechanismconsistent with a rolling-circle model. Extended restric-tion analysis of mtDNA supports this model of a circularstructure of the mt genome, since digestion by restrictionenzymes with a single recognition site within the genomerecovered a single linear DNA fragment, as expected forthe linearisation of circular molecules or multimericcircles (Schafer; unpublished data). In contrast, Bendichand co-workers found linear mtDNA molecules thatwere much larger than the genome size and suggestedthat the circles might result from incidental recombina-tion events between direct repeats within or betweentandemly arrayed genome units on linear mtDNA mol-ecules (Bendich 1993, 1996; Jacobs et al. 1996).

The replication of mtDNA in Sch. pombe seems to bestrictly controlled by the nucleus, since mtDNA syn-thesis ceases immediately after blocking nuclear proteinsynthesis (Del Giudice et al. 1981b). Furthermore, mtgrowth and DNA synthesis occur in the absence ofnuclear DNA replication (Sazer and Sherwood 1990).Mt and nuclear DNA replication are discontinuous andoccur at different points in the cell cycle (Del Giudiceand Wolf 1980; Del Giudice et al. 1981a, 1981b).

mt gene expression

Transcription of mtDNA in Sac. cerevisiae is initiatedwithin short conserved motifs: a motif reading 5¢-ATA-TAAGTA-3¢ was found upstream to rRNA andprotein-encoding genes, whereas a motif reading5¢-TTATAAGT-3¢ was identified upstream to tRNAgenes. In Sch. pombe, one consensus motif, 5¢-ATA-TATGTA-3¢, was found in front of rnl which could—inanalogy to Sac. cerevisiae—function as a transcript-initiation site. A second motif (5¢-AGATAAGTA-3¢)further upstream to rnl might serve as another transcrip-tion initiation site. An additional promoter upstreamfrom the cox3 genewas reported, but that is very probablynot an active promoter element. Capping experiments todefine the location of the mt promoters will revealexperimentally whether one of the motifs allows efficienttranscription on both adjacent rRNA genes, whereas theother might serve as initiation site for other transcripts(Schafer et al., manuscript in preparation).

Fig. 2 Gene map of the mtDNA from Schizosaccharomyces pombestrains. The inner circle shows the gene content of mtDNA from theintronless strain. The outer circle shows the mosaic structure of thegenes cox1, cox2 and cob (exons in black bars, group I introns inlight grey bars, group II introns in dark grey bars). rnl Geneencoding large ribosomal RNA, rns gene encoding small ribosomalRNA. tRNA genes are shown as dots in the intergenic regions.cox1, cox2, cox3 genes encoding subunits 1, 2 and 3 of cyto-chrome c oxidase. atp6, atp8, atp9 genes encoding subunits 6,8 and 9 of ATPase. rps3 Ribosomal protein of the small subunit ofthe mt ribosomes, formerly urf a. rnpB Gene encoding the RNAcomponent of mt RNaseP

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Recognition and selective initiation from the Sac.cerevisiae promoter consensus sequence depends on acombined holoenzyme built up by the mt RNApolymerase as a catalytic core and the specificity factorMtf1 (Karlok et al. 2002). A homologue of the buddingyeast MTF1 gene exists in the fission yeast genome,indicating a similar mechanism for initiation of tran-scription in Sch. pombe (Schafer et al., manuscript inpreparation).

Starting from one or more initiation sites, one strandof the complete genome is transcribed, followed bymaturation of the large RNA molecules. Similar to thesituation in mammalian mitochondria, tRNAs flankalmost every gene. The only exception is the intergenicregion between cox1 and cox3 that does not contain anytRNA gene. The processing signals upstream anddownstream to every gene remain to be defined in detail.Interestingly, transcript processing in fission yeast ismuch slower than in Sac. cerevisiae, since significantamounts of precursor RNA are detectable, even in wild-type mitochondria.

The transcript of rnl can be folded into a commonsecondary structure similar to that of Escherichia coli. A5.8S-like structure is present within the 5¢ terminalregion. In contrast to other fungi, no 4.5S-like structurecould be detected at the 3¢ end (Lang et al. 1987). TheRNA of the small ribosomal subunit encoded by the rnsgene exhibits a structure that is very similar to estab-lished secondary structure models (Gray et al. 1984), butlacks a highly conserved secondary structure in the 5¢region that is believed to be essential for the ribosomalfunction in pro- and eukaryotes (Trinkl et al. 1989). The25 tRNA genes encode tRNA molecules with a typicalclover-leaf structure.

The codon usage in the structural genes and in theintronic open reading frames (ORFs) is inconsistent infission yeast mitochondria:

1. Use of UGA. While the universal translation code isused in almost all protein coding genes, UGA codonsare found in the rps3 gene and in the intronic ORFswithin the mosaic genes cox1, cox2 and cob. How-ever, a tRNA reading this deviant codon (likelyUGA=Trp, as in Sac. cerevisiae) bearing the anti-codon UCA was not recovered. Bullerwell andco-workers propose that UGA codons in this systemare recognised by the trnW(cca) itself or after a yetunknown modification of C in the wobble position ofthe trnW(cca) anticodon (Bullerwell et al. 2003).

2. Use of AUA. Following protein sequence alignments,ATA codes for isoleucine in Sch. pombe instead ofmethionine as in most other mitochondria (Bullerwellet al. 2003).

3. Use of CGN. Although U in the wobble position ofthe anticodon in the tRNA encoding arginine makesit possible to read all CGN codons, CGA is exclu-sively used in the structural genes, whereas 41.5% ofthe arginine codons in the ORFs of group I andgroup II introns are CGT or CGC.

4. Use of rare codons. Compared with the homologousintronless alleles, the exon sequences in intron-containing versions of cox1, cox2 and cob showremarkable differences immediately upstream anddownstream to the intron insertion sites. These vari-ations in the intron-containing alleles often resultin rarely used codons, whereas the same sites in theintronless alleles exclusively contain codons that arecommonly used in fission yeast mitochondria. Theseobservations may be explained as being the result of amodern invasion of introns into an ancestral fungalmt genome (Wolf et al. 1987; Schafer et al. 1998;Schafer and Wolf 1999). This aspect is discussed inmore detail later.

Maintenance of mtDNA in fission yeast

Unlike in Sac. cerevisiae, the integrity of mtDNA isobviously important in fission yeast. While incubation ofSac. cerevisiae with ethidium bromide or euflavininduces mutants with grossly altered mtDNA or lackingthe entire mtDNA (vegetative petites abbreviated q) andqo), it was a surprise that for a long time no q) or qo

mutants could be isolated in fission yeast. Thus, fromstudies carried out by De Deken (1961, 1966) and Bulder(1964a, 1964b), yeasts were divided into petite-positiveand petite-negative species. In the original work ofBulder, yeast species from which no respiratory-deficientmutants were obtained after acriflavine treatment weretermed petite-negative yeasts, in contrast to the petite-positive species from which respiratory-deficient petitemutants could be easily obtained (Bulder 1964a, 1964b).Sch. pombe was defined as petite-negative.

Petite-negativity and the metabolic dependenceon functional mitochondria

At least two main factors are known to be involved inmaintaining the susceptibility to petite mutations in thepetite-negative yeast Sch. pombe: (1) the inability toachieve the necessary redox balance, even when growingby fermentative processes and (2) the inability to gen-erate a membrane potential in mitochondria lackingmtDNA.

Consequently, disruption of mt genome maintenancegenes, such as MSP1 (Pelloquin et al. 1998), is lethal infission yeast. Furthermore, screening for Sch. pombe q)

mutants, even in mutator mutants (for explanation, seesection on mt-encoded mutator mutants), recovered nomutants in the genes for the mt translation system(tRNA genes, rnl, rns). This suggests that maintenanceof mtDNA and functional protein expression areessential. Furthermore, we also failed to obtain muta-tions in the genes for the mt-encoded ATPase proteins(atp6, atp8, atp9).

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Likewise, in Kluyveromyces lactis, the susceptibility topetite mutation in fission yeast is obviously not theconsequence of insufficient fermentative capacity in theabsence of a functional electron transport chain, sincemutants disrupted for certain nuclear-encoded compo-nents of the respiratory chain do grow on glucose (Chenand Clark-Walker 1993; Mulder et al. 1994; Hikkel et al.1997). Interestingly, those nuclear genes which contrib-ute to the assembly of the ATPase complex or ADP/ATP translocators are found to play an exceptional rolein maintaining susceptibility to petite mutation in bothpetite-positive and petite-negative yeasts.

One reason for the strict requirement for functionalmtDNA in petite-negative organisms could be the needfor an active respiratory chain to dispose of somereducing equivalents released from the oxidoreductionreactions of basal metabolism. Similar to the situation infission yeast, qo derivatives in chicken embryo fibro-blasts (Desjardins et al. 1985), avian cells (Desjardinset al. 1986; Morais et al. 1988) and human cells (Kingand Attardi 1989) were isolated after long-term exposureto low concentrations of ethidium bromide. Interest-ingly, the qo cells from higher eukaryotes becamepyrimidine auxotrophs. Further experiments revealed atopochemical link of uridine synthesis to the functionalrespiration chain. While most of the enzymes of thepyrimidine de novo synthesis pathway are located in thecytoplasm, the enzyme dihydro-orotate dehydrogenasecatalysing the conversion of dihydro-orotate into oro-tate is located in the inner mt membrane. The enzymetransfers the reducing equivalents at the ubiquinone ofthe respiratory chain as the electron acceptor (Loffler etal. 1997). The qo mammalian cells have also becomepyruvate-dependent, whereby pyruvate presumablyserves as an alternative electron acceptor.

The dihydro-orotate dehydrogenase enzyme of fissionyeast is also located in the inner mt membrane; and theintegrity of the respiratory chain is also required for theenzyme to be active (Nagy et al. 1992). In addition,potassium acetate is required at least at an intermediatestage during qo induction (see below, in this section).

The homologous enzyme from the facultative anaer-obic and petite-positive yeast Sac. cerevisiae has acytoplasmic localisation and uses fumarate as a terminalelectron acceptor (Nagy et al. 1992). These results sug-gest that, in fission yeast (and in mammalian cells), theubiquinone pool is used which is accessible in an activerespiratory chain whereas, in the petite-positive yeastSac. cerevisiae, the fumarate serves as an electronacceptor to achieve the necessary redox balance, evenwhen growing by fermentative processes. Consistently,functional complementation in Sac. cerevisiae with the(mt-localised) fission yeast homologue is abolished eitherunder anaerobic growth conditions or in a cytochrome bmutant (Nagy et al. 1992).

The qo mutants in fission yeast are obtained by long-term incubation of cells in liquid medium containing lowconcentrations of ethidium bromide and potassiumacetate (Haffter and Fox 1992). Unlike the pyruvate-

dependency in the qo cells of higher eukaryotes, the qo

Sch. pombe strains do not require potassium acetate,uridine or any other growth factors once the qo state hadbeen established. This difference to higher eukaryotescould be explained by the activity of an alternativerespiratory pathway in fission yeast mitochondria thatmight be stimulated under these conditions and couldrender supplements superfluous (Labaille et al. 1977). Itshould be noted that there is no experimental proof forthe existence of a gene encoding a bona fide alternativeoxidase. However, appreciable antimycin A- andNaCN-insensitive oxidation is observed in glucose-grown cells; and this activity could be stimulated underconditions in which the Antimycin A-sensitive respira-tion has been abolished by mutation, by aerobic growthin the presence of chloramphenicol, or by the absence ofmolecular oxygen (Heslot et al. 1970; Goffeau et al.1973).

The induction of qo derivatives depends on mutationsin two unlinked nuclear genes, termed ptp1-1 and ptp2-1(ptp for petite-positive). These mutations occurred insome, but not all, of the initial cultures and allowedreproducible ethidium bromide induction of viable qo

strains (Haffter and Fox 1992). The ptp1-1 mutation hasalso been shown to be required for the viability of cellsdefective in the RNaseMRP RNA gene that participatesin primer RNA metabolism for mtDNA synthesis(Paluh and Clayton 1996).

But petite-negativity is not a one-way path. Besidesthe conversion of petite-negative into petite-positivespecies by gain-of-function mutations in nuclear genes,such as the ptp1-1 and ptp2-1mutations in Sch. pombe, orthe atp mutations (formerly mgi) in K. lactis, the sameeffect could be observed when genes were transferredfrom petite-positive yeasts into petite-negative species.An interesting example for this is YME1 from Sac.cerevisiae, encoding a protein associated with the innermt membrane (Kominsky and Thorsness 2000). Theheterologous expression of YME1 in Sch. pombe con-verts this yeast into a petite-positive yeast, while inacti-vation of Yme1p in Sac. cerevisiae converts this yeast to apetite-negative state. This is a remarkable finding, be-cause a homologue of YME1p was discovered recently inthe Sch. pombe genome and was annotated as ORFSpC965.04c on chromosome III in the genome sequenc-ing program. Recent investigations in Sac. cerevisiaesuggest that the ATP- and zinc-dependent AAA prote-ase, Yme1p, is in part responsible for assuring sufficientATPase activity to generate a membrane potential inmitochondria lacking mtDNA (Kominsky et al. 2002).Interestingly, the mutations suppressing qo-lethality inK. lactis map at the contact sites between subunits ofF1-ATPase. Moreover, ATP hydrolysing activity of theATPase is essential for the suppression of qo-lethality(Chen and Clark-Walker 1995, 1999a; Chen et al. 1998;Clark-Walker et al. 2000).

Also, the op1mutation in the AAC2 gene encoding anADP/ATP translocator in budding yeast converts Sac.cerevisiae into a petite-negative state (Kovacova et al.

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1968; Kovac et al. 1972). It turns out that the inhibitoryeffect on the nucleotide translocation across the mtmembrane is responsible for the op1 phenotype and it isproposed that a AAC2-mediated ADP/ATP transloca-tion plays a role in the maintenance of the membranepotential Dw across the mt inner membrane (Chen andClark-Walker 1999b). The Dw is essential for mt bio-genesis and is usually generated by coupled H+ andelectron transport in respiring q+ cells or by reversibleH+ translocation through the F1F0-ATPase, at the ex-pense of ATP hydrolysis under anaerobic conditions.Since both pathways are absent in q)- and qo cells, theATP/ADP exchange through the ADP/ATP translocatoraccompanied by F1-ATPase-catalysed ATP hydrolysis isbelieved to play a significant role in generation of Dw(Giraud and Velours 1997). Additional factors involvedin the conversion of Sac. cerevisiae into a petite-negativestate are reviewed by Chen and Clark-Walker (1999b).

Nuclear genes involved in repair and integrityof mtDNA

Although it is common knowledge that Sch. pombe andmost other eukaryotic cells require a functional, intactmt chromosome for viability, little is known about themaintenance and repair of mtDNA. Repair mechanisms,recombination between mtDNA molecules and mitoticsegregation of mtDNA contribute to the maintenance ofintact mtDNA in the mitochondria of eukaryotes.

Concluding from findings made with Mip1p fromSac. cerevisiae, a remarkable protection mechanismagainst mutations in mtDNA is certainly given by the3¢–5¢ exonuclease function included in the mtDNApolymerase of fission yeast (Hu et al. 1995; Ropp andCopeland 1995).

For the repair of UV-damaged DNA, Sch. pombepossesses an endonuclease-dependent excision repairpathway (UVER; UV excision repair) in addition to thenucleotide-excision repair pathway (Yasuhira and Yasui2000).While the nucleotide-excision repair system repairsdamage only in the nuclear genome, UVER efficientlyremoves cyclobutane dimers in both nuclear and mt ge-nomes. The ectopically expressed wild-type UV-damagedDNA endonuclease was localised to both nucleus andmitochondria, while modification of N-terminal methio-nine codons restricted its localisation to oneorother of theorganelles, suggesting an alternative usage of multipletranslation initiation sites for targeting the protein todifferent organelles. Yasuhira and Yasui (2000) suggest apossible UVER function in mitochondria as a back-upsystem for other UV damage-tolerance mechanisms.

Another interesting activity involved in mtDNAmaintenance is encoded by the gene for the cruciformcutting endonuclease, SpCCE1. This Holliday junction-resolvase in Sch. pombe was exclusively found insidemitochondria (White and Lilley 1997, 1998; Doe et al.2000). Similar to its homologue CCE1 in Sac. cerevi-siae, SpCCE1 resolves recombination junctions that are

formed during homologous recombination as thetypical four-way DNA junction (Holliday junction)between strands of homologous DNA molecules.SpCCE1 can complement the DNA-repair deficiency ofa ruvC rusA mutant of E. coli. However, anSpcce1::ura4+ mutant strain exhibits normal levels ofUV sensitivity and spontaneous or UV-induced mitoticrecombination. The Holliday junction-resolvase activityis necessary not only to resolve Holliday junctionsarising from recombination repair of mt genomes butalso to facilitate mtDNA segregation. The effect of theSpCCE1 protein on mtDNA segregation was explainedby the observation that the majority of the mtDNA ina mutant with a Spcce1::ura4+ allele is aggregated, dueto the interlinking of DNA molecules by recombinationjunctions (Doe et al. 2000). Consequently, the absenceof the SpCCE1 protein in an Spcce1::ura4+ mutantresults in fewer mtDNA nucleoids as independentlysegregating genetic units than in wild-type cells. InCCE1 mutants of Sac. cerevisiae, the absence of Cce1por Mgt1p (encoding another recombination junction-resolving enzyme) leads to a larger proportion ofmtDNA molecules that are linked by recombinationjunctions, resulting in the aggregation of mtDNA.Lockshon et al. (1995) proposed this change in mtDNAstructure could account for an increase in the mitoticloss of mtDNA and an altered pattern of mtDNAsegregation from zygotes, which finally leads to anincrease in the frequency of petite colonies (Kleff et al.1992; Lockshon et al. 1995).

A further nuclear-encoded endonuclease, SpNUC1,degrading single-stranded DNA and RNA, was isolatedfrom fission yeast mitochondria (Ikeda et al. 1996; Ikedaand Kawasaki 2001). Its role in the mt metabolism ofDNA and RNA is yet unknown.

Two further homologous proteins, Pmf1p (the pombemt factor 1) and Hpm1p (the homologous Pmf1p fac-tor 1), are involved in the maintenance of mtDNA inSch. pombe by a yet unknown mechanism. Pmf1p islocalised in mitochondria and cytoplasm. Both proteinsare members of a family of small multifunctional pro-teins, called the p14.5 family. Although there is not yet aclear biological function for members of this family,p14.5 proteins have been identified in bacteria and ineukaryotic cells, indicating a functional conservation(Marchini et al. 2002).

Disruption of Mmf1p (mt matrix factor 1), the Sac.cerevisiae homologue of the fission yeast proteins, resultsin a decrease in mtDNA stability at 30 �C in glucosemedium or at 37 �C in glycerol medium (Oxelmark et al.2000). Heterologous expression of the fission yeastPmf1p restores the wild-type phenotype when expressedin the Dmmf1 Sac. cerevisiae strain.

Mt-encoded mutator mutants

Another factor that is responsible for the integrity of themt genome in fission yeast was found to be of mt origin.

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Indications for the mt localisation of the gene came firstfrom genetic analysis of several mutator mutants infission yeast. The mutator strains are characterised by anincrease in the occurrence of respiratory-deficientmutants carrying point mutations and deletions anddrug-resistant mutants (Seitz-Mayr and Wolf 1982).Experiments in which mitochondria isolated from awild-type strain were transferred into a mutator strainrevealed that the mutator activity is really mt-encoded(Schafer, unpublished data). Further molecular analysisverified that the mutator activity is correlated with cer-tain mutations in the mt rps3 gene (Zimmer et al. 1991).

The N-terminal part of the protein exhibits homologywith the var genes of Sac. cerevisiae and Torulopsisglabrata and with S3 proteins of the small ribosomalsubunit (Neu et al. 1998; Bullerwell et al. 2000).Sequencing of alleles from other fission yeast wild strainsrevealed that base pair changes in the 5¢ region of thegene encoding this var1 domain of the protein have nophenotype. In contrast, the 3¢ part contains all themutational sites which confer the mutator property. Itcontains a motif that is very similar to the DNA-bindingdomain of the phage Mu transposase. Therefore, thisdomain was called the mutator domain (Neu et al.1998). A homologue of this gene was recently recoveredin the mtDNA of Sch. octosporus (Bullerwell et al. 2003).

In order to elucidate the function of the rps3 gene, Neuet al. (1998) made use of the mutant anar-6, which hadoriginally been isolated as a mt Antimycin A-resistantmutant. In this mutant, a deletion of an A residue leads toa frameshift and creates a new stop codon further down-stream. This, however, does not lead to complete respi-ration deficiency, but instead a culture of anar-6continuously segregates into subclones that differ in theirability to grow on glycerol (Zimmer et al. 1991). Afterreplica-plating, a proportion of the colonies show wild-type growth on glycerol, whereas others display reducedgrowth. Up to 8% of the colonies fail to grow on glycerol.The appearance of colonies with graded respiratoryimpairment may be a consequence of translationalframeshifting by mt ribosomes at a run of A residues.Frameshifting inmt genomes has been reported from Sac.cerevisiae (Fox and Weiss-Brummer 1980).

Two versions of the rps3 gene were used for ectopicexpression of the mt gene in the nucleus of mutant anar-6: the native mt rps3 gene with a UGA (trp) codon atposition 175 in the ORF and a codon-adapted version inwhich the UGA (trp) codon was replaced by UGG (trp).Both genes were then fused to a DNA sequence whichcoded for the mt import leader peptide from subunit 4of the cytochrome c oxidase in Sac. cerevisiae. Trans-formation with the mt code version cloned into theexpression vector pART1 resulted in a reduction of theportion of completely respiratory-deficient clones to lessthan 1% (compared with up to 10% in the controls).The weaker complementation is presumably due to theabsence of amino acid residues 176–227 in the gene withthe UGA (trp) codon at position 175. Furthermore, nocolonies with intermediate growth on glycerol appeared.

In experiments with the standard genetic code versionencoding the entire rps3 protein, only 0.07% of com-pletely respiratory-deficient mutants were found. Thisdemonstrates that the phenotype of rps3 mutant anar-6can be complemented by ectopic versions of the wild-type mt gene and that the truncated version (with UGAat codon position 175) appears to be able to partiallyrestore the wild-type phenotype, while the entire protein(including the 52 amino acid residues of the C-terminus)expressed by the standard genetic code version fullycomplements the mutation in anar-6.

It can be concluded that rps3 encodes a bifunctionalprotein which acts both in mt protein synthesis and inmaintaining the integrity of the mt genome. It has beenfound that many ribosomal proteins have a secondfunction in addition to their role in translation (Wool1996).

Variability of the genome in fission yeast mitochondria

The strong tendency to maintain intact mtDNA con-trasts with the great variability of the mt genomesamongst naturally occurring Sch. pombe strains. ThemtDNA in wild strains varies from 17.4 kb in Sch.pombe strain EF1 (Zimmer et al. 1984) to up to morethan 25.0 kb in strain EF2 (Schafer and Wolf 1998,1999; Schafer et al. 1998). The smallest mt genome froma respiratory-competent strain is that from strains P3and P4. Its artificial but fully functional mt genome of14.6 kb was constructed by successive deletion of introns(Schafer et al. 1991) and represents the smallest mtgenome known so far.

Unlike in Sac. cerevisiae mtDNA, GC clusters orAT-rich spacers are absent and non-coding intergenicsequences are very short in Sch. pombe mtDNA. Thus,most of the size variation is due to the presence orabsence of introns (Zimmer et al. 1987).

The contribution of mobile elementson mt genome evolution

Even in the small mt genome of the fission yeast Sch.pombe, a screen for mt introns revealed that up to 42%of the entire mt sequence is occupied by interveningelements. They belong to two classes of introns, group Iand group II introns, which can be distinguished mainlyby the characteristics of their RNA secondary structure.

Slonimski and co-workers assumed that both intronsand their encoded proteins could contribute to the highlevel of mt recombination and could stimulate mtgenome evolution (Kotylak et al. 1985). It should bementioned that crosses between fission yeast strainsyield about 20–30% recombination between two mtmarkers, a phenomenon that could not be detected inmammalian mitochondria or other naturally occurringintronless mt genomes in higher eukaryotes. If intronsare involved in mt recombination, we expected that the

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above-mentioned strain devoid of introns should mimicthe situation in the intronless mt genomes of highereukaryotes. However, we learned from two-factorcrosses using alleles conferring resistance to erythro-mycin or diuron that mt recombination is not abolishedin the absence of introns (Schafer et al. 1991). Fur-thermore, the loss of introns has no other phenotype inthese strains. Similar observations were made with aSac. cerevisiae strain devoid of introns, although in thiscase the strain contained some free-standing ORFs re-lated to group I-encoded proteins (Boulet et al. 1990).

Group I intron homing

Group I introns are widespread in many evolutionaryphyla because they are very efficient in invading targetDNAs either at homologous sites within an intronlessallele of the same gene (homing) or by ectopic insertion.Usually, the process is accompanied by extended co-conversion of sequences upstream and downstream tothe insertion site (for details, see Fig. 3).

Four of the mt fission yeast introns are members ofthis intron class and all of them are restricted to the geneencoding cox1 (Fig. 1). Two of them are extremelyshort, with 256 bp and 421 bp, respectively (Trinkl andWolf 1986). The other two group I introns (cox1I1b,cox1I2b; of 1,081 bp and 1,210 bp, respectively) areendowed with ORFs which use the ATG start codon ofthe upstream exon (Schafer et al. 1994). Both introns aremobile and have been detected in all Sch. pombe wild

strains screened so far. Interestingly, a homologue of thefission yeast intron cox1I1b is also present in Sch.octosporus (cox1I1) and homologues of cox1I2b arefound in Sch. octosporus (cox1I3) and Sch. japonicus var.japonicus (cox1I2; Bullerwell et al. 2003).

Both intron-encoded proteins are active endonuc-leases as members of the LAGLIDADG family with twoconsensus motifs. The capability of homing of a fissionyeast group I intron was first shown by a geneticapproach with a strain containing a mt genomeharbouring exclusively the cox1I1b intron as the donorand a strain with an intronless mt genome as therecipient. The two strains were marked with differentnuclear auxotrophic mutations (ura4, ade7) and differentmt drug-resistance markers. In the resulting zygote, themitochondria fused and the mt genomes recombinedand segregated, yielding ascospores which could be mthomo- or heteroplasmic. By replica-plating colonies ondrug-containing media, transmission of the mt allelesconferring resistance or sensitivity to a drug could bemeasured. Transmission of the intron could be measuredby restriction enzyme analysis of the mtDNA of theprogeny and hybridisation with intron probes. In con-trast to the biparental transmission of drug-resistancemarkers, the transmission values for the cox1I1b werebetween 85% and 90%. Similar experiments with thecox1I2b also recovered the uniparental inheritance ofthis intron. This shows that both group I introns withORFs very frequently undergo homing into theircognate sites in the intronless allele of the cox1 gene(Paschke et al. 1994; Schafer et al. 1994).

Fig. 3 Homing pathway formobile group I introns. Part 1Initially, the intron ORF isexpressed to obtain the homingendonuclease. Parts 2, 3 Thehoming endonuclease initiatesgroup I intron mobility by theintroduction of a double-strandbreak (DSB) at the target DNAsite. The LAGLIDADGenzymes of fission yeast usuallyrecognise a sequence at theintron-insertion site within theintronless cox1 allele and createa typical 3¢ overhang,4 nucleotides in length. Host-encoded exonuclease activitythen promotes the widening ofthe DSB. Part 4 Homologoussequence alignment and 3¢ endinvasion of the donor alleleoccur, whereby the donor alleleserves as a template for repairsynthesis. Part 5 A variety ofrecombination-dependent andindependent pathways are thenused to repair the DSB andresolve the homologous alleles,resulting in duplication of theelement

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The biochemical activities of the intron-encodedproteins were initially demonstrated with native proteinsfrom mt extracts (Schafer et al. 1994). Further analysisof the enzymatic properties involved in recognising theintron insertion sites within the recipient alleles wasperformed with artificially expressed versions of theproteins purified from E. coli extracts (Pellenz et al.2002). The enzyme I-SpomI encoded by the introncox1I1b is a site-specific endonuclease which recognises

a 20 bp sequence symmetrical around the intron inser-tion site within the intronless cox1 allele. This recogni-tion sequence has a pseudopalindromic symmetry. Thecleavage pattern of I-SpomI shows the typical 3¢ over-hang of four nucleotides, as already described for manyother endonucleases of the LAGLIDADG family(Pellenz et al. 2002). Interestingly, the protein stimulatedintra-chromosomal recombination in an E. coli-basedtest system (Manna et al. 1991). Unlike I-SpomI, theprotein encoded by the intron cox1I2b (I-SpomII)revealed no symmetry within its recognition site (Schaferet al., manuscript in preparation).

To find out which of the amino acid residues would beessential for endonuclease activity, the native form andtruncated versions of the I-SpomI expressed in E. coliwere compared. Although the native I-SpomI homingendonuclease was encoded as a larger presumptive pro-tein of 495 amino acids, a truncated protein consisting ofexclusively the codons located in loop eight of the intronRNA secondary structure was sufficient to gain homingendonuclease activity. Neither cutting-capability norsequence-specificity was affected in the truncated protein.This clearly indicates that the core of the homing endo-nuclease is encoded by codons in loop eight of the intronribozyme. This interesting finding can be explained bythe fact that the mobile intron is built up by a self-splicingcompetent group I intron and a DNA endonuclease ORFthat is integrated into loop eight as a peripheral loop notnecessary for autocatalytic activity of the ribozyme.Although evolutionary changes lead to the extension ofthe endonuclease ORF into the upstream exon, the

Fig. 4 Insertion sites for LAGLIDADG endonuclease ORFs ingroup I introns. The ORFs within group I introns are almostexclusively found in regions that represent peripheral loops in theintron RNA structure. This can be explained by the fact thatmobile introns are built up by a self-splicing competent group Iintron and a DNA endonuclease ORF that integrated into aperipheral loop not necessary for autocatalytic activity of theribozyme. In many cases, subsequent evolutionary changes mayhave led to the extension of the endonuclease ORF into theupstream exon. The following group I introns encoding LAGLI-DADG have been taken into consideration: Ce Chlamydomonaseugametos (LSU.5: Z17234, LSU.6: S15139), Cr C. reinhardtii(LSU.1: X01977), Cs C. smithii (cob.1: CSAPOCYTB), EnEmericella nidulans (cob.1: MBI1_EMENI, cox1.2: C22735,cox1.3: D22735), Kt Kluyveromyces thermotolerans (LSU.1: MI-KTRRNA), Nc Neurospora crassa (cob.1: K01181, cob.2: K01181,atp6.2: MINC03, nad1.1: S06367, nad5.1: S10841, nad5.2: S10842),Pa Podospora anserina (cox1.2: C48327, cox1.3: D48327, cox1.5:F48327, cox1.7a: H48327, cox1.7b: I48327, cox1.8: A38888, cox1.9:B38888, cox1.10: C38888, cox1.11: D38888, cox1.12: E38888,cox1.13: F38888, cox1.15: H38888, cob.1a: B48326, cob.3a:E48326, nad1.4a: S06059; nad3.1: YMN3_PODAN, nad5.1:S10841, nad5.2: S09143, nad5.3: S09144), Sc Sac. cerevisiae(cox1.3: YMX3_YEAST, cox1.4: QXBY34, cox1.5a: S27138,cob.4: YMC4_YEAST, LSU1: TRAM_YEAST), Sp Sch. pombe(cox1.1b: YMC1_SCHPO; cox1.2b: YMC2_SCHPO)

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ancient ORF of the endonuclease enzyme is still present(Pellenz et al. 2002). Known LAGLIDADG ORFs fromother sources are also inserted in loop structures that arenot required for autocatalytic activity of the group Iintron ribozyme (Fig. 4).

Analysis of several mutants with aberrant intronsequence revealed that the LAGLIDADG proteins ofcox1I1b and cox1I2b are not only homing endonuc-leases. Evolutionary changes led to bifunctional proteinsthat are also involved in splicing the intron RNA(maturase function). I-SpomI and I-SpomII of Sch.pombe, together with the recently purified I-ScaI fromSac. capensis (Monteilhet et al. 2000) and I-AniI encodedby a mt group I intron in Asp. nidulans (Ho et al. 1997),are the first reported proteins that have both latentendonuclease and maturase activity. Since none of thegroup I introns in Sch. pombe is capable of splicingautocatalytically in vitro (Schafer et al. 1991), theremight have been a driving force to adapt endonucleaseproteins to function in RNA-splicing during evolution.While most of the LAGLIDADG proteins are eitherhoming endonucleases or maturases, one might specu-late that enzymes bearing both endonuclease andmaturase properties can be regarded as an intermediatestage during the evolution of mobile group I introns.The proteins of ancestral group I introns with LAGLI-DADG ORFs are endonucleases, whereas the proteinsof more highly developed group I introns have maturaseactivity. A well known example is the protein I-ScaIencoded by the intron bI2 of the cob gene in Sac. cere-

visiae, where the latent maturase can be converted intoan endonuclease just by exchanges of two non-adjacentamino acids (Szczepanek and Lazowska 1996; Szcze-panek et al. 2000).

Retrohoming and retrotranspositionof group II introns

Group II introns were initially found in genomes of theorganelles of lower eukaryotes and plants and wererecently discovered in bacteria (Ferat and Michel 1993).These introns interrupt genes encoding proteins, tRNAsand ribosomal RNAs. Although many introns needaccessory proteins, group II intron RNAs self-splice viaa lariat mechanism similar to the splicing of nuclearprecursor mRNAs. In fact, self-splicing group II intronsare thought to be the evolutionary progenitors ofeukaryotic spliceosomal introns. The result of thesplicing reaction is a lariat RNA, comprising a circularcomponent and a short 3¢ tail. The RNA of group IIintrons shares a common secondary structure consistingof six domains (DI–DVI) radiating from a central corestructure, which is essential for the catalytic activity(Michel and Ferat 1995). According to further featuresof the intron secondary structure, group II introns aresubdivided into subgroup A (with subgroups A1, A2)and subgroup B (Michel et al. 1989).

Group II introns exist in ORF-less and ORF-con-taining forms. ORF-containing introns encode proteins

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that promote mobility of the genetic element. Themobility process leads either to an insertion into cognatealleles that lack the intron caused by a process calledhoming or by insertion into ectopic sites, named retro-transposition (Lambowitz and Belfort 1993; Belfort andPerlman 1995; Michel and Ferat 1995). Details of thegroup II intron retrohoming process are given in Fig. 5.

A systematic search within mt genomes of 45 Sch.pombe isolates revealed altogether three introns thatbelong to subgroup A1 of the group II introns. The2,526-bp group II intron inserted in the cob gene (cobI1)was originally recovered in the mt genome of strainade7–50 h). While most introns tend to occur in highlyconserved regions, cobI1 is inserted within a less con-served region 684 bp downstream to the 5¢ end of thecob gene. A similar intron has been found in the cob geneof Sch. octosporus (Bullerwell et al. 2003). The consensussequence at the 5¢ exon/intron border of the cobI1 isUUGCGC instead of the GUGYG motif found in othergroup II introns (Lang et al. 1985).

The intron is able to undergo retrohoming and totranspose into ectopic sites (Paschke et al. 1994; Schmidt

et al. 1994). The ability to be mobile was first shown incrosses between a donor strain that has cobI1 as the onlygroup II intron in its mtDNA and an intronless recipientstrain which has a functional mtDNA but lacks all mtintrons. The overall frequency of homing by the wildtype Sch. pombe intron is 70–84%, similar to the 80–90% homing obtained with the budding yeast intronsaI1 or aI2 (Paschke et al. 1994; Eskes et al. 1997, 2000).While homing is initiated by reverse-splicing of cobI1RNA into the DNA target site, illegitimate integrationinto a number of short sequences resembling the originaltarget site is also observed, resulting in ectopic integra-tion of the intron (Schmidt et al. 1994).

The long ORF within the intron encodes a proteinwith amino acid segments related to the reverse trans-criptase (RT), maturase and endonuclease domains ofother group II introns and appears to be translated fromthe cob pre-mRNA starting from the AUG start codonof cob exon 1 and terminating at the UAG stop codonnear the end of the intron. To learn whether the proteinencoded by the Sch. pombe cobI1 has activities that mayrelate to the capability to undergo retrohoming, theprotein was extracted from Sch. pombe mitochondriaand biochemically characterised.

The multidomain protein is active in a ribonucleo-protein complex that contains the excised intron RNAand the intron-encoded protein (Schafer et al. 2003) TheRNP particles have RT activity, even without adding anexogenous primer. Characterisation of the cDNAproducts of such reactions showed a strong preferencefor excised intron RNA as template. Two main regionsfor the initiation of cDNA synthesis were mapped withinthe intron, one near the DIVa putative high-affinitybinding site for the intron-encoded protein and the othernear domain VI. Adding exogenous primers comple-mentary to cob exon 2 sequences near the intron/exonboundary stimulated RT activity, but mainly for pre-mRNA rather than mRNA templates. Further in vitroexperiments demonstrated that cobI1 RNA in RNPparticle fractions can reverse-splice into double-strandedDNA substrates containing the intron homing site.Interestingly, the RNP particles appear to lack anendonuclease activity, suggesting that the very activehoming mechanism used by this intron may differ fromthat of the better characterised introns (Schafer et al.2003; see Fig. 5).

Two additional introns were found in the cox2 gene(cox2I1) and the cox1 gene (cox1I1a). It is worthmentioning that the latter two group IIA1 introns aremuch more abundant among fission yeast strains thanthe mobile cob intron. Although cobI1, cox1I1a andcox2I1 are all representatives of the IIA1 subgroup,they are no more related to each other than to othergroup IIA1 introns (Schafer et al. 1998, 2003;Zimmerly et al. 2001). The 2,492-bp intron cox1I1a isinserted at the same position within the cox1 ORF asthe intron aI2 in the cox1 gene of Sc. cerevisiae. UnlikeaI2, the cox1I1a is inserted between the second andthe third nucleotide, whereas the intron aI2 is located

Fig. 5 Alternative pathways for the retrohoming of group II introns(adapted according to Eskes et al. 2000). Both the intronlessrecipient alleles (shown as white bars) and the intron donor alleles(shown as black bars with the intron shown as thick grey bars)participate in the homing process (strand polarity is indicated byarrows). Data from two mt group II introns in budding yeast (aI1,aI2), the lactococcal group II intron LtrB and the cobI1 in fissionyeast show that group II intron mobility can be achieved by co-existing but distinguishable pathways that are variations of a majorroute. It is common for all pathways that the intron-encodedprotein first binds to the unspliced precursor RNA, promotessplicing of the intron RNA and then remains bound to the excisedintron RNA lariat, forming a RNP particle active in retrohoming.The DNA target site is then cleaved by partial or complete reversesplicing of the intron RNA into the sense strand and by the intron-encoded endonuclease in the antisense strand. Path 1 In a reactionknown as target DNA-primed reverse transcription (TPRT), thecleaved site at the antisense strand 9–10 nucleotides downstreamfrom the intron insertion site in the 3¢ exon of the DNA target sitelacking the intron serves as the priming site for synthesis of first-strand cDNA. Since the template here is fully reverse-spliced intronRNA, this process leads to full-length cDNA. The subsequentrepair process does not appear to involve recombination andresults in the insertion of the intron into the recipient DNA with noco-conversion of flanking exon sequences. Paths 2, 3 In thesepathways, partial cDNAs synthesised from fully or partiallyreverse-spliced intron RNA complete their retrohoming by usingthe mt double-strand break repair (DSBR) recombination system.The DSBR recombination system strand is initiated by invasion ofthe donor DNA by the cDNA and completion of the intron DNAsynthesis using the donor DNA as a template, with copyingbeginning in the intron and extending into the upstream exon,followed by strand exchange back to the recipient DNA. Theintron insertion is completed by removal of the intron RNA andsynthesis of the opposite DNA strand. These events result ininsertion of the intron with unidirectional co-conversion ofupstream exon sequences. Path 4 Intron insertion can also beachieved by a RT-independent pathway. Here, a cleaved DNAtarget site containing partially reverse-spliced intron RNA leads tohoming via gapping and strand invasion of the donor DNA. Thepathway is characterised by co-conversion of both upstream anddownstream exon sequences, as described for group I intronhoming

b

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between the first and the second position in the samecodon. The DNA sequence upstream of the insertionsite shows a high similarity between Sch. pombe andSac. cerevisiae. The 2,436-bp intron in the cox2 gene isunusual, since it was the first group II intron found in acox2 gene except for higher plants (Schafer and Wolf1998; Schafer et al. 1998). Bullerwell et al. (2003) re-cently recovered a related group II intron in the cox2gene of Sch. octosporus.

Horizontal intron transfer

Phylogenic evidence indicates that mobile group I andgroup II introns are capable of inserting into ectopicsites within genomes of different strains of the samespecies (transposition) and to undergo horizontaltransmission between unrelated organisms (Dujon 1989;Lambowitz and Belfort 1993).

Some observations make it likely that the Sch. pombegroup I intron cox1I2b could have been acquired byhorizontal transmission, because the intron is inserted atthe same position as the intron 3 within the cox1 gene ofAsp. nidulans and the amino acid sequences of bothintron proteins are very similar. The high degree ofhomology is just restricted to the introns, whereas theconservation rate of the cox1 exons betweenthese organisms is significantly lower (Trinkl and Wolf1986).

Also, a detailed look at the group II introns in Sch.pombe indicates that they may be of mixed origin afterhorizontal inheritance between unrelated organismsduring evolution. Consequently, the protein encoded inthe ORFs of cox2I1 is closely related to a liverwortgroup II intron protein, whereas the cobI1 and cox1I1aproteins are more related to fungal group IIA1 intronmaturases (Schafer and Wolf 1999; Zimmerly et al.2001). Interestingly, the sequences in the upstream exonand to a lesser extent in the downstream exon differsomewhat in all intron-containing alleles, whereas theintronless alleles are identical. The alterations in intron-containing alleles often result in changes from morefrequently used to rare codons (Trinkl et al. 1985;Schafer et al. 1998; Schafer and Wolf 1999) and might bethe result of late acquisition of the introns.

Intron DNA excision

Following on from the addition of introns either byhoming or transposition, the precise excision of bothgroup I and group II introns at the DNA level (referredto as intron DNA-splicing) was discovered. In fact, weeliminated the two group I introns and the cobI1 fromthe mtDNA of a wild strain by initiating subsequentintron DNA-excision events (Schafer et al. 1991). Preciseloss of introns was also reported from Sac. cerevisiae(Gargouri et al. 1983) and from Podospora anserina(Kuck et al. 1985).

Intron-dependent genomic instability

Group II introns are also known to be involved in mtgenomic instability and the senescence of mycelia inP. anserina. In fission yeast, group II introns are apotential source for the formation of extended deletionswithin the mtDNA. Ahne et al. (1988) reported variousmutants in the cob gene with a large deletion extendingfrom the 3¢ splice junction of the cob intron into theuntranslated region (UTR) 62 bp downstream to thetermination codon. Interestingly, the 3¢ end of the dele-tion in the 3¢UTR exhibits a striking homology with theintron-binding site motifs (IBS 1/2). These short motifs,usually located adjacent to the 5¢ exon/intron border, areessential for correct splicing and for intron homing.Accordingly, Schmidt et al. (1994) were able to show bypolymerase chain reaction techniques that duplicativetransposition, twintron formation and insertions ofcobI1 occur in the motif at the 3¢UTR. This supports thehypothesis that the deletion in these mutants could haveresulted from a transient RNA-mediated insertion of theintron into the ectopic site, resembling the native splicesite, followed by recombination between the introncopies (Schmidt et al. 1994).

Factors involved in size variation amongst membersof the Schizosaccharomycetales

Although the gene content is quite similar to that ob-served in Sch. pombe, the genomes in mitochondria ofSch. octosporus (44,227 bp) and Sch. japonicus var.japonicus (>80,000 bp), as two other representatives ofthe genus Schizosaccharomyces, are larger (Bullerwellet al. 2003). One reason is the extent of highly A+T-richintergenic regions (11.1% in Sch. pombe; 49.4% in Sch.octosporus; 76.5% in Sch. japonicus var. japonicus),which are well known targets for genomic rearrange-ments.

However, Bullerwell et al. (2003) found five double-hairpin elements in the mtDNA of Sch. octosporus thatare absent in the mtDNAs of Sch. pombe and Sch.japonicus var. japonicus, suggesting a late acquisition inSch. octosporus.

Future prospects

Mitochondria are rather fascinating organelles that areessential in almost all eukaryotes known so far. Genome-wide systematic approaches have accelerated the discov-ery of novel mt proteins; and the observations made inmodel systems might even make it possible to gain newinsights into the molecular mechanisms of various mthereditary disorders in human. In particular, the proteinsinvolved in mt morphogenesis and movement have dras-tically changed our view on mitochondria.

This review summarises the current knowledge abouthow functional mitochondria can be maintained under

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different physiological conditions, how the cells canmanage changes in mt morphology and intracellularlocalisation and how mitochondria are inherited duringmitosis and meiosis in Sch. pombe as a model organism.Furthermore, the molecular basis of maintaining afunctional, intact mt chromosome for viability is dis-cussed.

Finally, a close look at the mobile group I and groupII introns within the small mt genome of fission yeastshows that homing and transposition events and intronDNA excision and other intron-dependent genomicinstabilities might have shaped the mt genome duringevolution.

Acknowledgement Research in this laboratory was supported bythe Deutsche Forschungsgemeinschaft.

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