Figure 1. Initiation of L1 reverse transcription by native L1 RNPs.

(A) Outline of the experimental procedure. LEAP, L1 element amplification protocol; DLEA, Direct L1 Extension Assay (B) Immunoblotting of human ORF1p (top 2 panels) or murine ORF1p (panel 3 from the top) in RNPs (16 µg) prepared from cells transfected with empty vector (lane 1), RT* hL1 (lane 2), wild-type hL1 (lane 3), RT* mL1 (lane 4), wild-type mL1 (lane 5). Ribosomal S6 protein was detected using an anti-S6 antibody and was used as an RNP loading control (bottom panel). (C) Detection of L1 RT activity by LEAP (top panel) and of L1 RNA by conventional RT-PCR (middle panel) in RNP preparations. GAPDH RNA is a cellular RNA used as a loading control for all RNPs (bottom panel). Annotations are the same as in (B). ct1, a control for the PCR step without cDNA; ct2, a control for the RT step without RNP or RNP-extracted RNA. The LEAP product is a diffuse smear starting from 207 bp (bracket). (D) Standard curve of murine (black square) or human (black circles) L1 RNP DNA polymerase activity, showing linear conditions, compared to vector control RNP (empty circles). Note that the intrinsic activities of mL1 and hL1 RNPs cannot be directly compared due to potential differences in their levels of expression. (E) Direct L1 extension assay (DLEA) with or without a (dT)₁₈ primer in the presence of α-³²P-dTTP (even and odd lanes, respectively). Sucrose cushion fractions prepared from human (lanes 5–8) or murine (lanes 9–12) L1-transfected cells or vector-transfected cells prepared in parallel (lanes 3–4) were used as a source of RNPs. Trace amounts of a 14-nt 5′ end-labeled oligonucleotide was added after the reaction as a recovery control (denoted RC). RT*, RT-defective L1 RNP; WT, wild-type L1 RNP.