1 Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-1, 842 15 Bratislava, Slovak Republic
2 Department of Genetics, Faculty of Natural Sciences, Comenius University, Mlynská dolina CH-1, 842 15 Bratislava, Slovak Republic
3 Institute of Genetics and Microbiology, University of Paris XI, 91 405 Orsay, France
Correspondence
Jozef Nosek
nosek{at}fns.uniba.sk
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ABSTRACT |
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The EMBL/GenBank accession numbers for the sequences reported in this paper are X76196, X76197, X75676, X75681 and AY391843AY391852.
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INTRODUCTION |
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The three groups of C. parapsilosis have been defined without any knowledge regarding structural differences in their mtDNA. In this report, we investigated the variability of mtDNA in the strains from different C. parapsilosis groups and compared their molecular profiles with the closely related species, Lodderomyces elongisporus. The analysis revealed a group-specific pattern at the level of restriction fragments of C. parapsilosis mtDNA. More importantly, the results indicate the striking differences between groups regarding the molecular architecture of the mitochondrial genophore. The presence of variant molecular forms of mtDNA in strains belonging to the same species opens up the unique possibility to analyse the structural differences between linear- and circular-mapping mtDNA, specifically questions concerning the evolutionary origin and replication strategy of linear mitochondrial genomes. Therefore, we developed a strategy for identification of strains with rearrangements in the telomeric region of the mtDNA. This effort uncovered several strains that might be considered as mutants in the mitochondrial telomeres.
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METHODS |
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Comparison of ITS rDNA sequences.
Total cellular DNA was isolated from 5 ml yeast cultures as described by Phillippsen et al. (1991). Subsequently, the ITS region of the rDNA was amplified using ITS oligonucleotide primers (Table 2
) in a reaction with a 10 : 1 mixture of Taq DNA polymerase (Gibco-BRL) and Vent DNA polymerase (New England Biolabs). The resulting PCR products were cloned into the pDrive cloning vector (Qiagen) and their sequences were determined as indicated below.
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PCR analysis.
The analysis was performed as described previously (Nosek et al., 2002) with modifications in the primer pairs (Table 2
). Briefly, amplification reactions (2050 µl) were performed using Taq DNA polymerase (Gibco-BRL) on cell lysates or isolated total cellular DNA (see above). Reactions were done in a thermal cycler (Biometra) using a standard three-step programme: 3 min at 95 °C, followed by 2530 cycles of 45 s at 94 °C, 1 min at 4349 °C and 1 min at 72 °C, with a final step of 3 min at 72 °C. Samples were then separated by agarose gel electrophoresis (1·01·5 %, w/v, agarose containing 0·5 µg ethidium bromide ml1) at 510 V cm1 for 4560 min in 45 mM Tris/borate1 mM EDTA buffer.
Miscellaneous.
Enzymic DNA manipulations, Southern blot analysis and cloning procedures were performed as described by Sambrook & Russell (2001). The DNA sequence was determined using the BigDye terminator sequencing kit and an ABI 310 automatic analyser (Applied Biosystems). Sequence assembly and analysis was performed using the Vector NTI package (Informax) and BLAST (http://www.ncbi.nlm.nih.gov/blast/). The sequence alignments and tree calculation were done using the AlignX program (Informax).
Reproducibility of results.
All PCR and PFGE analyses were repeated at least twice with the same results.
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RESULTS |
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To study the genetic organization of mtDNA in strains from groups II and III, the plasmid libraries of EcoRV and HindIII fragments of mtDNA from strains MCO 456 and MCO 448 were constructed. Next, the gene contents in both mtDNAs were inferred by sequencing the termini of the restriction fragments inserted within the plasmid vector. The data were then compared with the GenBank database as well as with the complete sequence of mtDNA of the group I strain CBS 7157 (Nosek & Fukuhara, 1994a, b
; Nosek et al., 1995
; J. Nosek, M. Novotna, Z. Hlavatovicova, D. W. Ussery, J. Fajkus & L. Tomaska, unpublished). This approach led to the identification of nad1-5, cytb, atp6 and rrnL genes in MCO 456 and nad1-5, cytb, cox1, cox3, atp6 and rrnL in MCO 448. Subsequently, these sequences were localized on the restriction enzyme maps of both mtDNAs. The results indicate that mitochondrial genomes in group II and III strains have the same gene order as found in group I strains (Fig. 4
). In several cases the coding regions were fully sequenced and the data show a significant degree of homology when compared with their counterparts from group I (Table 3
).
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Mitochondrial telomeres differ between strains belonging to the variant groups
DNA sequence analysis of terminal regions uncovered that linear mitochondrial genomes from strains in groups I (CBS 7157) and III (MCO 448) differ in the sequence of their telomeric regions. While the mitochondrial telomeres in the strain belonging to group I consist of arrays of tandem repeats of a 738 bp unit (Nosek et al., 1995), the telomeric repeat in the group III strain is remarkably shorter (620 bp). However, the two sequences possess highly conserved regions and overall identity is 72 % (for details see the GenBank/EMBL database entries AY391851, AY391852, X76196 and X76197).
Survey of C. parapsilosis strains
To analyse the frequency of linear- and circular-mapping mtDNA in this species, we employed the PFGE method for screening of linear mitochondrial genomes (Fukuhara et al., 1993). This approach revealed a distinct band corresponding to linear mtDNA molecules in almost all strains from groups I and III, but only in one group II strain (MCO 471; Table 1
).
Mitochondria of strain CBS 7157 harbour extragenomic minicircular DNA molecules derived solely from the mitochondrial telomere repeat motif that seem to play a key role in the dynamics of the linear mtDNA terminal structures (Tomaska et al., 2000; Nosek & Tomaska, 2002
). Therefore, we examined their occurrence in strains from the three groups by probing Southern blots of electrophoretically separated undigested total cellular DNA with an [
32P]dCTP-labelled telomeric probe. The results show that the presence of the minicircles (Fig. 5
) correlate with the linear form of mtDNA (Table 1
), supporting the hypothesis that minicircles are involved in mitochondrial telomere maintenance.
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Restriction enzyme mapping using EcoRV and HindIII localized the polymorphic region between genes atp6 and nad3 (Fig. 7b). The 1·1 kbp HindIII fragment of MCO 456 varies in size from 0·45 to 1·3 kbp in strains MCO 457, MCO 462 and PL 452. Interestingly, four non-stoichiometric bands produced by HindIII (0·9, 1·0, 1·2 and 1·3 kbp) that map in this region were observed in strain MCO 462, indicating either instability of this region or a heteroplasmic state of the cells (Fig. 7a
, lane 3). Furthermore, the circular-mapping mtDNA of strain PL 448 lacks telomeres and possesses a restriction fragment corresponding to the fusion of the left and right termini (Fig. 7c
). This indicates that, similar to group II strains, mitochondrial genomes identified in group III strains have identical genetic organization and differ only in their linear/circular state.
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DISCUSSION |
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Evolutionary implications
The existence of linear genophores in mitochondria of diverse phylogenetic taxa evokes questions concerning their evolutionary origin and relatedness to circular-mapping mtDNAs. In several cases it has been observed that the form of mitochondrial genome may differ among closely related species (Bridge et al., 1992; Fukuhara et al., 1993
; Martin, 1995
; Laflamme & Lee, 2003
). Investigations of mtDNA-binding proteins indicate that similar machineries are responsible for the maintenance of both forms (Miyakawa et al., 1995
, 1996
; Nosek et al., 1999
; Kaufman et al., 2000
; Tomaska et al., 2001
). The occurrence of linear- and circular-mapping genomes in different strains of the same species indicates that both types of mitochondrial genome do not differ radically in their life styles (Fukuhara et al., 1993
). One such example has been reported in two strains of the saturn-spored yeast, Williopsis suaveolens. While mitochondria of strain CBS 1670 with a linear-mapping genome harbour mtDNA molecules terminating with inverted repeats closed by a single-stranded hairpin loop, strain CBS 255 possesses a mitochondrial genome with similar genetic organization except that its map is circular (Fukuhara et al., 1993
; Drissi et al., 1994
). Here we report another example of intraspecies variability in mtDNA architecture in several strains of C. parapsilosis. The structure of the linear mitochondrial genome found in this species substantially differs in the type of mitochondrial telomere from that of W. suaveolens (Nosek et al., 1995
). While nearly all strains in C. parapsilosis groups I and III possess a linear mtDNA that terminates with arrays of tandem repeats, only one group II isolate (MCO 471) has a linear genome in its mitochondria. However, most of the strains in group II and PL 448 from group III display a circular map of their mtDNAs. Moreover, the linear mtDNAs in groups I and III differ in the sequences of their telomeric motifs.
The genetic organization and homology of the coding regions appear to be highly conserved among linear- and circular-mapping mitochondrial genomes found in C. parapsilosis strains, indicating that both forms originated from a common ancestor via a relatively simple mechanism. At present it is not possible to make any definitive conclusion as to whether a circular-mapping mtDNA represents either an ancestral form or a rearranged derivative (mutant) that lost the mitochondrial telomeres and solved the end-replication problem (Olovnikov, 1971,1973
; Watson, 1972
) by circularization of the genophore, similar to bacteriophage
(reviewed by Taylor & Wegrzyn, 1995
) or nuclear chromosomes in the telomerase-deficient mutant of the fission yeast Schizosaccharomyces pombe (Nakamura et al., 1998
).
Mitochondrial telomeres play essentially the same biological role(s) as their nuclear counterparts, i.e. they have to (i) ensure the complete replication of the linear genophore, (ii) mask the ends from DNA repair machinery and (iii) protect them from exonucleolytic degradation and/or end-to-end fusions. Identification of a specific mitochondrial telomere-binding protein (mtTBP) (Tomaska et al., 1997; Nosek et al., 1999
) and mitochondrial telomeric loop (t-loop) structures (Tomaska et al., 2002
) similar to the t-loops present at the ends of mammalian chromosomes (Griffith et al., 1999
) substantiates the analogy. Observation of extragenomic telomeric minicircles in mitochondria of several yeast species (Tomaska et al., 2000
) uncovered yet another general theme in telomere biology. Recently, we have demonstrated that the minicircles replicate via a rolling-circle mechanism which generates tandem arrays of the telomeric sequence that may recombine with linear molecules of mtDNA to lengthen the telomeres (L. Tomaska, A. M. Makhov, J. D. Griffith & J. Nosek, unpublished). This may parallel alternative mechanisms of nuclear telomere maintenance involving telomeric small polydisperse circular DNAs detected in several telomerase-negative tumour cell lines (Regev et al., 1998
) and Xenopus oocytes (Cohen & Mechali, 2002
), or an elongation of telomeres by recombination with DNA circles as demonstrated in the yeast Kluyveromyces lactis (Natarajan & McEachern, 2002
).
Recently, we proposed a hypothesis that mitochondrial telomeres might have evolved from mobile genetic elements (e.g. transposons, plasmids, telomeric minicircles) that invaded mitochondria, integrated into a circular-mapping mtDNA and eventually resulted in the formation of linear mtDNA molecules of defined length, terminating with specific telomeric structures (Nosek & Tomaska, 2002, 2003
). Insertions and excisions of such elements may represent a simple mechanism for switching between the two forms of the mitochondrial genophore. Some support for this idea comes from the correlation between the occurrence of the linear-/circular-mapping genome and the presence/absence of extragenomic telomeric minicircles (Fig. 5
, Table 1
). One possibility, although speculative, is that some drugs might interfere with the replication of mitochondrial telomeres and/or telomeric minicircles thus selecting for the formation of a circular-mapping genome without any apparent changes in mitochondrial physiology and/or maintenance of the mtDNA. Hence, the variability in the telomeric region might be generated by antifungal treatment of patients infected with C. parapsilosis. The identification of a collection of strains with altered mtDNA architecture among C. parapsilosis isolates opens the unique possibility of uncovering molecular mechanism(s) that trigger the alteration of the mtDNA form and may be instrumental in experimental testing of the above hypotheses.
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ACKNOWLEDGEMENTS |
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Received 12 December 2003;
revised 14 January 2004;
accepted 15 January 2004.