Conclusions The four retrogenes examined here share a number of sequence features and properties: (1) all are retrogenes, derived from a parent gene on the X chromosome, (2) all have a maternally methylated CpG island at their promoter and are surrounded by a “host” gene, (3) all are imprinted in mouse, and in human if present. However, evolution acts upon genes at the protein level, and we have shown here that from this perspective each retrogene has followed a distinct evolutionary path. The Mcts2 gene/protein has been maintained under strong se- lective pressure just like its parent gene/protein. Selective pressure upon the Inpp5f_v2 lineage was relaxed compared with its parent lineage. The Nap1l5 lineage has undergone a major truncation of its coding sequence. The U2af1-rs1 lineage shows evidence for positive selection, which is synonymous with protein functional shift. Genome-wide modeling of evolutionary properties of retro- genes would doubtless have missed this individuality. Similarly, the expression patterns of these gene families do not follow the “classical” trend for a more widely expressed parent gene and retrogene expression is mostly confined to the testes. Our case-by-case evolutionary analysis of four imprinted retrogenes has revealed their evolutionary trajectories. This in- formation can direct further studies, particularly into the potential oncogenic properties of the Mcts2 retrogene, and the changes in protein function predicted for U2af1-rs1. ACKNOWLEDGMENTS RBM was supported by a Harris Studentship. RJO and RBM acknowl- edge the Wellcome Trust for funding. MJO’C and NBL were supported by the Irish Research Council for Science, Engineering and Technol- ogy (Embark Initiative Postgraduate Scholarship to NBL, grant number RS/2006/1016). The authors would like to thank the SFI/HEA Irish Centre for High-End Computing (ICHEC) for computational power and techni- cal support. MJO’C is funded by Science Foundation Ireland (EOB2673). LPF was supported by a BBSRC studentship. We thank R. Schulz for helpful comments on the manuscript and A. Wood for ideas and discussion. LITERATURE CITED Chapman, M. A., J. H. Leebens-Mack, and J. M. Burke. 2008. Positive se- lection and expression divergence following gene duplication in the sunflower CYCLOIDEA gene family. Mol. Biol. Evol. 25:1260–1273. Choi, J. D., L. A. Underkoffler, A. J. Wood, J. N. Collins, P. T. Williams, J. A. Golden, E. F. Schuster, Jr., K. M. Loomes, and R. J. Oakey. 2005. A novel variant of Inpp5f is imprinted in brain, and its expression is correlated with differential methylation of an internal CpG island. Mol. Cell. Biol. 25:5514–5522. Chotalia, M., S. A. Smallwood, N. Ruf, C. Dawson, D. Lucifero, M. Frontera, K. James, W. Dean, and G. Kelsey. 2009. Transcription is required for establishment of germline methylation marks at imprinted genes. Genes. Dev. 23:105–117. Clamp, M., J. Cuff, S. M. Searle, and G. J. Barton. 2004. The Jalview Java alignment editor. Bioinformatics 20:426–427. I M P R I N T E D R E T RO G E N E E VO L U T I O N Conant, G. C., and K. H. Wolfe. 2008. Turning a hobby into a job: how duplicated genes find new functions. Nat. Rev. Genet. 9:938–950. Cusack, B. P., and K. H. Wolfe. 2007. Not born equal: increased rate asymme- try in relocated and retrotransposed rodent gene duplicates. Mol. Biol. Evol. 24:679–686. dbEST. http://www.ncbi.nlm.nih.gov/dbEST/. Emerson, J. J., H. Kaessmann, E. Betran, and M. Long. 2004. Extensive gene traffic on the mammalian X chromosome. Science 303:537–540. Ensembl. http://www.ensembl.org/index.html. Esnault, C., J. Maestre, and T. Heidmann. 2000. Human LINE retrotrans- posons generate processed pseudogenes. Nat. Genet. 24:363–367. Feinberg, A. P. 2007. An epigenetic approach to cancer etiology. Cancer J. 13:70–74. Force, A., M. Lynch, F. B. Pickett, A. Amores, Y. L. Yan, and J. Postlethwait. 1999. Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151:1531–1545. Gayral, P., P. Caminade, P. Boursot, and N. Galtier. 2007. The evolutionary fate of recently duplicated retrogenes in mice. J. Evol. Biol. 20:617–626. Hsu, H. L. , B. Shi, and R. B. Gartenhaus. 2005. The Mct-1 oncogene product impairs cell cycle checkpoint control and transforms human mammary epithelial cells. Oncogene 24:4956–4964. Hsu, H. L., C. O. Choy, R. Kasiappan, H. J. Shih, J. R. Sawyer, C. L. Shu, K. L. Chu, Y. R. Chen, H. F. Hsu, and R. B. Gartenhaus. 2007. Mct-1 oncogene downregulates p53 and destabilizes genome structure in the response to DNA double-strand damage. DNA Repair (Amst) 6:1319–1332. Hughes, A. L. 2007. Looking for Darwin in all the wrong places: the misguided quest for positive selection at the nucleotide sequence level. Heredity 99:364–373. ———. 2008. The origin of adaptive phenotypes. Proc. Natl. Acad. Sci. USA 105:13193–13194. Humphrey, W., A. Dalke, and K. Schulten. 1996. VMD: visual molecular dynamics. J. Mol. Graph. 14:33–38, 27–28. Jones, D. T., W. R. Taylor, and J. M. Thornton. 1992. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8:275–282. Kaessmann, H., N. Vinckenbosch, and M. Long. 2009. RNA-based gene duplication: mechanistic and evolutionary insights. Nat. Rev. Genet. 10:19–31. Kellis, M., B. W. Birren, and E. S. Lander. 2004. Proof and evolutionary analy- sis of ancient genome duplication in the yeast Saccharomyces cerevisiae. Nature 428:617–624. Kent, W. J. 2002. BLAT–the BLAST-like alignment tool. Genome Res. 12:656–664. Kielkopf, C. L., N. A. Rodionova, M. R. Green, and S. K. Burley. 2001. A novel peptide recognition mode revealed by the X-ray structure of a core U2AF35/U2AF65 heterodimer. Cell 106:595–605. Kleene, K. C. 2001. A possible meiotic function of the peculiar patterns of gene expression in mammalian spermatogenic cells. Mech. Dev. 106:3– 23. Kondrashov, F. A., I. B. Rogozin, Y. I. Wolf, and E. V. Koonin. 2002. Selection in the evolution of gene duplications. Genome Biol. 3:RESEARCH0008. Levasseur, A., P. Gouret, L. Lesage-Meessen, M. Asther, E. Record, and P. Pontarotti. 2006. Tracking the connection between evolutionary and functional shifts using the fungal lipase/feruloyl esterase A family. BMC Evol. Biol. 6:92. Marti-Renom, M. A., A. C. Stuart, A. Fiser, R. Sanchez, F. Melo, and A. Sali. 2000. Comparative protein structure modeling of genes and genomes. Annu. Rev. Biophys. Biomol. Struct. 29:291–325. Massey, S. E., A. Churbanov, S. Rastogi, and D. A. Liberles. 2008. Charac- terizing positive and negative selection and their phylogenetic effects. Gene 418:22–26. EVOLUTION MAY 2011 1 4 2 5