Diversity in Organization and the Origin of Gene Orders in the Mitochondrial DNA Molecules of the Genus Saccharomyces

Casper Groth1,, Randi Føns Petersen and Jure Piskur2,

Department of Microbiology, Technical University of Denmark, Lyngby, Denmark


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Sequencing of the Saccharomyces cerevisiae nuclear and mitochondrial genomes provided a new background for studies on the evolution of the genomes. In this study, mitochondrial genomes of a number of Saccharomyces yeasts were mapped by restriction enzyme analysis, the orders of the genes were determined, and two of the genes were sequenced. The genome organization, i.e., the size, presence of intergenic sequences, and gene order, as well as polymorphism within the coding regions, indicate that Saccharomyces mtDNA molecules are dynamic structures and have undergone numerous changes during their evolution. Since the separation and sexual isolation of different yeast lineages, the coding parts have been accumulating point mutations, presumably in a linear manner with the passage of time. However, the accumulation of other changes may not have been a simple function of time. Larger mtDNA molecules belonging to Saccharomyces sensu stricto yeasts have acquired extensive intergenic sequences, including guanosine-cytosine–rich clusters, and apparently have rearranged the gene order at higher rates than smaller mtDNAs belonging to the Saccharomyces sensu lato yeasts. While within the sensu stricto group transposition has been a predominant mechanism for the creation of novel gene orders, the sensu lato yeasts could have used both transposition- and inversion-based mechanisms.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The genetic material of a Saccharomyces cerevisiae cell consists of 16 nuclear chromosomes and several copies of mitochondrial DNA (mtDNA). While the primary structures of the nuclear and mitochondrial genomes have now been elucidated (Goffeau et al. 1996Citation ; Foury et al. 1998Citation ), so far very little is known about origins and the processes which have rearranged the genomes during evolution.

The mitochondrial genome of S. cerevisiae consists of 85 kb and has a low gene density and extensive intergenic regions, comprising two thirds of the genome (de Zamaroczy and Bernardi 1986aCitation ; Foury et al. 1998Citation ). The coding part of mtDNA contains protein-coding genes (for cytochrome c oxidase subunits I, II, and III [COX1, COX2, and COX3], ATP synthase subunits 6, 8 and 9 [ATP6, ATP8, and ATP9], apocytochrome b [COB], and a ribosomal protein [VAR1]) and RNA genes (for 21S and 15S ribosomal RNAs [LSU and SSU], 24 tRNAs, and the 9S RNA component of RNAse P [RPM1]). In addition, several intron-related and additional open reading frames were reported for some strains (Foury et al. 1998Citation ). All analyzed S. cerevisiae strains exhibited the same gene order (Dujon 1981Citation ; Evans, Oakley, and Clark-Walker 1985; de Zamaroczy and Bernardi 1986bCitation ; Foury et al. 1998Citation ). Mitochondrial intergenic regions are composed of adenosine-thymine (A+T) stretches of several hundred base pairs interrupted by over a hundred guanosine-cytosine (G+C)–rich clusters, which are less than 100 bp in length (de Zamaroczy and Bernardi 1986aCitation ). A vast majority of G+C clusters are represented by various direct and indirect repeats involved in generation and transmission of respiratory-deficient petite mutants (Bernardi 1979Citation ; Piskur 1995Citation ). Apart from site-specific recombination operating through short repeats (Bernardi 1979Citation ), homologous recombination events also occur with high frequency (Dujon, Slonimski, and Weill 1974Citation ). Both of these recombination mechanisms are involved in the generation of mutant mtDNA molecules characterized by intergenic deletions and/or a novel gene order (Piskur 1988Citation ; Clark-Walker 1989Citation ). These mutants are respiratory-competent, but the mutations are transmitted only poorly to progeny of genetic crosses with the wild-type molecule (Piskur 1989Citation ; Skelly and Clark-Walker 1990Citation ). Thus, even if the S. cerevisiae mtDNA molecule is very recombinogenic, the wild-type configuration is preferentially inherited. Apparently, in addition to the coding function, the sequence of mtDNA also determines the "fittest" structure, i.e., the form which is preferentially transmitted from generation to generation (Piskur 1995Citation ). We would like to know how this structure and organization has evolved and whether the progenitor mtDNA molecule can be deduced.

Evolutionary processes can often be postulated from comparisons of the structures of modern genomes belonging to closely related species. The Saccharomyces genus contains several species (Barnett 1992Citation ), which can be divided into a group of petite-positive and a group of petite-negative yeasts, including S. kluyveri (Piskur et al. 1998Citation ). Petite-positive Saccharomyces yeasts can be further divided into sensu stricto yeasts, including S. cerevisiae, and sensu lato yeasts. The evolutionary constraint on the respiratory functions encoded by the mtDNA genes has likely been the same for all Saccharomyces petite-positive yeasts, because the modern species share the same fermentation and respiration patterns.

On separation of the different yeast lineages, the coding parts of the genome accumulated point mutations, possibly as a linear function of time (Kurtzman and Robnett 1998Citation ). On the other hand, changes in organization, for example, size and gene order, do not necessarily accumulate in a linear fashion with time. In the present paper, we analyze the phylogeny and organization of mtDNA molecules belonging to the modern Saccharomyces yeasts and discuss their possible evolutionary history.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Yeast Strains
The following Saccharomyces yeasts were used in our studies. Sensu stricto yeasts: S. cerevisiae T3/3 (Evans, Oakley, and Clark-Walker 1985Citation ), S. bayanus CBS 380T, S. bayanus CBS 395 (syn. S. uvarum), S. paradoxus NRRL Y-17217T, S. paradoxus CBS 2908 (syn. S. douglassii), S. pastorianus CBS 1538T, S. pastorianus CBS 1513 (syn. S. carlsbergensis), and S. pastorianus CBS 1503 (syn. S. monacensis). Sensu lato yeasts: S. castellii NRRL Y-12630T, S. dairenensis NRRL Y-12639T, S. exiguus NRRL Y-12640T, S. servazzii NRRL Y-12661T, and S. unisporus NRRL-1556T. The superscript "T" indicates a type strain. CBS isolates are from the Centraalbureau voor Schimmelcultures, Delft, the Netherlands, and NRRL isolates are from the Agricultural Research Service, Northern Regional Research Center, Peoria, Ill. Yeasts were grown overnight in YPD medium (1% yeast extract, 2% bacto peptone, and 2% glucose) at 25°C.

Purification of mtDNA and Direct Sequencing
mtDNA was purified by bisbenzimide/CsCl buoyant density centrifugation (Piskur et al. 1998Citation ). The dideoxy sequencing procedure was modified to allow sequencing directly on CsCl-purified mtDNA without subcloning or PCR amplification. The double-stranded mtDNA, ~100–1,000 ng, was sequenced using the commercial Thermo sequenase cycle sequencing kit from Amersham based on the polymerase dideoxy chain termination method. Primers for sequencing the SSU and ATP9 genes were designed directly from the conserved regions between S. cerevisiae and Hansenula wingeii sequences. SSU YM-19 is a reverse primer, 5'-GCA GGT TCC CCT ACG GTA ACT GTA-3', covering positions 1630–1653 of the S. cerevisiae SSU gene. The primer allows sequencing of the last ca. 400 bp at the 3' end of the SSU gene. OLI1 YM-1 is a forward primer, 5'-GCA ATT AGT ATT AGC AGC TAA ATA TAT TGG-3', covering positions 3–32 of the S. cerevisiae ATP9 gene and allows the sequencing of most of the gene, but not the extreme 5' region of the open reading frame. This part was sequenced by species-specific reverse primers OLI1 YM-2-CAS (5'-GTGGCTTCACTTAATGCAAAACC-3') for S. castellii, OLI2 YM-2-DAI (5'-GTGGCTTCAGATAATGCGAAACC-3') for S. dairenensis, OLI2 YM-2-EXI (5'-GTTGCTTCTGATAAAGCCAT-3') for S. exiguus and S. unisporus, OLI2 YM-2-SER (5'-GTTGCTTCACTTAAAGCTATACC-3') for S. servazzii, and OLI1 YM-4 (5'-AATAAGAATGAAACCATTAAACAGA-3') for Saccharomyces sensu stricto.

Sequence Analysis
Sequences from the mitochondrial SSU (positions 1227–1625) and the open reading frames of ATP9 (positions 1–231) genes were aligned using the multiple-sequence alignment program CLUSTAL W (Thompson, Higgins, and Gibson 1994Citation ). Phylogenetic analyses were performed using the neighbor-joining method included in the CLUSTAL W package. Nucleotide distances for this analysis were computed according to the Kimura two-parameter model (for review, see Kimura 1983Citation ). The phylogenetic trees were displayed using the NJplot program by Manolo Gouy (University of Lyon, France). The stability of individual branches was assessed using the bootstrap method (Felsenstein 1989Citation ).

Restriction and Gene Mapping
Restriction analyses of mtDNAs were performed using several enzymes, in single or pairwise digestions. Fragments were separated by agarose gel electrophoresis and blotted on a membrane. Hybridization was performed at 55°C overnight in 5 x SSC (standard saline concentration), 5 x Denhardt's solution (Sambrook, Fritsch, and Maniatis 1989Citation ), 0.2% SDS (sodium dodecyl sulphate), 20 µl/ml of denatured salmon sperm DNA, and ca. 100 ng of a labeled probe. A description of the S. cerevisiae mtDNA probes used can be found in table 1 and elsewhere (Groth 1998Citation ; Petersen 1998Citation ). They originated from petite yeast strains or plasmid inserts and were labeled with 32P using the Boehringer/Roche random priming kit. The membranes were washed in 1 x SSC, 0.1% SDS twice for 20 min at room temperature and once for 20 min at 55°C.


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Table 1 Saccharomyces cerevisae Mitochondrial Probes Used in the Hybridization Experiments

 

    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Phylogenetic Relationship
It has previously been shown that the size and content of G+C clusters of mitochondrial genomes vary considerably among Saccharomyces yeasts (Piskur et al. 1998Citation ). In this study, initially, two mitochondrial genes, SSU and ATP9, from several Saccharomyces petite-positive yeasts were analyzed in order to propose a phylogenetic relationship among their mtDNA molecules (table 2 ). The sequences of both regions were identical among the S. pastorianus strains (table 3 ), and in addition, these yeast isolates gave identical restriction patterns when cut, alone, or in pair combinations with ClaI, EcoRI, HindIII, PvuII, and SalI (data not shown). The sequences and restriction patterns were also identical for both S. bayanus strains. While the S. paradoxus strains gave identical sequences, their restriction patterns were slightly different (for details, see Groth 1998Citation ). However, the sequences and restriction patterns were different among the four sensu stricto species, S. bayanus, S. cerevisiae, S. pastorianus, and S. paradoxus (tables 2 and 3 ). The sensu lato type strains each gave very distinctive sequences (tables 2 and 3 ). A phylogenetic tree based on analyses of the ATP9 and SSU genes is shown in figure 1 , and the percentages of identity for both genes are summarized in table 3 . Saccharomyces sensu stricto isolates contained mtDNA molecules which represented a well-defined and phylogenetically closely related group. While the sensu stricto yeasts showed more than 95% sequence identity in the two studied regions, the identity percentage was much lower for sensu lato yeasts (table 3 ). A couple of subgroups are apparent among the sensu lato yeasts. The identity percentage for S. castellii and S. dairenensis mtDNA for the SSU region was 91.3%, and that for the ATP9 region was 87.3% (table 3 ), suggesting a relatively close phylogenetic relationship between these two mtDNA molecules. Also, S. servazzii and S. unisporus seemed to be closely related on the basis of the SSU sequences (table 3 and fig. 1 ). The ATP9 results suggest that S. exiguus may also be closely related to S. unisporus (table 3 ). In general, the relationship among the members of any of the sensu lato subgroups was not as close as the relationship among sensu stricto yeasts.


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Table 2 Accession Numbers of the Mitochondrial SSU and ATP9 Genes Deposited in GenBank or EMBL

 

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Table 3 Relatedness Among the SSU and ATP9 Genes Belonging to Different Saccharomyces Yeasts is Shown as Percentages of Identical Nucleotides Among the Studied Sequences

 


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Fig. 1.—Phylogenetic tree of Saccharomyces yeasts based on the (A) SSU and (B) ATP9 sequences. The bar represents 2% difference between sequences. Schizosaccharomyces pombe was used as an outgroup. The stability of branches is represented by percentage bootstrap values (100 cycles were performed)

 
Restriction and Gene Mapping
While neutral point mutations in the coding areas are commonly thought to accumulate in a linear fashion with time, gross changes in genome structure may occur sporadically and abruptly. Two useful parameters of genome structure are size and gene order. Therefore, we mapped different Saccharomyces mtDNA molecules for the position of restriction sites and genes and subsequently determined their size and gene order (fig. 2 ). All mtDNA molecules are circular. Approximately a dozen different restriction enzymes were used initially to map each mtDNA molecule, but only those restriction enzymes whose sites were precisely determined are shown in figure 2 . Further details can be found elsewhere (Groth 1998Citation ; Petersen 1998Citation ). A majority of S. cerevisiae–based gene probes hybridized to their homologous counterparts in all species, except the RPM1 and large tRNA cluster probes, which did not hybridize to DNA from the sensu lato species. Presumably, these genes are too short to provide enough homology for heterologous hybridization and/or they are scattered in the sensu lato mtDNA molecules. In short, the coding potential of all Saccharomyces species seems similar to that of S. cerevisiae.



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Fig. 2.—Detailed restriction and gene maps and the sizes of the mtDNA molecules belonging to the analyzed Saccharomyces species. Note that S. cerevisiae (for review, see Dujon 1981Citation ), S. paradoxus syn. S. douglassii (Tian et al. 1991Citation ), S. bayanus syn. S. uvarum (Cardazzo et al. 1998Citation ), and S. exiguus (Clark-Walker, McArthur, and Sriprakash 1983Citation ) were previously determined

 
Size and Gene Order
All sensu stricto yeasts contain a mtDNA molecule which is larger than 60 kb, and the gene order of several pairs of genes is preserved (fig. 2 ); for example, COX1-ATP8-ATP6 represents one conserved block, ATP9-VAR1 represents another one, and LSU–large tRNA cluster–COXII is also conserved as a block. The first two blocks are transcribed as a single unit in S. cerevisiae (Grivell 1989Citation ). It is likely that these three blocks of genes also existed in the sensu stricto progenitor mtDNA molecule. Only three gene order types were found among sensu stricto yeasts (fig. 2 ). Note that the gene orders of S. cerevisiae (for review, see Dujon 1981Citation ), S. douglassii (=S. paradoxus syn. S. douglassii; Tian et al. 1991Citation ), and S. uvarum (=S. bayanus syn. S. uvarum; Cardazzo et al. 1998Citation ) have previously been determined. Apparently, all S. bayanus and S. pastorianus isolates have the same gene order. The gene order of S. paradoxus syn. S. douglassii is the same as that of S. paradoxus. Both of these gene orders, bayanus-like and paradoxus-like, are different from S. cerevisiae only in a limited number of rearrangements. Apparently, in all three types, a majority of genes seem to have orientations similar to that in S. cerevisiae (see the restriction maps in fig. 2 ). Therefore, they are expected to be coded almost entirely on one strand of DNA. However, this observation should be further confirmed by sequencing of the mtDNA molecules.

The sizes of the sensu lato mtDNA molecules are smaller (<50 kb) than in the sensu stricto group. In addition, diversity in size and organization is more extensive than that among the sensu stricto yeasts. While the coding potentials of all Saccharomyces species are likely to be the same, the extent of intergenic sequences in sensu lato yeasts is substantially lower than that in sensu stricto yeasts. The gene order of four sensu lato species is shown in figure 2 , while the gene order of S. exiguus can be found elsewhere (Clark-Walker, McArthur, and Sriprakash 1983Citation ). Only one block, ATP6-ATP8-COX1, seems to be preserved in all sensu lato yeasts, as well as the sensu stricto yeasts (fig. 3). It is likely, therefore, that these three genes were already physically linked in the progenitor mtDNA molecule and possibly also transcribed as a single message. On the other hand, another S. cerevisiae transcriptional unit covering two open reading frames, ATP9-VAR1, is not preserved in sensu lato yeasts. Thus, at least some of the modern transcriptional units originated after separation of the sensu stricto and sensu lato lines. Alternatively, some multigenic transcription units present in progenitor lines may subsequently have been split up during the formation of some new species.

Surprisingly, even if S. servazzii and S. unisporus are not as closely related to each other as, for example, S. bayanus to S. cerevisiae (fig. 1 ), they posses the same gene order. Also, the sizes of both mtDNA molecules (~28 kb) are similar. When these two yeast lines separated in evolution, they started accumulating point mutations (table 3 ), but the overall mtDNA structures have remained very similar (fig. 2 ). Another subgroup, castellii-dairenensis, which is likely to have diverged at approximately the same time as the servazzii-unisporus subgroup, also contains a very similar gene order (fig. 2 ). A majority of genes show the same physical linkage, except the ATP9-COX2 pair, which has inverted position in the two mtDNA molecules (fig. 2 ). However, the sizes of S. castellii (26 kb) and S. dairenensis (48 kb) are significantly different.

Saccharomyces exiguus exhibits a gene order completely different from that of the servazzii-unisporus subgroup, but the size, ~23 kb (Piskur et al. 1998Citation ), is similar. On the other hand, a block of genes, ATP6-ATP8-COX1-COB, is preserved between S. exiguus and the castellii-dairenensis subgroup. The gene order of S. exiguus also shows similarities to that of Candida glabrata (Clark-Walker, McArthur, and Sriprakash 1985Citation ), which has recently been shown to be a likely member of the genus Saccharomyces and a relatively close relative of S. exiguus (Kurtzman and Robnett 1998Citation ). Apparently, if two species are relatively closely related, at least some of the gene order is still preserved within their mtDNA molecules (figs. 1 and 2 ).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
When two yeasts become sexually isolated from one another, they start accumulating different mutations. Point mutations which are selectively neutral are commonly believed to accumulate in a clocklike manner (Kimura 1983Citation ). Based on this assumption, a likely phylogenetic relationship among Saccharomyces nuclear genomes has been deduced by comparison of the rDNA locus sequences (reviewed in Kurtzman 1994Citation ; Kurtzman and Robnett 1998Citation ). However, because of possible horizontal transfer of genetic material among Saccharomyces yeasts (Marinoni et al. 1999Citation ) and because of the very different inheritance patterns of nuclear and mitochondrial DNA (reviewed in Piskur 1995Citation ), the modern mtDNA molecules in diverse species may not have the same evolutionary histories as their nuclear counterparts. Therefore, several Saccharomyces petite-positive yeasts were analyzed for two mitochondrial genes, SSU and ATP9, to determine a likely phylogenetic relationship among their mtDNA molecules (tables 2 and 3 ). This relationship could be converted into a simple timescale, which could be used to determine the timing of evolutionary events other than accumulation of point mutations. A possible evolutionary history of a few Saccharomyces mitochondrial genomes has already been proposed, but it is based on a very limited number of mapped mtDNA molecules (Clark-Walker 1989, 1992Citation ; Cardazzo et al. 1998Citation ). In addition, the mapping of several Dekkera/Brettanomyces/Eeinella mitochondrial DNA molecules provided an idea on the evolution of the mitochondrial genome in these particular yeasts (Hoeben and Clark-Walker 1986Citation ). Here, on the basis of new data, a more likely path of the evolution of present-day Saccharomyces mitochondrial genomes was derived and discussed.

mtDNA molecules of the petite-positive Saccharomyces yeasts fall into two groups: the sensu stricto mtDNA molecules, which are phylogenetically closely related (their size is over 60 kb, and they show similar gene order configurations), and the sensu lato mtDNA molecules, which are smaller in size and show a higher degree of phylogenetic and gene order diversity (table 3 and figs. 1 and 2 ). The large size of the sensu stricto mtDNAs is primarily due to extensive intergenic sequences. Presumably, the sensu stricto progenitor mtDNA molecule, upon separation from the sensu lato mtDNAs, acquired G+C-rich clusters and a relatively large size (Piskur et al. 1998Citation ; fig. 2 ). Afterward, a relatively high number of rearrangements seems to have taken place on a short timescale. When the three modern gene order types are compared (fig. 2 ), it is possible to deduce the progenitor gene order, which, through a minimal number of rearrangements, gives the three modern gene order types (fig. 3 A ). The proposed original gene order type would generate novel types through transposition events. According to this hypothesis, upon sexual isolation of newly arisen yeast lines (bayanus-like, cerevisiae-like, and paradoxus-like) a single jump of a fragment containing one or more genes to the novel locus would have rearranged the common progenitor gene order into the contemporary gene order types (fig. 3A ). The progenitor of the S. cerevisiae–like and S. paradoxus–like lines would initially still contain the sensu stricto progenitor configuration, and the transposition events would first take place after separation of the two lines. The "transposition events" could be a product of illegitimate recombination mediated through GC-rich repeats, and this mechanism could preserve the orientation of the transposed genes.



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Fig. 3.—The origin of the Saccharomyces sensu stricto (A) and sensu lato (B) mitochondrial genome gene order. The sensu stricto progenitor molecule has given novel gene order configurations by a transposition mechanism. The "transposed" genes are underlined. The relationships among some sensu lato mtDNAs suggest that rearrangements have taken place via transpositions and/or inversions. However, note that while transpositions can preserve the orientation of the transposed transcription units, inversions always change it. The genes involved in "transpositions" and/or "inversions" are underlined. Note that ATP6, ATP8, and COX1 genes (shown in red) are physically linked in all examined species

 


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Fig. 3 (Continued)

 
Another interesting observation is that the gene order is preserved within the same species. If two isolates of the same species having different gene orders should mate, a potentially harmful situation arises. Homologous recombination in the zygote would create a number of respiratory-deficient mtDNA molecules, and a large number of the progeny would lack a complete set of mitochondrial genes. Thus, a change in the gene order may be the first step in sexual isolation and the generation of new species.

The situation within the sensu lato group is more complex, and the modern sensu lato yeasts seem to have diverged too much to enable one to characterize a putative progenitor of petite-positive Saccharomyces mtDNA molecules (fig. 2 ). However, among more closely related species pairs, some similarities are apparent and can be used to construct hypothetical evolutionary intermediate mtDNA molecules. For the servazzii-unisporus subgroup, which has small mtDNAs (29 kb), the gene order is the same. Even if the data presented in figure 1 suggest that these two species diversified a relatively long time ago, the mtDNA gene order has been preserved (figs. 2 and 3 ). One explanation could be that if the small size of mtDNA was preserved, accumulation of rearrangements was slow. A similar situation can also be found within the castellii-dairenensis subgroup. These two yeasts apparently diverged from each other at the same time as the S. servazzii and S. unisporus pair (fig. 1 ). A similar gene order has been preserved except for a simple inversion of the COX2 and ATP9 gene pair (figs. 2 and 3 ). In analogy, the small S. castellii mtDNA is likely to have preserved the progenitor gene order, while the larger S. dairenensis has undergone one inversion or, alternatively, a single transposition of ATP9 between the SSU and COX2 genes of the progenitor mtDNA. In any case, it is likely that illegitimate, but not homologous, recombination was responsible for the rearrangement. Another small mtDNA molecule, S. exiguus, shows similarity to S. castellii. In this case, two inversion events, or, alternatively, two transpositions, can rearrange one gene order into the other one. Even if S. exiguus is not a very distant relative of S. servazzii and S. unisporus (fig. 1 ), the gene orders are very different. While rearrangements within the sensu stricto group have taken place using a transposition mechanism, an inversion- or transposition-based strategy could have been employed within the sensu lato group (fig. 3 ). However, because there is a tendency for all yeast mitochondrial transcription units to have the same orientation (Foury et al. 1998Citation ; unpublished data), it is more likely that sensu lato yeasts have also been rearranged using a transposition-based mechanism.

The comparison of S. cerevisiae transcriptional units (reviewed in Grivell 1989Citation ) with the gene maps of other yeasts (fig. 2 ) demonstrates that at least some of S. cerevisiae transcription units are likely to be of recent origin. While the COX1-ATP8-ATP6 block is preserved in all species, and possibly represents an ancient transcriptional unit, another gene pair, ATP9-VAR1, does not represent a common transcriptional unit. Thus, it can be concluded that transcriptional units encompassing several open reading frames do not necessary represent a barrier for genome rearrangements during the process of speciation.

In conclusion, modern Saccharomyces mitochondrial genomes are highly diverse in structure and organization as a result of large rearrangements, accumulation of intergenic sequences, and accumulation of point mutations. At least among some closely related yeasts, it is possible to elucidate the molecular events which took place during evolution and to reconstruct the progenitor molecule.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The authors would like to thank A. Kahn, C. Roos, M. B. Pedersen, and T. Nilsson-Tillgren for their suggestions and interest in this work and C. Kurtzman for providing some of the yeast strains. This research was supported by grants from the Danish Research Council, the Novo Nordisk Foundation, the Carlsberg Foundation, and the Leo Foundation. C. G. and R. F. P. contributed equally to this work.


    Footnotes
 
Pekka Pamilo, Reviewing Editor

1 Present address: Department of Genetics, University of Adelaide, Australia. Back

1 Keywords: evolution gene order mitochondrial genome yeast recombination Back

2 Address for correspondence and reprints: Jure Piskur, Department of Microbiology, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark. E-mail: imjp{at}pop.dtu.dk Back


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 Introduction
 Materials and Methods
 Results
 Discussion
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Accepted for publication August 3, 2000.