Two conflicting in vitro observations suggest that retroviral recombinations are temperature dependent. Ouhammouch & Brody (Nucleic Acids Research 20, 5443–5450, 1992) suggested that retroviral recombination rates should increase as temperature increases. However, Shimomaye & Salvato (Gene Analysis Techniques 6, 25–28, 1989) and Brooks et al. (Biotechniques 19, 806–812, 814–815, 1985) found that at low temperature the tightly folded structure of RNAs may hinder reverse transcription proceeding along the RNA template, which increases its chance of dissociating from the template; therefore, raising the reaction temperature was the simplest way to overcome template secondary structure and prevent premature termination of cDNA synthesis. In this report, two vectors based on murine leukaemia virus (MLV) were constructed. The first contained two mutated gfp genes in tandem positions. The upstream gfp gene encoded a mutation at its 3′ end, while the downstream gfp gene encoded a mutation at its 5′ end. The recombination that occurred between the two mutated gfp genes restored a functional gfp gene. The cells that contained the functional gfp gene were green when observed under a fluorescence microscope. The second MLV vector contained a functional gfp gene with two identical sequences flanking either end. A recombination that occurred between the two identical sequences resulted in deletion of the gfp gene. Cells containing the vector with the gfp deletion were colourless or clear when observed under the microscope. Using these two vectors, we have demonstrated that retroviral recombination is temperature dependent and the rate of recombination decreases as temperature is raised from 31 to 43 °C.
Infectious in vivo and in vitro transcripts from a full-length cDNA clone of PVY-N605, a Swiss necrotic isolate of potato virus Y.
