FIG. 6.

Hybrid LTR of ACE-GFP-At remains unchanged through multiple serial passages. A PCR-based assay designed to assess the stability of the hybrid LTRs by amplification and sequencing of the 5′ LTR from proviral DNA was utilized. Amplification of a cloned proviral copy of ACE-GFP-At as a positive control resulted in products of approximately 720 and 510 bp (left gel). As depicted schematically, sequencing demonstrated that these products corresponded to the full-length, intact At LTR and an artifactual form of the At LTR missing one of the two ARRs, respectively. ACE-GFP-At was subjected to seven cell-free serial passages through LNCaP cultures, and genomic DNA from each culture was used as the template in PCR amplification and sequencing of the LTR (right gel). Sequencing of the two resulting products from selected passages demonstrated that their sequences were identical to those of the products generated by amplification of the positive control template. Abbreviations: L, 100-bp ladder; P, amplification using a plasmid containing a cloned proviral copy of ACE-GFP-At as the template; N, amplification using DNA from uninfected cells.