Determination of the frequency of retroviral recombination between two identical sequences within a provirus.

Abstract

Retroviruses use RNA as their genetic material within viral particles and DNA (provirus) as their genetic material within cells. The rate of recombination during reverse transcription between two identical sequences within the same RNA molecule is very high. In this study, we have developed a sensitive system to study recombination occurring within the proviral sequence. This system includes a murine Moloney leukemia virus vector which contains a neomycin resistance gene (neo) and two mutated green fluorescent protein genes (gfp) in tandem positions. The 3' end of the first gfp and the 5' end of the second gfp gene are both mutated, so that neither of these two gfp genes is functional. However, if recombination occurs between the two gfp genes it will create a functional gfp protein. Cells containing such a functional recombinant gfp appear green under fluorescence microscopy. The rate of recombination between the two gfp sequences during a single round of replication is as high as 51%. Green cells appear during proliferation of a clonal clear-cell population and allow a small portion of these recombinations between sequences of proviral DNA to be detected. The frequency of recombination at the proviral DNA level is about 10(-5) events/cell division, which is very low compared with the frequency of recombination (51%) caused by reverse transcriptase and/or RNA polymerase II.