BMC Genomics 2005, 6:136 http://www.biomedcentral.com/1471-2164/6/136 symbiosis. Naturally, this simplification in the mitochon- drial genome has taken different routes as life diverged into different kingdoms. Vertebrate mitochondrial genomes are among the most compact, gene-rich genomes, while some plant mitochondria have evolved to have a low percentage of coding region similar to that of nuclear DNA [2]. Features of the mitochondrial genomes that have persisted through the divergent evolution of eukaryotic life are likely to be due to fundamental limita- tions on the variation of that genome. In this paper we discuss three such features that are preserved across eukaryotic species. Because of the relatively small size of mitochondrial DNA, it is ideally suited for analysis by n-dimensional DNA walks. One dimensional pyrimidine-purine walks were first used to find long-range correlations in nucleotide sequences [3]. Recently multi-fractal walks of mitochon- drial DNA were used to find a nonlinear organization in the mitochondrial genome [4]. Combining this informa- tion with pyrimidine-purine walks and walks of G-C ver- sus A-T content [5] gives a better understanding of the nucleotide organization of the genome. Using these techniques we demonstrate certain features of mtDNA sequences which have been preserved by evolu- tion. Greater understanding of the evolutionary selection pressures on mtDNA will allow the construction of more accurate phylogenetic trees based upon mtDNA gene sequences [6,7] as well as a better grasp of the root causes of mitochondrial DNA mutations responsible for many human diseases. Results A pyrimidine (C and T) – purine (A and G) walk of the (+) strand of human mtDNA (commonly called the "light" strand in vertebrates) is shown in Figure 1. For each pyri- midine in the sequence a step up is taken and for each purine a step down is taken. In vertebrates, all mitochon- drial genes except ND6 and many tRNAs are encoded on the heavy strand. Therefore the mRNA species are pre- dominantly (+) light strand synonymous. The first 3-kilo- base section of the human mtDNA encodes two ribosomal RNAs and the pyrimidine-purine walk slopes downward in this region, indicating that the ribosomal RNAs are slightly purine-rich. The remainder of the genome predominately encodes mitochondrial oxidative phosphorylation proteins with small tRNAs interspersed between them. Each mitochondrial protein transcript (except ND6) is pyrimidine-rich giving the remainder of the graph an upward slope. Within this overall rise, small almost flat sections where tRNA genes are located can be seen between the protein coding regions. A particularly large group of these tRNAs is contained in the section of mtDNA around the origin of light strand replication (OL) (shown as an inset to Figure 1). The OL is dramatically clear in this plot as a large run of pyrimidines on one side of the OL followed by a long run of purines on the other side. This section is thought to form a stem-loop structure as an initiation event for light strand DNA synthesis [8,9]. However, we should note that a stem-loop structure does not require a dramatic separation of pyrimidines and purines as is seen here. The ND6 gene has a slightly upward slope in the walk indicating that the human ND6 mRNA is slightly purine-rich in contrast to the other pro- tein-coding mRNAs encoded on the opposite strand. Clear pyrimidine and purine rich genome segments can also be seen in the mtDNA from other eukaryotic species. The pyrimidine-purine walks of the mitochondrial genome of seven diverse species are shown in Figure 2. In these species many of the genes, the gene order, and the gene distribution over the two DNA strands are different. Figure 2A shows a pyrimidine-purine walk of the mito- chondrial genome of the red algae Chondrus crispus (Irish Moss). The genes are color-coded based upon whether they encode membrane proteins, soluble proteins, or rRNA and upon the strand in which they are encoded. Fig- ure 2B shows a mitochondrial genome walk of the red algae, Porphyra purpurea. The similar gene order in these two red algae species (Figures 2A and 2B) gives the walks a similar overall shape. Metazoan mitochondrial genomes such as those shown from Drosophila (Figure 2C) and sea urchin (Figure 2D) are pyrimidine rich overall (+ strands), especially in the sea urchin where all but one of the genes are encoded on the same strand. Plant, fungal, and protist genomes that are gene-poor, which encode oxidative phosphorylation proteins on both strands, and also encode soluble proteins are generally purine-rich. Small genomes (<25 kB), such as that of Schizosaccharomyces pombe (fission yeast) (Figure 2E), are pyrimidine-rich because they are gene-rich and encoded entirely on one strand. No matter which strand encodes the genes, or whether the entire mitochondrial genome is pyrimidine or purine- rich, there are highly conserved features in these walks. Places in the genome where the DNA walk went down were locations where rRNA or soluble proteins were encoded on the (-) (heavy) strand or where membrane proteins were encoded on the (+) (light) strand. Locations of membrane proteins on the (-) strand or rRNA or solu- ble proteins on the (+) strand were associated with an upward slope in the pyrimidine-purine walk. Exceptions to these rules are indicated on the figure with an asterisk. The most notable exception that we found was the mtDNA from the slime mold Dictyostelium discoideum (Figure 2G), which has a very strand-asymmetric genome, being very purine-rich on the (+) strand (60%). Unlike other species, in Dictyostelium almost all oxidative-phos- Page 2 of 12 (page number not for citation purposes)