the confusing presence of ZRSR1 in the human genome. Human ZRSR1 is likely to have been formed by an independent retropo- sition event of the ancestral U2af1-rs2, because it is located in a different genomic position compared to mouse U2af1-rs1 (Zhang et al. 2006; Wood et al. 2007). It is possible to date the retroposi- tion event that lead to these retrogenes being generated, by exam- ining the host gene intron for presence of a retrogene ortholog, as in Wood et al. (2007). We have extended this analysis further here using more recent sequence data from a wider variety of species. Inpp5f_v2 is derived from Tmem114a. Recently it has emerged that Tmem114a is the murine homolog of human VMA21, an essential assembly chaperone for the V-ATPase complex, which is the main mammalian proton pump (Ramachandran et al. 2009). We will henceforth refer to Tmem114a as Vma21. The parentage of Nap1l5 is less clear. Two paralogues, Nap1l2 and Nap1l3, exist on the X chromosome. Both are mono- exonic, and are likely to be retrogenes derived from one of the multiexonic genes Nap1l4 or Nap1l1. Previously, Nap1l2 was identified as the most likely parent for Nap1l5 through phyloge- netic reconstruction (Wood et al. 2007). Both Nap1l2 and Nap1l3 have been examined here as putative parents. For information on the gene families and nomenclature used in this study see Table 1. To examine the evolution of these four parent-imprinted ret- rogene families at the protein level, we have used codon-based models of evolution in a maximum likelihood framework to test for heterogeneity in selective pressures across parent and retro- genes (Yang 1997; Yang and Nielsen 2002; Zhang et al. 2005). We note that results using computational analyses alone can be mis- informative when not examined in the context of the underlying biology of the proteins concerned (Hughes 2007, 2008). Here, we have suggested biological reasons for the selection pressures pre- dicted, and treat the results as a tool for generating hypotheses on the function of the proteins in question, to be tested empirically. Positive selective pressure resulting in amino acid substitu- tions has been definitively linked to changes in protein function (Levasseur et al. 2006). Hence, prediction of positively selected amino acids gives an important insight into potential functional changes in the proteins coded for in this study. Positive selection in the retrogene lineage alone could indicate neofunctionalization. We were able to differentiate between the parent genes and retro- genes by identifying the emergence of each retrogene individually using phylogenetics. Then using site-specific, lineage-specific, and combined site and lineage-specific models of codon evolution, we examined the evolutionary rate heterogeneity of these proteins. Materials and Methods SEQUENCE RETRIEVAL AND INVESTIGATION OF RETROGENE AGE To identify retrogene orthologs from as many species as possible, the mouse retrogene was compared in a sequence similarity search 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 to the host gene intron in the species of interest using blat (Kent 2002). For the following species, this was done using the UCSC genome browser, (UCSC). Chimp (Pan troglodytes) = panTro2, orangutan (Pongo pygmaeus abelii) = ponAbe2, rhesus (Macaca mulatta) = rheMac2, marmoset (Callithrix jacchus) = calJac1, rat (Rattus norvegicus) = rn4, guinea pig (Cavia porcellus) = cav- Por3, cat (Felis catus) = felCat3, dog (Canis lupus familiaris) = canFam2, horse (Equus caballus) = equCab2, cow (Bos taurus) = bosTau4, opossum (Monodelphis domestica) = monDom5, platy- pus (Ornithorhynchus anatinus) = ornAna1, and chicken (Gallus gallus) = galGal3. For the sloth (Choloepus hoffmanni) and the lesser hedgehog tenrec (Echinops telfairi), chained alignments are not available. In the ensembl genome browser, gene scaffolds were identified containing the host gene and were then compared using blat to the mouse genome to identify a retrogene ortholog. For the elephant, Loxodonta africana, armadillo, Dasypus novemcinctus, and the opossum species, Monodelphis domesticus, the gene in question was located in the human genome, and then identified in the species of interest using the pre-existing BLASTZ alignments from the Ensembl website (Ensembl). BLASTN searches of the NCBI trace archive (NCBITraceArchive), or the dbEST database (dbEST) in the case of opossum (Trichosurus vulpecula) was used when data were not available in Ensembl. To identify parent gene orthologs, the UCSC chained align- ments and Ensembl BLASTZ alignments were used, together with comparisons using blat (Kent 2002). Homologous sequence alignments were checked for quality by eye and manually edited as necessary. See Supplementary File 1 for a full list of the nucleotide sequences used. PCR EXPRESSION STUDIES RNA was extracted from frozen C57BL-6 tissues using the Qia- gen (Crawley, UK) RNeasy Mini kit (cat. no. 74104) according to the manufacturer’s instructions. RNA was quantified on an Agi- lent (Wokingham, UK) Bioanalyzer and 5 μg was used for each cDNA synthesis reaction. Invitrogen (Paisley, UK) SuperScript First-Strand kit (cat. no. 12371-019) was used to generate cDNA, according to the manufacturer’s instructions. cDNA was diluted 1 in 4 for PCR. PCR was carried out with 1 μl diluted cDNA template, 0.5 μl 20 μM primer (Supplementary File 2), and 1.1X Abgene (Epsom, UK) ReddyMix PCR Master Mix (cat. no. ab- 0575/LD/B), using 28 PCR cycles at an annealing temperature of 60◦C. PCRs were visualized with UV light on 1% agarose gel stained with ethidium bromide. Primers used are listed in Sup- plementary File 2. To ensure primers were amplifying only the required gene (no cross-contamination), bands were sequenced using Applied Biosystems (California, USA) BigDye Terminator version 3.1 Cycle Sequencing kit. EVOLUTION MAY 2011 1 4 1 5