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 Figure 1. Timing of retroposition events within mammalian evolution. Arrows indicate the time of insertion of the retrogene indicated, given the sequence data available to date. refine the time of emergence of Mcts2 compared with informa- tion in Wood et al. (2007). U2af1-rs1 was formed from the most recent retroposition event of the four. We have examined the relevant intron of the host gene Commd1 in the kangaroo rat (Dipodomys ordii) and guinea pig (C. porcellus) and found no similarity with mouse U2af1-rs1. We also searched the deer mouse (Peromyscus maniculatus) for sequences with similarity to mouse U2af1-rs1. We discovered an incomplete sequence, but with no flanking host exons it is impossible to verify as an U2af1-rs1 ortholog. Thus at this time the U2af1-rs1 retroposition event is confined to mouse and rat, and may in future be confirmed in the deer mouse. EXPRESSION OF ALL GENES IS WIDESPREAD AND IS ROUGHLY SIMILAR WITHIN FAMILIES We were interested in whether the retrogenes might be differ- ent from their parents in terms of their expression patterns. We discovered that all the genes in question are expressed in numer- ous tissues and at many stages of embryonic development in the mouse (Fig. 2). There are specific tissues where only one member of a gene family is expressed. These are highlighted in Figure 2. Vma21 is ubiquitously expressed (although the expression in new- born kidney is difficult to see but present at low levels). This is expected as it is an essential assembly chaperone, thought to be required in all mammalian cells (Ramachandran et al. 2009). Al- though the retrogenes all have a methylated promoter in oocytes and an unmethylated promoter in sperm, this does not necessarily correlate with their expression in ovary and testis, which contain somatic tissues as well as germ cells. Indeed, transcription through the retrogene locus in oocytes could be required for methylation establishment (Chotalia et al. 2009). MAJOR CHANGES IN THE NAP1L GENE FAMILY We performed an MSA of the amino acid sequences for the Nap1l family across 11 mammalian species. We focused on the three retrogenes Nap1l5, Nap1l2, and Nap1l3. The multiexonic genes Nap1l1 and Nap1l4 sequences were also included for some species (Supplementary File 6). Nap1l5 orthologs are the youngest family members, as they lack the region of homology shared by all other family members at residue 432 to 540 in the alignment. The alignment shows that large changes have taken place since the retroposition events that produced Nap1l2, 3 and 5 orthologs. For example, Nap1l5 orthologs are truncated compared to Nap1l2 and Nap1l3, mouse Nap1l5 having 158 amino acids compared with 546 amino acids for mouse Nap1l3 and 462 for mouse Nap1l2. The Nap1l3 orthologs have a protein region composed almost en- tirely of serine residues from residue 38 to 82 of the alignment that is unique among the Nap1l family. The Nap1l gene family members have undergone major structural changes during or af- ter the duplication events that produced the gene family members, and these likely impacted protein function. EVOLUTION MAY 2011 1 4 1 7