The Alu family is a highly successful group of non-LTR retrotransposons ubiquitously found in primate genomes. Similar to the L1 retrotransposon family, Alu elements integrate primarily through an endonuclease-dependent mechanism termed target site-primed reverse transcription (TPRT). Recent studies have suggested that, in addition to TPRT, L1 elements occasionally utilize an alternative endonuclease-independent pathway for genomic integration. To determine whether an analogous mechanism exists for Alu elements, we have analyzed three publicly available primate genomes (human, chimpanzee and rhesus macaque) for endonuclease-independent recently integrated or lineage specific Alu insertions. We recovered twenty-three examples of such insertions and show that these insertions are recognizably different from classical TPRT-mediated Alu element integration. We suggest a role for this process in DNA double-strand break repair and present evidence to suggest its association with intra-chromosomal translocations, in-vitro RNA recombination (IVRR), and synthesis-dependent strand annealing (SDSA).

Gibbons (Hylobatidae) are small, arboreal apes indigenous to Southeast Asia that diverged from other apes 15-18 Ma. Extant lineages radiated rapidly 6-10 Ma and are organized into four genera (Hylobates, Hoolock, Symphalangus, and Nomascus) consisting of 12-19 species. The use of short interspersed elements (SINEs) as phylogenetic markers has seen recent popularity due to several desirable characteristics: the ancestral state of a locus is known to be the absence of an element, rare potentially homoplasious events are relatively easy to resolve, and samples can be quickly and inexpensively genotyped. During radiation of primates, one particular family of SINEs, the Alu family, has proliferated in primate genomes. Nomascus leucogenys (northern white-cheeked gibbon) sequences were analyzed for repetitive content with RepeatMasker using a custom library. The sequences containing Alu elements identified as members of a gibbon-specific subfamily were then compared with orthologous positions in other primate genomes. A primate phylogenetic panel consisting of 18 primate species, including 13 gibbon species representing all four extant genera, was assayed for all loci, and a total of 125 gibbon-specific Alu insertions were identified. The resulting amplification patterns were used to generate a phylogenetic tree. We demonstrate significant support for Symphalangus as the most basal lineage within the family. Our findings also place Nomascus as a derived lineage, sister to Hoolock, with the NomascusHoolock clade sister to Hylobates. Further, our analysis groups N. leucogenys and Nomascus siki as sister taxa to the exclusion of the other Nomascus species assayed. This study represents the first use of SINEs to determine the genus level phylogenetic relationships within the family Hylobatidae. These relationships have been resolved with robust support at most internal nodes, demonstrating the utility of SINE-based phylogenetic analysis. We postulate that hybridization and rapid radiation may have contributed to the complex and contradictory findings of the previous studies. Our findings will aid in the conservation of these threatened primates and inform future studies of the biogeographical history and distribution of modern gibbon species.

Retrotransposons, specifically Alu and L1 elements, have been especially successful in their expansion throughout primate genomes. While most of these elements integrate through an endonuclease-mediated process termed target primed reverse transcription, a minority integrate using alternative methods. Here we present evidence for one such mechanism, (which we term internal priming) and demonstrate that loci integrating through this mechanism are qualitatively different from “classical” insertions. Previous examples of this mechanism are limited to cell culture assays, which show that reverse transcription can initiate upstream of the 3′ polyA tail during retrotransposon integration. To detect whether this mechanism occurs in vivo as well as in cell culture, we have analyzed the human genome for internal priming events using recently integrated L1 and Alu elements. Our examination of the human genome resulted in the recovery of twenty events involving internal priming insertions, which are structurally distinct from both classical TPRT-mediated insertions and non-classical insertions. We suggest two possible mechanisms by which these internal priming loci are created and provide evidence supporting a role in staggered DNA double-strand break repair. Also, we demonstrate that the internal priming process is associated with inter-chromosomal duplications and the insertion of filler DNA.

BackgroundL1s are one of the most successful autonomous mobile elements in primate genomes. These elements comprise as much as 17% of primate genomes with the majority of insertions occurring via target primed reverse transcription (TPRT). Twin priming, a variant of TPRT, can result in unusual DNA sequence architecture. These insertions appear to be inverted, truncated L1s flanked by target site duplications.ResultsWe report on loci with sequence architecture consistent with variants of the twin priming mechanism and introduce dual priming, a mechanism that could generate similar sequence characteristics. These insertions take the form of truncated L1s with hallmarks of classical TPRT insertions but having a poly(T) simple repeat at the 5' end of the insertion. We identified loci using computational analyses of the human, chimpanzee, orangutan, rhesus macaque and marmoset genomes. Insertion site characteristics for all putative loci were experimentally verified.ConclusionsThe 39 loci that passed our computational and experimental screens probably represent inversion-deletion events which resulted in a 5' inverted poly(A) tail. Based on our observations of these loci and their local sequence properties, we conclude that they most probably represent twin priming events with unusually short non-inverted portions. We postulate that dual priming could, theoretically, produce the same patterns. The resulting homopolymeric stretches associated with these insertion events may promote genomic instability and create potential target sites for future retrotransposition events.