Recombination between Two Identical Sequences within the Same Retroviral RNA Molecule

Abstract

As a consequence of being diploid viruses, members of the Retroviridae have a high recombination rate. To measure recombination between two identical sequences within the same RNA molecule per round of retroviral replication cycle, a murine leukemia virus based vector (JZ442 + 3′ Hyg) has been constructed. It carries a drug resistance gene, hyg , and a 290-bp repeat sequence of the 3′ hyg gene inserted into the 3′ untranslated region of the green fluorescent protein gene ( gfp ). Under fluorescence microscopy, Hyg r cells containing the recombinant proviruses were clear, while a green color was observed in the drug-resistant cells carrying the parental proviruses. The rate of recombination was determined by the ratio of the number of clear colonies to the total number of Hyg r colonies (green and clear colonies). The rate of recombination was found to be 62% by this method. The intermolecular recombination rate between an infectious virus bearing two copies of the 290-bp segment and a noninfectious chimeric RNA virus containing only a single copy of this sequence was also measured.