When nucleotide (G, C, T and A) contents were plotted against each nucleotide, their relationships were clearly expressed by a linear formula, y = alphax + beta in the coding and non-coding regions. This linear relationship was obtained from the complete single-stranded DNA. Similarly, nucleotide contents at all three codon positions were expressed by linear regression lines based on the content of each nucleotide. In addition, 64 codon usages were also expressed by linear formulas against nucleotide content. Thus, the nucleotide content not only in coding sequence but also in non-coding sequence can be expressed by a linear formula, y = alphax + beta, in 145 organisms (112 bacteria, 15 archaea and 18 eukaryotes). Based on these results, the ratio of C/T, G/T, C/A or G/A one can essentially estimate all four nucleotide contents in the complete single-stranded DNA, and the determination of any ratio of two kinds of nucleotides can essentially estimate four nucleotide contents, nucleotide contents at the three different codon positions and codon distributions at 64 codons in the coding region. The maximum and minimum values of G content were approximately 0.35 and approximately 0.15, respectively, among various organisms examined. Codon evolution occurs according to linear formulas between these two values.

Codon evolution is influenced by an organelle-specific bias existing within cellular compartments. The discovery of evidence that codon evolution is controlled by different factors among nuclei, mitochondria and chloroplasts, indicates the existence of biases based on what we term "organelle biases". In plant chloroplasts and mitochondria, nucleotide substitutions, based on the correlations among nucleotides, were governed by the linear formula, y = ax + b, where y and x represent nucleotide contents, and a and b are constants. In animal mitochondria, with respect to the correlations between each nucleotide, only the correlations between purines (A versus G) or pyrimidines (C versus T) were almost linear, while the other correlations between purine and pyrimidine (A or G versus T or C) were not. However, nucleotide substitutions, based on the correlations of the same nucleotide between complete single-strand DNA and its coding or non-coding region, were always linear, y = ax + b, in any organelle. Linear correlations of the same nucleotide were also obtained between coding and non-coding region DNAs. The present results clearly reveal evidence that genome evolution is governed by universal linear formulas, although different rules are apparently observed in the different organelle fields formed by nuclei, mitochondria and chloroplasts. This report quantitatively demonstrates the existence of factors controlling genome evolution that are expressed by linear formulas.

Summary. High performance liquid chromatography was used to analyze the amino acid composition of cells. A total of 17 amino acids was analyzed. This method was used to compare the amino acid compositions of the following combinations: primary culture and established cells, normal and transformed cells, mammalian and bacterial cells, and Escherichia coli and Staphylococcus aureus. The amino acid compositions of mammalian cells were similar, but the amino acid compositions of Escherichia coli and Staphylococcus aureus differed not only from mammalian cells, but also from each other. It was concluded that amino acid composition is almost independent of cell establishment and cell transformation, and that the amino acid compositions of mammalian and bacterial cells differ. Thus, it is likely that changes in amino acid composition due to cell transformation or species differences between mammalian cells are negligible compared with the differences between mammalian and bacterial cells, which are more distantly related.

Cluster analyses using the amino acid content predicted from the coding regions (13 genes) of complete vertebrate mitochondrial genomes as traits grouped selected vertebrates into two clusters, terrestrial and aquatic vertebrates. Exceptions were the hagfish (Eptatretus burgeri), thought to be an early ancestor of vertebrates, and the black spotted frog (Rana nigromaculata), which is terrestrial as an adult and aquatic as a larva. These two species fall into the terrestrial and aquatic clusters, respectively. Using the nucleotide (G, C, T and A) content in the coding and non-coding regions, and in the complete genome as traits, similar results were obtained but with some additional exceptions. In addition, phylogenetic analyses of 16S rRNA sequences produced a consistent result. The results of this study indicated that vertebrate evolution is controlled by natural selection under both an internal bias as a result of nucleotide replacement genomic rules, and an external bias caused by environmental biospheric conditions.