BMC Genomics 2005, 6:136 http://www.biomedcentral.com/1471-2164/6/136 The relative contribution of C versus T, and A versus G throughout the transcripts can be seen in the 2-dimen- sional walks of the genes using A-G on one axis and C-T on the other (additional file 1: Figure S2). The percentage of C vs. T has been shown to vary greatly in different mammalian lineages [7]. The greater abundance of C over T on the mitochondrial light (+) strand first appears evolutionarily in reptiles and is accompanied by a slightly more G-C rich mitochondrial genome (37 % in Xenopus compared to 44 % in humans (additional file 1: Table S1)). The development of GC-rich isochores also first occurred in the nuclear DNA of reptiles and may be one of the factors allowing the evolution of warm-blooded birds and mammals [19]. Based on the data presented here, some of the same selective pressures may be affecting both the nuclear and mitochondrial genomes. Mitochondrial-encoded soluble protein transcripts are purine-rich It has been suggested that purine-loading of transcripts may have evolved to prevent detrimental RNA-RNA inter- actions [20]. However this hypothesis does not explain the codon-specific pattern of purine-richness in mito- chondrial soluble protein-coding transcripts. An A in the second codon position of nuclear-encoded transcripts often encodes relatively hydrophilic amino acids [12]. These amino acids have been shown to be abundant in the aperiodic secondary structure of soluble proteins. However, in the mitochondrial genome, purines (see Fig- ure 3C), specifically A, in the first codon position (not the second) mainly drives the purine-richness of soluble pro- teins (see Table 2). To the best of our knowledge, purine abundance in the first codon position has not previously been associated with the hydrophilic nature of soluble proteins, even though this signature does occur in the vast majority of nuclear-encoded transcripts [21,22]. One hypothesis that could be tested is that ribosomes translate more efficiently when purines are present at the first codon position. Additionally, increased levels of specific tRNAs in the mitochondrial matrix may select for such a trend. However A in the second codon position also con- tributes to the signal. A decrease in T nucleotides accom- panies the increase in A nucleotides in both positions. The result of this A for T substitution in the first two codon positions is the greater abundance of the hydrophilic amino acids lysine and asparagine (codon AAX) in soluble mitochondrial proteins and the decreased abundance of the hydrophobic amino acids phenyla- lanine and leucine (codons UUX and CUX). In fact much of the purine-rich signal in the soluble proteins is due to the 3–4 fold increase in positively charged lysine residues in these proteins compared to membrane proteins (data not shown). Mitochondrial ribosomal proteins use these residues to bind the negatively charged phosphate back- bone of ribosomal RNA [23,24]. Mitochondrial ribosomal RNA is purine-rich The selective pressure that maintains the slight purine richness of mitochondrial ribosomal RNA is not entirely clear [25,26]. It is known that ribosomal RNA interacts with ribosomal protein through hydrophobic interactions of unpaired A residues in the RNA loop regions with hydrophobic protein side chains [27,28]. Purine nucle- otides are more hydrophobic than pyrimidine nucleotides [13,29]. Therefore this slight purine abundance in the loop regions may be conserved to facilitate this interac- tion. Mitochondrial introns are also purine-rich (addi- tional file 1: Figure S3), likely conserving a hydrophobic interaction between splicing proteins and the loop struc- tures in the RNA. It is difficult to hypothesize how such small magnitutudes of purine and pyrimidine base skew can be conserved over the billion years of mitochondrial evolution. Skewed ribonucleoside triphosphate pools (highest in ATP) [30] may select for a high level of A (purine) in ribosomal RNA and soluble protein transcripts while the need for hydro- phobicity in membrane proteins may overcome this pres- sure, resulting in pyrimidine-rich transcripts. The selective pressure to contain charged hydrophilic amino acids in soluble proteins may also contribute to the maintenance of the purine-rich signal in soluble protein transcripts as well as the abundance of hydrophobic A residues in the loop regions of ribosomal RNA. A better understanding of these mitochondrial selection pressures may be gained in the future by comparing the pyrimidine-purine transcript asymmetries with that of non-coding mtDNA. Methods Mitochondrial gene sequences, amino acid sequences and genomes were downloaded from the NCBI website. Java (JDK 1.50) programs were written to analyze the sequences. The programs or software details are available from the authors upon request. Other websites such as the OGRe database of mitochondrial genomes [31] also allow analysis and graphing of base composition at the codon positions as well. The calculations were performed on a 2.8 GHz desktop computer and typically took less than a few seconds to run. The gene sequences analyzed are the mRNA synonymous sequences as available in PubMed. The mitochondrial genome strand labels (+) and (-) fol- low PubMed convention. Authors' contributions PB generated the data, constructed Figures 3, 4, 5, 6 and drafted the manuscript. AR wrote the majority of the computer code for sequence analysis, generated the data for and constructed Figures 1 and 2. DC designed and supervised the study. Page 11 of 12 (page number not for citation purposes)