Recognition and prediction of biological function of proteins based on amino acid sequences is a simple method employed in so many software and operators. However, the sequence similarity does not always imply to similarity of biological function. The aim of this study was to determine the semantic similarity of gene ontology (GO) of six pluripotency factors, Oct4, Sox2, C-Myc, Klf-4, Lin28 and Nanog in six species and evaluate their conformity with their protein sequence similarity and phylogenetic distance. C-myc factor exhibited a significant correlation between phylogenetic distance and protein similarity. The other factors like Sox2, Klf-4 and Lin-28 showed the correct changes of phylogenetic distance and protein similarity, but Nanog and Oct4 factors did not display a correct correlation between two indices because, the increase of protein similarity was not followed with the decrease of phylogenetic distance. Following the study, the protein or nucleotide similarity was assumed as dependent variable and GO similarity in three categories of biological process (BP), molecular function (MF) and, cell component (CC) were expected as the independent variables. With this assumption, regression analysis was accomplished to determine the best model for protein and nucleotide similarity estimation. The protein or nucleotide similarity also displayed a significant regression with GO similarity for C-myc factor and category of BP and CC were selected to estimate protein or nucleotide similarity by model, but a significant regression was not observed for other pluripotency factors for estimation of protein or nucleotide similarity. It means that except of C-myc, GO similarity of other studied pluripotency factors didn’t reflect the protein or nucleotide similarity. It is suggested that related data for five pluripotency factors, including Oct-4, Sox2, Klf4, Lin28 and Nanog in the six studied species should be reviewed.

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