Department of Microbiology, Technical University of Denmark, Lyngby, Denmark
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Abstract |
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Introduction |
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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 1986a
; Foury et al. 1998
). 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. 1998
). All analyzed S. cerevisiae strains exhibited the same gene order (Dujon 1981
; Evans, Oakley, and Clark-Walker 1985; de Zamaroczy and Bernardi 1986b
; Foury et al. 1998
). 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 1986a
). 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 1979
; Piskur 1995
). Apart from site-specific recombination operating through short repeats (Bernardi 1979
), homologous recombination events also occur with high frequency (Dujon, Slonimski, and Weill 1974
). 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 1988
; Clark-Walker 1989
). These mutants are respiratory-competent, but the mutations are transmitted only poorly to progeny of genetic crosses with the wild-type molecule (Piskur 1989
; Skelly and Clark-Walker 1990
). 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 1995
). 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 1992
), which can be divided into a group of petite-positive and a group of petite-negative yeasts, including S. kluyveri (Piskur et al. 1998
). 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 1998
). 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.
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Materials and Methods |
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Purification of mtDNA and Direct Sequencing
mtDNA was purified by bisbenzimide/CsCl buoyant density centrifugation (Piskur et al. 1998
). The dideoxy sequencing procedure was modified to allow sequencing directly on CsCl-purified mtDNA without subcloning or PCR amplification. The double-stranded mtDNA,
1001,000 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 16301653 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 332 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 12271625) and the open reading frames of ATP9 (positions 1231) genes were aligned using the multiple-sequence alignment program CLUSTAL W (Thompson, Higgins, and Gibson 1994
). 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 1983
). 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 1989
).
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 1989
), 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 1998
; Petersen 1998
). 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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Results |
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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 1983
). 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. 1998
), 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 1985
), 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 1998
). 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
).
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Discussion |
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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. 1998
; 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. cerevisiaelike and S. paradoxuslike 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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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. 1998
; 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 1989
) 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.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Genetics, University of Adelaide, Australia.
1 Keywords: evolution
gene order
mitochondrial genome
yeast
recombination
2 Address for correspondence and reprints: Jure Pikur, Department of Microbiology, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark. E-mail: imjp{at}pop.dtu.dk
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literature cited |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barnett, J. A. 1992. The taxonomy of the genus Saccharomyces Meyen ex Rees: a short review for non-taxonomists. Yeast 8:123
Bernardi, G. 1979. The petite mutation in yeast. Trends Biochem. Sci. 4:197201[ISI]
Cardazzo, B., S. Minuzzo, G. Sartori, A. Grapputo, and G. Carignani. 1998. Evolution of mitochondrial DNA in yeast: gene order and structural organization of the mitochondrial genome of Saccharomyces uvarum. Curr. Genet. 33:5259
Clark-Walker, G. D. 1989. In vivo rearrangements of mitochondrial DNA in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci USA 86:88478851
. 1992. The evolution of mitochondrial genome in fungi. Int. Rev. Cytol. 141:89127[ISI][Medline]
Clark-Walker, G. D., C. R. McArthur, and K. S. Sriprakash. 1983. Order and orientation of genic sequences in circular mitochondrial DNA from Saccharomyces exiguus. J. Mol. Evol. 19:333341[ISI][Medline]
. 1985. Location of transcriptional control signals and transfer RNA sequences in Torulopsis glabrata mitochondrial DNA. EMBO J. 4:465473[Abstract]
de Zamaroczy, M., and G. Bernardi. 1986a. The GC clusters of the mitochondrial genome of yeast and their evolutionary origin. Gene 41:122
. 1986b. The primary structure of the mitochondrial genome of Saccharomyces cerevisiaea review. Gene 47:155177
Dujon, B. 1981. Mitochondrial genetics and functions. Pp. 505635 in J. N. Strathern, E. W. Jones, and J. R. Broach, eds. The molecular biology of the yeast Saccharomyces. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
Dujon, B., P. P. Slonimski, and L. Weill. 1974. Mitochondrial genetics IX: a model for recombination and segregation of mitochondrial genomes in Saccharomyces cerevisiae. Genetics 78:415437
Evans, R. J., K. M. Oakley, and G. D. Clark-Walker. 1985. Elevated levels of petite formation in strains of Saccharomyces cerevisiae restored to respiratory competence. I. Association of both high and moderate frequencies of petite formation with the presence of aberrant mitochondrial DNA. Genetics 111:389402
Felsenstein, J. 1989. PHYLIPphylogeny inference package (version 3.2). Cladistics 5:164166
Foury, F., T. Roganti, N. Lecrenier, and B. Purnelle. 1998. The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae. FEBS Lett. 440:325331
Goffeau, A., B. G. Barell, H. Bussey et al. (15 co-authors). 1996. Life with 6000 genes. Science 274:562567
Grivell, L. A. 1989. Nucleo-mitochondrial interactions in yeast mitochondrial biogenesis. Eur. J. Biochem. 182:477493[ISI][Medline]
Groth, C. 1998. Saccharomyces sensu stricto yeasts. Characterization of mitochondrial DNA. M.Sc. thesis, University of Copenhagen, Copenhagen
Hoeben, P., and G. D. Clark-Walker. 1986. An approach to yeast classification by mapping mitochondrial DNA from Dekkera/Brettanomyces and Eeinella genera. Curr. Genet. 10:371379[ISI][Medline]
Kimura, M. 1983. The neutral theory of molecular evolution. Cambridge University Press, Cambridge, England
Kurtzman, C. P. 1994. Molecular taxonomy of the yeasts. Yeast 10:17271740
Kurtzman, C. P., and C. J. Robnett. 1998. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73:331371
Marinoni, G., M. Manuel, R. F. Petersen, J. Hvidtfeldt, P. Sulo, and J. Piskur. 1999. Horizontal transfer of genetic material among Saccharomyces yeasts. J. Bacteriol. 181:64886496
Petersen, R. F. 1998. Genome dynamics and evolution of the mitochondrial and nuclear genomes in Saccharomyces sensu lato species. Ph.D. thesis, University of Copenhagen, Copenhagen
Piskur, J. 1988. Transmission of yeast mitochondrial loci to progeny is reduced when nearby intergenic regions containing ori/rep sequences are deleted. Mol. Gen. Genet. 214:425432[ISI][Medline]
. 1989. Transmission of the yeast mitochondrial genome to progeny: the impact of intergenic sequences. Mol. Gen. Genet. 218:161168[ISI][Medline]
. 1995. Inheritance of the yeast mitochondrial genome. Plasmid 31:229241
Piskur, J., S. Smole, C. Groth, R. F. Petersen, and M. B. Pedersen. 1998. Structure and genetic stability of mitochondrial genomes vary among yeasts of the genus Saccharomyces. Int. J. Syst. Bacteriol. 48:10151024
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
Skelly, P. J., and G. D. Clark-Walker. 1990. Conversion at large intergenic regions of mitochondrial DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 10:15301537
Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:46734680[Abstract]
Tian, G. L., C. Macadre, A. Kruszewska et al. (11 co-authors). 1991. Incipient mitochondrial evolution in yeasts. I. The physical map and gene order of Saccharomyces douglasii mitochondrial DNA discloses a translocation of a segment of 15,000 base-pairs and the presence of new introns in comparison with Saccharomyces cerevisiae. J. Mol. Biol. 218:735746[ISI][Medline]