Evolution of linear chromosomes and multipartite genomes in yeast mitochondria.

15 MB·English

by  Valach, Matus

4202–4219 Nucleic Acids Research, 2011, Vol. 39, No. 10 Published online 25 January 2011
doi:10.1093/nar/gkq1345
Evolution of linear chromosomes and multipartite
genomes in yeast mitochondria
Matus Valach1, Zoltan Farkas2, Dominika Fricova1, Jakub Kovac3, Brona Brejova3,
Tomas Vinar4, Ilona Pfeiffer2, Judit Kucsera2, Lubomir Tomaska5, B. Franz Lang6 and
Jozef Nosek1,*
1Department of Biochemistry, Comenius University, Mlynska dolina CH-1, 842 15 Bratislava, Slovak republic,
2Department of Microbiology, Faculty of Sciences and Informatics, University of Szeged, Kozep fasor 52,
H-6726 Szeged, Hungary, 3Department of Computer Science, 4Department of Applied Informatics, Faculty of
Mathematics, Physics and Informatics, Comenius University, Mlynska dolina M, 842 48 Bratislava, 5Department
of Genetics, Faculty of Natural Sciences, Comenius University, Mlynska dolina B-1, 842 15 Bratislava, Slovak
republic and 6De´partement de Biochimie, Universite´ de Montre´al, 2900 Boul. Edouard-Montpetit, Montre´al,
Que´bec H3T 1J4, Canada
Received September 23, 2010; Revised December 20, 2010; Accepted December 22, 2010
ABSTRACT genomes in prokaryotes and organelles vary substantially
(1). For instance, certain animal mitochondrial DNAs
Mitochondrial genome diversity in closely related
(mtDNAs) are monomeric circles (2), kinetoplastid
speciesprovidesanexcellentplatformforinvestiga-
protists have networks of catenated circles (3,4), and
tion of chromosome architecture and its evolution most plants and fungal mitochondria contain linear (cir-
by means of comparative genomics. In this study, cularly permuted) concatemers that are heterogeneous in
wedeterminedthecompletemitochondrialDNAse- size (termed polydisperse linear DNA) (5–7). Finally,
quences of eight Candida species and analyzed uniform linear mtDNAs terminating with defined
their molecular architectures. Our survey revealed terminal structures (mitochondrial telomeres) are found
a puzzling variability of genome architecture, in a number of phylogenetically diverse taxa (8–28). In
addition, mitochondria of numerous organisms contain
including circular- and linear-mapping and multi-
multipartite genome; i.e. fragmented into multiple (from
partite linear forms. We propose that the arrange-
few to several hundred) circular or linear chromosomes
ment of large inverted repeats identified in these
(29–39).
genomes plays a crucial role in alterations of their
The predominant genome architecture may even differ
molecular architectures. In specific arrangements,
in closely related organisms, i.e. conceptually different
theinvertedrepeatsappeartofunctionasresolution (monomeric linear versus circular-mapping and linear
elements, allowing genome conversion among dif- polydisperse) or containing varying proportions of topo-
ferent topologies, eventually leading to genome logically different mtDNA molecules. For example,
fragmentation into multiple linear DNA molecules. mitochondria of Candida glabrata and Saccharomyces
We suggest that molecular transactions generating cerevisiae contain polydisperse linear DNA molecules,
linear mitochondrial DNA molecules with defined with a minor fraction of circles and lariat structures
generated by rolling circle replication (40). In contrast, a
telomeric structures may parallel the evolutionary
recentstudyrevealedthatmitochondriaofC.albicanslack
emergence of linear chromosomes and multipartite
significant amounts of circular mtDNA molecules, con-
genomes in general and may provide clues for the
tainingpredominantlyanetworkofbranchedDNAstruc-
originoftelomeresandpathwaysimplicatedintheir
tures with linear polydisperse mtDNA molecules—
maintenance.
interpreted as recombination-driven replication (41,42).
Analternativeinterpretationwouldbemitochondrialrep-
lication just like in S. cerevisiae, and a reduced level of
INTRODUCTION
circular replicative DNA molecules, due to more effective
Genome fragmented into multiple linear chromosomes recombinationthatisalsoresponsibleforbranchedstruc-
terminating with telomeric arrays is a hallmark of the eu- tures. Whatever the replication mechanism, physical
karyotic cell. In contrast, molecular architectures of mapping approaches such as restriction mapping, DNA
*To whom correspondence should be addressed. Tel:[phone]; Fax:[phone]; Email: [email protected]
(cid:2)TheAuthor(s)2011.PublishedbyOxfordUniversityPress.
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttributionNon-CommercialLicense(http://creativecommons.org/licenses/
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NucleicAcidsResearch,2011,Vol.39,No.10 4203
sequencing or PCR amplification indicate that any of reveals that their molecular forms vary dramatically
these populations of linear polydisperse mtDNA mol- providingauniqueopportunityforidentificationofstruc-
ecules have predominantly circularly permuted sequences tural elements and molecular mechanisms affecting the
(i.e. can be reasonably well represented assingle sequence genome architecture. At the same time, our analysis
records as deposited in GenBank, but should not be provides an insight on the evolution of linear chromo-
labeledcircularasenforcedbythedatabasemanagement). somes and their telomeric structures.
Such genomearchitectures arein mostcases illustrated as
circular maps. In contrast, species containing linear
mtDNA molecules terminating with specific telomeric MATERIALS AND METHODS
structures are best depicted in linear maps (43). Yeast strains and cultivation
Accordingly, we term mitochondrial genomes as
Yeast strains analyzed in this study are listed in Table 1.
circular- or linear-mapping.
YeastsweregrowninliquidYPDGmedia(1%(w/v)yeast
However, the picture is more complex in some yeast
extract, 1% (w/v) peptone, 0.5% (w/v) glucose and 3%
species with multiple forms of mitochondrial genomes.
In Pichia pijperi and Williopsis mrakii, the linear (v/v) glycerol), with constant shaking at 25–30(cid:2)C until
the late exponential phase.
mtDNA molecules terminating with telomeric hairpins
(t-hairpins) coexist with monomeric and dimeric circles
Pulsed field gel electrophoresis
and linear polydisperse mtDNAs (11,44,45). Two types
of DNA replicons occur in mitochondria of Screening for linear mitochondrial genomes was per-
C. parapsilosis. Namely, the linear mtDNA molecules formed by pulsed field gel electrophoresis (PFGE)
with telomeric arrays (t-arrays) of tandem repeats and approach (10,11). Briefly, whole-cell DNA samples were
multimeric minicircles derived exclusively from the preparedinagaroseblocks,andseparatedina1.5%(w/v)
sequence of mitochondrial telomeres (telomeric circles, agarose gel using a CHEF Mapper XA Chiller System
t-circles) implicated in the telomere maintenance (Biorad) with pulse switching set at 5–20s (linear
pathway (16,20,46–48). ramping and 120(cid:2) angle) for 42h at 5V/cm. All separ-
At present, little is known about the biological roles of ations were performed in 0.5(cid:3) TBE buffer (45mM Tris–
different mtDNA forms and molecular mechanisms borate, 1mM EDTA, pH 8.0) at 10(cid:2)C.
leading to architectural alterations of the mitochondrial
genomes. Our ambition is to identify these mechanisms DNA sequencing and mitochondrial genome annotation
and to uncover their role in the evolution of linear
ThemtDNAusedforDNAsequencingwaspurifiedfrom
chromosomes and corresponding telomeric structures.
isolated mitochondria. Procedures for mtDNA prepar-
Therefore, we initiated a large-scale comparative study
ation, DNA sequence analysis and contig assembly were
of the mitochondrial genomes in yeast species closely
described previously (10,46,51,58–60). Genome annota-
related to C. parapsilosis, whose mitochondrial telomeres
tions were performed using the MFannot tool (http://
share a number of structural features with their counter-
megasun.bch.umontreal.ca/cgi-bin/mfannot/mfannot
parts at the ends of nuclear chromosomes (49,50). In
Interface.pl), and manually adjusted according to the
previous reports, we described that strains of
sequence alignments of deduced protein products with
C. metapsilosis and C. orthopsilosis possess either
their homologs from closely related yeast species. Intron
linear-mappingmitochondrialgenome,withsimilararchi-
sequences were identified using MFannot, RNAweasel
tecture as found in C. parapsilosis, or a circularized
(61) and Rfam (62). The precise boundaries were con-
(mutant) form of the genome (51,52). Moreover, we
firmed by alignments of the corresponding sequence with
found that C. subhashii contains yet another type of
anintron-lessgenefromarelatedspecies.Putativeprotein
linear mitochondrial genome, which does not come with
products encoded by intronic open reading frames were
any detectable circular or concatemeric form. Instead, its
identified by searching the Pfam database (63) and classi-
linear mtDNA terminates with invertron-like telomeres,
fied accordingly.
with a protein covalently bound to both 50 termini (10).
The four Candida species containing linear mitochondrial
Phylogenetic analysis
genomes are classified within the monophyletic ‘CTG
clade’ofHemiascomycetes(53,54).Thesamephylogenetic For phylogenetic analysis, we have used amino acid se-
group also contains species with circular-mapping mito- quences of a protein set (Atp6–Atp8–Atp9–Cob–Cox1–
chondrialgenomessuchasC.albicans(55),Debaryomyces Cox2–Cox3–Nad1–Nad2–Nad3–Nad4–Nad4L–Nad5–
hansenii (56) and Pichia sorbitophila (57). The occurrence Nad6) encoded by 23 mitochondrial genomes. The se-
of closely related organisms or even strains of the same quences were translated using translation table 4 (mold,
species with different mitochondrial genome architecture protozoan and coelenterate mitochondrial code), except
is in line with the hypothesis that linear- and for S. cerevisiae, where translation table 3 (yeast mito-
circular-mapping mitochondrial genomes do not exhibit chondrialcode)applies.Themultiplealignmentswereper-
aradicaldifference,butthatthegenomeformsmayspor- formed by MUSCLE (64) and concatenated to one
adically interconvert via currently unknown molecular alignment. Alignment columns with >50% of gaps were
mechanism(s) (11,52). filteredout, resulting in analignment with3932 sites. The
In this study, we analyze the complete mtDNA se- phylogenetic tree was built with three different programs:
quences of eight additional Candida species. Our survey PhyloBayes with the CAT substitution model (65),
4204 NucleicAcidsResearch,2011,Vol.39,No.10
cReference ThisstudyThisstudy(85)BroadinstituteThisstudyThisstudyThisstudyThisstudyThisstudySangerInstitute,thisstudyThisstudyThisstudyBroadInstitute ThisstudyThisstudyThisstudy(51,52)(52,60)(60),thisstudy(51,52)(52)(52,60)(60)(20,46)Thisstudy(20),thisstudyThisstudy(10),thisstudyThisstudyThisstudyBroadinstituteThisstudyThisstudyBroadInstitute,thisstudy(56),thisstudy BroadInstitute,(52)Thisstudy heterminalnucleotidesofthemappinggenomesaredefined www.broad.mit.edu/)andthe
SummaryofthemitochondrialgenomemappinginyeastspeciesinvestigatedinthisstudyTable1. SpeciesStrainMitochondrialgenome aGenomeMitochondrialGenomesize(bp)%G+CGenBankabformtelomeresAcc.No. Td,eeCandidaalaiNRRLY-27739C3036820.9HQ267968NTdCandidaalbicansCCBS562ee4042032.2AF285261SC5314Ce32.6WO-1n.d.55284(unfinished)TTdCandidablackwellae(AS2.3639)CCBS10843TdCandidabohiensisCNRRLY-27737TdCandidabuenavistaensisCNRRLY-27734TdCandidachauliodesCNRRLY-27909TdCandidacorydaliCNRRLY-27910TTd,eeCandidadubliniensis(CBS7987)C3473230.6CD36Td,eeCandidafrijolesensis3xL1t-hairpins37215(masterchromosome)21.6HM594866NRRLY-48060TdCandidagigantensisCNRRLY-27736TTfeCandidaguilliermondii(CBS566)n.d.23890(unfinished)25.7ATCC6260(Pichiaguilliermondii)TTd,eeCandidajiufengensisCBS10846(AS2.3688)C2967228.4GU136397Tdd(cid:4)Candidalabiduridarum3xL1n.d.38000(masterchromosome)NRRLY-27940Td,ebCandidamaltosaC6294922.0CBS5611Td,eeCandidametapsilosisL2t-arrays+t-circles23062+2nx62025.1AY962591MCO448d,ee2217525.8AY391853PL448CTTd,eeCandidaneerlandica(CBS434)C3214124.4EU334437NRRLY-27057d,eeCandidaorthopsilosis2252825.0AY962590MCO456CTdd(cid:4)C25000MCO457d,eet-arrays+t-circles24697+2nx77725.0DQ026513MCO471L2TTd,eeCandidaparapsilosis(CBS604)L2t-arrays+t-circles30928+2nx73823.8DQ376035CLIB214d,eet-arrays+t-circles30923+2nx73823.8X74411SR23(CBS7157)L2TTdCandidapseudojiufengensis(AS2.3693)CCBS10847Td,eeCandidasalmanticensisL2t-arrays+t-circles25718+2nx10420.5HQ267969CBS5121Td,eeCandidasojaeC3941529.1EF468347CBS7871TTd,eeCandidasubhashii(CBS10753)L3t-proteins2979552.7GU126492FR-392-06TdCandidatetrigidarumCNRRLY-48142Td,eCandidatropicalisCCBS94ee5030437.3MYA-3404Cgd$(cid:4)CandidaviswanathiiCL1palindrome/t-hairpins39000CBS1924Td,ee$CL1palindrome/t-hairpins3924226.2EF536359CBS4024TTdeClavispora(Candida)lusitaniae(ATCC42720)C18942(unfinished)27.4CBS6936Td,eeDebaryomyceshanseniiC2946227.0DQ508940CBS767(Candidafamata)TTd,eeLodderomyceselongisporusNRRLYB-4239(CBS2605)C3560128.8TTfdPichiaguilliermondii(ATCC46036)CCBS2030(Candidaguilliermondii) aGenomeformandsizewerededucedfromPFGEanalysisandDNAsequencing(seealsoFigures1–3andSupplementaryFigureS1formoredetails).NotethattC.salmanticensisshortestlinearmtDNAmoleculeswerenotpreciselymappedinthecaseof;C,circular-mapping;L1,L2,L3,linear-mapping,thetypesoflinear-accordingtotheirterminalstructures(10,11,20);3xL1,tripartitetypeIlinear-mapping.betal.MitochondrialtelomeresclassifiedaccordingtoTomaska(50).cmtDNAmappingorsequencedataweredeterminedinthisstudy,publishedpreviouslyordownloadedfrompublicdatabasesoftheBroadInstitute(http://WellcomeTrustSangerInstitute(http://www.sanger.ac.uk/).dPFGEanalysis.eDNAsequencing.fCandidaguilliermodniiPichiaguilliermondiiATCC6260istheanamorphicstrainof.gTCandidalodderaeC.viswanathiiCBS1924isthetypestrainof,itsmtDNAdisplayssimilarrestrictionenzymeprofileasthemtDNAfromCBS4024.n.d.—notdone.TNTandindicatethetypeandtheneotypestrainofthespecies,respectively.
NucleicAcidsResearch,2011,Vol.39,No.10 4205
MrBayes (66) with JTT model of amino acid substitution polynucleotide kinase has been performed essentially as
(67) and g-distributed rate variation between sites, and described previously (20).
PhyML (68) with JTT model. Application of all three
programs gives the same tree topology. The only differ- DNA hybridization probes
ences occur within C. metapsilosis–C. orthopsilosis–
Southern hybridization of PFGE separated yeast DNA
C. parapsilosis clade due to high sequence similarity
samples(Figure1)wasperformedwithaprobecontaining
among these species. In the rest of the tree, all branches
anequimolarmixtureofPCRproductsderivedfromcox2
are highly supported by posterior probabilities (above 0.9
(345bp) and nad4 (374bp) of corresponding species. The
in MrBayes and PhyloBayes), and most branches have
following PCR primers were used: 50-TAGATGT
bootstrap value of 100 in PhyML. Placement of C. alai
NCCWACWCCWTGAG-30 and 50-AYTCRTATTTTC
inthetreehashighposteriorprobabilityinbothBayesian
AATATCATTG-30 (cox2); 50-AGGTATHWTGG
programs, but low bootstrap values in PhyML.
TWAARACACC-30 and 50-CAGGWGAWACDAAWC
CATG-30 (nad4). For C. subhashii, the equivalent PCR
Gene order comparison primers were 50-CGTCCCAACACCATGAGG-30 and
50-ACTCGTACTTCCAGTACCACTG-30 (cox2); 50-AG
To infer possible ancestral gene order, we used protein
GGATCATGGTCAAGACG-30 and 50-CTGGTGAGA
coding, rRNA and tRNA genes of 16 mitochondrial
CTAGCCCGTG-30 (nad4). In subsequent experiments,
genomes from the ‘CTG clade’. Non-conserved genes
we used the following probes: P-668 (668bp fragment
that occur only in some species (i.e. trnM3 in D. hansenii
amplified by PCR from the C. frijolesensis mtDNA
and P. sorbitophila; dpoBa, dpoBb and orf756 in
using primers 50-ATAATGGGTCAGTGAGTT-30 and
C.subhashii)wereomittedinthisanalysis.Wehaverecon-
50-ACGTTCTCTAGCAGTTGA-30), EH-1350 (1350bp
structed a possible history of rearrangements using a
EcoRV-HindIII fragment from C. frijolesensis mtDNA),
simple double cut and join model (DCJ) (69). The DCJ
H-1030 (1030bp HindIII fragment from C. neerlandica
modelisbasedonparsimonyandincludescommonlycon-
mtDNA), and Oligo-32 (32nt oligonucleotide 50-AATG
sidered rearrangement operations, such as reversal, trans-
AGATGAGGAAGTAAAGGGATAAGGATAA-30,
location, chromosome circularization, linearization,
corresponding to a palindrome sequence in C. viswanathii
fusion and fission. There is an efficient algorithm to
mtDNA).
compute the parsimonious distance of two genomes in
DCJmodel(69);however,anexactalgorithmforinferring
DNA sequence accession numbers
ancestral gene orders for a given set of present day
genomes is not known. To infer the ancestral gene order Mitochondrial DNA sequences described in this work
under DCJ, we have implemented a local optimization were deposited in the GenBank nucleotide sequence data
procedure that in each iteration attempts to improve the library under following accession numbers: HQ267968
solution by choosing new ancestral gene orders from a (C. alai NRRL Y-27739), HM594866 (C. frijolesensis
local neighborhood using a dynamic programming algo- NRRL Y-48060), GU136397 (C. jiufengensis CBS10846),
rithm(70).TheDCJmodeldoesnothandlegenomeswith EU267175 (C. maltosa CBS5611), EU334437
duplicated genes. To resolve recent duplications in some (C. neerlandica NRRL Y-27057), EF468347 (C. sojae
of the genomes (C. albicans, C. maltosa, C. sojae, CBS7871), EF536359 (C. viswanathii CBS4024),
C. viswanathii), we removed duplicated genes, and HQ267969 (C. salmanticensis CBS5121).
included both possible forms of the genomes as alterna-
tives in the corresponding leaves. Similarly, both isomers
are allowed in the genomes that include long inverted RESULTS AND DISCUSSION
repeats (C. alai, C. albicans, C. maltosa, C. neerlandica,
PFGE analysis of yeast mitochondrial genomes
C. sojae, L. elongisporus). In each leaf, one ofthe alterna-
tiveordersischosenasarepresentative,soastominimize We employed the PFGE approach (11,40) to distinguish
the overall parsimony cost. Finally, we penalize occur- between polydisperse and uniform linear mtDNA mol-
rence of multiple circular chromosomes, or combinations ecules in samples from 24 yeast species (Table 1 and
of linear and circular chromosomes in ancestral genomes. Figure 1). In experimental conditions used for the
screening (see ‘Materials and Methods’ section), the
uniform linear mtDNA molecules from C. subhashii (10)
Enzymatic mapping of termini
migrateasadiscretebandof(cid:4)30kb(Figure1,lane8).In
Approximately 1mg mtDNA aliquots were treated with contrast, a smear is typical for C. albicans [Figure 1, lane
exonuclease III (ExoIII; New England Biolabs) or 10; (42)] and most other yeast species, with
BAL-31 nuclease (New England Biolabs) according to mtDNA-derived probes revealing a strong signal
the manufacturer’s instructions, for increasing time between (cid:4)20 and 50kb (Figure 1 and Table 1). This indi-
periods. After enzyme inactivation (ExoIII for 20min at cates that most examined species contain polydisperse
70(cid:2)C; BAL-31 for 10min at 65(cid:2)C in the presence of linear mtDNAs. On average, their lengths are larger
EDTA), the mtDNA was precipitated with ethanol, than the genome unit, apparently containing more than
dissolved in water, digested with a restriction endonucle- one genome equivalent per molecule. However, in
ase and electrophoretically separated in a 0.9% agarose C. labiduridarum and C. frijolesensis we detected three
gel. The labeling of mtDNA termini with T4 discrete bands migrating in the region between 15 and
4206 NucleicAcidsResearch,2011,Vol.39,No.10
Figure 1. PFGEanalysisoftheyeastmtDNAs.Thewhole-cellDNAsampleswereseparatedbyPFGEusingaCHEFMapperXAChillerSystem
(Biorad), blotted onto a nylon membrane and hybridized with mtDNA-derived probes as described in ‘Material and Methods’ section. Lane 1—
C.viswanathiiCBS4024;lane2—C.sojaeCBS7871;lane3—C.maltosaCBS5611;lane4—C.neerlandicaNRRLY-27057;lane5—C.alaiNRRL
Y-27739;lane6—C.labiduridarumNRRLY-27940;lane7—C.frijolesensisNRRLY-48060;lane8—C.subhashiiCBS10753;lane9—C.jiufengensis
CBS 10846; lane 10—C. albicans CBS 562. Note that three discrete bands migrating in the region <50kb represent three linear mitochondrial
chromosomesinC.labiduridarumandC.frijolesensis(lanes6and7).Incontrast,fourbandsinC.alai(lane5)donothybridizewithmtDNAprobes
and correspond to linear DNA plasmids (data not shown).
50kb(Figure1,lanes6–7).Thispointstoapossibilitythat the identification of isomers by restriction enzyme
these species contain three uniform chromosomes in analysis inconclusive.
mitochondria (see below). Four distinct bands were also Physical mapping uncovered genome isomers also in
found in C. alai. However, these bands did not hybridize mitochondria of C. viswanathii. However, in this case we
withthemtDNAprobe (Figure1,lane5)and subsequent observed two BamHI ((cid:4)7 and (cid:4)3.5kb) and Eco91I ((cid:4)5
sequence analysis revealed that they correspond to linear and (cid:4)2.5kb) bands, with sizes corresponding to a
DNA plasmids (to be described elsewhere). monomer (lower faint band) and a dimer (upper band).
Southern hybridization indicates that the two fragments
Mitochondrial genome isomers in species with have the same sequence (Figure 3A), and that the ratio
polydisperse mtDNA between them varied in different preparations (data not
shown). In this case, the complete mtDNA sequence of
Since the mitochondrial genome of C. albicans occurs in
C. viswanathii contains LIRs arranged as a large palin-
twoisomers (42,71),weexaminedthepresence ofgenome
drome (see below), suggesting that thepalindrome (repre-
isomers also in other species with polydisperse mtDNAs.
sented by the upper band) could be resolved into the
Restriction enzyme analysis of the mtDNAs from
smaller fragment (the lower band), i.e. a linear mtDNA
C. maltosa, C. neerlandica and C. sojae identified four
minor mtDNA fragments (e.g. (cid:4)15, (cid:4)13, (cid:4)9 and (cid:4)7kb with defined terminal sequences/structures. To support
inthecaseofC.neerlandicamtDNAdigestedwithBamHI this idea we treated isolated mtDNA with BAL-31
and PvuII) indicating that they contain a nuclease prior to restriction enzyme analysis. This experi-
circular-mapping genome with large repeated regions mentdemonstratedthatthelowerfaintbandwastheonly
generatingtwoisomersthatarepresentinastoichiometric mtDNA fragment sensitive to BAL-31 nuclease activity
ratio (Figure 2A–C). Subsequent sequence analysis con- (Figure 3B). On the other hand, this fragment seems to
firmed that all three genomes contain large inverted be refractory to both exonuclease III and T4 polynucleo-
repeats (LIRs) that could be involved in the flip-flop re- tide kinase (data not shown). This indicates that the
combination generating genome isomers. This is in line terminiofresolvedlinearmtDNAmoleculesareprotected
with the observation that the LIRs represent recombin- by a special arrangement. We presume that, similar to
ation hotspots in C. albicans mtDNA (42). The LIRs species from the genera Williopsis and Pichia (44), the
were also detected in the C. alai mtDNA sequence, but linear mtDNA molecules terminate with single-stranded
the presence of contaminating linear plasmids rendered covalently closed telomeric hairpins (t-hairpins).
NucleicAcidsResearch,2011,Vol.39,No.10 4207
Figure 2. Restrictionenzymeanalysisrevealscircular-mappinggenomeisomers.Candidaneerlandica(A),C.maltosa(B)andC.sojae(C)mtDNAs
were digested with the restriction enzyme combinations BamHI+PvuII, ApaLI+MluI and AgeI+ApaLI, respectively, and electrophoretically
separated in 0.9% (w/v) agarose gel. Black arrows indicate the DNA fragments present in both isomers, grey arrows label the pair of fragments
specific to the isomers I or II. Schemes illustrate both isomers with the position of inverted repeats (shown bold within the inner circle) and
corresponding restriction enzyme fragments (the outer circle).
In contrast, we did not identify genome isomers in bands (Figure 1, lane 6–7), suggesting that the longest
C. jiufengensis, which lacks LIRs. molecules might represent a master chromosome split
into two non-identical fragments. Therefore, we
analyzed PFGE-separated mtDNA molecules of
Multipartite (fragmented) linear-mapping genomes
C. labiduridarum and C. frijolesensis by Southern hybrid-
Asmentionedabove,PFGEanalysisrevealedthepresence ization using two probes derived from distant regions of
of three distinct mtDNA bands in C. labiduridarum and chromosome I (Figure 4A). The probe P-668 hybridized
C. frijolesensis. The size of the largest band (chromosome with chromosomes I and III and also detected some
I) corresponds approximately to the sum of the middle mtDNA in wells and smears. The pattern detected by
(chromosome II) and the smallest (chromosome III) the probe H-1030 was similar, except that it hybridized
4208 NucleicAcidsResearch,2011,Vol.39,No.10
Figure 3. Circular- and linear-mapping genome isomers in mitochondria of C. viswanathii. (A) The mtDNA samples were digested with BamHI
(lane1)orEco91I(lane2)andseparatedin1%(w/v)agarosegel.TheSouthernblotwashybridizedwithradioactivelylabeledoligonucleotideprobe
Oligo-32derivedfromthelargepalindrome(shownasdashedarrows).Thesolidarrowsshowpositionsofthepalindromeandthepresumedterminal
fragments of resolved linear molecules capped with t-hairpins. Scheme shows presumed circular- (I) and linear-mapping (II) genome isomers.
(B) Isolated mtDNA was treated or untreated with BAL-31 nuclease (0.2U for 5min). The mtDNA was then extracted from the reaction,
digested with BamHI or Eco91I endonuclease, and electrophoretically separated. Note that the fragments containing presumed t-hairpins were
sensitive to BAL-31 nuclease (indicated by asterisk).
NucleicAcidsResearch,2011,Vol.39,No.10 4209
Figure 4. Multipartite linear-mapping genomes in C. labiduridarum and C. frijolesensis (A) PFGE separated samples of C. labiduridarum NRRL
Y-27940 (lane 1) and C. frijolesensis NRRL Y-48060 (lane 2) were blotted onto a nylon membrane and hybridized with the radioactively labeled
probes P-668 and H-1030 (regions hybridizing with both probes are shown as dashed lines). Presumed master (I) and two smaller chromosomes
(II and III) are indicated. Note that the master chromosome occurs in four isomers (i.e. L (cid:5)R (cid:5)L (cid:5)R (shown in the scheme),
III III II II
L (cid:5)R (cid:5)R (cid:5)L , R (cid:5)L (cid:5)L (cid:5)R and R (cid:5)L (cid:5)R (cid:5)L . ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The
III III II II III III II II III III II II
C. frijolesensis mtDNA ((cid:4)1mg) was digested with BAL-31 nuclease (B) or exonuclease III (ExoIII) (C) as indicated. After nuclease inactivation,
theDNAwasdigestedwithEcoRV,separated in0.9%(w/v)agarosegel.TheSouthernblotswerehybridized withthe P-668andEH-1350probes
specificfortheleftandtherightarmofthemasterchromosome,respectively(see‘MaterialsandMethods’section).Arrowsshowthepositionsofthe
left(L)andright(R)terminalfragmentsandtheirfusions(R+R,R+LandL+L).NotethatafterExoIIItreatmentthetelomericfragmentsform
twosubpopulationsthatdifferintheirsensitivitytotheExoIIItreatment.ThisindicatesthatthelinearmtDNAmoleculespossessanopenstructure
with50overhangorbluntendorcovalentlyclosedt-hairpin.(D)TheC.frijolesensismtDNAwastreatedwithantarcticphosphataseandlabeledwith
[g32P]ATP and T4 polynucleotide kinase. The mtDNA was then digested with restriction endonuclease EcoRV (lane 1) or BglII (lane 2) and
separated in 0.8% (w/v) agarose gel (left panel). The gel was fixed in 10% (v/v) methanol/10% (v/v) acetic acid for 30min, dried overnight and
autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.
4210 NucleicAcidsResearch,2011,Vol.39,No.10
Figure 4. Continued
with chromosome II instead of chromosome III. To opened and thus accessible to exonuclease III and T4
confirm that all three chromosomes are linear, we polynucleotide kinase (Figure 4D). Southern hybridiza-
treated C. frijolesensis mtDNA with BAL-31 nuclease, tion revealed four faint restriction fragments of the
exonuclease III, and T4 polynucleotide kinase. We mtDNA (designated as L +L , R +R , L +R ,
II III II III II III
observed that the presumed terminal restriction enzyme L +R ) that are refractory to all three enzymes
III II
fragments were all sensitive to BAL-31 nuclease (Figure 4B–D). The fragment sizes correspond to junc-
(Figure 4B). Interestingly, the treatment with exonuclease tions between chromosomes II and III, and their
IIIrevealedtwosubpopulationsofterminalfragmentsdif- presence shows that the master chromosome occurs in
fering in their accessibility to the enzyme activity four flip-flop isomers (i.e. L (cid:5)R (cid:5)L (cid:5)R ,
III III II II
(Figure 4C). This indicates that the ends of the linear L (cid:5)R (cid:5)R (cid:5)L , R (cid:5)L (cid:5)L (cid:5)R and
III III II II III III II II
mtDNA molecules might adopt a covalently closed struc- R (cid:5)L (cid:5)R (cid:5)L ). This suggests that fragmented
III III II II
turesuchasat-hairpin,whichinafractionofmoleculesis linear-mapping genomes (i.e. uniform linear mtDNAs
NucleicAcidsResearch,2011,Vol.39,No.10 4211
Table 2. Genes present in the mitochondrial genomes sequenced in this study
Species Protein subunits of oxidative phosphorylation complexes Protein synthesis and RNA processing
I III IV V Ribosomal rRNA tRNA RNase
protein P RNA
nad1 nad2 nad3 nad4 nad4L nad5 nad6 cob cox1 cox2 cox3 atp6 atp8 atp9 rps3 rns rnl trn rnpB
Candida alai + + + + + + + + + + + +a +a + (cid:5) + + 24+1a (cid:5)
Candida frijolesensis + + + + + + + + + + + + + + (cid:5) + + 24 (cid:5)
Candida jiufengensis + + + + + + + + + + + + + + (cid:5) + + 24 (cid:5)
Candida maltosa + + + + + + + + + + + + + + (cid:5) + + 24+2a (cid:5)
Candida neerlandica + + + + + + + + + + + + + + (cid:5) + + 24 (cid:5)
Candida salmanticensis+ + + + + + + + + + + + + + + + + 24 +
Candida sojae + +a +a + + + + +a + +a + + + +a (cid:5) +a + 24+5a (cid:5)
Candida viswanathii + + + + + + + + + + + + + + (cid:5) + + 24+1a (cid:5)
aDuplicated within LIRs.
with resolved termini corresponding to chromosomes recognized as tryptophan and leucine, respectively. The
I–III) may coexist with circular-mapping genome forms codon assignment was verified by multiple alignments of
(i.e. polydisperse linear mtDNAs lacking homogeneous protein sequences, which led us to the conclusion that
terminal structures that correspond to the smear UGA(Trp) is the only deviation from the standard
observed in PFGE). genetic code.
The occurrence of multipartite genomes raises a Introns were identified in cob, cox1, nad5 and rnl genes
questionconcerningthedistributionofmtDNAmolecules (Table 3). Their number and distribution among species
duringcelldivision.Sincewedonothaveanyevidencefor vary from one (in C. alai) to 10 (in C. frijolesensis). The
a specific segregation machinery analogous to the mitotic introns predominantly belong to group I and in many
spindle ensuring the proper segregation of individual cases contain an open reading frame (ORF) coding for
chromosomes in mitochondria, we propose that putative LAGLIDADG and GIY-YIG type endonucle-
presumed circular-mapping genome and/or chromosome ases (group I) or reverse transcriptases/maturases (group
I may represent the ‘master copies’ playing a key role in II introns).
the genome transmission. Our previous studies (46,51) revealed that
C. metapsilosis, C. orthopsilosis and C. parapsilosis have
Genetic organization of the mitochondrial genomes the same gene order in their mtDNAs. Likewise,
C. frijolesensis, C. neerlandica and C. viswanathii have
With the aim to investigate the mitochondrial genome
the same genetic organization, except that trnM1 has
architecture in more detail and to identify sequence and/
been duplicated and inverted in the latter species
or structural features involved in the genome architecture
(Figure 5A). All other species examined exhibit unique
alterations, we determined the complete mtDNA se-
gene arrangements, with synteny reduced to four
quences of eight yeast species; C. alai, C. frijolesensis,
conserved gene clusters (i.e. trnN-atp6, cox1-atp9,
C. jiufengensis, C. maltosa, C. neerlandica,
cob-nad3 and rnl-cox2) in C. jiufengensis versus
C. salmanticensis, C. sojae and C. viswanathii
C. parapsilosis (Figure 5B).
(Supplementary Figure S1). The sizes of sequenced
mtDNAs range from 25.7 (C. salmanticensis) to 62.9kb
LIRs and the genome architecture
(C. maltosa), and their G+C content varies from 20.5
(C. salmanticensis) to 29.1% (C. sojae) (Table 1). The Analysis of the collected yeast mtDNA sequences reveals
genomes contain essentially the same set of conserved that the most prominent feature of the genome architec-
genes including the genes for subunits of ATP synthase ture is the presence of relatively long duplications,
(atp6,8,9), apocytochrome b (cob), cytochrome c oxidase arranged as inverted repeats (LIRs). These elements are
(cox1,2,3), NADH:ubiquinone oxidoreductase present in all but one (C. jiufengensis) mtDNA, and their
(nad1,2,3,4,4L,5,6), large and small ribosomal RNA (rnl lengths vary from 109bp (present as the subterminal
and rns) and a complete set of transfer RNAs (trn). The repeats in the linear mtDNA of C. salmanticensis) to
C. salmanticensis mtDNA has two additional genes: i.e. 14379bp (in C. maltosa) thus substantially expanding
rps3/var1 coding for a subunit of the mitochondrial the genome length (Supplementary Figure S1). In most
ribosome and rnpB/rpm1 for the RNA subunit of RNase cases, LIRs comprise non-coding sequences or contain
P (Table 2). One or more genes are duplicated in only a few genes or gene fragments. In C. sojae, the
C. maltosa, C. sojae and C. viswanathii mtDNAs as they 8658bp LIRs represent a block duplication of 11 genes
are localized within the LIRs. (i.e. trnA, cox2, trnM1, cob, trnM2, rns, trnI, atp9,
ThepresenceoftRNATrpwithanUCAanticodon,and trnR1, nad2, nad3). In most cases, the pairs of LIRs are
the absence of an S. cerevisiae homolog of the abnormal separated by long unique regions. However, we noticed
tRNAThr1with8-ntintheanticodonloop(decodingCUN two special arrangements of LIRs: (i) in C. viswanathii
as threonine), indicate that UGA and CUN codons are identical copies of 4162bp inverted repeats separated by