Fig. 2

Human SVAs are spliced in the context of the mneoI reporter cassette. a Schematic representation of the cell-based retrotransposition assay. The element of interest is tagged with a reporter cassette containing a neomycin phosphotransferase (neo) coding region driven by the SV40 promoter and polyadenylated at an HSV TK poly A site in antisense. The neo open reading frame is interrupted by an intron in sense direction. Following transcription of the VNTR composite from the 5′ CMV promoter, the intron is spliced out and the RNA is polyadenylated at the downstream SV40 pA site. Mediated by the L1 proteins encoded on a cotransfected vector the RNA is then reverse transcribed and the cDNA copy inserted into the genome. A functional neomycin phosphotransferase can now be generated from the uninterrupted coding region – giving rise to G418 resistant (G418^R) cells once retrotransposition has occurred. SD – splice donor; SA – splice acceptor; G418^S – G418 sensitive (b) Retrotransposition assay of mneoI-tagged human (H19_27, H8_43) and orangutan (OU3, OU4) SVA elements. Retrotransposition rates +/− SEM are shown relative to H19_27 (100%). Average colony counts are given on top of each column. n ≥ 3 (c) Northern blot analysis of mneoI-tagged SVA transcripts. In case of the human SVA (H8_43) splicing between the VNTR and the mneoI cassette generates additional mature RNAs schematically depicted on the right. Lengths are given in the order of loading on the gel. d Structure of the H8_43 VNTR-neo splice variants as determined by RT-PCR. Nucleotides important for splicing are bold and underlined; intron sequence is in lowercase