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Evolution of linear chromosomes and multipartite genomes in yeast mitochondria.

Valach M, Farkas Z, Fricova D, Kovac J, Brejova B, Vinar T, Pfeiffer I, Kucsera J, Tomaska L, Lang BF, Nosek J - Nucleic Acids Res. (2011)

Bottom Line: Our survey revealed a puzzling variability of genome architecture, including circular- and linear-mapping and multipartite linear forms.We propose that the arrangement of large inverted repeats identified in these genomes plays a crucial role in alterations of their molecular architectures.We suggest that molecular transactions generating linear mitochondrial DNA molecules with defined telomeric structures may parallel the evolutionary emergence of linear chromosomes and multipartite genomes in general and may provide clues for the origin of telomeres and pathways implicated in their maintenance.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Comenius University, Mlynska dolina CH-1, 842 15 Bratislava, Slovak republic.

ABSTRACT
Mitochondrial genome diversity in closely related species provides an excellent platform for investigation of chromosome architecture and its evolution by means of comparative genomics. In this study, we determined the complete mitochondrial DNA sequences of eight Candida species and analyzed their molecular architectures. Our survey revealed a puzzling variability of genome architecture, including circular- and linear-mapping and multipartite linear forms. We propose that the arrangement of large inverted repeats identified in these genomes plays a crucial role in alterations of their molecular architectures. In specific arrangements, the inverted repeats appear to function as resolution elements, allowing genome conversion among different topologies, eventually leading to genome fragmentation into multiple linear DNA molecules. We suggest that molecular transactions generating linear mitochondrial DNA molecules with defined telomeric structures may parallel the evolutionary emergence of linear chromosomes and multipartite genomes in general and may provide clues for the origin of telomeres and pathways implicated in their maintenance.

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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. LIII − RIII − LII − RII (shown in the scheme), LIII − RIII − RII − LII, RIII − LIII − LII − RII and RIII − LIII − RII − LII. ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease (B) or exonuclease III (ExoIII) (C) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. (D) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ32P]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 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.
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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. LIII − RIII − LII − RII (shown in the scheme), LIII − RIII − RII − LII, RIII − LIII − LII − RII and RIII − LIII − RII − LII. ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease (B) or exonuclease III (ExoIII) (C) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. (D) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ32P]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 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.

Mentions: As mentioned above, PFGE analysis revealed the presence of three distinct mtDNA bands in C. labiduridarum and C. frijolesensis. The size of the largest band (chromosome I) corresponds approximately to the sum of the middle (chromosome II) and the smallest (chromosome III) bands (Figure 1, lane 6–7), suggesting that the longest molecules might represent a master chromosome split into two non-identical fragments. Therefore, we analyzed PFGE-separated mtDNA molecules of C. labiduridarum and C. frijolesensis by Southern hybridization using two probes derived from distant regions of chromosome I (Figure 4A). The probe P-668 hybridized with chromosomes I and III and also detected some mtDNA in wells and smears. The pattern detected by the probe H-1030 was similar, except that it hybridized with chromosome II instead of chromosome III. To confirm that all three chromosomes are linear, we treated C. frijolesensis mtDNA with BAL-31 nuclease, exonuclease III, and T4 polynucleotide kinase. We observed that the presumed terminal restriction enzyme fragments were all sensitive to BAL-31 nuclease (Figure 4B). Interestingly, the treatment with exonuclease III revealed two subpopulations of terminal fragments differing in their accessibility to the enzyme activity (Figure 4C). This indicates that the ends of the linear mtDNA molecules might adopt a covalently closed structure such as a t-hairpin, which in a fraction of molecules is opened and thus accessible to exonuclease III and T4 polynucleotide kinase (Figure 4D). Southern hybridization revealed four faint restriction fragments of the mtDNA (designated as LII + LIII, RII + RIII, LII + RIII, LIII + RII) that are refractory to all three enzymes (Figure 4B–D). The fragment sizes correspond to junctions between chromosomes II and III, and their presence shows that the master chromosome occurs in four flip-flop isomers (i.e. LIII − RIII − LII − RII, LIII − RIII − RII − LII, RIII − LIII − LII − RII and RIII − LIII − RII − LII). This suggests that fragmented linear-mapping genomes (i.e. uniform linear mtDNAs with resolved termini corresponding to chromosomes I–III) may coexist with circular-mapping genome forms (i.e. polydisperse linear mtDNAs lacking homogeneous terminal structures that correspond to the smear observed in PFGE).Figure 4.


Evolution of linear chromosomes and multipartite genomes in yeast mitochondria.

Valach M, Farkas Z, Fricova D, Kovac J, Brejova B, Vinar T, Pfeiffer I, Kucsera J, Tomaska L, Lang BF, Nosek J - Nucleic Acids Res. (2011)

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. LIII − RIII − LII − RII (shown in the scheme), LIII − RIII − RII − LII, RIII − LIII − LII − RII and RIII − LIII − RII − LII. ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease (B) or exonuclease III (ExoIII) (C) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. (D) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ32P]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 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.
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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. LIII − RIII − LII − RII (shown in the scheme), LIII − RIII − RII − LII, RIII − LIII − LII − RII and RIII − LIII − RII − LII. ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease (B) or exonuclease III (ExoIII) (C) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. (D) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ32P]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 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.
Mentions: As mentioned above, PFGE analysis revealed the presence of three distinct mtDNA bands in C. labiduridarum and C. frijolesensis. The size of the largest band (chromosome I) corresponds approximately to the sum of the middle (chromosome II) and the smallest (chromosome III) bands (Figure 1, lane 6–7), suggesting that the longest molecules might represent a master chromosome split into two non-identical fragments. Therefore, we analyzed PFGE-separated mtDNA molecules of C. labiduridarum and C. frijolesensis by Southern hybridization using two probes derived from distant regions of chromosome I (Figure 4A). The probe P-668 hybridized with chromosomes I and III and also detected some mtDNA in wells and smears. The pattern detected by the probe H-1030 was similar, except that it hybridized with chromosome II instead of chromosome III. To confirm that all three chromosomes are linear, we treated C. frijolesensis mtDNA with BAL-31 nuclease, exonuclease III, and T4 polynucleotide kinase. We observed that the presumed terminal restriction enzyme fragments were all sensitive to BAL-31 nuclease (Figure 4B). Interestingly, the treatment with exonuclease III revealed two subpopulations of terminal fragments differing in their accessibility to the enzyme activity (Figure 4C). This indicates that the ends of the linear mtDNA molecules might adopt a covalently closed structure such as a t-hairpin, which in a fraction of molecules is opened and thus accessible to exonuclease III and T4 polynucleotide kinase (Figure 4D). Southern hybridization revealed four faint restriction fragments of the mtDNA (designated as LII + LIII, RII + RIII, LII + RIII, LIII + RII) that are refractory to all three enzymes (Figure 4B–D). The fragment sizes correspond to junctions between chromosomes II and III, and their presence shows that the master chromosome occurs in four flip-flop isomers (i.e. LIII − RIII − LII − RII, LIII − RIII − RII − LII, RIII − LIII − LII − RII and RIII − LIII − RII − LII). This suggests that fragmented linear-mapping genomes (i.e. uniform linear mtDNAs with resolved termini corresponding to chromosomes I–III) may coexist with circular-mapping genome forms (i.e. polydisperse linear mtDNAs lacking homogeneous terminal structures that correspond to the smear observed in PFGE).Figure 4.

Bottom Line: Our survey revealed a puzzling variability of genome architecture, including circular- and linear-mapping and multipartite linear forms.We propose that the arrangement of large inverted repeats identified in these genomes plays a crucial role in alterations of their molecular architectures.We suggest that molecular transactions generating linear mitochondrial DNA molecules with defined telomeric structures may parallel the evolutionary emergence of linear chromosomes and multipartite genomes in general and may provide clues for the origin of telomeres and pathways implicated in their maintenance.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Comenius University, Mlynska dolina CH-1, 842 15 Bratislava, Slovak republic.

ABSTRACT
Mitochondrial genome diversity in closely related species provides an excellent platform for investigation of chromosome architecture and its evolution by means of comparative genomics. In this study, we determined the complete mitochondrial DNA sequences of eight Candida species and analyzed their molecular architectures. Our survey revealed a puzzling variability of genome architecture, including circular- and linear-mapping and multipartite linear forms. We propose that the arrangement of large inverted repeats identified in these genomes plays a crucial role in alterations of their molecular architectures. In specific arrangements, the inverted repeats appear to function as resolution elements, allowing genome conversion among different topologies, eventually leading to genome fragmentation into multiple linear DNA molecules. We suggest that molecular transactions generating linear mitochondrial DNA molecules with defined telomeric structures may parallel the evolutionary emergence of linear chromosomes and multipartite genomes in general and may provide clues for the origin of telomeres and pathways implicated in their maintenance.

Show MeSH
Related in: MedlinePlus