Origin of Split Gene Structure Figure 5. Specific generation of non-conforming ORFs due to exon splicing. (A) Human genes with a complete coding sequence (spliced- exons) .2000 bases and a gene sequence devoid of ORFs .750 bases were selected. The ORF and ARF lengths were computed in all three RFs of each of these genes, and their combined frequencies were plotted. The frequencies of the lengths of exons from this set of genes were also plotted. Next, the exons from each gene were spliced to form its coding sequence and the frequency distribution of ORF and ARF lengths from the spliced sequences were plotted. The X-axis was broken into two parts: from 0–749 bases and from 750–10000 bases. The Y-axis scales corresponding to 0– 749 bases are shown on the left, and those corresponding to 750–10000 bases are shown on the right. The frequencies corresponding to 0–749 bases were binned for every 6 consecutive ORF/ARF/exon lengths and the frequencies corresponding to 750–10000 bases were binned every 100 consecutive ORF/ARF lengths. (B) Frequency distribution of ORF lengths in prokaryotic genes. All the genes from the E. coli K12 genome, each of whose coding sequence length was at least 2000 bases, were selected. The ORF lengths in all three RFs of each of these genes were computed and their combined frequencies plotted. The ORF length frequency from the spliced sequences of the .2000 base human gene set (Figure 3A) was overlaid for comparison. The methods used for line break, binning and plotting are the same as in Figure 3A. doi:10.1371/journal.pone.0003456.g005 found, the vast majority (,92%) differed from the three stop codons TAA, TAG, and TGA only by a single base. Secondly, we found that one of the stop codons (TAG) occurred at a high frequency bordering the 59 end of the exons, and only three other codons (CAG, GAG and AAG) with a single base difference from TAG occurred at this position. The probability for any one of the three stop codons to occur at a position with respect to the splice junction (the +2 position in the donor splice signal) is practically zero, (m+n)Cn6(3/64)n6(61/64)m (where n is the number of donor splice signals containing stop codons (200,091) and m is the number of donor splice signals with no stop codons (73,336) in the human genome (Text S1). It is even more improbable for stop codons to fortuitously occur on both ends of the exons as observed here. A plausible explanation is that since the exons were selected from ORFs, the stop-codons at exon ends originated from ORF ends within random DNA sequences. Perhaps, these stop codon containing regions later evolved as the splicing signals for supporting the splicing machinery. These observations made from 273,427 exons from the complete human genome are essentially consistent with our previous analysis, which was conducted with a very small number of about 1000 exons [30,31]. An interesting corollary to this is the fact that splice signal sequences for tRNA and rRNA genes that are not based on protein-coding sequences do not include stop codons [32,33] – further suggesting that the stop codons at the ends of coding exons may have arisen from the ORF ends in random DNA sequences. The fact that introns are far longer than exons may be a result of the low probability of the precise combination of splicing signals in random DNA that are required for the elimination of introns. The secondary selection of genes based on the biological usefulness of their encoded proteins could be another reason for this. Our preliminary analysis indicates that, while the split coding sequences for given proteins occur at a far higher probability compared to non- split genes in random DNA sequences, the introns in such genes are far longer than the exons, as typically observed in human genes. Discussion Random-sequence origin of eukaryotic split coding genes The initial analysis based on the random-sequence origin of protein-coding genes [27,30,31] with limited data provided evidence that the DNA of eukaryotic organisms may be highly random. Given that primordial DNA was long and contained PLoS ONE | www.plosone.org 7 October 2008 | Volume 3 | Issue 10 | e3456