BMC Genomics 2009, 10:435 http://www.biomedcentral.com/1471-2164/10/435 FDiigsturribeu5tions of Ka/Ks for TPADDs (blue bar), and TPANDDs (red bar) Distributions of Ka/Ks for TPADDs (blue bar), and TPANDDs (red bar). Also shown are: the distribution of Ka/Ks for simulated sequences that have randomly mutated without coding-sequence selection pressures (green curve; derived as described in Methods), and the Ka/Ks distribution for orthologous pairs of known protein-coding genes from rhesus monkey and human. The Ka/Ks results suggest that these transcribed pseudo- genes are relaxing to higher Ka/Ks values, since origina- tion from their parents. But why do they not have Ka/Ks values of ~1.0? We suggest that this is chiefly because: (i) there may be some inaccuracy in modelling the expected frequency for the different possible nucleotide substitu- tions, which varies for different genomic areas (as noted in the previous section); (ii) in some cases, the transcribed pseudogenes were originally protein-coding, and became disabled subsequently in multiple lineages; (iii) some of them maintain an imprint of the original coding sequence because of selection pressure for regulation of homolo- gous genes via antisense interference (e.g., through genesis of siRNAs); (iv) selection pressures on non-synonymous codon substitution rates in protein-coding genes, may have relaxed in the pseudogenes, contributing to an apparent relative increase in Ks; (v) it is also possible that some of these sequences are currently protein-coding, and have evolved through multiple coding-sequence disable- ments, as discussed previously [4]. To examine these data more closely, we calculated whether the Ka/KsΨ-ortho values are significantly less than would be expected for mutation without coding-sequence selection pressures (using the simulational analysis described in the Methods section). Several cases with such significant values (that may indicate purifying selection typical of protein-coding sequences), are observed (repre- sented by circles in the Figure 6 plots). These Ka/Ks values (that apparently indicate protein-coding ability) may arise for the reasons listed in the preceding paragraph. In addition, we examined whether the TPAs contain a pro- tein domain of known three-dimensional structure, that is disabled by a frameshift or a premature stop codon (denoted 'TPADDs'; see Methods for details of annotation of such domains). The TPADDs are indicated by unfilled symbols in parts (a) and (b) of Figure 6. Interestingly, in the human-dog comparisons, there are three cases of TPA orthologous pairs that have such a disabled protein domain, despite Ka/Ks values that indicate apparent puri- Page 8 of 14 (page number not for citation purposes)