No abstract

Analysis of mitochondrial (mt)DNA size polymorphism in the form of variable number tandem repeats (mtVNTRs) has become an increasingly popular methodology for addressing questions in molecular ecology. When detected by PCR, mtVNTR analysis can provide a sensitive, rapid, and cost-effective measure of genetic variability that may be exploited in studies of population differentiation and biogeography. Despite the emergence of this approach, there has been little critical evaluation of its success or utility as a practical tool. In this review, we identify problematic methodological, theoretical and interpretive factors that can influence the utility of mtVNTR analysis. The reliability of the procedure is considered in terms of both detection of alleles and scoring of intra-individual allele frequencies. While many of the potential technical problems of the technique do not raise serious practical concerns, this rapid and sensitive methodology is seriously compromised by the difficulty of reliably assessing allele frequencies, of assaying only germline tissue, and in our ignorance of the mechanisms generating mtVNTR diversity. Thus, although there is a considerable potential for mtVNTR pilot studies to assess genetic diversity, the utility of the technique to resolve broader questions in molecular ecology should be treated cautiously until such a time as the system is better understood.

Rare syntenic conservation, sequence duplication, and the use of both DNA strands to encode genes are signature architectural features defining mitochondrial genomes of enoplean nematodes. These characteristics stand in contrast to the more conserved mitochondrial genome sizes and transcriptional organizations of mitochondrial DNAs (mtDNAs) derived from chromadorean nematodes. To address the frequency of gene rearrangement within nematode mitochondrial DNA (mtDNA), mitochondrial genome variation has been characterized within a more confined enoplean taxonomic unit, the family Mermithidae. The complete nucleotide sequences of the mosquito parasitic nematodes Romanomermis culicivorax, R. nielseni, and R. iyengari mtDNA have been determined. Duplicated expanses encompassing different regions of the mitochondrial genomes were found in each of these congeners. These mtDNA shared few rRNA and protein gene junctions, indicating extensive gene rearrangement within the Romanomermis lineage. Rapid structural changes are also observed at the conspecific level where no two individual nematodes carry the same haplotype. Rolling circle amplification was used to isolate complete mitochondrial genomes from individuals in local populations of Thaumamermis cosgrovei, a parasite of terrestrial isopods. Mitochondrial DNA length variants ranging from 19 to 34 kb are observed, but haplotypes are not shared between any two individuals. The complete nucleotide sequences of three haplotypes have been determined, revealing a constant region encoding most mitochondrial genes and a hypervariable segment that contains intact and pseudogene copies of several mitochondrial genes, duplicated to different copy numbers, resulting in mtDNA size variation. Constant rearrangement generates new T. cosgrovei mtDNA forms.

Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.

Characterization of mitochondrial genomes from individual Thaumamermis cosgrovei nematodes, obligate parasites of the isopod Armadillidium vulgare, revealed that numerous mtDNA haplotypes, ranging in size from 19 to 34 kb, are maintained in several spatially separated isopod populations. The magnitude and frequency of conspecific mtDNA size variation is unprecedented among all studied size-polymorphic metazoan mitochondrial genomes. To understand the molecular basis of this hypervariation, complete nucleotide sequences of two T. cosgrovei mtDNA haplotypes were determined. A hypervariable segment, residing between the atp6 and rrnL genes, contributes exclusively to T. cosgrovei mtDNA size variation. Within this region, mtDNA coding genes and putative nonfunctional sequences have accumulated substitutions and are duplicated and rearranged to varying extents. Hypervariation at this level has enabled a first insight into the life history of T. cosgrovei. In five A. vulgare hosts infected with multiple nematodes, four carried nematodes with identical mtDNA haplotypes, suggesting that hosts may become infected by ingesting a recently hatched egg clutch or become parasitized by individuals from the same brood prior to dispersal of siblings within the soil.