Table 1. Frequency of ORFs, Exons, ARFs (between amino-acid codons GTT, ACA, and CTG) & R-ORFs (ORFs computed from a Random Sequence) of increasing lengths in different genomes. Organism (length of genome in million bases) Average no. of introns per gene 1 Frequency of Exons, ORFs, ARFs & R-ORFs (in percentages) , = 750 bases 750–1500 bases 1500–2250 bases 2250–3000 bases H. sapiens (3300) C. elegans (100) A. thaliana (120) D. melanogaster (118) P. falciparum (22) S. pombe (12.5) S. pneumoniae (2.2) E. coli (5) 7.4 6.5 5 3 1 1 0 0 97.73 99.97 99.99 99.99 97.75 99.95 99.98 99.99 94.65 99.91 99.99 100 87.08 99.88 99.99 99.99 69.01 99.87 99.91 100 67.34 99.77 99.99 100 51.32 99.54 99.99 100 43.05 99.18 99.99 100 1.63 0.02 0.002 161025 1.87 0.04 0.01 761026 4.12 0.07 0.003 0 9.58 0.09 0.003 561025 12.32 0.06 0.09 0 17.81 0.12 0.006 0 37.17 0.35 0.01 0 43.14 0.63 0.003 0 0.38 0.002 0.0002 0 0 0.24 0.004 0.002 0.91 0.015 0.0001 0 2.23 0.02 0.003 0 6.01 0.02 0.004 0 8.42 0.06 0.0002 7.88 0.08 9.58 0.13 0 0 0 0 0 0.15 0.0005 361025 0 0.06 0.002 0.0005 0 0.24 0.004 161025 0 0.64 0.006 561025 0 3.86 0.015 961025 0.0004 0 3.14 0.02 0 2.3 0.02 2.93 0.04 0 0 0 0 Origin of Split Gene Structure .3000 bases 0.12 0.0003 761026 0 0.08 0.002 0.0006 0 0.07 0.002 261025 0 0.47 0.007 0.0001 0.0007 861025 0 8.8 0.04 0 3.3 0.02 0 1.3 0.01 1.3 0.02 0 0 0 0 Exons ORFs ARFs R-ORFs Exons ORFs ARFs R-ORFs Exons ORFs ARFs R-ORFs Exons ORFs ARFs R-ORFs Exons ORFs ARFs R-ORFs Exons ORFs ARFs R-ORFs Exons ORFs ARFs R-ORFs Exons ORFs ARFs R-ORFs doi:10.1371/journal.pone.0003456.t001 medium intron-rich (Drosophila), intron-poor (Plasmodium and yeast) and intron-less (E. coli) genes. The eukaryotic genomes were also chosen to represent organisms across different phyla: Homo sapiens (vertebrata), Arabidopsis thaliana (Plantae, Magnoliophyta), Caenorhabditis elegans (nematoda), Drosophila melanogaster (Arthropo- da), Plasmodium falciparum (Apicomplexa), Schizosaccharomyces pombe and Saccharomyces cerevisiae (Ascomycota). The frequencies of ORFs ,750 bases within all the genomes followed a NED, similar to that in random sequences (Figure 3B, 3C and 3D). Furthermore, .99% of all ORFs in these genomes were ,750 bases. The human genome contained the lowest percentage of non-conforming ORFs. The order of the frequencies of ORFs .750 bases among the genomes were as follows: E. coli (0.82%).S. pneumoniae (0.46%).S. ceresiviae (0.33%).S. pombe (0.22%).P. falciparum (0.13%).D. melanogaster (0.12%).A. thaliana (0.09%).C. elegans.(0.05%).H. sapiens (0.03%) (Table 1). The ARFs in these genomes also followed a NED similar to that in random DNA. The markedly lesser fraction of ARFs .750 bases relative to ORFs in these genomes indicate that the long non-conforming ORFs in these genomes arose through a selective biological process. Non-conforming exons could have originated through intron-loss Long ORFs must be synthesized from originally random DNA via splicing of nascent exons in order to encode biological proteins. The frequencies of varying lengths of exons and ORFs are shown in Table 1 for eight genomes. The vast majority of exons (97.73%) and ORFs (99.97%) in the human genome did not exceed the length of ORFs in random DNA. Only a small minority (,2%) of exons were .750 bases, which must be contained within a subset PLoS ONE | www.plosone.org 4 October 2008 | Volume 3 | Issue 10 | e3456