Figure 1.

Potentially important RUNX sites in the 5′UTR for L1Hs retrotransposition. (A) Construction of backbones for random mutagenesis of L1_RP 5′UTR. L1Hs contains 5′ and 3′UTRs and two ORFs. Unique PacI and NaeI sites were created at +465 to +472 and +869 to +874 in the L1_RP 5′UTR, respectively. An EGFP cassette to detect L1Hs retrotransposition is inserted within the 3′UTR in the antisense direction and consists of the CMV immediate early promoter (pCMV), the EGFP gene disrupted by a sense direction oriented γ-globin intron (intron) and the TK poly(A) signal (pA). (B) The created PacI (left) and NaeI (right) sites have no effect on retrotransposition. Retrotransposition activities of the random mutagenesis backbones for the 5′UTR, i.e. L1_RP-PacI and L1_RP-PacI–NaeI (empty triangle), the wild-type L1_RP (solid triangle), a negative control of L1_RP(JM111) (solid circle) and a positive control of L1.3 (empty circle) were tested by FACS on days 4, 6 and 8 post-transfection (n = 6). The original sequence and created mutations are shown in the upper left region of the corresponding retrotransposition activity curve. (C) Two single-nucleotide mutations, 100T/C and 84G/A, identified by random mutagenesis at a potential RUNX site and their retrotransposition activities in 143B cells (n = 6). One representative FACS result of each sample is shown. (D) Three potential RUNX sites in the L1_RP 5′UTR. Each site consists of 19 nt, and the consensus core RUNX sequences are shown (boxed). The first two RUNX sites are on the sense strand, and the third on the antisense strand.