Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada
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Abstract |
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Introduction |
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To date, mitochondrial genomes from eight taxa representing diverse lineages of green algae (Chlorophyta sensu Sluiman 1985
) have been sequenced to completion (reviewed by Lang, Gray, and Burger 1999
; Turmel, Otis, and Lemieux 2002
). Seven of these taxa have circular mapping mtDNAs, whereas the remaining taxon Chlamydomonas reinhardtii, a member of the "Volvox clade" (sensu Nakayama et al. 1996
) of the class Chlorophyceae (sensu Mattox and Stewart 1984
), has a linear 15.8-kb mtDNA with a 580- or 581-bp sequence at one terminus that is repeated in an inverted orientation at the other terminus (Vahrenholz et al. 1993
). Mitochondrial genomes from other members of the Volvox clade that have been characterized by gel electrophoresis are also linear mtDNAs (Moore and Coleman 1989
; unpublished data). To date, there is no evidence among the green algae of a mitochondrial gene being associated with a subgenomic mtDNA such as found in some lineages outside of this group.
The genus Polytomella is composed of a morphologically and physiologically homogeneous group of colorless and wall-less unicells (Pringsheim 1955
) which appear to have arisen from a green ancestor within the Volvox clade (Nakayama et al. 1996
) of chlorophycean green algae. The absence of both a cell wall and thylakoid membranes in Polytomella has facilitated the purification of mitochondrial respiratory proteins and therefore made this an attractive taxon for mitochondrial studies (Gutiérrez-Cirlos et al. 1994
; Atteia, Dreyfus, and González-Halphen 1997
). Sequences have been reported for the mitochondrial genes, cox1 and cob, from Polytomella strain SAG 198.80 (Antaramian et al. 1996
, 1998
).
In this study we describe two linear mtDNA components of P. parva, a 13.5-kb mtDNA which contains most of the standard mitochondrial-coding sequences present in C. reinhardtii mtDNA and a 3.5-kb mtDNA which contains only one gene, nad6, a gene which is absent from the 13.5-kb mtDNA. Both DNA components contain long-terminal inverted repeat sequences which are almost identical between the 13.5- and 3.5-kb linear mtDNAs but show no similarity with the terminal inverted repeat sequences in the linear mtDNA of C. reinhardtii. This is the first report of subgenomic mtDNA in green algae.
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Materials and Methods |
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The isolation of DNA followed the method of Ryan et al. (1978) with the following exceptions. Whole cell and mitochondrial-enriched pellets were lysed in 2% sarkosyl, 1% SDS, 1 mg/ml proteinase K (Boehringer Mannheim) at 50°C for 1 h. After the RNase treatment step and the final extraction with chloroform-isoamyl alcohol, some remaining non-DNA materials were removed by precipitation at room temperature in the presence of 2.5 M ammonium acetate. DNA was then precipitated twice with ethanol and redissolved in 10 mM Tris-HCl (pH 8.0)-1 mM EDTA. There was no further DNA purification step employing preparative CsCl gradient centrifugation as described by Ryan et al. (1978).
DNA Amplification
PCR experiments were performed in a thermal cycler (Geneamp PCR System 2400, Perkin-Elmer) using total cellular DNA as the template and reagents from MBI Fermentas. DNA was initially denatured at 94°C for 3 min and amplified by 40 cycles, each involving denaturation at 94°C for 45 s, annealing at 50°C for 30 s, and extension at 72°C for 2 min; there was a final extension period at 72°C for 7 min. PCR products were purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Pharmacia Biotech).
DNA Transfer and Southern Blot Hybridization
After fractionation by agarose (1%) gel electrophoresis (6 V/cm), DNA was transferred to a nylon membrane (Hybond N+, Amersham Pharmacia Biotech) using the capillary transfer method (Sambrook, Fritsch, and Maniatis 1989
, pp. 9.349.35) with 0.5 M NaOH as the transfer solution. The Alkphos Direct Labelling and Detection System kit (Amersham Pharmacia Biotech) was used for DNA probe labeling, and the subsequent hybridization followed the recommended protocol, except that hybridization was carried out overnight at 60°C. Chemiluminescent detection was achieved by exposing the autoradiographic film to the membrane. For reprobing, the membrane was stripped in 0.5% SDS and checked for completeness of signal removal.
MtDNA Cloning
DNA isolated from a mitochondrial-enriched preparation was fractionated by agarose (1%) gel electrophoresis. After being stained with ethidium bromide, the 13.5- and 3.5-kb bands, presumed to be mtDNAs, were cut from the gel, and the DNA was recovered using the GFX PCR DNA and Gel Band Purification kit (Amersham Pharmacia Biotech). The recovered 13.5 and 3.5 kb DNAs were digested with HindIII and EcoRI, respectively, and then ligated into the HindIII or EcoRI site of the vector pBluescript II SK+ (Stratagene). The ligation mixture was used to transform Escherichia coli strain XL1-Blue MCF'(Stratagene). Recombinant plasmids were extracted from the host cells by the alkaline lysis preparation method (Sambrook, Fritsch, and Maniatis 1989
, pp. 1.251.28), and the recombinant plasmids containing inserts of the 13.5- and 3.5-kb mtDNAs were identified by Southern blot hybridization using these DNAs as probes.
DNA Sequencing
Cloned, and in some cases PCR amplified, mtDNA segments were sequenced commercially (Dalhousie UniversityNRC Institute for Marine Biosciences Joint Laboratory, Halifax, or Center for Applied Genomics, Hospital for Sick Children, Toronto) on both strands using LICOR 4200 (LICOR; dye primers) or ABI 373 or 377 (PE-Applied Biosystems; dye terminators) automated DNA sequencers. The sequence of DNA amplified by PCR was obtained with the PCR product or two independent clones of the product.
Data Analysis
The BLAST network services (Altschul et al. 1990
) provided at the National Center for Biotechnology Information were used for sequence similarity searches. The program Gene Runner (Hastings Software) was used for sequence editing and compiling. Multiple DNA and protein sequence alignments were performed using the program CLUSTAL W, version 1.7 (Thompson, Higgins, and Gibson 1994
). The program RRTree, version 1.1.10 (Robinson-Rechavi and Huchon 2000
) was employed for nucleotide substitution analyses and relative rate tests.
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Results |
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Polytomella parva 13.5-kb mtDNA shares the feature of discontinuous and scrambled LSU and SSU rRNA coding regions with all other well characterized chlorophycean green algal mtDNAs (Nedelcu et al. 1996
, 2000
and references therein). In the case of P. parva, the LSU and SSU rRNA genes are disrupted into at least eight (rnl_a through _h) and four (rns_a through _d) modules, respectively (table 1
and fig. 2
). Interestingly, rns_a and rnl_g are located transcriptionally opposite to the rest of the LSU and SSU rDNA modules and thus show a feature not previously reported in the mtDNA of green algae; however, rRNA-coding regions in the apicomplexans Plasmodium and Theileria are distributed on both DNA strands (Feagin 1994
).
Southern blot hybridization experiments of P. parva total cellular and mitochondrial-enriched DNA preparations with P. parva mtDNA probes confirm the homology between the termini of the 13.5- and 3.5-kb mtDNA maps as well as the absence of nad6 from the 13.5-kb mtDNA. The clone containing the 13.5-kb mtDNA fragment H54, which contains cob, rns_a, and part of rnl_g, detected the 13.5- but not the 3.5-kb mtDNA. The clone containing the 3.5-kb mtDNA fragment E12 (fig. 2 ), which contains part of the two inverted repeat regions and nad6, detected both the 13.5- and the 3.5-kb mtDNA components. Finally, the PCR product, derived from nad6, detected the 3.5- but not the 13.5-kb mtDNA (fig. 3 ). The last two probes gave additional discrete signals in the mitochondrial-enriched DNA preparation (and in more exposed blots of total cellular DNA) at positions corresponding to linear DNA molecules of about 2.1 and 1.8 kb. These results, which have been observed consistently with independent DNA samples, imply the existence of additional small mtDNA molecules that harbor nad6 sequence.
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Discussion |
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Examples of subgenomic mtDNAs, most of which are circular mapping, have been described in other eukaryotic lineages, with varying degrees of completeness. Polytomella parva, however, seems to offer the only clear example of a mitochondrial genome containing subgenomic linear DNA molecules that harbor standard mitochondrial-coding regions and that share homologous inverted repeat ends. Certain hydrozoan taxa have been shown to contain two ca. 8-kb linear mtDNA molecules, in contrast to the single 14- to 17-kb linear mtDNA found in most hydrozoans (Warrior and Gall 1985
; Bridge et al. 1992
); nevertheless, except for a 3.2-kb sequence at an end of one of the two linear mtDNAs from Hydra attenuata (Pont-Kingdon et al. 2000
), these genomes are not well characterized.
The gene content and discontinuous structure of the rRNA-coding regions identified in the P. parva mitochondrial genome is typical of the reduced-derived type of mtDNA (Gray, Burger, and Lang 1999
) identified in C. reinhardtii (Boer and Gray 1988a,
Michaelis, Vahrenholz, and Pratje 1990
), Chlorogonium capillatum (= C. elongatum SAG 12-2e) (Kroymann and Zetsche 1998
), and C. moewusii (UTEX 9) (Denovan-Wright, Nedelcu, and Lee 1998
), except for two tRNA genes, trnW(cca) and trnQ(uug), not identified in the P. parva mtDNA; a reverse transcriptaselike coding region (rtl) (Boer and Gray 1988b
), possibly a degenerate group-II intron (Nedelcu and Lee 1998
), so far identified only in the mtDNA of C. reinhardtii has also not yet been detected in the P. parva mtDNA. The missing tRNA-coding regions could not be identified in the sequenced portion of the two P. parva mtDNAs using the program tRNAscan SE 1.21 (Lowe and Eddy 1997
), and there appears to be no remaining space outside of the inverted repeat sequence regions of the 13.5- and 3.5-kb mtDNAs that could accommodate the expected ca. 75 bp coding regions. Moreover, we suggest that the short DNA segments currently unsequenced at each end of the two identified P. parva mtDNAs are also part of the terminal inverted repeats and have no coding function. Although transfer of trnW(caa) and trnQ(uug) to the nucleus in P. parva is possible, an alternative explanation is that they are encoded in one or two additional as yet unidentified mtDNA(s).
On the basis of the available evidence, we cannot decide conclusively at the present time whether or not the 13.5- and 3.5-kb mtDNAs of P. parva replicate autonomously. In our Southern blot hybridization experiments (fig. 3 ), probes specific to the 13.5- or 3.5-kb mtDNAs both revealed signals in the well regions of the gel almost equivalent in intensity to those of the migrating 13.5 and 3.5 kb components. These signals could have resulted from (1) 13.5- and 3.5-kb linear mtDNA molecules that were trapped in the well regions possibly by nuclear DNA or impurities (or both), or (2) one or more larger replicative forms of mtDNA from which the 13.5- and 3.5-kb sequences are normally excised. We favor the former possibility because of our inability to obtain PCR products connecting the 13.5- and 3.5-kb mtDNAs or bridging the ends of each of these DNA components.
If the 13.5- and 3.5-kb linear DNAs are not derived from some larger replicative form(s) and they replicate autonomously as linear molecules, they would require a mechanism to replicate their 5'-ends like any other linear DNA capable of replication. mtDNA telomeres from a variety of organisms have evolved a diversity of mechanisms aimed at solving this problem as revealed by their distinct structures (reviewed by Nosek et al. 1998
). The available information does not enable us to propose a specific telomeric mechanism that might be employed by the mtDNAs of P. parva, and it is unclear as to the possible role in this potential process, if any, that could be played by the direct subrepeat sequences common to the four copies of the inverted repeat sequence. In the absence of sequence at the very termini of the 13.5- and 3.5-kb mtDNAs, we cannot rule out the possibility that these direct subrepeat sequences share sequence identity with the outermost termini of the P. parva mtDNAs and have a role in telomere maintenance, as proposed for the internal 86 bp repeat of the outermost inverted repeat sequence in C. reinhardtii mtDNA (Vahrenholz et al. 1993
; Duby et al. 2001
).
Elevated Evolutionary Rate
On an average, the nonsynonymous substitution rate in the mitochondrial protein-coding genes is about 3.3 times greater in the P. parva lineage compared with the C. reinhardtii lineage. Polytomella parva mitochondrial protein-coding genes, therefore, in terms of nucleotide substitutions that cause amino acid change, seem to be evolving conspicuously faster than those in C. reinhardtii. Interestingly, the same trend is observed in phylogenetic trees based on 18S rDNA sequences (Nakayama et al. 1996
); therefore, this suggests that the higher evolutionary rate is characteristic of the P. parva lineage rather than a particular genetic compartment of the lineage. Such a lineage effect could be explained by (1) a greater number of mutations, potentially because of a greater number of generations, or (2) a higher probability of mutation fixation, possibly because of a smaller population size (Pringsheim 1955
), or both, relative to the C. reinhardtii lineage. It is noteworthy that an accelerated rate of evolution in rRNA genes residing in the nuclear, mitochondrial, and plastid compartments has also been observed in some nonphotosynthetic holoparasitic plants (Wolfe et al. 1992
; Duff and Nickrent 1997
and references therein).
Nonstandard Start Codon
On the basis of the DNA sequence, a nonstandard start codon, GTG, is predicted in nad5 of P. parva 13.5-kb mtDNA. Evidence has been reported that mitochondria in several lineages use nonstandard initiation codons, and in many cases this includes GTG, as, for example, in the protist Tetrahymena pyriformis (Edqvist, Burger, and Gray 2000
). Although there is no previous report for the use of unusual start codons in green algal mitochondria, other nonstandard codons appear to be used (Hayashi-Ishimaru et al. 1996
; Turmel et al. 1999
; Kück, Jekosch, and Holzamer 2000
; Nedelcu et al. 2000
). The possibility that G to A editing could modify the GTG codon to ATG in the nad5 transcript of P. parva has not been formally eliminated; however, this seems unlikely at the present time because RNA editing has not yet been reported in green algal mitochondria, and G to A editing is rare, having only recently been detected in HIV-1 viral transcripts (Bourara, Litvak, and Araya 2000
).
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Keywords: Polytomella parva
green algae
subgenomic mitochondrial DNA
nonstandard start codon
evolutionary rate
Address for correspondence and reprints: Robert W. Lee, Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1. robert.lee{at}dal.ca
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