Recombination between two identical sequences within the same retroviral RNA molecule

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 const ructed. It carries a drug resistance gene, hyg, and a 290-bp repeat sequenc e 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 co lonies to the total number of Hyg(r) colonies (green and clear colonies). T he rate of recombination was found to be 62% by this method. The intermolec ular recombination rate between an infectious virus bearing two copies of t he 290-bp segment and a noninfectious chimeric RNA virus containing only a single copy of this sequence was also measured.