Mitochondrial Genome of the Colorless Green Alga Polytomella parva: Two Linear DNA Molecules with Homologous Inverted Repeat Termini

Jinshui Fan and Robert W. Lee

Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Most of the well-characterized mitochondrial genomes from diverse green algal lineages are circular mapping DNA molecules; however, Chlamydomonas reinhardtii has a linear 15.8 kb unit mitochondrial genome with 580 or 581 bp inverted repeat ends. In mitochondrial-enriched fractions prepared from Polytomella parva (=P. agilis), a colorless, naturally wall-less relative of C. reinhardtii, we have detected two linear mitochondrial DNA (mtDNA) components with sizes of 13.5 and 3.5 kb. Sequences spanning 97% and 86% of the 13.5- and 3.5-kb mtDNAs, respectively, reveal that these molecules contain long, at least 1.3 kb, homologous inverted repeat sequences at their termini. The 3.5-kb mtDNA has only one coding region (nad6), the functionality of which is supported by both the relative rate at which it has accumulated nonsynonymous nucleotide substitutions and its absence from the 13.5-kb mtDNA which encodes nine genes (i.e., large and small subunit rRNA [LSU and SSU rRNA] genes, one tRNA gene, and six protein-coding genes). On the basis of DNA sequence data, we propose that a variant start codon, GTG, is utilized by the P. parva 13.5-kb mtDNA-encoded gene, nad5. Using the relative rate test with Chlamydomonas moewusii (=C. eugametos) as the outgroup, we conclude that the nonsynonymous nucleotide substitution rate in the mitochondrial protein-coding genes of P. parva is on an average about 3.3 times that of the C. reinhardtii counterparts.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Mitochondrial genomes in diverse lineages, despite their proposed, shared {alpha}-proteobacterial ancestry, show extensive variability in size and structural organization (Gray, Burger, and Lang 1999Citation ). For example, mitochondrial genomes in land plants encode about 50–70 genes, excluding unique and intron-encoded open reading frames (Lang, Gray, and Burger 1999Citation ; Kubo et al. 2000Citation ), range in size from about 180 to 2,400 kb, and have a very complex and not well understood in vivo structural organization (Bendich 1993Citation ; Backert, Nielsen, and Börner 1997Citation ; Oldenburg and Bendich 2001Citation ), despite the presence of physical maps which suggest that master circular forms can give rise to subgenomic circular forms by intramolecular recombination (Palmer and Shields 1984Citation ; Fauron et al. 1995Citation ; Unseld et al. 1997Citation ). In comparison, mitochondrial genomes in the protozoan genus Plasmodium, the causative agent of malaria, encode only five genes in a 6-kb element which is repeated in variably sized tandem arrays (Feagin 1994Citation ; Wilson and Williamson 1997Citation ). Moreover, examples of subgenomic, presumably autonomously replicating, circular or linear mitochondrial DNA (mtDNA) forms that encode standard mitochondrial genes have been identified in some mesozoan (Watanabe et al. 1999Citation ) and metazoan (Bridge et al. 1992Citation ; Armstrong, Blok, and Phillips 2000Citation ; Pont-Kingdon et al. 2000Citation ) animals. These mtDNAs differ from the special classes of small circular mtDNA molecules found in the single mitochondrion (kinetoplast) of trypanosomal protozoa that encode only guide RNAs used to edit transcripts produced by the main mtDNA (Shapiro and Englund 1995Citation ) and the senDNA or other subgenomic circular mtDNA forms associated with senescent or particular mutant strains of filamentous fungi, respectively (Griffiths 1992Citation ).

To date, mitochondrial genomes from eight taxa representing diverse lineages of green algae (Chlorophyta sensu Sluiman 1985Citation ) have been sequenced to completion (reviewed by Lang, Gray, and Burger 1999Citation ; Turmel, Otis, and Lemieux 2002Citation ). Seven of these taxa have circular mapping mtDNAs, whereas the remaining taxon Chlamydomonas reinhardtii, a member of the "Volvox clade" (sensu Nakayama et al. 1996Citation ) of the class Chlorophyceae (sensu Mattox and Stewart 1984Citation ), 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. 1993Citation ). Mitochondrial genomes from other members of the Volvox clade that have been characterized by gel electrophoresis are also linear mtDNAs (Moore and Coleman 1989Citation ; 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 1955Citation ) which appear to have arisen from a green ancestor within the Volvox clade (Nakayama et al. 1996Citation ) 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. 1994Citation ; Atteia, Dreyfus, and González-Halphen 1997Citation ). Sequences have been reported for the mitochondrial genes, cox1 and cob, from Polytomella strain SAG 198.80 (Antaramian et al. 1996Citation , 1998Citation ).

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.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Strain, Culture Conditions, Mitochondrial Isolation, and DNA Isolation
Polytomella parva (UTEX L 193) was obtained from the University of Texas at Austin culture collection and routinely checked to ensure the absence of microbial contaminants. Cells were cultured at 25°C in the medium of Sheeler, Cantor, and Moore (1968), with shaking for small cultures (100–250 ml) or mild aeration for larger cultures (5–15 liter) and harvested in the late logarithmic phase of growth (OD750 nm = 0.45) by centrifugation (2,000g) at 4°C. Mitochondrial-enriched fractions were prepared and treated with DNase I (code DPRF, Worthington) following procedure B of Ryan et al. (1978).

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 1989Citation , pp. 9.34–9.35) 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 1989Citation , pp. 1.25–1.28), 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 University—NRC 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. 1990Citation ) 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 1994Citation ). The program RRTree, version 1.1.10 (Robinson-Rechavi and Huchon 2000Citation ) was employed for nucleotide substitution analyses and relative rate tests.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Identification and General Features of the Two MtDNA Components
After fractionation by agarose gel electrophoresis, DNA from the mitochondrial-enriched fraction of P. parva revealed two prominent components that were barely visible or not visible, respectively, in the total cellular DNA preparation. The two components consistently corresponded to sizes of 13.5 and 3.5 kb, relative to linear DNA size markers when the concentration of agarose was either 1% (fig. 1 ) or 0.6% (data not shown). These results support the linear conformation of the two DNA species (Johnson and Grossman 1977Citation ). By means of genomic DNA cloning, and in some cases PCR amplification, DNA segments collectively spanning 97% and 86% of the 13.5- and 3.5-kb DNAs, respectively, were recovered and sequenced, thereby yielding two partial physical and gene maps (fig. 2 ). The inverted repeat structure of the termini of the two partial maps, together with our inability to recover either clones or PCR products bridging the ends of the 13.5- or 3.5-kb molecules, further argue that these DNAs are linear molecules with unique ends. Considering the source of the two DNAs and the coding regions they contain, it is concluded that the 13.5- and 3.5-kb DNAs are components of the P. parva mitochondrial genome.



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Fig. 1.—Agarose (1%) gel electrophoresis of P. parva DNA isolated from total cellular (T) and mitochondrial-enriched (M) fractions. The DNA sizes indicated are based on lambda DNA HindIII and BstEII fragments (MBI Fermentas)

 


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Fig. 2.—Partial physical and gene maps of P. parva 13.5- and 3.5-kb mtDNAs based on sequences obtained from 13,135 bp of the 13.5-kb mtDNA and 3,018 bp of the 3.5-kb mtDNA. Most of these sequences were obtained from cloned HindIII or EcoRI restriction fragments of the two mtDNAs, indicated above and below the respective maps. The specific linkage of H54, H11, H50, and H24 in the 13.5-kb mtDNA is based on gene continuity between the fragments. The sequences of the two internal uncloned regions of the 13.5-kb mtDNA were obtained from PCR products that were produced with primers designed from the flanking cloned fragments. The sequences flanking the left terminus of the 13.5-kb mtDNA and the two termini of the 3.5-kb mtDNA were obtained from PCR products produced with primer pairs, including in each case an outside primer designed from the outermost region of fragment H51 and an inner sequence of the closest cloned region. For gene abbreviations see table 1 . Half arrows indicate directions of gene transcription. Thick solid arrows near the ends of the maps denote terminal inverted repeats; the two flags within these regions represent two direct subrepeats. Shading depicts the homologous feature of the repeat sequences between the two mtDNAs. Dashed arrows at the very ends of the maps represent the predicted and unsequenced termini of the 13.5- and 3.5-kb mtDNAs. Restriction sites shown are: A, AvaI; B, BglI; E, EcoRI; H, HindIII; S, SalI

 
Coding regions identified in the two P. parva mtDNAs include seven respiratory chain protein–coding genes, one tRNA gene, and LSU and SSU rRNA genes (table 1 ). All these coding regions are in the 13.5-kb mtDNA, except for nad6, which is in the 3.5-kb mtDNA. As in the mtDNA of C. reinhardtii (reviewed by Michaelis, Vahrenholz, and Pratje 1990Citation ), the coding regions in the P. parva 13.5-kb mtDNA are compactly organized, intron-free, and arranged into two unequally sized clusters, one of which is in the opposite transcriptional orientation from the other.


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Table 1 Coding Regionsa Identified in the Mitochondrial Genome of P. parva

 
Although most of the protein-coding genes in the two identified mtDNAs of P. parva appear to have a standard ATG start codon, we infer from the DNA sequence data that mitochondria of this taxon utilize an unusual start codon for nad5. Two initiation codons have been proposed for C. reinhardtii nad5, with the one of Boer and Gray (1986)Citation being 63 nucleotides downstream of the one proposed by Vahrenholz et al. (1985)Citation . Pairwise alignment of the derived amino acid sequences of P. parva and C. reinhardtii nad5 (data not shown) is consistent only with the downstream start codon position of this gene in C. reinhardtii; however, the P. parva gene revealed a GTG rather than an ATG codon at the corresponding position.

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. 1996Citation , 2000Citation 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 1994Citation ).

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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Fig. 3.—Southern blot hybridization analysis of the P. parva 13.5- and 3.5-kb mtDNAs. A, ethidium bromide staining pattern and B, C, and D, Southern blot analysis with clone H54, clone E12, and a PCR product of nad6, respectively. DNA markers derived from plasmid digests (MBI Fermentas) (lane 1), total cellular DNA (lane 2), and DNA extracted from a mitochondrial-enriched pellet (lane 3) were fractionated by agarose (1%) gel electrophoresis. Homology between the vector sequence of clones H54 and E12 and the plasmid-derived DNA markers accounts for the hybridization signals associated with these markers in B and C

 
Flanking Sequences of the 13.5- and 3.5-kb MtDNAs
The alignment of the available terminal sequences derived from the 13.5- and 3.5-kb mtDNAs reveals a homologous inverted repeat sequence of almost 1.3 kb in the two DNAs (fig. 4 ). The left and right repeat sequences in the 13.5-kb mtDNA and the right repeat sequence in the 3.5-kb mtDNA start immediately downstream of cob, nad1, and nad6, respectively. The left repeat of the 3.5-kb mtDNA starts 43 bp upstream of nad6, and a stem-loop structure (not shown) can be modeled from this 43 bp sequence. The four copies of the repeat sequence show only occasional differences in sequence, and a 44-bp sequence present in the right repeat of the 13.5-kb mtDNA is missing from the other three copies of the repeat. It is noteworthy that single copies of the 7-bp sequence 5'-TGCGCAC-3' are located at one end of and immediately following the other end of this extra 44 bp sequence, thereby suggesting its loss from the remaining three terminal repeat regions by unequal crossing over, intrastrand deletion, or slipped-strand mispairing (Graur and Li 2000, pp. 32–35). Interestingly, all four copies of the terminal repeat sequence contain two copies of a 42-bp direct subrepeat which are separated from each other by 197 bp. No open reading frame having a potential coding capacity of more than 70 amino acids was detected in the sequenced part of the inverted repeat regions. After BLAST searches, no sequence in any of these regions was found to be significantly similar to any sequence in the GenBank at the level of either protein or DNA.



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Fig. 4.—Multiple alignment of the available flanking sequences in the 13.5- and 3.5-kb mtDNAs of P. parva. Sequences begin immediately (a) downstream of cob in the 13.5-kb mtDNA, (b) downstream of nad1 in the 13.5-kb mtDNA, (c) upstream of nad6 in the 3.5-kb mtDNA, and (d) downstream of nad6 in the 3.5-kb mtDNA. A 43-bp sequence immediately upstream of nad6, that is missing from the other three repeat regions, can be modeled into a stem-loop structure. A 44-bp sequence is present in the repeat region downstream of nad1, that is missing from the other three repeat regions; single copies of a 7-bp sequence located at one end of and immediately following the other end of this extra 44 bp sequence are underlined. Shaded areas indicate two direct repeats within each copy of the inverted repeat sequence. Asterisks indicate positions in which an identical nucleotide appears in all the four sequences

 
Evolutionary Rate Analysis
Nucleotide substitution levels for seven protein-coding genes encoded in mtDNA were estimated for all pairwise comparisons between homologs of P. parva, C. reinhardtii, and Chlamydomonas moewusii (=C. eugametos UTEX 9). Levels of synonymous substitution were saturated between all homologous gene sequences and therefore could not be calculated. Differences in the number of nonsynonymous substitutions were estimated (table 2 ), and these were used to calculate the rate of nonsynonymous substitution between homologous mitochondrial genes in the P. parva lineage relative to the C. reinhardtii lineage using C. moewusii as the outgroup (Buchheim et al. 1996Citation ; Nakayama et al. 1996Citation ). The value of K13 (number of nonsynonymous substitutions between P. parva and C. moewusii) - K23 (number of nonsynonymous substitutions between C. reinhardtii and C. moewusii), for each of the mitochondrial genes compared, is consistently positive and more than five times the standard error, indicating that the nonsynonymous substitution rate difference between the P. parva and C. reinhardtii lineages for these genes is highly significant. The nonsynonymous substitution rate of the protein-coding genes in the P. parva lineage averages about 3.3 times that of the homologs in the C. reinhardtii lineage, with the lowest value being 2.5 times for cob and the highest value being 4.4 times for nad1. Interestingly, nad6, the only gene identified in the 3.5-kb mtDNA, has a nonsynonymous substitution rate ratio of 2.8 between the two lineages which is not remarkable compared with the other mitochondrial genes characterized.


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Table 2 Differences in the Number of Nonsynonymous Substitutions per 100 Sites and the Relative Rates of Nonsynonymous Substitutions in Mitochondrial Genes Between P. parva (species 1) and C. reinhardtii (species 2) with C. moewusii (species 3) as a Reference

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
Genome Structure
We have identified and partially characterized two linear mtDNA components from P. parva which have sizes of 13.5 and 3.5 kb. This represents the first description of subgenomic mtDNAs from a green alga. The standard, apparently required gene, nad6, is the only gene identified in the 3.5-kb mtDNA; the absence of nad6 from the 13.5-kb mtDNA argues for the function of this DNA. Moreover, for nad6, the ratio of the number of nonsynonymous substitutions in the P. parva lineage to that in the C. reinhardtii lineage is within the range of the other mtDNA-encoded genes, thus supporting the view that nad6 is under normal evolutionary constraints and therefore functional. A potential stem-loop structure in the region immediately upstream of nad6 might play some role in the initiation of nad6 transcription because similar potential structures have been identified upstream of genes in the minicircular mtDNAs of the mesozoan animal Dicyema misakiense (Watanabe et al. 1999Citation ). Interestingly, no similar potential structure was identified in the 13.5-kb mtDNA molecule of P. parva. We did, however, identify two potential promoter sequences for bidirectional transcription initiation between rnl_g and cox1 in P. parva 13.5-kb mtDNA. One, 5'-ATATTCTTA-3', is located nine nucleotides upstream of cox1 and the other, 5'-GTATTGCTG-3', is located five nucleotides upstream of rnl_g. These sequences show similarity with the consensus promoter sequence in the mtDNA of fungi (Tracy and Stern 1995Citation ) as well as the potential promoters identified upstream of cox1 and nad5 (Duby et al. 2001Citation ) in the region of bidirectional transcription initiation proposed for C. reinhardtii mtDNA (Gray and Boer 1988Citation ).

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 1985Citation ; Bridge et al. 1992Citation ); 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. 2000Citation ), 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 1999Citation ) identified in C. reinhardtii (Boer and Gray 1988a,Citation Michaelis, Vahrenholz, and Pratje 1990Citation ), Chlorogonium capillatum (= C. elongatum SAG 12-2e) (Kroymann and Zetsche 1998Citation ), and C. moewusii (UTEX 9) (Denovan-Wright, Nedelcu, and Lee 1998Citation ), except for two tRNA genes, trnW(cca) and trnQ(uug), not identified in the P. parva mtDNA; a reverse transcriptase–like coding region (rtl) (Boer and Gray 1988bCitation ), possibly a degenerate group-II intron (Nedelcu and Lee 1998Citation ), 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 1997Citation ), 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. 1998Citation ). 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. 1993Citation ; Duby et al. 2001Citation ).

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. 1996Citation ); 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 1955Citation ), 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. 1992Citation ; Duff and Nickrent 1997Citation 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 2000Citation ). 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. 1996Citation ; Turmel et al. 1999Citation ; Kück, Jekosch, and Holzamer 2000Citation ; Nedelcu et al. 2000Citation ). 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 2000Citation ).


    Supplementary Material
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
The partial sequences of the P. parva 13.5- and 3.5-kb mtDNAs are registered under GenBank accession numbers AY062933 and AY062934, respectively.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 
We thank Murray Schnare for suggestions, assistance with experiments, encouragement throughout the course of this work, and for his detailed editing of an earlier version of this paper. We also acknowledge the helpful discussions with Gertraud Burger, Michael Gray, Mark Laflamme, and Michael Reith, and the preparation of figures by Mark Laflamme. This work was supported by a research grant to R.W.L. from the Natural Sciences and Engineering Research Council of Canada. Partial financial support was provided to J.F. by Dalhousie University and Patrick Lett Scholarships.


    Footnotes
 
Ken Wolfe, Reviewing Editor

Keywords: Polytomella parva green algae subgenomic mitochondrial DNA nonstandard start codon evolutionary rate Back

Address for correspondence and reprints: Robert W. Lee, Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1. robert.lee{at}dal.ca Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Supplementary Material
 Acknowledgements
 References
 

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Accepted for publication January 24, 2002.