Figure 1.

A cell-culture SVA retrotransposition assay. (A) A full-length ‘canonical' SVA in the human genome, with the individual domains in order from 5′ to 3′. (i) CCCTCT hexamer; (ii) the Alu-like domain consisting of two antisense-spliced Alu fragments and a sequence of unknown origin; (iii) VNTR; and (iv) SINE-R (env sequence and right LTR from an extinct HERV-K), terminating in a polyA tail (AAA_n), with the entire insertion flanked by a TSD (black horizontal arrows). (B) The SVA.10 mneoI construct. A ‘master' SVA locus, SVA.10, from the SVA_F1 (MAST2) subfamily containing both 5′ (5′ TR, Alu) and 3′ (Alu, 3′ TR) transductions (TR) marked with the mneoI retrotransposition cassette cloned into the pCEP-Pur plasmid backbone. (C) The rationale of the trans-complementation assay is illustrated. Only if the SVA containing the mneoI reporter undergoes a round of transcription, followed by reverse transcription and integration presumably mediated by a full-length L1 (shown), will the reading frame of the neomycin phosphotransferase reporter be restored, conferring G418 resistance (G418^R). (D) G418^R foci formation is observed in HeLa HA cells when SVA.10 mneoI is co-transfected with the highly active L1 driver construct, pcDNA.L1-RP. The mean number of clones per well ± SEM and the range of clones across the wells are displayed below. The number of wells (n) assayed for this experiment is shown. The retrotransposition frequency (mean number of clones/number of transfected cells) for SVA.10 mneoI is listed below the range.