Figure 4. TAP tagged ORF2p and RT activity detection in RNP preparation.

A. Schematic representation of the amino acid mutation positions in L1 sequence: The names of plasmids containing L1s with mutations in the ORF1p coiled-coil domain (CC-LZ), the ORF1p RNA recognition motif (RRM), and the ORF1p carboxyl-terminal (CTD) domain are indicated below the schematic. The names of plasmids containing mutations in the ORF2p endonuclease domain (EN), reverse transcriptase domain (RT) or cysteine-rich domain (C) also are shown. pADL/R is a double mutant that contains a putative leucine zipper mutation and a carboxyl-terminal domain mutation in ORF1p. pADL/C is a double mutant that contains a putative leucine zipper mutation in ORF1p and a C-domain mutation in ORF2p. The flags indicate the epitope tag present on ORF1 and ORF2. B. Detection of ORF1p and ORF2p from mutant L1 constructs: RNPs from HeLa cells transfected with a RC-L1 (pAD2TE1) or the indicated mutant L1 constructs (see Figure 4A) were analyzed by western blotting [16]. Tagged L1 proteins were detected as in Figure 3; ORF2p (top panel), ORF1p (middle panel). Ribosomal S6 protein detection was used as a loading control (bottom panel). Molecular weight markers (Invitrogen) are indicated at the left of the image. C. L1 RT activity of RNP fractions detected by LEAP: An aliquot from each of the indicated RNP preparations noted above was used to perform LEAP assays (see Figure 3) [17]. RNPs from pAD2TE1 served as a positive control. RNPs from untransfected HeLa cells or pAD135 (D₇₀₂A; RT mutant) transfected cells served as negative controls. Reactions without RNPs (No RNP/RNA) or template (No Template) also were used as negative controls. Top panel: LEAP reactions (LEAP-L1). Middle panel: L1 RT-PCR reactions conducted with M-MLV reverse transcriptase control for the presence of L1 RNA in the RNP fractions (M-MLV-L1). Bottom panel: GAPDH RT-PCR reactions conducted with M-MLV reverse transcriptase assess RNP RNA quality and serve as a RT-PCR internal control (M-MLV-GAPDH). DNA size markers (Invitrogen) are indicated at the left of the image. All constructs in panel B and C contain the mneoI retrotransposition indicator cassette.