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Background  Many protein families have undergone functional divergence after gene duplications such that current subgroups of the family carry out overlapping but distinct biological roles. For the protein families with known functional subtypes (a functional split), we developed the software,  SplitTester , to identify potential regions that are responsible for the observed distinct functional subtypes within the same protein family.    Results  Our software,  SplitTester , takes a multiple protein sequences alignment as input, generated from protein members of two subgroups with known functional divergence.  SplitTester  was designed to construct the neighbor joining tree (a split cluster) from variable-sized sliding windows across the alignment in a process called  split-clustering .  SplitTester  identifies the regions, whose split cluster is consistent with the functional split, but may be inconsistent with the phylogeny of the protein family. We hypothesize that at least some number of these identified regions, which are not following a random mutation process, are responsible for the observed functional split. To test our method, we used reverse transcriptase from a group of  Pseudoviridae  retrotransposons: to identify residues specific for diverged primer recognition. Candidate regions were then mapped onto the three dimensional structures of reverse transcriptase. The locations of these amino acids within the enzyme are consistent with their biological roles.    Conclusion   SplitTester  aims to identify specific domain sequences responsible for functional divergence of subgroups within a protein family. From the analysis of retroelements reverse transcriptase family, we successfully identified the regions splitting this family according to the primer specificity, implying their functions in the specific primer selection.

SplitTester : software to identify domains responsible for functional divergence in protein family