To enable investigation of nematode mitochondrial DNA evolution, methodology has been developed to amplify intact nematode mitochondrial genomes in preparative yields using a rolling circle amplification strategy. Successful reactions were generated from whole cell template DNA prepared by alkaline lysis. Rolling circle amplification products can be used as templates for standard PCR reactions, restriction enzyme reactions, and direct DNA sequencing.

Eukaryotic cells are divided into many compartments, enabling spatial and temporal regulation in higher cells. Mitochondria are unique among these compartments in animal cells, as they harbor a DNA genome separate from the nuclear genome, the mitochondrial DNA (mtDNA). Defects in the replication, transcription, translation or assembly of mtDNA encoded subunits cause severe diseases in humans. Deleterious mtDNA mutations may be introduced by several routes, particularly replication error. A better understanding of the mechanism of mtDNA replication is required to generate hypotheses as to how to remedy the introduction of mtDNA mutations as a possible avenue to reduce the incidence of mitochondrial myopathies. To further understand the mechanism of mtDNA replication as well as the spectrum of mtDNA mutations present across eukaryotic species, I initiated computational and biochemical studies addressing mtDNA replication processes using the genetic nematode model, Caenorhabditis elegans. I used a computational approach to analyze transition and transversion mutations which alter the distribution of nucleotides across mtDNA strands. The development of custom scripts allowed an expansion of this work to include all metazoan mitochondrial genomes publicly available. I identified lineage-specific variation in mtDNA mutation patterning that suggested differences in the mechanism of mtDNA replication between animal phyla. Furthermore, I examined mtDNA topoisomers isolated from the nematode C. elegans. A detailed analysis by two-dimensional agarose gel electrophoresis and transmission electron microscopy revealed that the mtDNA of C. elegans is not replicated by these mechanisms, but rather rolling circle replication, a fourth mechanism novel among animals. My results indicate that generalizations regarding mtDNA replication modes cannot be derived by studying model organisms alone. Multiple mtDNA replication mechanisms have evolved to protect replicating mtDNA from a vulnerable, single-stranded state which is thought to be highly mutable.

Evolutionary relationships based on nucleotide variation within the D3 26S rDNA region were examined among a collection of seven Meloidogyne hapla isolates and seven isolates of M. arenaria, M. incognita, and M. javanica. Using D3A and D3B primers, a 350-bp region was PCR amplified front genomic DNA and double-stranded nucleotide sequence obtained. Phylogenetic analyses using three independent clustering methods all provided support for a division between the automictic M. hapla and the apomictic M. arenaria, M. incognita, and M. javanica. A nucleotide sequence character distinguishing M. hapla from the three apomictic species was a 3-bp insertion within the interior of the D3 region. The three apomictic species shared a common D3 haplotype, suggesting a recent branching. Single M. hapla individuals contained two different haplotypes, differentiated by a Sau3AI restriction site polymorphism. Isolates of M. Javanica appeared to have only one haplotype, while M. incognita and M. arenaria maintained more than one haplotype in air isolate.