FIG. 3.
L1s retrotranspose in cis. (A) Results of the retrotransposition assay. RD-L1s containing the mneoI indicator cassette were cotransfected into 2 × 105 HeLa cells with an RC-L1 lacking the cassette (JM101/L1.3 Δneo). G418r foci were fixed and stained with Giemsa for visualization. Samples cotransfected with JM101/L1.3 Δneo and representative mutants in ORF1 (JM111/L1.3), the endonuclease or RT domains of ORF2 (JM116/L1.3 or JM105/L1.3), or a double mutant (JM124/L1.3) are shown. Cells transfected with JM101/L1.3, as well as 1/10 (2 × 104) and 1/100 (2 × 103) dilutions of transfected cells are indicated as positive controls. Cells transfected with JM105/L1.3 are shown as a negative control. (B) The coexpression of RD-L1s does not inhibit RC-L1 retrotransposition. A RC-L1 containing the mneoI indicator cassette (JM101/L1.3) was cotransfected into 2 × 104 HeLa cells with RD-L1s lacking the cassette, and retrotransposition was determined as described above. An experiment using a 1:9 (RC-L1 to RD-L1) molar ratio of transfected DNAs is shown. Cells transfected with JM101/L1.3 and an empty expression vector (CEP4) yielded G418r foci at roughly the same levels as cells that were cotransfected with JM101/L1.3 and RD-L1s lacking the indicator cassette (i.e., there was less than a 20% difference between respective samples). JM105/L1.3 was used as a negative control. Notably, RD-L1s whose transcription is driven from either the CMV promoter or the CMV promoter and L1 5′ UTR are complemented to similar extents (not shown).