Origin of Split Gene Structure ORF length constraints in genomes The ORF length frequencies in a stretch of 100,000 bases (Figure 2) from the human genome were strikingly similar to that in random DNA. While ORF frequency distribution from the whole human genome (Figure 3A) was essentially similar to that in random DNA, the vast majority of the ORFs (99.97%) were ,750 bases, leaving only a very minute percentage of ORFs (0.027%) that exceeded the 750-base limit (Table 1). Statistical analyses on the data set comprising lengths ,750 bases indicated that the human genome sequence had essentially random characteristics (R = 0.9986). The lengths between any three of the 61 amino acid codons (amino acid reading-frames, ARFs) in the human genome followed a negative exponential distribution (NED), having far fewer ARFs that were .750 bases in length than the ORFs (Figure 3A and Table 1). This finding was replicated for many three amino acid codon combinations (other than those containing CG dinucleotides, which are deficient in eukaryotic DNA) [27,29]. Thus the presence of any long ORFs in an otherwise strikingly random human genomic sequence must be the result of some biologically driven process. We then compared the ORF length distributions in the complete genomes of seven eukaryotic organisms (Homo sapiens, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Plasmodium falciparum, Schizosaccharomyces pombe, and Saccharomyces cerevisiae) and two prokaryotic organisms (Escherichia coli and Streptococcus pneumoniae). The genomes were chosen to span a wide-range of intron density: from the highly intron-rich (human), Figure 3. Frequency distributions of ORF lengths in complete eukaryotic and prokaryotic genomes. The ORF lengths in the six RFs of the complete eukaryotic genomes H. sapiens (A), A. thaliana, D. melanogaster, and C. elegans (B), P. falciparum, S. pombe and S. cerevisia (C), and the prokaryotic genomes E. coli K12 and S. pneumoniae R6 (D) were computed and the frequency distributions were plotted. Only frequencies up to 750 bases in length were plotted. The ARF lengths were computed using the amino-acid codons GAC, ACT, CTG (A). The ORF length frequencies were also computed in a computer generated random DNA of length and base composition (See Methods) that matched the respective genomes (B, C, D). doi:10.1371/journal.pone.0003456.g003 PLoS ONE | www.plosone.org 3 October 2008 | Volume 3 | Issue 10 | e3456