The structure and function of transfer RNA (tRNA) genes have been extensively studied for several decades, yet the general mechanisms controlling tRNA gene family evolution remain unclear, primarily because previous phylogenetics-based methods fail to distinguish between paralogs and orthologs that are highly similar in sequence. We have developed a system for identifying orthologs of tRNAs using flanking sequences to identify regions of conserved synteny and used it to annotate sets of orthologous tRNA genes across the 12 sequenced species of Drosophila. These data have allowed us to place the gains and losses of individual tRNA genes on each branch of the Drosophila tree and estimate rates of tRNA gene turnover. Our results show extensive rearrangement of the Drosophila tRNA gene complement over the last 60 My. We estimate a combined average rate of 2.18 ± 0.10 tRNA gene gains and losses per million years across the Drosophila lineage. We have identified 192 tRNAs that are ancestral to the genus, of which 157 are “core” tRNAs conserved in at least 11 of 12 extant species. We provide evidence that the core set of tRNA genes encode a nearly complete set of anticodons and have different properties from other “peripheral” tRNA genes, such as preferential location outside large tRNA clusters and higher sequence conservation. We also demonstrate that tRNA isoacceptor and alloacceptor changes by anticodon shifts have occurred several times in Drosophila, annotating 16 such events in functional tRNAs during the evolution of the genus.

Mitochondrial pseudogenes in nuclear chromosomes (numts) have been detected in the genomes of a diverse range of eukaryotic species. However, the numt content of different genomes and their properties is not uniform, and study of these differences provides insight into the mechanisms and dynamics of genome evolution in different organisms. In the genus Drosophila, numts have previously only been identified on a genome-wide scale in the melanogaster subgroup. The present study extends the identification to 11 species of the Drosophila genus. We identify a total of 302 numts and show that the numt complement is highly variable in Drosophilids, ranging from just 4 in D. melanogaster to 67 in D. willistoni, broadly correlating with genome size. Many numts have undergone large-scale rearrangements in the nucleus, including interruptions, inversions, deletions and duplications of sequence of variable size. Estimating the age of the numts in the nucleus by phylogenetic tree reconstruction reveals the vast majority of numts to be recent gains, 90% having arisen on terminal branches of the species tree. By identifying paralogs and counting duplications among the extant numts we estimate that 23% of extant numts arose through post-insertion duplications. We estimate genus average rates of insertion of 0.75 per million years, and a duplication rate of 0.010 duplications per numt per million years.

Pathogenic deviations (PDs) in humans are disease-causing missense mutations. However, in some cases, these disease-associated residues occur as the wild-type residues in functionally equivalent proteins in other species and these cases are termed 'Compensated Pathogenic Deviations' (CPDs). The lack of pathogenicity in a non-human protein is presumed to be explained in most cases by the presence of compensatory mutations, most commonly within the same protein. Identifying structural features of CPDs, and detecting specific compensatory events, will help us to understand traversal along fitness landscape valleys in protein evolution.We divided mutations listed in the OMIM database into PD and CPD datasets and performed two independent analyses: (i) we searched for potential compensatory mutations spatially close to the CPDs and (ii) using our SAAPdb database, we examined likely structural effects to try to explain why mutations are pathogenic, comparing PDs and CPDs. Our datasets were obtained from a set of 245 human proteins of known structure and contained a total of 2328 mutations of which 453 (from 85 structures) were seen to be compensated in at least one functionally equivalent protein in another (non-human) species.Structural analysis results confirm previous findings that CPDs are, on average, 'milder' in their likely structural effects than uncompensated PDs and tend to be on the protein surface. We also showed that the residues surrounding the CPD residue in the folded protein are more often mutated than the residues surrounding an uncompensated mutation, supporting the hypothesis that compensation is largely a result of structurally local mutations.

Embedded in the sequence of each transfer RNA are elements that promote specific interactions with its cognate aminoacyl tRNAsynthetase. Although many such "identity elements" are known, their detection is difficult since they rely on unique structural signatures and the combinatorial action of multiple elements spread throughout the tRNA molecule. Since the anticodon is often a major identity determinant itself, it is possible to switch between certain tRNA functional types by means of anticodon substitutions. This has been shown to have occurred during the evolution of some genomes; however, the scale and relevance of "anticodon shifts" to the evolution of the tRNA multigene family is unclear. Using a synteny-conservation-based method, we detected tRNA anticodon shifts in groups of closely related species: five primates, 12 Drosophila, six nematodes, 11 Saccharomycetes, and 61 Enterobacteriaceae. We found a total of 75 anticodon shifts: 31 involving switches of identity (alloacceptor shifts) and 44 between isoacceptors that code for the same amino acid (isoacceptor shifts). The relative numbers of shifts in each taxa suggest that tRNA gene redundancy is likely the driving factor, with greater constraint on changes of identity. Sites that frequently covary with alloacceptor shifts are located at the extreme ends of the molecule, in common with most known identity determinants. Isoacceptor shifts are associated with changes in the midsections of the tRNA sequence. However, the mutation patterns of anticodon shifts involving the same identities are often dissimilar, suggesting that alternate sets of mutation may achieve the same functional compensation.