Most retroviral recombinations occur during minus-strand DNA synthesis

Retroviral RNA molecules are plus, or sense in polarity, equivalent to mRNA , During reverse transcription, the first strand of the DNA molecule synthe sized is minus-strand DNA. After the minus strand is polymerized, the plus- strand DNA is synthesized using the minus-strand DNA as the template. In th is study, a helper cell line that contains two proviruses with two differen t mutated gfp genes was constructed. Recombination between the two frameshi ft mutant genes resulted in a functional gfp. If recombination occurs durin g minus-strand DNA synthesis, the plus-strand DNA will also contain the fun ctional sequence. After the cell divides, all of its offspring will be gree n. However, if recombination occurs during plus-strand DNA synthesis, then only the plus-strand DNA will contain the wild-type gfp sequence and the mi nus-strand DNA will still carry the frameshift mutation. The double-strande d DNA containing this mismatch was subsequently integrated into the host ch romosomal DNA of D17 cells, which were unable to repair the majority of mis matches within the retroviral double-strand DNA, After the cell divided, on e daughter cell contained the wild-type gp, sequence and the other daughter cell contained the frameshift mutation in the gfp sequence. Under fluoresc ence microscopy, half the cells in the offspring were green and the other h alf of the cells were colorless or clear. Thus, we demonstrated that more t han 98%, if not all, retroviral recombinations occurred during minus-strand DNA synthesis.